Genetic and metabolic engineering approaches were implemented to increase the precursor availability required for the biosynthesis of surfactin, a lipopeptide produced by Bacillus subtilis. This study is based on the overproduction of surfactin through enhancement of intracellular pool of leucine. A metabolic network of branched chain amino acids (BCAA: isoleucine, leucine and valine) metabolism in B. subtilis was first formalized through biocomputing tools. Predictions were based on genes which are directly or indirectly involved in the regulation of the promoter (P ilv-leu ) activity of the ilv-leu operon in the final step of leucine biosynthesis or influx of pyruvate (glycolytic pathway). A markerless gene deletion strategy (PopIn-PopOut technique) was adopted to carry out multiple deletions in a single strain and avoid antibiotic limitation. Various single knockout strains were developed with the deletion of bcd, codY, lpdV and tnrA genes. Besides these single knockout genes, a double knockout strain with the deletion of codY and tnrA was developed too. Strains with the deletion of the binding region of CodY (site II) and a leucine T-box regulator were also studied. It was observed that the deletion of these genes have varied positive impact on surfactin production, quantitatively and qualitatively. The role of extracellular metabolites in determining the production of surfactin isomers was analyzed. Genes (pgcA, pgi, pfkA and pykA) which encode various enzymes in the glycolytic pathway were also deleted, to study the role of pyruvate metabolic flux in the surfactin production. Gene yvbW, a potential candidate for leucine with a hypothetical function as leucine transporter was also studied for the effect on surfactin production. Moreover, the surfactin production of all these mutants was analyzed in different environmental conditions (medium composition and aeration). Depending on the environmental conditions and the gene knockout, the surfactin production was enhanced with a factor from 2 to 21. This work establishes that the precursor limitation problem of lipopeptide biosynthesis can be overcome by using this integrative approach.

The journey towards a PhD is by itself an adventure. You keep falling but always to strike back stronger. This strength comes from the wonderful people that were in many ways an integral part of my journey. Of course, this started with the guidance, support and constant encouragement of my supervisor and mentor, the director of my thesis, Prof. Philippe Jacques. His motivations and insightful guidance are one of the main reasons leading to the successful completion of the work. I would like to express my heartfelt gratitude for believing in me, which was one of the main factors, which always kept me going.

My other driving force was Dr. François Coutte, my co-supervisor, whom I am immensely grateful to for his unconditional support throughout the three years. He was always available for insightful discussions whenever I needed his presence. His dynamic attitude towards work has kept inspiring me even during my failures and motivated me to thrive further. I would like to thank him for helping me right from the day I arrived in this Matthieu Jules for accepting to be the jury members for my defense.

I would also like to thank the entire staff at ProBioGEM, for providing me with a friendly environment to work in. I cherish the valuable time they spent with me. Especially I would like to thank Dr. Max Béchet for helping me in the molecular biology experiment in the early days. I am really grateful to Corine for helping me every time I needed her help for the experiments. I am thankful to Cathy for clearing my bills every time I travelled. I really appreciated all of their help. I have learnt from them that if you have the eagerness to help, language cannot be a barrier.

Besides the staff, I am thankful to Juliette, Remi, Antoine, Luiz, Qassim, Oumayma, Mickael, Kalim, Sandy, Delphine, Alaa, Ameen and all other members in C124 for their time, each time I needed them. Alongside my lab members in Lille, I am grateful to all the fellows (Laura, Transi, Jad, Nora, Paola, Joana, Nicolas, Luminita and Maria del Carmen) in AMBER project for their discussion on facebook and whenever we met. This journey wouldn't have been the same without the help, support and company of my dearest friends Narinder, Mayank, Mohit, Samadhan, Pushpendra and Neetu for being amazing friends and keeping company with me in everything. I really enjoyed the friendly chats with my coffee partners Yazeen and Thibault as well as for their help in experiments and many academic related matters. Enjoyment in Lille would not have been the same without the party couple Koyal and François; I really had some memorable moments with them.

Joana

I would like to express my deepest love and gratitude to Ma and Papa for being there and bearing me in all times. Their shower of love and affection helped to maintain positivity in my life during this period. You are the reason for whatever little things I have achieved in life. I dedicate my thesis to the both of you. Shreya has been the closest person to me in my life besides my parents. She has been a source of encouragement, knowledge and intellect. I am really thankful to have her as my life partner. She helped me a lot during this entire journey. I am really grateful to her for the constant support and especially bearing with my temper all the time. I am thankful to European Union and Marie Curie ITN AMBER (317338) for funding this project.

Lastly, I am thankful to Lille, it is my home away from home.

RESUME

Dans ces travaux des approches de génie génétique et métabolique ont été mises en oeuvre pour augmenter la fourniture des précurseurs nécessaire à la biosynthèse d'un lipopeptide produit par Bacillus subtilis. Cette étude a porté plus particulièrement sur la surproduction de la surfactine grâce à l'augmentation du pool intracellulaire de leucine. Le réseau métabolique du métabolisme des acides aminés branchés (BCAA: isoleucine, leucine et valine) chez B. subtilis a d'abord été formalisé et modélisé par des outils bio-informatique.

Puis, grâce à l'utilisation de la programmation par contrainte et à l'interprétation abstraite des interruptions de gènes ont été prédites dans ce réseau. Les prédictions se sont basées sur des gènes qui sont directement ou indirectement impliqués dans la régulation de l'activité du promoteur de l'opéron ilv-leu (P ilv-leu ), dans les voies de dégradations des BCAA, ainsi que dans le métabolisme central du carbone. Une stratégie de suppression de gènes (knockout) sans l'utilisation de marqueur antibiotique (technique du PopIn-PopOut) a été entreprise pour mener à bien ces multiples délétions dans une même souche et en évitant ainsi une limitation par les antibiotiques. Différent mutants contenant de simple knockout des gènes bcd, codY, lpdV, tnrA, ainsi que du site de fixation de CodY sur P ilv-leu et de son atténuateur à leucine (T-box) ont été construits. Un mutant contenant le double knockout des gènes codY et tnrA a aussi été développé. Les résultats obtenus suite à la délétion de ces gènes ont montré un impact positif sur la production du lipopeptide tant d'un point de vue quantitatif que qualitatif. Les gènes (pgcA, pgi, pfkA et pykA) qui codent pour diverses enzymes de la glycolyse ont également été supprimés afin de confirmer le rôle du flux métabolique en pyruvate dans la production de la surfactine. Par ailleurs, la délétion du gène yvbW, qui est responsable du transport extracellulaire de la leucine a également montré un impact significatif sur la production de la surfactine. Enfin, la production de surfactine de tous ces mutants a été analysée dans différentes conditions environnementales (composition du milieu de culture et aération). En fonction des mutants et du milieu de culture utilisés la production surfactine a été améliorée d'un facteur de 2 à 21. Ce travail a établi que la limitation de la biosynthèse de la surfactine par la fourniture en précurseur peut être surmontée en utilisant cette approche intégrée.

Figure 2 Overview of regulatory pathways affecting competence development as well as positive and negative regulation of comK and related genes are indicated by arrows and Tbars [START_REF] Hamoen | Controlling competence in Bacillus subtilis: shared use of regulators[END_REF] (Mootz et al., 2002) ............ 19 Figure Assembly and mode of synthesis of iterative NRPSs (Mootz et al., 2002) ........ 20 Figure Assembly and mode of synthesis of nonlinear NRPSs ( Mootz et al., 2002) ..... 21 Figure Role of glycolytic pathway and production of various metabolites as a result of this pathway [START_REF] Tojo | Heavy involvement of stringent transcription control depending on the adenine or guanine species of the transcription initiation site in glucose and pyruvate metabolism in Bacillus subtilis[END_REF] Figure 45 Specific surfactin yield obtained with BBG258 and its derivative strains in Landy medium with phosphate buffer at 37 0 C in shake flask condition with 250 rpm. Results are expressed as mean value with standard deviation and statistical analysis of these data was made using one-way ANOVA with Tukey's multiple comparisons test .. 112

LIST OF TABLES

Table 1 An overview of enzymes and actives molecules along with their corresponding Table 2 Substrate that enhance the synthesis of specific fatty acids in B. subtilis [START_REF] Kaneda | Fatty acids of the genus Bacillus: an example of branched-chain preference[END_REF]

GENERAL INTRODUCTION

Motivation Bacillus subtilis is a soil bacterium which serves as a model Gram-positive microorganism. This microorganism is considered as "micro-factory" for the production of wide range of industrially beneficial products. The strain B. subtilis 168 has been widely studied as well as the entire genome was published in 1997. Depending on the strains, B. subtilis can produce various lipopeptides. These peptides are extensively studied for various applications from medicinal to environmental. These biologically synthesized peptides have the potential to replace their chemical counterparts. But the main limitation of its application is it's low-level of production, which currently limits their industrial application. Various studies have been carried out to enhance the production of lipopeptides, such as genetic engineering of the strains or bioprocess development for their production and purification. Previous work carried-out in the Research Institute for Food and Biotechnology -Charles Viollette from the University of Lille, Sciences and Technologies, revealed that there might be lack of precursor molecules for their mechanism of biosynthesis, which can have a role in improving the production further.

Thus, an intensive and multi-disciplinary approach was required to increase the production of lipopeptides through the enhancement of its precursor concentration. This project was a part of multidisciplinary European project AMBER.

Objective

The main objectives of this work were:

1. To analyze the precursor metabolism and define a strategy of gene knockout using computational modelling to increase their intracellular concentration.

2. To optimize the precursor biosynthesis of lipopeptide (surfactin) to increase its production in B. subtilis.

3.

To analyze the impact of environmental conditions on the surfactin production in the different genetic engineered mutant strains of B. subtilis

BACILLUS SUBTILIS: IN A NUTSHELL

Bacillus subtilis and its genome

Bacillus subtilis is the best-characterized Gram-positive bacteria. It's a rod shaped, spore forming and an aerobic bacterium which is profoundly found in the upper layers of the soil and in association with plants. Under aerobic conditions, it can differentiate into heatresistant spores. In addition, it can utilize various carbon sources which include many plant-derived molecules. They are widely used for the synthesis of broad range of agricultural, pharmaceutical and chemical compounds, which includes amino acids, antibiotics and biosurfactants. B. subtilis has a simple and competent gene transfer system which has led to the construction of genetic map of its genome.

In 1997, Kunst et al. published the entire genome of B. subtilis with a genome of 4 million base pairs with an average G+C ratio of 43%, consisting of 4,100 protein-coding genes and later in 2009, the sequence was updated [START_REF] Barbe | From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later[END_REF]. B. subtilis CDS can be divided into three different classes. Class I includes mainly genes of sporulation, Class II consists of genes expressed during exponential growth conditions i.e. genes related to transcription and translation machineries, core intermediary metabolism, stress proteins, and one-third of genes of unknown function. Lastly, Class 3 includes genes of unidentified function as well as this class has enriched A+T residues codon [START_REF] Hénaut | Uneven Distribution of GATC Motifs in the Escherichia coli Chromosome, its Plasmids and its Phages[END_REF]. It has been found that ATG as start codon for 78% genes, TTG for 13% and 9% with GTG in comparison to 85%, 3% and 14% for E.coli [START_REF] Blattner | The complete genome sequence of Escherichia coli K-12[END_REF]. RNA synthesis and degradation; protective functions and unknown represented in .

The complex regulation of the lipopetpide synthesis

Two-component signal-transduction pathways comprise a sensor protein kinase and a response regulator. In B. subtilis 34 genes encode response regulator, largely having neighboring genes encoding histidine kinases. The ComP/ComA two component system is required for the expression of various peptide pheromones, which are involved in various signal transduction pathways [START_REF] Lazazzera | An autoregulatory circuit affecting peptide signaling in Bacillus subtilis[END_REF]. It has an important role in competence development and regulation of the secondary metabolites biosynthesis, as the lipopeptide surfactin [START_REF] Hamoen | Controlling competence in Bacillus subtilis: shared use of regulators[END_REF].

B. subtilis can develop a physiological condition which makes them to uptake exogenous DNA in macromolecular form under defined growth condition, this process is known as competence. [START_REF] Hamoen | Controlling competence in Bacillus subtilis: shared use of regulators[END_REF] suggested five genetic loci involved for the transport of DNA: comC, comE, comF, comG and nucA. After transport into the cell, recombination proteins i.e. RecA and DNA helicase AddAB help in integration of the foreign DNA into the genome [START_REF] Haijema | Regulated expression of the dinR and recA genes during competence development and SOS induction in Bacillus subtilis[END_REF]. ComK is a major candidate for the development competence as all the above genes are regulated by this competence factor [START_REF] Van Sinderen | comK encodes the competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis[END_REF]. The crucial step for competence development is the activation of comK promoter by ComK [START_REF] Hamoen | Controlling competence in Bacillus subtilis: shared use of regulators[END_REF]. Sporulation gene spoOA can also regulate competence as phosphorylated spoOA (spoOA ~P) can inhibit the expression of abrB, which is a potential inhibitor of comK expression (Green et al., 1991). Besides inhibition by abrB, based on GTP levels as well as concentration of branched chain amino acids codY can bind to the RNA polymerase binding site of comK promoter, thus inhibiting its expression [START_REF] Serror | CodY is required for nutritional repression of Bacillus subtilis genetic competence[END_REF].Various competence stimulating factors have

CONFIDENTIAL DOCUMENT

direct or indirect effect on the positive expression of comK. As it can be seen from Figure 2, expression of comQ is required for post-translational modification and secretion of comX, which is the main competence stimulating factor. ComX acts as an environmental signal for the phosphorylation of ComA by its protein kinase partner ComP [START_REF] Solomon | Convergent sensing pathways mediate response to two extracellular competence factors in Bacillus subtilis[END_REF]. Phosphorylated ComA (ComA~P) is required for the expression of srfA operon [START_REF] Hahn | Growth stage signal transduction and the requirements for srfA induction in development of competence[END_REF]. It has been found that this operon positively regulates comK through comS, which is located with the srfA operon but out of its ORF [START_REF] Hamoen | A small gene, designated comS, located within the coding region of the fourth amino acidactivation domain of srfA, is required for competence development in Bacillus subtilis[END_REF].

There are several phr genes (7 genes), which encode regulatory peptides that might have a role in quorum sensing mechanism [START_REF] Carrera | Aspartyl-phosphate phosphatases deactivate the response regulator components of the sporulation signal transduction system in Bacillus subtilis[END_REF]. PhrC, one of the pheromone which forms another half of competence stimulating factor can also induce competence through srfA transcription via ComP and ComA activation. An oligopeptide permease, SpoOK senses this pheromone and facilitates its entry [START_REF] Lazazzera | An exported peptide functions intracellularly to contribute to cell density signaling in Bacillus subtilis[END_REF]. PhrC can inhibit the activity RapC. This protein can dephosphorylate ComA~P, thus reducing the expression of srfA and finally hindering competence development [START_REF] Solomon | Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis[END_REF].

B. subtilis during transition state expresses various extracellular proteases and other degrading enzymes, transport function and chemotaxis to allow maximum utilization of nutrients as well as it excretes various antimicrobial compounds to defeat competing microbial species [START_REF] Phillips | Bacillus subtilis sporulation and stationary phase gene expression[END_REF]. There are various transition state regulators which inhibit the expression of genes that are only needed during stationary phase [START_REF] Strauch | A positive feedback loop controls transcription of the spoOF gene, a component of the sporulation phosphorelay in Bacillus subtilis[END_REF]. This transition state regulator includes AbrB, ScoC, SinR, CodY and Abh.

AbrB, ScoC and SinR overexpression can restrain sporulation [START_REF] Gaur | Characterization of a cloned Bacillus subtilis gene that inhibits sporulation in multiple copies[END_REF] while AbrB is involved in the production and resistance to various antibiotics [START_REF] Hahn | The major role of Spo0A in genetic competence is to downregulate abrB, an essential competence gene[END_REF].

Both SpoOA and AbrB regulate the expression of scoC [START_REF] Perego | Sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in Bacillus subtilis[END_REF]. SinR represses the genes (spoIIA, E and G) responsible for sporulation, it is a global regulator for degrading enzymes synthesis, motility, competence and sporulation [START_REF] Gaur | The Bacillus subtilis sin gene, a regulator of alternate developmental processes, codes for a DNA-binding protein[END_REF].

CodY is activated in the presence of amino acids (valine and isoleucine) and GTP; it represses competence and dipeptide permeases as well as genes involved in the utilization of alternate carbon and nitrogen sources [START_REF] Serror | CodY is required for nutritional repression of Bacillus subtilis genetic competence[END_REF]. [START_REF] Kunst | The complete genome sequence of the gram-positive bacterium Bacillus subtilis[END_REF].

Industrial applications

Bacillus species have been the "protagonists" in applied microbiology for industrial applications for the last 20 years and more. In 1982, B. subtilis (natto) was first used for the solid state fermentation of soyabeans in Japan. They have been drawing attention towards industrial application due to the following characteristics: high growth rates, ability to secrete proteins and various important compounds into extracellular medium and generally recognized as safe (GRAS) organisms for food and drug administration. The availability of the genomic information related to various pathways have facilitated reconstruction of this pathway through gene modification, as role expression studies can be carried out for the overproduction of various industrially important compounds along with

CONFIDENTIAL DOCUMENT

the characteristics of development of competent state to uptake foreign DNA and sporulation. There has been extensive proteome analysis providing a panoramic view of B.

subtilis proteome and its ability to adopt environmental changes [START_REF] Becher | From the genome sequence to the protein inventory of Bacillus subtilis[END_REF].

There are various genes which are responsible for the production of lipid, carbohydrate and protein degrading enzymes as well as responsible for secretary function, thus creating an industrial interest. B. subtilis secreted proteins have N-terminal signal peptides, which are important feature for transport from the cytoplasm [START_REF] Dalbey | Membrane proteases in the bacterial protein secretion and quality control pathway[END_REF] and the best studied translocation pathway for secretory proteins are secretory (Sec) pathway and twinarginine translocation pathway [START_REF] Antelmann | The extracellular proteome of Bacillus subtilis under secretion stress conditions[END_REF]. [START_REF] Reuß | Complete Genome Sequence of Bacillus subtilis subsp. subtilis Strain ∆6[END_REF].

Industrial enzymes market will be worth 6.2 billion $US by 2020, B. subtilis can be the major workhorse for this market. Its importance comes from its ability to produce these substances at gram per litre concentrations. B. subtilis produces α-amylase, which cleaves internal α-1, 4-linkages in an endo fashion and it is used in food, fermentation, textile and paper industries [START_REF] Pandey | Advances in microbial amylases[END_REF]. The enzyme β-glucanase is used for enzymatic modification of barley. It can produce various alkaline proteases which have pH optima in the range of 9-12, is thermostable as well as can be used as commercial detergents [START_REF] Gupta | Bacterial alkaline proteases: molecular approaches and industrial applications[END_REF]. Two novel phytases from B. subtilis were cloned and biochemically characterized [START_REF] Tye | Molecular cloning and the biochemical characterization of two novel phytases from Bacillus subtilis 168 and B. licheniformis[END_REF]. [START_REF] Braaz | Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis[END_REF] found that B. subtilis was an efficient host for the overexpression of poly (3-hydoxybutyrate) depolymerase from P. lemoignei. B. subtilis can be a great host for the production of heterologous proteins as it has the potential to secrete proteins into extracellular medium but it requires various genetic signals for effective transcription and translation of foreign genes [START_REF] Braaz | Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis[END_REF]. Production of heterologous eukaryotic proteins in B. subtilis is problematic due to the presence of large number of extracellular proteases [START_REF] Wang | Expression and secretion of human atrial natriuretic alpha-factor in Bacillus subtilis using the subtilisin signal peptide[END_REF]. About 4% of the B. subtilis genomes code for large multifunctional enzymes which include genes for loci i.e. Srf, Pps and Pks. Due to presence of these genes, it can produce compounds with antibiotic activity. Bacitracin gene cluster was transferred from B. licheniformis to B. subtilis; it was able to produce the antibiotic in high levels [START_REF] Eppelmann | Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis[END_REF]. Production of riboflavin at the

CONFIDENTIAL DOCUMENT

concentration of 30 g/l in 3-day fermentation was obtained using mutant strain of B.

subtilis [START_REF] Dauner | Intracellular carbon fluxes in riboflavin-producing Bacillus subtilis during growth on two-carbon substrate mixtures[END_REF].

Table 1 An overview of enzymes and active molecules along with their corresponding genes in B. subtilis B. subtilis spores can also be used as an agent for vaccine delivery [START_REF] Duc | Bacterial spores as vaccine vehicles[END_REF].

System and synthetic biology approach along with fundamental and applied approach can convert B. subtilis into micron-scale incomparable factories.

It can be seen that B. subtilis genome is properly studied to be used for genetic modification for the synthesis of various molecules. In the next section, the focus is on non-ribosomal peptide synthesis (NRPS) mechanism in B. subtilis, which is essential for the production of particular class of industrially important molecules, such as lipopeptidic biosurfactants.

INSIGHTS INTO UNIQUE NON-RIBOSOMAL PEPTIDE SYNTHESIS

Introduction

In 1928, Alexander Fleming returned to his laboratory after a month and saw his culture of Staphylococcus aureus had become contaminated with a mold. He was unaware of the fact that this incident will act as a catalyst in the world of medicine with the discovery of first antibiotic (which he called "mold juice", later renamed as penicillin) and the mold was Penicillin notatum. Penicillin is a peptide derivative from Penicillin notatum. Lipmann and his co-workers (1971) communicated enzymatic synthesis of tyrocidine and gramicidin S from Bacillus sp. It was reported that their synthesis was independent on nucleic acid and was carried out by a large multifunctional enzyme complex analogous to fatty acid synthetases [START_REF] Lipmann | Polypeptide synthesis on protein templates: the enzymatic synthesis of gramicidin S and tyrocidine[END_REF].

Non-Ribosomal Peptide Synthetase (NRPS) is a multifunctional, multienzyme complex found in various bacteria, fungi and lower eukaryotes. In comparison to conventional ribosomal synthesis, it does not require mRNA or ribosome for the synthesis of the peptides. Non ribosomal peptides (NRPs) display more than 500 different monomers and can be linear, cyclic or branched in structure. NRPs can be classified into lipopeptides, chromopeptides, glycopeptides etc. There is another class of multifunctional enzymes known as PKS (Poly Ketide Synthetases). The difference between PKSs and NRPs is that, the former uses acetate and propionate as builiding blocks while the latter uses monomers [START_REF] Mootz | An improvement of surfactin production by Bacillus subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor[END_REF]. NRPs represent a large class of bioactive compounds with distinctive structural features like D-and L-amino acids; heterocyclic elements which can be methylated or glycosylated. Non-ribosomal peptides are generally produced by microorganisms present in soil e.g. Bacilli, Actinomycetes or Pseudomonas. Although, it has been found that marine microorganisms can be a resource for these multifunctional secondary metabolites [START_REF] Faulkner | Marine natural products[END_REF]. These peptides display various functions i.e.

antibiotic activity (Tyrocidine A; Bacitracin A; Vancomycin), biosurfactant (Surfactin, Iturin), antifungal (Iturin, Fengycin), elicitor (Surfactin, Fengycin), anti-inflammatory (Surfactin) and act as immunosuppressive agent (Cyclosporin) as well as can function as

CONFIDENTIAL DOCUMENT

siderophore (Bacillibactin; Enterobactin; Vibriobactin) [START_REF] Maget-Dana | Working outside the protein-synthesis rules: Insights into nonribosomal peptide synthesis[END_REF]. They can also be divided into classes based on their structural conformation: linear product (vancomycin, etc.) and cyclic product (surfactin, cyclosporine, etc.) following intramolecular nucleophilic reaction.

NRPS is organized into various modules which forms the template for the synthesis of various linear and cyclic peptides. Modules are further divided into domains, which are responsible for specific catalytic activity during synthesis (Felnagle et al., 2008). Each module determines the nature of the monomer and inclusion of the monomer in the growing peptide [START_REF] Caboche | NORINE: a database of nonribosomal peptides[END_REF] whereas the domains are responsible for substrate binding, activation, modification, elongation and release of the complete peptide (Sieber, 2005). The length of the final peptide is generally determined by the size of the NRPS.

Studies have revealed the presence of linker regions (~15 amino acids) between domains in a randomly distributed fashion. These regions being flexible in nature can be used for synthesizing hybrid NRPS template without disturbing the enzymatic integrity [START_REF] Weber | Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases[END_REF]. A database consisting of all non-ribosomal peptides have been created called NORINE [START_REF] Caboche | NORINE: a database of nonribosomal peptides[END_REF]. The biological activity of NRPS depends on their structure, which is defined by the peptide sequence and the length and isomery of the fatty acid chain for the lipopeptidic compounds. Sometimes, the peptide products can become problematic for the producer strain, so they acquire several defensive mechanism i.e. modification of the target region within the strain, temporary inactivation of the product and efflux pumps to escape the toxic nature [START_REF] Crosa | Genetics and assembly line enzymology of siderophore biosynthesis in bacteria[END_REF].

Primary domains

The elongation module (C-A-PCP) is made of at least three essential domains i.e.

Adenylation (A) domain, Peptidyl Carrier Protein (PCP) domain and Condensation (C) domain, for the selection and activation of the monomer, covalent binding to 4'phosphopantetheine (PPan) and peptide bond formation, respectively. At the end, the final peptide release is catalyzed by thioesterase (TE) domain. These four domains make the primary domains of Non-Ribosomal Peptide Synthetase.

CONFIDENTIAL DOCUMENT

Adenylation (A) domain

Adenylation (A) domain is responsible for the selection and conversion of the monomer (amino or carboxylic acid). Upon binding, the domain converts the substrate into aminoacyl adenylate and release PPi in the presence of ATP and Mg 2+ Figure 3 ( ). The amino acids present in the core motifs are responsible for the ATP binding and hydrolysis as well as adenylation intermediate formation [START_REF] Stachelhaus | The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases[END_REF]. The Adenylation domain is composed of ~500 amino acids [START_REF] Mootz | The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains[END_REF]. domain [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF] This domain belongs to the superfamily known as adenylate-forming enzymes. Its functional requirement of ATP is similar to aminoacyl-tRNA-synthetases [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF]. The hydrophobic active site is between the large size N-terminal subdomain and C-terminal subdomain, which is small and flexible. The latter subdomain adopts different positions relative to N-terminal subdomain to facilitate the initial catalytic reaction. This orientation is favored by a rotation of 140 ° during open state conformation (substrate loading) relative to the closed state conformation (Adenylation reactions) [START_REF] Yonus | Crystal structure of DltA. Implications for the reaction mechanism of non-ribosomal peptide synthetase adenylation domains[END_REF].

Peptidyl carrier protein (PCP) domain

PCP domains consist of PPan (4'-phosphopantatheine) prosthetic arm by the activity of a specific PPan-transferase [START_REF] Mootz | 4'-phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis[END_REF] [START_REF] Koglin | Conformational switches modulate protein interactions in peptide antibiotic synthetases[END_REF]. NMR studies reveal PCP domain consists of a large loop region (contains the serine residue which is attached to PPan) between two helical regions.

The PCP cofactor was modified by Sfp-Glu151. This residue of Sfp deprotonates the serine-hydroxyl group of PCP domain (Figure 4) and the subsequent nucleophilic reaction on the β-phosphate of CoA leads to the formation of holo-PCP and 3',5'-ADP [START_REF] Mofid | Structure-based mutational analysis of the 4'-phosphopantetheinyl transferases Sfp from Bacillus subtilis: carrier protein recognition and reaction mechanism[END_REF]. PCP domain has a four-helix bundle structure and Sfp binds within an extended loop between the first two helices [START_REF] Weber | Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases[END_REF].

Figure 4 Role of product for the attachment of prosthetic arm [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF] Sfp monomers have a pseudo-2-fold symmetry and coenzyme A binds within a pocket formed by Sfp bent conformation [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF]. It was observed that sfp gene product can efficiently phosphopantetheinylate not only apo-PCP (NRPS templates) but also acyl carrier proteins (fatty acid) of polyketide synthases [START_REF] Kealey | Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts[END_REF].

Phosphopantetheine (PPan) transferase expressed by sfp validated the findings that PCP domain's protein-protein interaction depends on its conformation state [START_REF] Lai | Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection[END_REF].

CONFIDENTIAL DOCUMENT

Figure 5 Activated amino-acyl adenylate forming a covalent bond with the free thiol of PCP domain [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF] Initiation module is formed by both adenylation (A) and PCP domain as they are required for the initial activation and covalent bond formation for the subsequent peptide synthesis.

The reactive intermediate (amino-acyl adenylate) from A domain is then transferred to the thiol moiety of holo-PCP domain (Figure 5) by a nucleophilic reaction between the free thiol-PPan cofactor and amino acid -O-AMP, with the formation of a thioester [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF]. The holo-PCP domain composed of 80-100 amino acids. It acts as a swinging arm for the transfer of thioester bound substrate and elongation intermediate from A domain to C domain.

Condensation (C) domain

Condensation domain is responsible for the formation of peptide bond between the PCP intermediates. This domain is composed of ~450 amino acids. The peptide bond is formed by a nucleophilic reaction between the acceptor site α-amino group of the aminoacyl-S-PCP and peptidyl-S-PCP substrate in the donor site (Figure 6) i.e. PCP-bound acceptor amino acid downstream and upstream PCP activated donor substrate [START_REF] Stachelhaus | Peptide bond formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain[END_REF]. In vitro analysis revealed C-domain has a high substrate specificity at the acceptor in comparison to the donor site [START_REF] Belshaw | Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis[END_REF]. The C-domain has a V-shaped conformation for the binding of two PCP substrates and an active site having a conserved core motif located in close proximity to the binding site [START_REF] Samel | Structural and functional insights into a peptide bond-forming bidomain from a nonribosomal peptide synthetase[END_REF]. Studies on the active site pK value revealed that the peptide bond formation depends on electrostatic interactions. The C-domain has been replaced by heterocyclic (Cy) domain in synthesis of vibractin, as it can perform similar functions like peptide elongation and heterocyclization in a similar way as C-domain [START_REF] Maget-Dana | Working outside the protein-synthesis rules: Insights into nonribosomal peptide synthesis[END_REF].

Thioesterase (TE) domain

Thioesterase (TE) domain is composed of ~280 amino acids [START_REF] Maget-Dana | Working outside the protein-synthesis rules: Insights into nonribosomal peptide synthesis[END_REF]. Its structure consists of a fold of α/β hydrolases and a lid region which covers the active site consisting of a conserved catalytic triad (Ser, His and Asp). The lid region has both open and closed conformations for preventing the active site from water [START_REF] Samel | The thioesterase domain of the fengycin biosynthesis cluster: a structural base for the macrocyclization of a nonribosomal lipopeptide[END_REF]. It is only found in the termination module of NRPS.

Figure 7 Role of TE domain for peptide release [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF] This domain is responsible for the termination of synthesis (Figure 7) by two-step reaction: or hydrolysis (linear product) [START_REF] Trauger | Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase[END_REF]. TE domains are responsible for cyclization of diverse nonribosomal peptides. The diverse nature can be distributed on the basis of two predominant different cyclization strategies: 1 lactone (Surfactin, Enniatin, Fengycin, Syringomycin etc.); 2 lactam (Iturin A, Mycosubtilin, Tyrocidine, Cyclosporin A, Gramicidin S etc.). Lactone and lactam cyclization strategies can be differentiated on the basis of processing, while in the former ketone is reduced to hydroxyl group and in the latter, it is converted to amino group by head to tail condensation [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF]). An alternative pathway of termination can take place by the aid of reductase (R) domain in the terminal by the reduction of peptidyl-S-PCP final product [START_REF] Kopp | Where chemistry meets biology: the chemoenzymatic synthesis of nonribosomal peptides and polyketides[END_REF]. This domain has been discussed later in the chapter.

Analysis of TE domain of SrfA towards substrate tolerance, showed the significance of both N-and C-terminal monomers forming the peptide. This result revealed that leucine (6 and 7 position) and glutamic acid (1 st

In addition to natural synthesis of non-ribosomal peptides, chemoenzymatic approaches have also been carried out utilizing the versatile nature of TE domains. In order to carry position) in the peptide moiety of surfactin are essential monomers for TE domain for substrate recognition [START_REF] Trauger | Cyclization of backbone-substituted peptides catalyzed by the thioesterase domain from the tyrocidine nonribosomal peptide synthetase[END_REF].

CONFIDENTIAL DOCUMENT

out the synthesis, TE domains were provided with substrate attached to PCP domain or an artificial solid support or as soluble thioester intermediates [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF].

Secondary domains

There are five types of secondary domains, which include epimerization (E) domains, Nmethyltransferase (Mt) domain, Formylation (F) domains, and oxidation (Ox) and reduction (Red) domains.

Epimerization (E) domain

This domain transforms the L-isoforms (aminoacyl and peptidyl thioesters) attached to PCP domain into corresponding D-isoforms [START_REF] Maget-Dana | Working outside the protein-synthesis rules: Insights into nonribosomal peptide synthesis[END_REF]. In few cases, epimerization can also be catalyzed by an external reaction e.g. in the synthesis of cyclosporin, A domain includes D-Ala provided by en external racemase [START_REF] Hoffmann | Purification and characterization of eucaryotic alanine racemase acting as key enzyme in cyclosporin biosynthesis[END_REF]. [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF].

CONFIDENTIAL DOCUMENT

N-methyltransferase (N-Mt) & C-methylation (C-Mt) domain

N-methyltransferase (Mt) domain is placed adjacent to PCP domain and is responsible for transferring an electrophilic methyl group from S-adenosylmethionine (SAM) to the αamino group (Figure 9) of the thioesterified amino acid, peptide, thus forming Sadenosylhomocysteine [START_REF] Schauwecker | Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides[END_REF]. N-methylated peptide bonds are present in peptides i.e. cyclosporin, enniatin, actinomycin and pristinamycin [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF]. There is another secondary domain which is responsible for methylation at the C-terminal.

C-Mt domain catalyzes the Cα-atoms of cysteine residue into α-methyl-thiazoline. This domain also depends upon SAM for the methyl group. This domain is accountable for cyclization of the peptide and located at the C-terminal of PCP domain [START_REF] Maget-Dana | Working outside the protein-synthesis rules: Insights into nonribosomal peptide synthesis[END_REF].

Formylation (F) domains

This domain is responsible for the transfer of formyl group to the peptide chain during initiation (Figure 10) [START_REF] Maget-Dana | Working outside the protein-synthesis rules: Insights into nonribosomal peptide synthesis[END_REF]. [START_REF] Maget-Dana | Working outside the protein-synthesis rules: Insights into nonribosomal peptide synthesis[END_REF]. In vancomycin, six of the seven amino acids are nonproteinogenic; this modification can be carried out by the oxidation domain [START_REF] Vaillancourt | Nature's inventory of halogenation catalysts: oxidative strategies predominate[END_REF]. Certain peptides i.e. surfactin and fengycin have fatty acid in their N-terminal. It is assumed that acyl transferase provides the acyl to donor site of C-domain to have this modification [START_REF] Steller | Structural and functional organization of the fengycin synthetase multienzyme system from Bacillus subtilis b213 and A1/3[END_REF].

Classification

Based on the interaction between the modules or domains, NRPSs mode of biosynthesis can be classified into three different categories [START_REF] Mootz | An improvement of surfactin production by Bacillus subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor[END_REF]:

1. Linear NRPS (type A)

2. Iterative NRPS (type B)

3. Nonlinear NRPS (type C)

Linear Synthesis (type A)

In this mode of biosynthesis; the final peptide contains n amino acids, which is incorporated by n modules with a domain arrangement A-PCP-(C-A-PCP) n-1 Figure 12 -TE ( ), while the secondary domains are placed in their respective modules (Henning D. [START_REF] Mootz | An improvement of surfactin production by Bacillus subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor[END_REF]. The initiation (A-PCP) module lacks (except for some lipopeptide) the C-domain whereas C-A-PCP forms the elongation and TE domain and in some cases modified C-domain (peptides of fungal origin) forms the termination module. In this type of NRPS, the order and number of modules determines the sequence of the final peptide [START_REF] Keating | Chain termination steps in nonribosomal peptide synthetase assembly lines: directed acyl-S-enzyme breakdown in antibiotic and siderophore biosynthesis[END_REF]. Surfactin, cyclosporin, tyrocidine, actinomycin, bacitracin, fengycin, etc are examples of linear NRPSs [START_REF] Mootz | An improvement of surfactin production by Bacillus subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor[END_REF].

Figure 12 Assembly and mode of synthesis of linear NRPSs [START_REF] Mootz | An improvement of surfactin production by Bacillus subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor[END_REF] 20 CONFIDENTIAL DOCUMENT

Iterative Synthesis (type B)

In this typeof biosynthesis, the modules or domains are used more than once for the synthesis of final peptide. It contains reduced number of modules relative to the sequence of the final peptide. One set of repetition is provided by the initiation module, while the other module is responsible for the cyclization of the product by oligomerization process as per example (Figure 13). The repeated cycles as well as the bond nature between the monomers are controlled by PCP-C didomain and TE domain [START_REF] Mootz | An improvement of surfactin production by Bacillus subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor[END_REF].

Figure 13 Assembly and mode of synthesis of iterative NRPSs [START_REF] Mootz | An improvement of surfactin production by Bacillus subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor[END_REF] Examples of this type of synthesis include enterobactin produced by E.coli and enniatin produced by Fusarium scirpi [START_REF] Haese | Molecular characterization of the enniatin synthetase gene encoding a multifunctional enzyme catalysing Nmethyldepsipeptide formation in Fusarium scirpi[END_REF].

Nonlinear Synthesis (type C)

The modular arrangement in nonlinear synthesis is different in comparison to linear NRPSs (Figure 14). This structural difference leads to unusual internal conformation.

Syringomycin, yersniabactin, vibriobactin, mycobactin, bleomycin etc. are examples of this type of biosynthesis [START_REF] Mootz | An improvement of surfactin production by Bacillus subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor[END_REF].

CONFIDENTIAL DOCUMENT

Glycolytic pathway

B. subtilis displays Embden-Meyerhof-Parnas glycolytic pathway, linked to a functional tricarboxylic acid cycle. The pathway comprises of two sections: the upper section is responsible for the conversion of hexose carbon source to triose and the bottom portion is responsible for pyruvate synthesis using the product from upper section as substrate. There are numerous enzymes involved in the import and conversion of glucose into pyruvate and finally pyruvate is used as a substrate for TCA cycle or other pathways for the production of various metabolites required for metabolism, cell wall synthesis and synthesis of secondary metabolites. A summarized overview of this pathway is provided in Figure 15. CcpA has a positive effect on gapA operon [START_REF] Sonenshein | Control of key metabolic intersections in Bacillus subtilis[END_REF]. There are two transcriptional repressor ccpN and cggR in the glycolytic pathway. The former is expressed constitutively; it binds in the promoter region and can repress the expression of gapB. While the latter is controlled by the concentration of fructose-1,6-bisphosphate and can repress gapA operon [START_REF] Servant | CcpN (YqzB), a novel regulator for CcpAindependent catabolite repression of Bacillus subtilis gluconeogenic genes[END_REF].

The regulon ptsGHI is the first set of gene clusters involved in the glycolytic pathway. The gene ptsG (glucose permease) enables transport of glucose into the cell and phosphorylates glucose to glucose-6-phosphate via HPrK/P (Bifunctional kinase/phosphatase). The activity of HPrK/P is stimulated by ATP and fructose-1,6-biphospahte. The transfer of phosphoryl group from phosphoenol pyruvate (PEP) to permease is carried out by ptsH and ptsI. This system only produces glucose-6-phosphate (Ludwig et al., 2001). The RNA binding protein glcT, acts as a transcriptional antiterminator, which in the presence of glucose binds stem-loop structure of ptsGHI mRNA, thus preventing formation of a

CONFIDENTIAL DOCUMENT

terminator structure and allowing transcription, but in the absence of glucose it cannot bind to ptsGHI mRNA and no transcription takes place (Commichau and Stülke, 2008).

Glucose-6-phosphate can also be obtained from glucose-1-phosphate by pgcA (αphosphoglucomutase). Glucose-1-phosphate is generally produced by other sugars except glucose [START_REF] Weart | A metabolic sensor governing cell size in bacteria[END_REF]. Glucose-6-phosphate is then converted into fructose-6phosphate by a reversible reaction carried out by pgi (glu-6-phosphate isomerase), which is constitutively expressed and does not depend on the presence of glucose [START_REF] Macek | The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis[END_REF]. The conversion of fructose-1,6-biphosphate to fructose-6-phosphate can be carried out by two types of fructose-1, 6-biphosphatase fbp or glpX [START_REF] Fujita | Isolation and properties of a Bacillus subtilis mutant unable to produce fructose-bisposphatase[END_REF]) [START_REF] Fujita | Identification and expression of the Bacillus subtilis fructose-1, 6-bisphosphatase gene (fbp)[END_REF]. AMP and PEP concentrations regulate the expression of fbp while only PEP regulates the expression of glpX [START_REF] Jules | The Bacillus subtilis ywjI (glpX) gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the class III Fbp enzyme[END_REF] is responsible for the reversible conversion of 1,3-bisphosphateglycerate and 3phosphateglycerate as well as it requires magnesium and manganese ions as cofactors [START_REF] Biochem | Phosphoglycerate Kinase of Bacillus stearothermophilus 2 . The enzyme was composed of a single polypeptide chain of molecular weight 42 , 000 . The thermophilic enzyme had many properties in common with enzymes from mesophilic sources , e . g[END_REF]. The reversible conversion of 3-phosphateglycerate to 2phosphateglycerate is catalyzed by pgm (2,3-biphosphoglycerate-independent phosphoglycerate mutase). The interconversion of 2-phosphateglycerate to phosphoenol pyruvate is catalyzed by eno (enolase). It interacts with pfkA in vitro and increases the flux in glycolytic pathway [START_REF] Newman | Dissection of the network of interactions that links RNA processing with glycolysis in the bacillus subtilis degradosome[END_REF]. The final reaction of glycolytic pathway, conversion of phosphoenol pyruvate to pyruvate is carried out by pykA.

The essential genes in the glycolytic pathway are eno (enolase; responsible for the conversion of 2-phospho-D-glycerate to phosphoenolpyruvate), gapA (glyceraldehyde 3phosphate dehydrogenase; converts D-glyceraldehyde 3-phosphate to 3-phospho-Dglyceroyl phosphate) and pgm (phosphoglycerate mutase; conversion of 2-phospho-D-glycerate to 3-phospho-D-glycerate) as the mutants lacking these genes were unable to grow on LB-medium [START_REF] Commichau | Essential genes in Bacillus subtilis: a reevaluation after ten years[END_REF].

Pyruvate produced from the glycolytic pathway along with threonine is directed towards the synthesis of branched chain amino acids i.e. valine, leucine and isoleucine. The metabolism of branched chain amino acids (BCAAs) is described in the following section.

Branched chain amino acid pathway

The branched chain amino acids (BCAA), i.e. leucine (Leu), valine (Val), and isoleucine (Ile) are produced by this metabolic pathway with complex regulatory mechanisms.

Threonine (Thr), pyruvate (Pyr) and L-2-amino-acetoacetate (Akb) are used by the cell for the production of leucine, valine, and isoleucine. The ilv-leu operon contains the main genes involved in the production of branched chain amino acids. The ilv-leu operon is subjected to multiple forms of regulation on its promoter P Ilv-Leu , as studied in a series of articles. There are two proteins that down-regulate the activity of P Ilv-Leu by binding to different sites upstream the promoter and inhibiting the RNA polymerase (Belitsky and Sonenshein, 2011). These are CodY (transcriptional pleiotropic regulator, which regulates the transcription of early-stationary-phase genes) and TnrA (nitrogen pleiotropic transcriptional regulator). In addition, the down-regulation of the promoter P Ilv-Leu

Figure 16 is also contributed by Leu in terms of ribosome-mediated attenuation mechanism, Tbox [START_REF] Brinsmade | Dissecting complex metabolic integration provides direct genetic evidence for CodY activation by guanine nucleotides[END_REF]. The expression of different genes involved in the production of branched chain amino acids is shown in .

Figure 16 Complex regulation of branched chain amino acid pathway [START_REF] Brinsmade | Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools[END_REF] As is shown in Figure 17, the protein IlvA activates the conversion of Thr into Akb. IlvBH (a cluster of IlvB and IlvH), IlvC and IlvD activate the transformation of Akb to Keta. The proteins LeuA and LeuBCD (a cluster of LeuA, LeuB, and LeuC) perform a sequence of reactions justifying the transformation of Ketb to Ketc [START_REF] Mäder | SubtiWiki--a comprehensive community resource for the model organism Bacillus subtilis[END_REF]. The genes coding the proteins IlvBH, IlvC, LeuA, and LeuBCD are co-located in the same operon (ilv-leu), so they are under the dependence of the same promoter P Ilv-Leu [START_REF] Tojo | Elaborate transcription regulation of the Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids through global regulators of CcpA, CodY and TnrA[END_REF].

Expression of ilvA, ilvD, and ybgE is dependent on their own promoters and is downregulated by CodY at the transcriptional level. The expression of ilvA is deactivated by both Ile and Val (Mäder et al., 2004a). The transcription of all proteins IlvC, IlvBH, LeuA, and LeuBCD starts through the activation of their promoter P Ilv-Leu The intermediates, Ket a ; Ket b and Ket c are used for fatty acid synthesis and transformed by protein BkL, this metabolic pathway has been described in the next section.

Branched chain fatty acids synthesis pathway

B. subtilis contains a high percentage of branched chain fatty acids in comparison to other fatty acids and does not depend on the medium condition [START_REF] Klein | Cold shock response of Bacillus subtilis: CONFIDENTIAL DOCUMENT isoleucine-dependent switch in the fatty acid branching pattern for membrane adaptation to low temperatures[END_REF]. The fatty acid profile of B. subtilis is largely dominated by iso-and anteiso-C 15:0 and C 17:0 The fatty acid species produced depends on the availability of various substrates that affect the chain isomery and length as shown in the Table 2. In B. subtilis branched-chain amino acid amino transferase (BCAT) enzyme uses various α-keto substrates as primers and forms the determining feature of the fatty acid isoforms produced. The reactivity order of the α-keto substrates are as follows α-Keto-β-methyl-valerate ≥ α-Keto-iso-caproate ≥ α -Keto-iso-valerate [START_REF] Naik | Biosynthesis of branched long-chain fatty acids by species of Bacillus: relative activity of three alpha-keto acid substrates and factors affecting chain length[END_REF].

CONFIDENTIAL DOCUMENT

Table 2 Substrates that enhance the synthesis of specific fatty acids in B. subtilis [START_REF] Kaneda | Fatty acids of the genus Bacillus: an example of branched-chain preference[END_REF] In B. subtilis, there are seven genes present in bkd operon that encode proteins which are responsible for the synthesis of branched chain fatty acids through utilization of the respective amino acids. These genes include ptb, bcd, buk, lpd (lpdV), bkdA1, bkdA2, and bkdB, which encode for phosphate butyryl coenzyme A transferase, leucine dehydrogenase, butyrate kinase, and four components of the branched-chain keto acid dehydrogenase complex: E3 (dihydrolipoamide dehydrogenase), E1α(dehydrogenase), E1β (decarboxylase), and E2 (dihydrolipoamide acyltransferase), respectively (Debarbouille et al., 1999). The maximum affinity of this operon is towards isoleucine and valine in comparison to leucine for its activation (Belitsky, 2015).

Branched chain amino acid transporter

Besides the use of branched chain amino acid (BCAA) for the synthesis of their respective fatty acids, these amino acids can also be transported out into the environment. There are various transporters which possess the ability to export as well as import branched chain amino acids from/to the environment (Figure 19). The operon azlBCDEF is responsible for both import and export of branched chain amino acids. The gene azlB negatively regulates the expression of this operon (Belitsky et al., 1997b). Two genes (azlC and azlD) of this operon are responsible for the export of branched chain amino acids while azlE (brnQ) is responsible for the uptake of valine and isoleucine. The export efficiency of AzlC and AzlD is higher for leucine and isoleucine in comparison to valine [START_REF] Kennerknecht | Export of L-isoleucine from Corynebacterium glutamicum: a two-gene-encoded member of a new translocator family[END_REF]. The protein, BrnQ depends on Na + for the import of amino acids. Homologues of these proteins were also reported in C. glutamicum [START_REF] Ebbighausen | Transport of branched-chain amino acids in Corynebacterium glutamicum[END_REF]. Although different in functions, the genes brnQ and azlCD are co-transcribed in B. subtilis (Belitsky et al., 1997a).

BCAAs

In addition to BrnQ, BcaP and BraB are also responsible for the uptake of valine, leucine and isoleucine (ILV), while BcaP is the most efficient permease and allows B. subtilis to utilize isoleucine and valine as nitrogen sources. In absence of brnQ, bcaP and braB, the activity of CodY was strongly reduced as intracellular concentrations of these amino acids are insufficient to completely activate its regulating activity (Belitsky, 2015). Although the DNA binding activity depends on both GTP and ILV, but prior interaction to ILV is essential for its activity (Belitsky and Sonenshein, 2011). These permeases are adequate enough to provide the cells with ILV from the environment in liv mutants of B. subtilis which are deprived of ILV biosynthesis when grown in TSS glucose-ammonium medium containing ILV and no growth defects were observed in comparison to the control (Belitsky, 2015). It was reported that the permeases have high affinity towards isoleucine and valine uptake in comparison to leucine, which relates the existence of another

CONFIDENTIAL DOCUMENT

permease which has high affinity towards leucine as a leuB mutant strain lacking three permease was able to grow in a minimal medium containing leucine (40 µg/ml) (Belitsky, 2015). A key candidate for this type of permease was found to be YvbW, a putative permease for amino acid which is regulated by T-box mechanism specific to leucine limitation [START_REF] Rollins | Induced Transcriptional Expression of Bacillus subtilis Amino Acid Permease yvbW in Response to Leucine Limitation[END_REF]. This permease is responsible for both import and export of leucine.

Interest of the NRPS peptides produced by B. subtilis

Nonribosomal peptides have diverse applications as well as being beneficial for the producer during nutritional starvation and defense mechanism. The amphiphilic lipopeptides i.e. surfactin, fengycin, iturin produced by B. subtilis are potential antimicrobial agents, as these compounds have the ability to penetrate and disrupt the cell membrane [START_REF] Duitman | The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase[END_REF]. Toll-like receptors (TLR) are transmembrane proteins, which act as therapeutic agents for the treatment of infectious diseases. Lipopeptides can behave as TLR [START_REF] Gay | Structure and function of Toll receptors and their ligands[END_REF]. Lipopeptides have been used to prepare vaccine against hepatitis B virus [START_REF] Vitiello | Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection. I. Induction of a primary cytotoxic T lymphocyte response in humans[END_REF]. The lipopeptide C 16 -KTTS can be used in skincare application as an antiwrinkle agent (Karl Lintner, 2004). Surfactin, fengycin and mycosubtilin have the potential to be used as biopesticide [START_REF] Ongena | Bacillus lipopeptides: versatile weapons for plant disease biocontrol[END_REF].

Besides their function in biocontrol, lipopeptides are involved in bacterial motility and swarming behavior as well as helps in attachment to surfaces [START_REF] Raaijmakers | Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics[END_REF].

SURFACTIN: EXPLORING FROM GENE TO PRODUCT

Discovery

Arima et al. [START_REF] Arima | Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation[END_REF]) isolated an extracellular compound from the growth medium inoculated with B. subtilis. Later in the same year, Kakinuma et al. (Kakinuma et al., 1968) elucidated the structure of this compound that was named as surfactin. Surfactin is an amphiphilic cyclic lipopeptide consisting of seven amino acids (Glu-Leu-Leu-Val-Asp-Leu-Leu) in its peptide moiety with a chiral conformation of LLDLLDL for the respective amino acids.

Structure

Surfactin is a branched lactone lipopeptide while the cyclic heptapeptide is linked to βhydroxy fatty acids with chain length 12-16 carbon atoms. Surfactin has a "horse saddle"

topology also known as β-sheet structure. The fatty acid chain can be in n, iso or anteiso configuration while the most predominant chain lengths are C14 and C15. The former is present mainly in iso-form or a mixture of iso/n while the latter is predominant in anteisoform and iso-form is present in fewer amounts [START_REF] Kowall | Separation and Characterization of Surfactin Isoforms Produced by Bacillus subtilis OKB 105[END_REF]. The peptide moiety, comprises hydrophobic amino acids at positions 2, 3, 4, 6 and 7 while polar amino acids are at the remaining positions, thus imparting two negative charges to the molecule (Figure 20).

CONFIDENTIAL DOCUMENT

The three-dimensional structure of surfactin was established using 1 H NMR studies along with molecular imaging. This study revealed the presence of polar residues like glutamic acid (Glu 1 ) and aspartic acid (Asp 5 ) on opposite sides of the molecule. Hydrophobic amino acid (Val 4 ) faces the lipidic chain, which forms the major hydrophobic domain as well as Asp 5 and Leu 3 forms the side chain of Val 4 . In addition to L-isoform amino acids, the peptide moiety also includes two residues of D-Leu at position 3 and 6 [START_REF] Bonmatin | Production, isolation and characterization of [Leu4]-and [Ile4]surfactins from Bacillus subtilis[END_REF]. Among all the amino acids in the peptide ring the D-amino acid are highly conserved and plays an important role in structural integrity (Peypoux et al., 1999a).

Surfactin has a β-sheet arrangement with a typical horse saddle arrangement, this structural feature accounts for wide range of biological activities [START_REF] Bonmatin | Solution three-dimensional structure of surfactin: a cyclic lipopeptide studied by 1H-NMR, distance geometry, and molecular dynamics[END_REF]. It has an aggregation number of ~170 and appears as rod-like micelles [START_REF] Heerklotz | Detergent-like action of the antibiotic peptide surfactin on lipid membranes[END_REF].

Properties

Surfactin are surface-active compounds that can be used as biosurfactants, which are biodegradable and effective in broad range of temperature and pH. It can reduce the surface tension (measure of surface free energy per unit area) of water from 72 mN.m -1 to 27 mN.m -1 at a concentration of 20µM (Peypoux et al., 1999a). It is the most powerful biosurfactant known till now. Surfactin which is soluble in both polar and non-polar solvents tends to precipitate at lower pH (< 6) as well as in the presence of high salt and organic compounds. In addition to lowering the surface tension, it has an effect on the interfacial rheological behavior by reducing the free energy of the system as well as can influence mass transfer. Microorganisms use this property of the biosurfactant to increase the bioavailability of nutrients through swarming on solid media by attaching and detaching from the substrate. It was observed previously that B. subtilis swarming motility is based on flagella and depends on the production of surfactin but B. subtilis 168 lacks this motility due to truncated sfp, which is essential for surfactin biosynthesis. There are various swarming motility genes i.e. swrA, swrB, swrC (yerP) and efp whose activation can lead to swarming motility in the laboratory strains. This phenomenon was observed when reversibility of the swrA mutation occurred, which enabled swarming motility in B.

subtilis 168 [START_REF] Kearns | A field guide to bacterial swarming motility[END_REF].

CONFIDENTIAL DOCUMENT

Biosynthesis

Kluge et al. (1988) suggested a multienzyme and, thiotemplate based mechanism known as non-ribosomal peptide synthesis (NRPSs) that catalyzes the biosynthesis of surfactin. A strong regulated operon srfA (25kb) is responsible for the synthesis of surfactin [START_REF] Hamoen | Controlling competence in Bacillus subtilis: shared use of regulators[END_REF]. whereas the activation, transfer and carboxythioester bond formation is performed by the respective domains of each domain (Seydlová and Svobodová, 2008). The step-wise biosynthesis mechanisms for the synthesis of non-ribosomal peptides have been discussed

in the previous section. The protein SrfAA gene is responsible for the activation of Glu, Leu and Leu; SrfAB contains the activation domain for Val and Asp, SrfAC is responsible for incorporation of L-Leu and SrfAD encodes protein, which is structurally similar to thioesterases and belongs to the family of α/β hydrolase (Figure 21).

The initial ORFs (SfAA and SrfAB) are responsible for the elongation through multiple thioester bond cleavage and transpeptidation reactions simultaneously. Besides normal L-isoform amino acids, there are two D-Leu residues. These residues are isomerized by the epimerization (E) domain adjacent to C domain present in both the subunit (Peypoux et al., 1999a). The ORF SrfAC is responsible for the condensation reaction of the last amino acid as well as release of the final lipoheptapeptidyl intermediate. The final reaction for peptide release is contributed by the thioesterase domain (TE) which is fused to SrfAC by intramolecular lactonization involving a nucleophilic reaction as shown in Figure 22. The macrolactonization process is a stereo-and regiospecific reaction involving the hydroxyl group of the fatty acid and oxygen of 3-(R)-3-hydroxyacyl heptapeptidyl thioester on the Leu 7 carbonyl group [START_REF] Bruner | Structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain SrfTE[END_REF].

Figure 22

Modular and domain representation for surfactin biosynthesis [START_REF] Bruner | Structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain SrfTE[END_REF] The last ORF SrfAD (40 kDa) plays a crucial role of initiation reaction in the biosynthesis mechanism. This protein initiates the biosynthesis mechanism through the formation of βhydroxyacyl-glutamate by transferring the β-hydroxy-fatty acid substrate to the Glumodule [START_REF] Steller | Initiation of surfactin biosynthesis and the role of the SrfD-thioesterase protein[END_REF]. This protein also consists of a thioesterase activity (TE), which plays the role of proof-reading and eliminates incorrect transfer of Ppant in the reaction module of the synthetase [START_REF] Schwarzer | Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases[END_REF]. Thus, it can be concluded that synthesis of surfactin requires two bond formations at the extreme ends of the peptide ring:

i) carboxyl group of the fatty acid is attached through an amide bond to the amino group of Glu 1 ; ii) hydroxyl group of the fatty acid is linked through an ester bond to the carboxyl group of Leu 7 .

A comparative study between the activation domain of Surfactin synthetase and Gramicidin S synthetase was carried out. This study revealed that the clusters of amino acid residues responsible for identifying Leu, or Val are similar in all domains as well as Glu-binding region was similar to Asp-and Orn-binding region. It was also found that the Pro-binding domain is also related to Phe-binding domain [START_REF] Cosmina | Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis[END_REF]. This might be the reason for the variability in the surfactin or lipopeptide structure giving rise to various isoforms.

Table 3 Various isoforms of surfactin based on its peptidic moiety along with surfactin (S) native structure

The adenylation (A) domain present in SrfAA and SrfAB for the activation of L-Glu, L-Leu, L-Asp and L-Leu are highly specific and in vitro studies revealed that the A domain for other positions i.e. 2, 4 and 7 can accept different aliphatic amino acids as substrates (Jacques, 2011). However for the thioesterase domain, it was found that an alteration in the amino acid at positions 2, 3, 4 and 5 are tolerated while position Glu in position 1, and Leu in position 6 and 7 are essential for its acceptance of the substrate [START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF]. Although a study based on structural content of peptide moiety revealed that position 1, 3, 5 and 6 denoting Glu, Leu, Asp and Leu are usually conservative in nature and the amino acid present in position 2, 4 and 7 are replaceable [START_REF] Bonmatin | Diversity among microbial cyclic lipopeptides: iturins and surfactins. Activity-structure relationships to design new bioactive agents[END_REF].

There can be variation from the native peptidic structure of surfactin giving rise to various isoforms as shown in Table 3 .

Regulators

The genetic loci mainly responsible for the synthesis of surfactin are sfp and srfA.

Surfactin operon (srfAA-AD) expression is regulated by various genes belonging to different phase of microbial growth. The most essential gene regulating its production is sfp, this gene encodes an enzyme (224 amino acids) related to the superfamily of 4'phosphopantetheinases. Both sfp and srfA are related genetically but are expressed by different genetic loci. It appeared that Sfp regulates the surfactin biosynthetic pathway post-translationally [START_REF] Cosmina | Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis[END_REF]. It acts as a swinging arm between the A and C domain. It is also responsible for the conversion of apo-synthetase to holo-synthetase in B.

subtilis 168 and triggers surfactin production [START_REF] Lambalot | A new enzyme superfamily -the phosphopantetheinyl transferases[END_REF].

During the transition phase, srfA expression is induced by two-component system ComP-

ComA based on quorum sensing mechanisms triggered by various pheromones. During stationary phase, the expression is regulated by SpoOA-AbrB based on nutritional availability. Another competence related gene comS, which is located within srfA operon but out of reading frame also regulates its expression [START_REF] Hamoen | Controlling competence in Bacillus subtilis: shared use of regulators[END_REF]. It was found that a null mutant of spoOH (encoding σ H ) resulted in a 50% decrease in β-galactosidase activity which relates to srfA expression in comparison to spo + cells. Thus, it can be inferred that σ H According to the is responsible for the srfA regulation through promoter expression [START_REF] Nakano | Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis[END_REF]. SpoOK has negative influence on srfA expression as influx of PhrC is prevented which leads to inhibition of ComA by RapC as this inhibition can be blocked by PhrC [START_REF] Solomon | Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis[END_REF]. Several other transcriptional factors i.e., DegU, PerR and

CodY have an influence over srfA expression [START_REF] Serror | CodY is required for nutritional repression of Bacillus subtilis genetic competence[END_REF]) [START_REF] Duitman | Novel methods for genetic transformation of natural Bacillus subtilis isolates used to study the regulation of the mycosubtilin and surfactin synthetases[END_REF]. Surfactin self resistance and efflux is contributed by yerP, which has a highest expression at the end of logarithmic phase. Although there is a lack of direct evidence, it was observed that null mutant strain of yerP showed growth inhibition at a concentration of 100µg/ml of surfactin as well as surfactin production was decreased in comparison to control. The expression of this gene only depends on the growth phase and not on the extracellular surfactin concentration. YerP have homology to RND (resistance, nodulation and cell division) family, which can use a proton-motive force efflux pump for the secretion of surfactin. These families of proteins are responsible for oligosaccharides transport, export of heavy metals and solvent tolerance (Tsuge et al., 2001). Recently, a study carried with B. subtilis THY-7 a surfactin producer, revealed that ycxA, a gene located downstream srfA operon might be a putative lipopeptide exporter [START_REF] Li | Overexpression of specific proton motive force-dependent transporters facilitate the export of surfactin in Bacillus subtilis[END_REF] .

Structure and activity relationship

Owing to its amphiphilic nature, it can form dimer with phospholipid bilayer and thus can destabilizing membrane integrity [START_REF] Carrillo | Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin[END_REF]. It can interact with the membrane via hydrophobic interactions, thus affecting the order of hydrocarbon chain as well as membrane thickness [START_REF] Maget-Dana | Interactions of surfactin with membrane models[END_REF]. Its interaction with phospholipid bilayer is concentration dependent. At low concentration it can penetrate the cell membrane and form mixed micelles with phospholipids; moderate level leads to the formation of ion conducting pores in the membrane through segregation of bilayer; further increase in concentration can result in disruption of membrane displaying its detergent nature [START_REF] Grau | A study on the interactions of surfactin with phospholipid vesicles[END_REF]. Below critical micelle concentration (CMC), the lipidic moiety extends freely in solution but above CMC, the lipidic moiety involves in hydrophobic interaction as micelle or oligomers at the interface of two phases (Peypoux et al., 1999a).

The fatty acid branching types have an impact on the surfactant property of surfactin. This property is enhanced in the order normal > iso > anteiso. Based on fatty acid chain length, it was found that increase in chain length leads to an increment in hydrophobicity by a certain factor thus increasing the anti-viral activity. The anti-viral activity differs on the basis of the fatty acid chain length; it was found that C13 fatty acid surfactin isoform showed very less antiviral property in comparison to C14 and C15 isoforms. Monomethyl ester of surfactin C15 was able to inactivate Semliki Forest Virus (SFV) while it was resistant to surfactin isoform mixture [START_REF] Kracht | Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives[END_REF]. However, it was found that the surfactant activity of nC 14 was higher in comparison to iso or anteiso C 15

The presence of acidic residues within the peptide moiety can assist in membrane penetration. Glutamic acid and Aspartic acid form a claw shaped structure which stabilizes (Peypoux et al., 1999a).

CONFIDENTIAL DOCUMENT

surfactin-Ca +2

The nitrogen source within the nutrient determines the amino acid occurrence in the peptide ring. The position 4 within the peptide ring has most substrate variation. The use of L-leucine or L-isoleucine leads to 5-10% production of [Leu complex thus enabling deeper penetrations into the membrane. It can form complex with both monovalent as well as divalent cations, but presence of divalent cations drives complete neutralization [START_REF] Maget-Dana | Working outside the protein-synthesis rules: Insights into nonribosomal peptide synthesis[END_REF]. When position 2 is occupied by isoleucine in the surfactin variant [Ile2, Ile4, Ile7], It enhances the affinity for calcium in comparison to leucine in position 2 in the variant [leu2, Ile4, Ile7] as former conformation increases the accessibility of the cation for the acidic residues in comparison to the latter conformation [START_REF] Kowall | Separation and Characterization of Surfactin Isoforms Produced by Bacillus subtilis OKB 105[END_REF]. This cation chelation property enables surfactin to inhibit cyclic AMP phosphodiesterase activity [START_REF] Hosono | Acylpeptides, the inhibitors of cyclic adenosine 3',5'monophosphate phosphodiesterase. I. Purification, physicochemical properties and structures of fatty acid residues[END_REF].

The presence of both isoforms of amino acids (L-and D-) assists surfactin to have specific interaction with its cellular target (Seydlová and Svobodová, 2008). The haemolytic activity of surfactin can be enhanced by esterification of its anionic residues within peptide moiety [START_REF] Dufour | Hemolytic activity of new linear surfactin analogs in relation to their physico-chemical properties[END_REF]. It was observed that esterification of the glutamate residue resulted in 100% haemolysis at a concentration of 12 µM in comparison to 200 µM of nonesterified surfactin [START_REF] Kowall | Separation and Characterization of Surfactin Isoforms Produced by Bacillus subtilis OKB 105[END_REF]. But esterified derivatives show decrease in anti-tumoral activity [START_REF] Liu | Production and characterization of a C15-surfactin-O-methyl ester by a lipopeptide producing strain Bacillus subtilis HSO121[END_REF]. Methylation of the acidic residues enhanced the oil displacement activity by 20% as well as acid tolerance was increased but the anti-viral activity was lost [START_REF] Shao | Structural diversity of the microbial surfactin derivatives from selective esterification approach[END_REF]. Both mono-and di-methylated derivative of surfactin showed decrease and null anti-inflammatory activity respectively [START_REF] Tang | Characterization and online detection of surfactin isomers based on HPLC-MS n analyses and their inhibitory effects on the overproduction of nitric oxide and the release of TNF and IL-6 in LPS-induced macrophages[END_REF]. surfactin [START_REF] Menkhaus | Structural and functional organization of the surfactin synthetase multienzyme system[END_REF]. Presence of amino acid in position 4 can increase the hydrophobicity of surfactin in the order Ala < Leu < Val < Ileu which enhanced the surfactant nature of [Ile 4 ] surfactin variant. Replacement of hydrophobic amino acid in position 4 (L-Val) can result in a decline in CMC but an increase in monolayer stability at the air/water interface [START_REF] Bonmatin | Production, isolation and characterization of [Leu4]-and [Ile4]surfactins from Bacillus subtilis[END_REF]. Activity comparison based on fatty acid chain length, C14 shows high surface activity whereas the presence of C15 can increase the antiviral and hemolytic activity [START_REF] Bonmatin | Diversity among microbial cyclic lipopeptides: iturins and surfactins. Activity-structure relationships to design new bioactive agents[END_REF]. Surfactin isomers with Leu/Ile in position 7 in comparison to Val in the same position have strong inhibitory effect on the release of IL-6 as well as nitric oxide overproduction on LPS-induced murine macrophage cell RAW264.7 [START_REF] Tang | Characterization and online detection of surfactin isomers based on HPLC-MS n analyses and their inhibitory effects on the overproduction of nitric oxide and the release of TNF and IL-6 in LPS-induced macrophages[END_REF].

The surfactin variant [ΔAsp 5 ] has higher haemolytic activity, which led to an enhancement of antimicrobial activity but no zone of inhibition was found for filamentous fungi. Similar studies with [ΔLeu 3 ] and [ΔLeu 6 ] resulted in a decline of haemolytic activity in comparison to surfactin but both the variants inhibited filamentous fungi Fusarium moniliforme ATCC3893 with MICs of 200µg/ml and 50µg/ml respectively [START_REF] Jiang | Identification of novel surfactin derivatives from NRPS modification of Bacillus subtilis and its antifungal activity against Fusarium moniliforme[END_REF].

Analysis

Surfactin production can be detected through various methods, which include: i) culture dependent method which comprise haemolytic activity assay, blue agar plate method, atomized oil assay; ii) surface activity methods i.e. cell surface hydrophobicity, surface/interfacial tensions etc iii) on the basis of structure (TLC, HPLC, MS, ECI-MS, MALDI-ToF, NMR) [START_REF] Plaza | Detection of biosurfactants in Bacillus species: Genes and products identification[END_REF]. The most commonly used method for separation and analysis of surfactin is RP-HPLC as this method can easily discriminate between various homologs of surfactin with different hydrophilicity. Amino acids present within the peptide moiety based on their hydrophobic properties interact with the mobile phase and the portion with higher hydrophobicity will be eluted at the end i.

Application

Surfactin has a wide range of application due to its structural arrangement and existence of its monomers in different stereoscopic arrangement as well as presence of fatty acid chain in various lengths. Surfactin has antiviral activity, it can inhibit fibrin clot formation, due to its interaction with lipid bilayer it can form ion channels, it can hinder the activity cyclic adenosine mono phosphate as well as it displays antitumor activity (Seydlová and Svobodová, 2008). Surfactin production helps in pellicle formation for the producer strain at the air/water interface [START_REF] Chollet-Imbert | Differentiated pellicle organization and lipopeptide production in standing culture of Bacillus subtilis strains[END_REF], enable swarming [START_REF] Debois | In situ localisation and quantification of surfactins in a Bacillus subtilis swarming community by imaging mass spectrometry[END_REF] as well as biofilm formation on roots [START_REF] Bais | Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[END_REF]. Surfactin helps in biofilm formation which helps in communal development and survival in natural habitat [START_REF] Stanley | Environmental signals and regulatory pathways that influence biofilm formation[END_REF]. The haemolytic behavior of surfactin is the sole drawback characteristics for its medical application. Although it shows haemolytic behavior at concentrations of 40 -60µM [START_REF] Noudeh | Investigation of cellular hydrophobicity and surface activity effects of biosynthesed biosurfactant from broth media of PTCC 1561[END_REF], it can completely inhibit mycoplasma growth and proliferation of human colon cancer cells at a concentration of 25µM and 30µM respectively [START_REF] Kim | Surfactin from Bacillus subtilis displays antiproliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression[END_REF]. It can also be used for various environmental applications due to its strong surface and emulsification properties. The application includes bioremediation through degradation of hydrocarbons in contaminated sites [START_REF] Whang | Application of rhamnolipid and surfactin for enhanced diesel biodegradation--effects of pH and ammonium addition[END_REF]; for microbial enhanced oil recovery [START_REF] Schaller | Characterization of surfactin from Bacillus subtilis for application as an agent for enhanced oil recovery[END_REF]; heavy oil transportation [START_REF] Ghojavand | Production and properties of a biosurfactant obtained from a member of the Bacillus subtilis group (PTCC 1696)[END_REF]; xylene water decontamination [START_REF] Etchegaray | Production of Bacillus amyloliquefaciens OG and its metabolites in renewable media: valorisation for biodiesel production and p-xylene decontamination[END_REF], it also stimulates metal remediation by increasing nutrient availability [START_REF] Mulligan | Environmental applications for biosurfactants[END_REF].

In the following paragraphs, the application of surfactin are categorically explained.

Anti-inflammatory activity

Studies have revealed that surfactin can reduce the impact of inflammatory agent lipopolysaccharide (LPS) on eukaryotic cells. It suppresses LPS activity by inhibiting the expression of IL-1β and Inos (Hwang et al., 2005) and repress LPS-binding protein (LBP) interaction with lipid A thus hindering the transfer of LPS to its receptor [START_REF] Yoneda | Inhibition of lipopolysaccharide activity by a bacterial cyclic lipopeptide surfactin[END_REF]. It was also found that it reduces the levels of nitric oxide, TNF-α and plasma endotoxin in rats due to septic shock [START_REF] Hwang | Lipopolysaccharide-binding and neutralizing activities of surfactin C in experimental models of septic shock[END_REF].

Anti-microbial and Anti-viral activity

Among all the lipopeptides produced by NRPS mechanism, surfactin, iturin and fengycin have wide range of antifungal targets. Surfactin was active against rubberwood sapstain fungus, Lasiodiplodia theobromae as it can induce plasomolysis, shrinkage and lysis of fungal mycelium [START_REF] Sajitha | Identification and Characterization of Lipopeptides from Bacillus subtilis B1 Against Sapstain Fungus of Rubberwood Through MALDI-TOF-MS and RT-PCR[END_REF].

Mycoplasma is a causative agent for urogenital tract diseases, respiratory inflammation and also can act as a cofactor in AIDS. Antimycoplasmal activity of surfactin is more desirable in comparison to conventional antibiotics as conventional antibiotics have cytotoxic effects on cells as development of resistant cells. At higher concentrations it can cause leakage in plasma membrane which leads to influx of medium thus resulting in complete disintegration of the membrane. Surfactin has the ability to inhibit mycoplasma cells with MIC of 25µM independent of mycoplasma concentration [START_REF] Shaligram | Surfactin -a review on biosynthesis, fermentation, purification and applications[END_REF]. Due to low toxicity to mammalian cells, surfactin allows high specificity of inhibition of mycoplasma. Fassi et al. showed that when surfactin was used in combination with enrofloxacin it showed two times more activity in comparison to molecules used separately [START_REF] Fassi Fehri | Activities of antimicrobial peptides and synergy with enrofloxacin against Mycoplasma pulmonis[END_REF].

Surfactin has been found to inhibit biofilm formation for pathogenic organism by preventing them from adhering to solid surfaces or infection sites or on cathetars. These pathogenic organisms include S. typhimurium, S. enterica, E. coli and P. mirabilis. It was found that surfactin at concentrations between 5-50 mg/l can inhibit biofilm formation of Salmonella on catheter [START_REF] Mireles | Salmonella enterica serovar typhimurium swarming mutants with altered biofilm-forming abilities: surfactin inhibits biofilm formation[END_REF]. It can also restrict growth of P. syringae on roots [START_REF] Bais | Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[END_REF]. Surfactin surface-active property enabled the producing organism to spread over wood blocks after one1 week of inoculation even for inhibiting the growth of Lasiodiplodia theobromae [START_REF] Sajitha | Identification and Characterization of Lipopeptides from Bacillus subtilis B1 Against Sapstain Fungus of Rubberwood Through MALDI-TOF-MS and RT-PCR[END_REF].

Anti-viral activity of surfactin includes inactivation of membrane integrity by incorporating into lipid bilayer leading to disintegration of the envelope and it was found that surfactin was more efficient against enveloped viruses than non-enveloped viruses.

The reason behind such activity is the fact that antiviral activity depends on viral envelope lipid composition as well as glycoproteins encoded by the virus. The activity can be against wide range of viruses i.e. Semliki Forest Virus, herpes simplex virus, feline calicivirus, murine encephalomyocarditis virus, etc. [START_REF] Vollenbroich | Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis[END_REF]. Anti-viral study was carried out on three viruses: vesicular stomatitis virus (VSV), suid herpes virus type 1 (SHV) and Semliki Forest Virus (SFV). It was found that C13-surfactin has very less anti-viral activity only against SHV while C14-and C15-surfactin were highly active against both SHV and VSV. But all the three isoforms were inactive against SFV. When the isoforms were esterified and added in equimolar amount, it was found that C14 and C15 monomethyl ester were active against both SHV and VSV. While in the case of Semliki Forest Virus, it was observed that the monomethyl C14 and C15 showed anti-viral activity but no activity was observed for non-esterified surfactin mixture [START_REF] Kracht | Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives[END_REF].Thus, it can be concluded that the anti-viral activity depends on fatty acid chain length (hydrophobicity), peptide moiety charge as well as on virus species.

Biomedicine

It was reported that surfactin can act as antitumor agent against Ehrlich's ascite carcinoma cells [START_REF] Kameda | Antitumor activity of bacillus natto. V. Isolation and characterization of surfactin in the culture medium of Bacillus natto KMD 2311[END_REF]. It can inhibit human colon carcinoma cell proliferation by inducing apoptosis as well as by suppressing the signals of cell cycle [START_REF] Kim | Surfactin from Bacillus subtilis displays antiproliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression[END_REF].

It can inhibit fibrin clot formation both in vitro and in vivo by inhibiting the conversion of fibrin monomer to its respective polymer [START_REF] Arima | Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation[END_REF]. It increases the activity of prourokinase as well as changes the conformation of plasminogen at a concentration of 3-20 µM. It can also avoid platelet aggregation [START_REF] Kikuchi | Enhancement of plasminogen activation by surfactin C: augmentation of fibrinolysis in vitro and in vivo[END_REF]. This activity of surfactin is due to its effect on the downstream signaling pathways [START_REF] Kim | Surfactin C inhibits platelet aggregation[END_REF].

Surfactin display a notable antihypercholesterolemic activity [START_REF] Arima | Surfactin[END_REF].

Biocontrol

Around 50% of the biopesticides in world market are produced industrially by species of Bacillus [START_REF] Fravel | Commercialization and implementation of biocontrol[END_REF]. They oppose pathogens for niche and nutrients as well as can activate defensive mechanism of the host plant and direct them to produce antimicrobial compounds. B. subtilis can produce wide variety of bioactive compounds and surfactin is among one of those compounds. These compounds are highly promising in comparison to chemical counterparts for agricultural application because of their high biodegradability, low toxicity and eco-friendly properties. These compounds can hinder the growth of phytopathogens and pests causing plant diseases [START_REF] Ongena | Bacillus lipopeptides: versatile weapons for plant disease biocontrol[END_REF]).

Surfactin's role as biocontrol can be subdivided into two categories; firstly, its role to stimulate systemic resistance in plants [START_REF] Jourdan | Insights into the defense-related events occurring in plant cells following perception of surfactin-type lipopeptide from Bacillus subtilis[END_REF] and secondly, as a factor to enhance colonizing efficacy as plant growth-promoting rhizobacteria (PGPR) [START_REF] Gao | Swarming motility plays the major role in migration during tomato root colonization by Bacillus subtilis SWR01[END_REF].

Basal immune response in plants is triggered upon recognition of microbial motifs known as pathogen-associated molecular patterns (PAMPs). These motifs are generally recognized by high affinity pattern recognition receptors (PRR) located in the plasma membrane of plants [START_REF] Henry | The bacterial lipopeptide surfactin targets the lipid fraction of the plant plasma membrane to trigger immune-related defence responses[END_REF]. These receptors consist of extracellular ligandbinding domain, single transmembrane domain and an intracellular kinase-signaling domain. Induced systemic resistance is triggered by a local stimulus by a beneficial isolate/compound which results in extension of resistance in all the organs of the plant.

CONFIDENTIAL DOCUMENT

Surfactin can trigger defense-related responses in tobacco suspension through ROS accumulation both in the cytoplasm and extracellular environment. ROS formation is induced through plasma-membrane associated NADPH oxidase quenched either by cell wall or extracellular peroxydases. At a concentration of 20µM of surfactin, no cell death was observed but can develop signaling process which can activate biochemical pathways leading to development of defensive response [START_REF] Henry | The bacterial lipopeptide surfactin targets the lipid fraction of the plant plasma membrane to trigger immune-related defence responses[END_REF]. Surfactin can turn on oxylipin pathway in plants, which can produce various biologically active secondary metabolites resulting from unsaturated fatty acids. These metabolites may accumulate at the infection site and inhibit pathogen entry. Surfactin can induce the production of other secondary metabolites which resemble to phenylpropanoids deriving from PAL enzyme.

The plasma membrane requires specific composition and/arrangement of the lipid bilayer for the perception of surfactin. Surfactin is recognized as PAMPs at the cell surface and not as effector molecule transported into the cell. However in comparison to PAMPs which are recognized in nanomolar concentration, surfactin is needed in micromolar range [START_REF] Jourdan | Insights into the defense-related events occurring in plant cells following perception of surfactin-type lipopeptide from Bacillus subtilis[END_REF].

B. subtilis can stimulate plant growth and control plant diseases through root colonization.

Root colonization is facilitated through a swarming mechanism. Swarming mechanism in B. subtilis is directed by swrA operon [START_REF] Kearns | Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility[END_REF] and flagellum as well as surfactin is also required for this phenomenon. Surfactin promote swarming by reducing the surface tension between the bacterial cell and substrate as well as it favors grouping of cells into dendrites [START_REF] Kearns | A field guide to bacterial swarming motility[END_REF]. It was reported that surfactin is important for biofilm formation on Arabidopsis root surfaces by B. subtilis 6051 [START_REF] Bais | Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[END_REF]. Impairment of surfactin synthesis in mutant ΔsrfAC can obstruct swarming mechanism. which can lead to reduced root colonization efficacy [START_REF] Gao | Swarming motility plays the major role in migration during tomato root colonization by Bacillus subtilis SWR01[END_REF]. This suggest that profitable outcome of PGPR in agricultural use can be increased by boosting the swarming motility.

OVERPRODUCTION OF SURFACTIN

Present scenario

The surfactant industry is increasing by 9 million US$ every year. The surfactants mostly available in market are of petrochemical origin. The previous chapter concluded with the broad range of applications of surfactin (a biosurfactant) due to which it is gaining importance over chemical surfactants. A study was carried out to synthesize this molecule through a chemical process. Although it was revealed that the synthetic process requires multiple steps and ultimately increasing the production costs. Despite surfactin's advantage over its chemical origin counterpart, the use of surfactin for commercial application is very limited due to its low-level of production in comparison to high-level requirement in specific applications. Sigma-Aldrich markets surfactin at a price of 25.4€/mg for research purpose. Thus, the problem needs a biotechnological solution to make production cost economical and sustainable. Lipopeptide production in B. subtilis is closely linked to competence and sporulation, thus forming a three way metabolic network (section 3.5). It was inferred from various studies that surfactin production depends on rate of utilization of carbon source, oxygen availability and factors governing lipid formation (Peypoux et al., 1999b).

Role of various factors

Various strategies like optimizing the medium, using waste from agriculture or industrial origin to reduce raw material cost or bioreactor design have been utilized to reduce the production cost. Implementing various mutagenesis techniques for strain improvement has also been carried out to overproduce surfactin [START_REF] Kim | Surfactin from Bacillus subtilis displays antiproliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression[END_REF].

Nutrient composition

Various culture media are used for the production of surfactin i.e. Cooper's medium, Landy medium etc. The production of surfactin starts from exponential phase and maximum production is observed during transition phase. It has already been seen in the previous chapter (Chapter 1.3 -Regulators) that surfactin production depends on cell biomass and biomass requires proper nutrient composition for its growth. Thus, nutrient composition can have a profound effect on the surfactin production. B. subtilis ATCC 6633 can produce surfactin when grown on Landy medium [START_REF] Duitman | The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase[END_REF]. The dependence of nutrient composition is not only limited to carbon sources but also various nitrogen sources and various minerals have a significant impact on the production. An alteration of medium composition for enhancing surfactin production is an effective approach.

Carbon source: Glucose is the most preferred carbon source for surfactin production.

Mainly, glucose is used as carbon source with a concentration between 20 and 40 g/l. B.

subtilis C9 when grown in the presence of glucose, the specific surfactin yield was higher in comparison to sucrose as carbon source [START_REF] Kim | Surfactin from Bacillus subtilis displays antiproliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression[END_REF]. Various complex carbon sources i.e. diesel, starch, glycreol or feed stock have also been used for the study of surfactin production [START_REF] Etchegaray | Production of Bacillus amyloliquefaciens OG and its metabolites in renewable media: valorisation for biodiesel production and p-xylene decontamination[END_REF] [START_REF] Amin | Exponential fed-batch strategy for enhancing biosurfactant production by Bacillus subtilis[END_REF]. In the absence of carbon source surfactin production was not observed as the cells were not able to grow properly.

When cells were grown with an excess of glucose (80 g/l), the surfactin yield was significantly decreased, which can suggest that an excess of glucose can have a negative impact of the production [START_REF] Dauner | Bacillus subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture Bacillus subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture[END_REF]. Utilization of glucose as a sole carbon source can lead to a production of 80 mg/l of C 15

Nitrogen source: Cooper medium contains ammonium nitrate and classical Landy medium contains glutamic acid as nitrogen source. Both medium have been widely used for surfactin production. Thus, it can be revealed that there is no preference for nitrogen source for surfactin production. B. subtilis ATCC 21322 utilizes organic nitrogen for growth, while the inorganic source i.e. nitrate is utilized for switching on secondary metabolism. In aerobic condition, nitrate utilization can only occur when ammonium is depleted [START_REF] Davis | The production of Surfactin in batch culture by Bacillus subtilis ATCC 21332 is strongly influenced by the conditions of nitrogen metabolism[END_REF]. When a derivative strain of B. subtilis 168 was grown in Landy medium for 24 hours in Biolector -surfactin from B.amyloliquefaciens MB199 (Liu et al., 2012).

® device (48-well microtiter Flowerplates), it was observed that higher concentrations of glutamic acid can have a positive impact on surfactin production [START_REF] Mootz | An improvement of surfactin production by Bacillus subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor[END_REF]. When the nitrogen source of the cooper medium was substituted from NH 4 Cl to (NH 4 ) 2 SO 4 , an enhancement of surfactin production was observed [START_REF] Willenbacher | Enhancement of Surfactin yield by improving the medium composition and fermentation process[END_REF]. Addition of (NH 4 ) 2 SO 4 enhanced the C 15 -surfactin production from B.amyloliquefaciens MB199 upto 60 mg/l (Liu et al., 2012). Wie et al (2004) found that iron and manganese can boost the production of surfactin. It was determined that a critical molar ratio of 920:7.7:1 for

CONFIDENTIAL DOCUMENT

nitrogen/iron/manganese is required for continuous production [START_REF] Sheppard | The response of Bacillus subtilis ATCC 21332 to manganese during continuous-phased growth[END_REF].

Other nutrients: These nutrients include magnesium, manganese, potassium, iron etc.

Higher concentration of both Fe 2+ and Mn 2+ can stimulate surfactin production but the former can produce phthalic acid which can decrease the pH and surfactin solubility [START_REF] Wei | Identification of induced acidification in iron-enriched cultures of Bacillus subtilis during biosurfactant fermentation[END_REF], while the latter requires cooperation from nitrogen source and other essential factors to have a positive effect [START_REF] Wei | Using Taguchi experimental design methods to optimize trace element composition for enhanced surfactin production by Bacillus subtilis ATCC 21332[END_REF]. However addition of 4 mM of Fe 2+

along with pH adjustment can enhance the surfactin production to 3 g/l in minimal salt medium with 40 g/l glucose using B. subtilis ATCC 21322 [START_REF] Wei | Optimizing iron supplement strategies for enhanced surfactin production with Bacillus subtilis[END_REF]. When the same strain was cultivated in the presence of Mn 2+ concentration (0.001 to 0.1 mmol/l), the maximum production of 1.5 g/l of surfactin was obtained with 40 g/l of glucose. Upon increasing the Mn 2+ concentration to higher than 0.05 mmol/l, a shift of nitrogen utilization was observed as nitrate became the main nitrogen source instead of ammonium. Addition of Mn 2+ increases both glucose usage and nitrate reductase activity. The increase in nitrate reductase activity supports the nitrate utilization efficacy of the cells, thus leading to highest surfactin production during nitrate-limited conditions. Glutamate synthase (GOGAT) which is responsible for the synthesis of glutamate, the activity of this enzyme was enhanced in the presence of manganese ions. This process led to the generation of free amino acids for surfactin synthesis [START_REF] Huang | Surfactin C inhibits the lipopolysaccharide-induced transcription of interleukin-1beta and inducible nitric oxide synthase and nitric oxide production in murine RAW 264.7 cells[END_REF]. In the presence of glucose (40 g/l), both Mg 2+ and K + ions can have significant impact on B. subtilis ATCC 21322 for surfactin production. Upon decreasing the magnesium concentration from 0.6 mM to 0.04 mM, the surfactin concentration decreased from 0.69 g/l to 0.16 g/l. When K + concentration was increased from 0 to 10 mM, both increase in cell growth and surfactin concentration were observed. Maximum surfactin production of 3.34 g/l was obtained with potassium concentration of 10 mM. According to Taguchi and statistical analysis, it was found that Mg 2+ and K + ions play a greater role in surfactin production in comparison to Mn 2+ and Fe 2+ . The impact of Mg 2+ and K + can affect the cell as the former is a cofactor of Sfp protein which is essential for surfactin production, while the latter helps in surfactin secretion [START_REF] Wei | Using Taguchi experimental design methods to optimize trace element composition for enhanced surfactin production by Bacillus subtilis ATCC 21332[END_REF].

Oxygenation

Dissolved oxygen and mechanical agitation plays a determining role for surfactin production. Surfactin production is highly linked to oxygen volumetric mass transfer coefficient (K L a) under various agitation and aeration rate. The highest surfactin production (7g/l) was obtained in the foam using B. subtilis C9 in the presence of Cooper's nitrogen source and oxygen limiting conditions [START_REF] Kim | Surfactin from Bacillus subtilis displays antiproliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression[END_REF] but B. subtilis S499 requires high aeration condition (Jacques, 2011). Maximum surfactin production was obtained at a K L a value 47.52 h -1 . Since surfactin is a growth associated product, the production rate is directly proportional to the specific growth rate of the cell. So, aerobic bacteria like B. subtilis needs adequate oxygen concentration for growth and product i.e.

surfactin formation. At a fixed aeration rate, K L

The volumetric oxygen transfer co-efficicent in Biolectorwith each filling ratio was calculated with the following equation, a can be increased on increasing the agitation rate in bioreactor or flask culture. At a low aeration rate of 0.5 vvm, maximum surfactin production increased 1.5 folds on increasing the agitation rate from 200 rpm to 350 rpm during batch fermentation [START_REF] Yeh | Bioreactor design for enhanced carrier-assisted surfactin production with Bacillus subtilis[END_REF]. Thus, in a batch mode both aeration and agitation rates have a great impact on cell growth and surfactin production. subtilis (Jacques, 2011). As in shaking flasks, the overall oxygen absorption rate depends on the replacement of thin liquid layer or fresh liquid surface generation. It was found that poor oxygenation has a detrimental effect on surfactin production while K L a from 10.8 to 288 h -1 have beneficial impact on surfactin production. It was observed that a K L a > 216 h - 1 can significantly enhance the surfactin production of a derivative strain of B. subtilis ATCC 21322 (Fahim et al., 2012a). Based on filling volume, it was found that when the filling volume was increased there was a significant decrease in the surfactin production by B. subtilis ATCC6633 while the filling volume did not show significant impact on the production by a derivative strain of B. subtilis ATCC6633, where the native promoter was replaced by a constitutive one [START_REF] Guez | Respiration activity monitoring system (RAMOS), an efficient tool to study the influence of the oxygen transfer rate on the synthesis of lipopeptide by Bacillus subtilis ATCC6633[END_REF]. Lipopeptides especially surfactin can decrease K L a owing to its high surface activity as it forms a layer of stable film in the interfacial area prohibiting oxygen transfer into the medium [START_REF] Hbid | Influence of the production of two lipopeptides, iturin a and surfactin s1, on oxygen transfer during Bacillus subtilis fermentation[END_REF]. It can be hypothesized that if the inner diameter of the flask can be increased this surface activity of surfactin can be reduced, which can lead to higher K L a and thus increase the surfactin production.

Strain improvement

A surfactin overproducing mutant strain from a wild type B. subtilis (B.s-E-8) strain was obtained using ion beam implantation. This strain was cultivated in a modified bioreactor with cell/foam recycler. The surfactin production reached to a maximum value of 12.20 g/l after 32h cultivation (Gong et al., 2009). An improvement of 4-6 times and 4-25 times was obtained using random mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine on B.

subtilis ATCC 55033 and B. subtilis SD901, strain respectively. Implementing UV mutagenesis generated an overproducer strain of B. subtilis ATCC 21332. This mutant known as ATCC 5138 has 30 times low isocitrate dehydrogenase activity in comparison to its parent strain and can produce 1.1g/l of surfactin [START_REF] Carrera | Aspartyl-phosphate phosphatases deactivate the response regulator components of the sporulation signal transduction system in Bacillus subtilis[END_REF].

In the following sections, various genetic engineering approaches for strain improvement for overproduction of surfactin have been discussed through direct and indirect approaches.

CONFIDENTIAL DOCUMENT

Direct approach

Direct approach outlines distinct strategies used for strain improvement through modification of the native srfA gene or replacement of the native promoter and lastly a hypothesis has been developed on the basis of sfp.

Role of promoters

The promoter forms the most important component in an expression system as it can dictate the expression level of a gene. The activity of a promoter depends on the consensus sequence in the several core regions known as UP element. This UP element consists of -35 region, -15 region, -10 region and +1 region [START_REF] Cheng | Corresponding author : Zhemin Zhou and Weijiang Li[END_REF]. Till date, there are three categories of promoters used for the overproduction of a particular molecule. This includes autoinducible promoter, constitutive promoter and inducer specific promoter.

Autoinducible promoters can trigger expression of target gene based on the growth phase (late log phase to stationary phase) in the absence of an inducer. The promoters under this category includes P pst , P rpsF and P ppsA [START_REF] Cheng | Corresponding author : Zhemin Zhou and Weijiang Li[END_REF]. Constitutive promoters are those promoters which can activate expression of a gene irrespective of growth phase or any stimulus. P veg [START_REF] Lam | Construction of an efficient Bacillus subtilis system for extracellular production of heterologous proteins[END_REF], P repU (Coutte et al., 2010), P43 [START_REF] Ye | High-level secretory production of intact, biologically active staphylokinase from Bacillus subtilis[END_REF], P secA [START_REF] Herbort | Temporal expression of the Bacillus subtilis secA gene, encoding a central component of the preprotein translocase[END_REF], P yxiE (Zhang et al., 2007) etc. comes under this group of promoters. Lastly, the inducer specific promoters require inducer for the expression of gene under their control. This promoter is widely used for the production of various biological molecules but is not cost effective. The examples of this promoter are spac promoter, T7 promoter, groES-groEL promoter ,driven by IPTG; xylA induced by xylose [START_REF] Cheng | Corresponding author : Zhemin Zhou and Weijiang Li[END_REF]. Surfactin production of 50 g/l was obtained with B. subtilis strain having exogenous lpa-14 gene. The culture was carried out in a fermentation vessel and the surfactin was analyzed in the liquid medium without the removal of foam (Tadashi [START_REF] Yoneda | Inhibition of lipopolysaccharide activity by a bacterial cyclic lipopeptide surfactin[END_REF]. Replacement of native promoter with a constitutive promoter, P repU (Coutte et al., 2010) and with an inducible promoter, P spac

The role of each type of promoters for the overproduction of surfactin have been described precisely in the following paragraphs. has been studied [START_REF] Sun | NOTE / NOTE Enhancement of surfactin production of Bacillus subtilis fmbR by replacement of the native promoter with the Pspac promoter 1006[END_REF].

CONFIDENTIAL DOCUMENT

The promoter for surfactin operon P srfA Figure 23 is an autoinducible expression system activated by signal peptides i.e. ComX and CSF (

). These signal molecules are components of quorum sensing pathway leading to competence development. The promoter's activity depending on cell density was studied using GFP fluorescence, the expression was found maximum during the transition phase, constant during stationary phase and low during early and mid-exponential phase [START_REF] Guan | Construction and development of an auto-regulatory gene expression system in Bacillus subtilis[END_REF].

The promoter consists of 607bp and regulatory regions have already been determined located at -35 region and -10 region, which is the transcriptional start site [START_REF] Nakano | Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in Bacillus subtilis[END_REF]. Its 5'-flanking region spans from -607 to -40 bp. The expression of various truncated region of the 5'-flanking region was studied in fusion with GFP. This study revealed that the 5'-flanking region (-136 to -76) showed significant reduction in the GFP fluorescence intensity, which determines its significance for the expression of the promoter. The maximum expression was obtained with the mutant having the region (-136 -+291) in comparison to wild type and this promoter with this sequence (AATGTT……A……ATGACAATG) was termed as P04. Site-directed mutagenesis was carried out in the region between -35 and -10 of the promoter P04 (GTGATAAAAACATTTTTTTCATTTAAACT); this generated a promoter P10

(TTGACA) with a change in the consensus sequence at -35. The expression P10 was maximum among all derivatives of P04 as well as the expression was 1.5 times more than P04 [START_REF] Cheng | Corresponding author : Zhemin Zhou and Weijiang Li[END_REF].

Besides modification of the UP element, various studies have been performed where at least two promoters (same or different) were placed as tandem repetitive sequences upstream of the expressed gene. The productivity of TSaGT in B. subtilis was enhanced several folds by the alignment of HpaII promoter along with blma promoter or amyR2

promoter (Kang et al.,2010). Alignment of same promoter was studied by aligning

MCPtacs promoter clusters until it reached the critical number of five [START_REF] Li | Rational improvement of the engineered isobutanol-producing Bacillus subtilis by elementary mode analysis[END_REF].

This strategy enhances the window of expression of the system. [START_REF] Guan | Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis[END_REF] used two way strategies to extend the window of expression srfA operon in a non-sporulating B.

subtilis strain, where the σ F was deleted. First strategy was to link gsiB promoter to P srfA , where the former is under σ B and the latter is under σ A . This strategy did not enhance the activity in comparison to the control. Whereas the other strategy, P HpaII to P srfA , where both the promoters are under the control of σ A enhanced the activity to a significant amount.

The consensus sequence was also changed in both promoters resulting in higher expression activity in comparison to respective control [START_REF] Guan | Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis[END_REF]. Thus, it can be concluded that changing the core regions of the promoter can lead to overexpression of surfactin as well as it can help in tuning the synthetic genetic circuits. While the use of dual promoters can increase the expression window time of srfA operon.

In this section the role of constitutive promoter for the production of surfactin has been discussed. [START_REF] Radeck | The Bacillus BioBrick Box : generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis The Bacillus BioBrick Box : generation and evaluation of essential genetic building blocks for standardized work with B[END_REF]. This promoter was integrated in the upstream region of srfA operon in two strains B. subtilis 3A38 and B. subtilis DSM 10 T . The former originating from strain NCIB 3610 produces surfactin in small amount while the latter is a wild type, high surfactin producing strain. The mutant strain of 3A38 produces surfactin to a maximum concentration of 0.26 g/l (3.7 times higher than control) while the mutant of DSM 10 T could only produce a mere quantity of 0.04 g/l (15.5 times less than control). It was concluded that the surfactin biosynthesis regulation is different in dissimilar strain based on its ability to produce surfactin without modification. Introduction of the constitutive promoter in the high producer DSM 10 T Lastly in this segment, the role of inducible promoter for overproduction of surfactin has been discussed. Replacement of the native promoter by an inducible promoter was carried out in B. subtilis fmbR to generate a mutant strain fmbR-1. It was found that fmbR-1 was able to produce 1.56 g/l of surfactin without induction while with an induction of 300 led to a disturbance in quorum sensing mechanism which ultimately resulted a decline in surfactin production (Willenbacher et al., 2016a).

µmol/l of IPTG, it was able to produce 3.86 g/l and with lowest induction of 100µmol/l of IPTG fmbR-1 was able to produce 1.39 g/l of surfactin. In comparison to control, without induction, fmbR-1 produces 4 times higher while with induction 10-fold more with 300µmol/l of IPTG. Beyond 300 µmol/l of IPTG induction, the surfactin production was decreased as high induction might lead to spore formation before surfactin production. The mutant strain's antagonistic activity against B.cereus was also increased [START_REF] Sun | NOTE / NOTE Enhancement of surfactin production of Bacillus subtilis fmbR by replacement of the native promoter with the Pspac promoter 1006[END_REF].

Role of Sfp

Phosphopantetheine-transferase plays a crucial role in lipopeptide production through NRPS mechanism. Sfp encoded by genetic loci sfp, transfers 4'-phospopantetheine moiety from coenzyme A (CoA) onto the conserved serine residue of peptidyl carrier protein (PCP). In B. subtilis, there are several carrier proteins which include: acyl carrier protein (ACPs) for fatty acid synthesis, ACPs for polyketide synthesis, PCPs for nonribosomal peptide synthesis [START_REF] Walsh | Posttranslational modification of polyketide and nonribosomal peptide synthases[END_REF]. A comparative study was carried out between acyl carrier protein synthetase (AcpS) and Sfp on the basis of their specificity to transfer CoA to various protein partners ACP, DCP and PCP. AcpS was able to modify only ACP and DCP required for primary metabolism and not PCP which is required for secondary metabolism. On a contrary, Sfp recognizes all proteins and enables its modification [START_REF] Mootz | 4'-phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis[END_REF].

Overproduction of sfp gene has a negative effect on the expression of srfAA. This was reflected in a reduction of surfactin production too. Thus it can be inferred that sfp can regulate and interact directly in surfactin synthesis [START_REF] Nakano | Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in <i>Bacillus subtilis</i&gt[END_REF].

Thus, it can be hypothesized that modification of sfp or binding site of cofactor on ACP and DCP restricts its substrate specificity to PCP, and it might lead to higher Phosphopantetheine-transferase in NRPS, which results in overproduction of surfactin.

Modification of sfp promoter is required as it was found out that it is under the control of a weak constitutive promoter [START_REF] Nakano | Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in <i>Bacillus subtilis</i&gt[END_REF].

Other factors

Bacillus subtilis 168 can produce two families of lipopeptides namely surfactin and plipastatin (fengycin) (at very low concentration, under 50 mg/l) when sfp gene is integrated into its chromosome as B. subtilis harbors a mutated sfp gene. In order to direct the precursor flux towards the production of one lipopeptide, a study was carried out with disruption of ppsA gene which is responsible for plipastatin synthesis. The mutant strain produced lipopeptides 1.8 times in Landy medium and 1.03 times in Landy MOPS medium in comparison to control at 24 hours (Coutte et al., 2010).

Replacement of native A-PCP domains with A-PCP domains of different bacteria and fungus origin recognizing different amino acids lead to the production of different surfactin isoforms with variant properties, although there was a marked reduction in the production of surfactin due to the specificity of the C-domain acceptor site (Peypoux et al., 1999a).

Linkage between Direct and Indirect approaches

The expression of srfA operon is activated by a two component system ComA/ ComP and inhibited by CodY and AbrB through their binding to the promoter of srfA [START_REF] Hamoen | Controlling competence in Bacillus subtilis: shared use of regulators[END_REF].Through genome shuffling technology, an overproducing B. amyloliquefaciens mutant FMB38 strain was developed from parental strain ES-2-4 [START_REF] Zhao | Differential proteomics analysis of Bacillus amyloliquefaciens and its genome-shuffled mutant for improving surfactin production[END_REF]. The production of surfactin in the mutant strain was increased 3.5 times and 10.3 times in shaked flask and fermenter respectively. Expression analysis revealed that the srfA transcript number of mutant strain was 15.7 times higher than the parental strain. Through comparative proteomics methodology differentially expressed proteins were analyzed.

Three proteins ComA, DegU and CodY directly related to surfactin synthesis were differentially expressed. It was found that the ComA was unregulated which was obvious as ComA binds to srfAD promoting its expression and thus increasing surfactin production. Downregulation of DegU was observed. But a contradictory result was observed with the upregulation of CodY as it was found earlier that CodY acts as a transcriptional inhibitor of srfA expression (Serror and Sonenshein, 1996b) but codYknockout strain of B. subtilis 6633 showed no effect on surfactin production [START_REF] Duitman | Novel methods for genetic transformation of natural Bacillus subtilis isolates used to study the regulation of the mycosubtilin and surfactin synthetases[END_REF]. In order to enhance the surfactin production it was observed that not only enhancement of promoter activity but also overexpression of various proteins is required.

Thus proving the role of indirect approach can also lead to surfactin overproduction which includes surfactin monomer overproduced which has been discussed in the following section of the chapter.

CONFIDENTIAL DOCUMENT

Indirect approach

This approach deals with various aspects of overproduction of surfactin based on overproduction of its monomers. B. subtilis can utilize diverse carbon sources. Lipopeptide synthesis was carried out in vitro in the presence of amino acids, ATP, CoA-iso-3hydroxytetradecanoic acid [START_REF] Ullrich | Cell-free biosynthesis of surfactin, a cyclic lipopeptide produced by Bacillus subtilis[END_REF]. The expression of srfA depends upon the phosphorylation of ComA by ComP. The genetic loci pta is responsible for the conversion of acetyl-CoA to acetyl phosphate as well the reverse reaction of acka can also produce acetyl phosphate from acetate. This acetyl phosphate acts as a phosphate donor for the phosphorylation of ComA [START_REF] Kim | Involvement of acetyl phosphate in the in vivo activation of the response regulator ComA in Bacillus subtilis[END_REF]. As a result, pta and acka can indirectly activate the expression of srfA. In a surfactin overproducing strain, there was an upregulation of 2-Aminoethylphosphonate-pyruvate transaminase, which is involved in the synthesis of aspartate [START_REF] Zhao | Differential proteomics analysis of Bacillus amyloliquefaciens and its genome-shuffled mutant for improving surfactin production[END_REF]. The peptide moiety of surfactin contains aspartate, so an enhancement of aspartate can result in an increase of surfactin production.

So we can speculate that an abundance of key enzymes or specific enzymes can have a positive impact on surfactin production. Thus, it can be concluded from chapter 1 as follows:

] variant surfactin (Peypoux et al., 1999c). Leucine and valine are the precursors for odd and even iso-β amino acid and fatty acid, respectively while isoleucine is the precursor for odd anteiso-amino acid and fatty acid [START_REF] Hourdou | Fatty acid and beta-amino acid syntheses in strains of Bacillus subtilis producing iturinic antibiotics[END_REF]. When no amino acid was added to the culture medium the proportion of surfactin variants was found to be C15>C14>C13>C16 based on β-hydoxy fatty acid isoform.

Feeding with valine (1 g/l) maximum proportion of iso-C14 was observed in comparison to other surfactin variants. In the case of Leucine, maximum proportion of iso-C15 was observed in comparison to others but upon addition of Isoleucine the maximum proportion was found to be anteiso-C15 (Liu et al., 2012).

1. B. subtilis can be represented as a model for gram positive bacteria and is highly efficient for the production of high valued products.

CONFIDENTIAL DOCUMENT

2. NRPS is a RNA-independent mechanism which depends on various monomers for the assembling of various non-ribosomal peptides.

3. Surfactin is a highly-valuable end product produced by B. subtilis through NRPS mechanism with highly complex regulatory pathway.

4. Various approaches have been carried out to overproduce surfactin, but until now no evidence have been provided for surfactin overproduction based on intracellular increase of its precurssors, as for example leucine, which is one of the monomers required for non-ribosomal peptide synthesis of the petptide moety of this lipopeptide.

Here, a study was carried out based on the hypothesis that the intracellular pool of branched chain amino acids has determining effect on the production of surfactin. In this entire study, two wildtype strains of B. subtilis (BS 168 and BSB1) were used for the construction of various mutants and for the analysis of surfactin production. The strain BSB1 is a tryptophan prototrophic (tryp + ) obtained from BS168 strain, which is tryptophan auxotroph (tryp -). B. subtilis BSB1 strain (BaSysBio reference strain) was used for the transcriptomic study exposing it to various environmental and nutritional factors that it can confront in the living world [START_REF] Nicolas | Condition-Dependent Transcriptome Architecture in Bacillus subtilis[END_REF]. It can be deduced that both the strains have genomic similarity. The entire genome of BS 168 has already been sequenced and is easily accessible with accession no. NC_000964.3 (NCBI database). It possesses high specificity and efficiency of genetic transformation and is widely used as a genetic model.

57

The genomic analysis revealed, that contains two operons encoding for nonribosomal peptide synthetases: the surfactin and plipastatin operons. But it cannot produce the lipopeptides as it has mutated sfp gene, which is responsible for the conversion of aposynthetase to holo-synthetase). The sfp gene encodes a 4-phosphopantetheinyl transferase, which converts inactive apoenzyme peptide synthetases to their active holoenzyme forms by posttranslational transfer of the 4-phosphopantetheinyl moiety of coenzyme A to the synthetases. An intact sfp gene (844 bps) was transferred from B. subtilis ATCC 21322 to BS168 and BSB1 strains to convert them into lipopeptide producing strains BBG111 and BBG258 respectively (Coutte et al., 2010).

Bacillus subtilis BSB1N

Strain improvement through metabolic engineering is thought to be a well suited strategy for the overproduction of a particular metabolite. In order to carry out metabolic engineering various deletions and insertions of genes need to be carried out for appropriate flow of metabolite in a desired direction. Availability of selective markers forms one of the crucial limiting factors to carry out multiple modifications. In order to carry out multiple modifications, markerless strategies are required. There are various strategies which carry out modification in a markerless fashion i.e. temperature sensitive plasmid (pSC101 replicon) for gene replacement [START_REF] Hamilton | New method for generating deletions and gene replacements in Escherichia coli[END_REF]; gene replacement using pORI vectors [START_REF] Leenhouts | A general system for generating unlabelled gene replacements in bacterial chromosomes[END_REF]; gene replacement using auxotrophy based method [START_REF] Brans | New integrative method to generate Bacillus subtilis recombinant strains free of selection markers[END_REF]; Cre/lox system for genome engineering [START_REF] Yan | Cre/lox system and PCR-based genome engineering in Bacillus subtilis[END_REF].

Finally, a strategy based on Pop-in Pop-out technique using alternative neomycin and phleomycin selective marker [START_REF] Tanaka | Building the repertoire of dispensable chromosome regions in Bacillus subtilis entails major refinement of cognate largescale metabolic model[END_REF] was found impressive to carry out the modification. In order to carry out this technique, a strain TF8A lacking 231 kb of chromosome in comparison to the wildtype B. subtilis 168, which contains the prophage SPβ and PBSX as well as prophage-like element skin [START_REF] Westers | Genome Engineering Reveals Large Dispensable Regions in Bacillus subtilis[END_REF]. The upp gene of this strain was replaced by a neomycin-resistance gene under the control of Lamda Pr promoter (λPr-neo) resulting in strain TF8A λPr-neo::Δupp [START_REF] Tanaka | Building the repertoire of dispensable chromosome regions in Bacillus subtilis entails major refinement of cognate largescale metabolic model[END_REF]. The sequence of this region was amplified using λPr-neo primer pairs (λPr-neo1:

CGCTACGCCCAGAACACCGATTGAGCTGAT; λPr-neo2:CTTAGCTCCTGAAAATC TCGGATCC). The resulted PCR product was used for the transformation of BSB1 strain in order to achieve the master strain B. subtilis BSB1N (Figure 24), this strain is a prerequisite to carry out markerless deletion.

Escherichia coli JM109

This strain was used for the amplification and the conservation of the different plasmids.

JM109 is a high-efficiency competent strain ( ≥1 × 10 cfu/μg DNA) for transformations.

The genotype is recA1, endA1, gyrA96, thi, hsdR17 (rK-, mK+), relA1, supE44, Δ (lac-proAB), [F´,traD36,proAB,lacIqZΔM15]. It was supplied by Promega.

Plasmids

All the plasmids used in the study are stated below as well as in APPENDIX I.

Plasmid with sfp gene construct

The plasmid containing the gene sequence (amyE -sfp -Cm R -amyE -Spec R

) was constructed by the digestion of pBC16 (containing the sfp gene) with EcoRI.

Subsequently, pDG1661ΔPvuII circularised was digested with EcoRI for its linearization.

The former and later digested fragments were then ligated together to obtain pBG400 ( ). The plasmid concentration was amplified using JM109.

Figure 25

The integration plasmid pBG400 used for the integration of sfp gene

CONFIDENTIAL DOCUMENT

K7010 plasmid

The plasmid K7010 (Figure 26A) contains the cassette used for the construction of markerless deletion strains. This plasmid was derived from pUC19 plasmid. The cassette includes upp (uracil phosphoribosyltransferase gene) used previously for counter selection;

phleo (phleomycin-resistance gene) used as selective marker for cassette integration; cI gene (encodes cI protein which acts as a repressor in Lambda phage) was used as counter selection as it can inhibit the expression of λPr-neo by binding the Lamda promoter (λPr). The cassette upp-phleo-cI is abbreviated as K7 cassette. The amplification of the cassette (Figure 26B) was achieved using primer pairs (Phleo5:5'-CGACGGCCAGTGAATTCGAGC-3' and Phleo3: 5'-AGCTTGCATGCCTGCAGGTCG -3') [START_REF] Tanaka | Building the repertoire of dispensable chromosome regions in Bacillus subtilis entails major refinement of cognate largescale metabolic model[END_REF].

Plasmid with constitutive promoter P repU -neo construct

The plasmid containing the gene sequence (P repU -neo) was constructed using two plasmids pBG127 and pBG124 (APPENDIX I). The plasmid pBG127 contains hxlR sequence while the plasmid pBG124 contains the P repU -neo sequence along with HaeIII restriction site and srfAA sequence. Competent JM109 cells were transformed with pBG124 and pBG127 to obtain EBG132 and EBG128 respectively. Finally, both plasmids (pBG124 and pBG127)

were extracted. After the extraction, the plasmids were digested with the following restriction enzymes: pBG127 was digested with PaeI and BglII and pBG124 was digested with SphI and BglII. The digested samples were purified by agarose gel electrophoresis.

The required fragments were extracted from the gel and ligated to obtain as a product the pBG133 plasmid (Figure 27) (Coutte et al., 2010).

Figure 27

The integration plasmid pBG133 used for the integration of P repU -neo gene ). In addition to modified Landy medium, two different types of media have been also used to study the surfactin production analysis i.e. TSS medium supplemented with 16 amino acids [START_REF] Brinsmade | Dissecting complex metabolic integration provides direct genetic evidence for CodY activation by guanine nucleotides[END_REF] in order to analyze the impact of glucose concentration as a sole carbon source on surfactin production and AMBER medium in order to analyze the glycolytic mutants as it contains two carbon sources (Glucose and Malate) as well as to have common nutrient environment among all the partners of European project (AMBER) which is focused to determine the linkage between central carbon metabolism and various cellular processes (APPENDIX II). The buffer solution used in former was MOPS and in the latter was K-buffer. Selective media were prepared by adding various antibiotics in LB-agar medium. The following concentration of various antibiotics were added, Chloramphenicol (Cm) 5 µg/ml; Neomycin (Neo) 5 µg/ml, Erythromycin (Erm) 2 µg/ml; Phleomycin (Ph) 4 µg/ml; Spectinomycin (Spec) 100 µg/ml or Tetracycline (Tet) 20 µg/ml for the selection of the desired B. subtilis mutant strains where as Erythromycin (Erm) 300 µg/ml; Ampicillin (Ap) 100 µg/ml or Neomycin (Nm) 50 µg/ml for the desired E. coli mutant selection.

The pH of all culture media was adjusted to 7.0 using 3M KOH/1M H 2 SO 4 . All the chemicals used for the preparation of the culture media are of analytical grade.

Polymerase chain reaction

Polymerase chain reaction (PCR) was carried out using the PCR Master Mix (2X) (Thermo Scientific Fermentas, Villebon sur Yvette, France). A PCR mixture was prepared constituted of PCR Master Mix (1X), 0.5 µM forward and reverse primer, DNA (200 ng)

and the volume was made up with MQ water. The reaction was carried out under the following conditions: 5 min at 94°C; (30s at 94°C, 30s at 55°C, X min at 72°C) for 25 cycles; 10 min at 72°C, where X depends on the length of the sequence to be amplified.

Generally, the rate of amplification of Taq polymerase is 1kb/minute. After PCR, the desired fragment length was verified using gel electrophoresis on 0.8% agarose gel.

DNA purification

Recovery of DNA from agarose gels was performed with GeneJET Gel Extraction kit (Thermo Scientific Fermentas). This kit combines the convenience of spin-column technology with the selective binding properties of a uniquely-designed silica-gel membrane. Efficient recovery of DNA was carried out using elution buffer provided by the kit.

CONFIDENTIAL DOCUMENT

Ligation

Ligation of PCR products with pGEM-T Easy vector (Promega Corp.) was carried out using ligation mixture of pGEM-T Easy vector system. The reaction mix is constituited of ligation buffer (1X), pGEM-T Easy Vector (50 ng), PCR product (X µl), T4 DNA Ligase (3 Weiss units) and Nuclease-free water to make final volume 10 µl. The volume of PCR product was determined using following equation:

This equation was used for the ligation of inserts into different vectors and for this purpose the DNA Ligation Kit from Takara (Ozyme, Saint Quentin en Yvelines, France) was used.

All ligation reactions were incubated overnight at room temperature.

Plasmid extraction

Plasmid extraction was carried out using GeneJET Plasmid DNAPurification Kit (Thermo Scientific Fermentas). This kit also utilizes spin-column technology and selective binding to silica-gel membrane. The extraction procedure consists in three basic steps: preparation bacterial lysate, adsorption of DNA onto the membrane, washing and elution of plasmid DNA using an elution buffer. The concentration and purity (A 260 /A 280 ) were measured using NanoDrop (Thermo Scientific NanoDrop Lite Spectrophotometer, USA).

Restriction digestion

Restriction endonucleases were supplied by Thermo Scientific (Fermentas, Villebon sur Yvette, France). The restriction digestion was carried out at 37˚C for 15 min or 1 hr based on FastDigest or conventional restriction endonucleases. This procedure is followed by gel electrophoresis on 0.8% agarose gel along with DNA ladder (O'Gene Ruler Mix, Thermo Scientific) for the analysis of proper product.

In all cases, the instructions of the suppliers were followed to carry out the techniques.

A reaction network was formalized to analyze the metabolic pathway of branched chain amino acids (BCAAs). This reaction network was based on partial kinetic knowledge [START_REF] Coutte | Modeling leucine's metabolic pathway and knockout prediction improving the production of surfactin, a biosurfactant from Bacillus subtilis[END_REF]. A reaction network with partial kinetic knowledge consists is a finite set of species, a subset of inflow species, a subset of outflow species, and a finite set of reactions. A reaction is a multiset of pairs between species and roles. Here the following set of identifiers: reactant, product, activator, accelerator, or inhibitors have been used. A species is called a modifier of a reaction if its role is equal to activator, accelerator, or inhibitor. Modifiers are neither consumed nor produced, but the presence of a modifier affects the rate of the reaction. Note that the same species can be used several times in the same reaction and with different roles. The partial knowledge on the kinetics of a reaction is given by the roles that are assigned to its species. The intuition is as follows. The rate of a reaction is increased by reactants, activators, and accelerators, and it is decreased by inhibitors. A reaction is enabled only if all its reactants and activators are present, i.e. have non-zero concentrations, while some or the accelerators may be absent. Steady state semantics was followed in the reaction network.

In a deterministic semantics, a chemical solution is considered as function of species with non-negative real numbers including zero. For most kinds of species, this number represents the concentration of the species, but in other cases it may also denote the activity of a promoter or the activity of a promoter binding site. Reactions can be applied to chemical solutions. In this case, the reactants are consumed and the products are produced, respectively, as many times as they occur in the reaction. A reaction can only be applied if all its activators are present (have a non-zero concentration), while its accelerators may be absent. The higher the concentration of an accelerator or activator is the higher will be the rate of the reaction. A reaction is applicable even if some inhibitors are present, but the higher the concentration the slower will be the rate of the reaction.

Reaction networks are usually situated in a context, which may be adjacent reaction networks, or the "chemical medium" from which some species may inflow and to which some species may outflow during wet lab experimentation. The exchange with the context is modeled by the set of inflow and outflow species of the network: inflow species may inflow from the context, while outflow species may outflow into the context. The precise rate laws of inflows and outflows are unknown, except the assumption that the outflow rate must increase with the concentration of the outflow species.

Reaction networks in our modelling language are represented as graphs similar to Petri nets (the concrete syntax of our reaction networks is based on XML, from which the graphs are computed. The XML representation is also the input for the prediction algorithm). These graphs contain two kinds of nodes, round nodes for representing its species and boxed nodes for representing its reactions. More precisely, any species "S" is represented by a round node and any reaction with name "r" by a boxed node. Solid edges either link a reactant to its reaction, or a reaction to one of its products. There are three kind of dashed edges, which start at the three kinds of modifiers. An accelerator edge links an accelerator to a reaction, an activator edge links an activator to a reaction, and an inhibitor edge links an inhibitor to a reaction. An input edge points from the context to an inflow species S, while an outflow edge points from an outflow species S to the context.

For convenience, a last kind of edges was introduced as a shortcut for a product that is degraded by a hidden reaction. The species nodes were used with three different colors, which indicate their biological roles. The dynamics of the network is not concerned by the colors though. Metabolite is denoted by yellow and in blue proteins. There is a third color for "artificial species" that serve for modelling regulation, such as for instance the promoter of the ilv-leu operon. The node colors are irrelevant for the dynamics of the network (Figure 29). Table 6 Primers used for deletion mutant construction Two sets of primer pairs were designed for upstream and downstream region of the gene to be deleted (using the sequence from SubtiList World-Wide Web Server and supplied by Eurofins Genomics, Ebersberg, Germany). These two sets of primers were named (forward l , reverse l ) for the upstream gene and (forward 2 , reverse 2 Table 6 ) for the downstream gene, with respect to the gene of interest ( ). During the design of the primers sets, direct repeat sequence were either added to the primer sequence reverse l or forward 2 as well as complement sequence of 5' and 3' region of the K7 cassette (upp-phleo-cI) are also added to the primer sequence of reverse l and forward 2 . PCR was carried out using specific primer pair (forward 1 , reverse l ) and (forward 2 ,reverse 2

The PCR was carried out under the following conditions: 5 min at 94°C; (30s at 94°C, 30s at 55°C, 2 min at 72°C) for 25 cycles; 5 min at 72°C. The PCR products were purified using gel extraction kit (Qiagen, Hilden, Germany). The DNA fragments generated using (forward

) each with 0.5 μM final concentration and 200 ng of master strain chromosomal DNA under standard condition to produce DNA fragments of atleast 1.0 kb long.

1 ,reverse l ) and (forward 2 ,reverse 2 ) was mixed equally along with upp-cI-phleo cassette at a proportion 2:1 and a joining PCR reaction was conducted under the following conditions: 5 min at 94°C; (15 s at 94°C, 15 s at 55°C, 12 min at 65°C) for 12 cycles; (15 s at 94°C, 15 s at 55°C, 12 min at 65°C) for 25 cycles; 10 min at 72°C in the presence of forward 1 and reverse 2

Positive selection of deletion mutants were carried out through phleomycin resistance on LB medium plates, which were incubated at 37˚C up to 24 hours. Colonies, with proper cell morphology were streaked twice on the same medium along with on neomycin-LB medium plates in order to purify the colony. Each clone was checked for the absence of the deleted chromosomal region through PCR using primer pair forward (0.1 μM final). The resultant product was confirmed through electrophoresis on 0.8% agarose gel and further used to transform competent cells of the master strain (B. subtilis BSB1N).

1 and reverse 2

The Pop-In strain was inoculated in 1 ml liquid LB medium for 6-7 hours at 37˚C at 160 rpm. After incubation, the entire culture was lawned on neomycin-LB medium plates, which were incubated at 37˚C up to 24 hours. Colonies with proper morphology were streaked on neomycin-LB medium plates, phleomycin-LB medium plates and LB medium plates containing both neomycin and phleomycin and incubated at 37˚C up to 24 hours.

, respectively. Strains constituting the expected deleted chromosomal structure were preserved in glycerol stock at -80°C. These strains with resistance to phleomycin and sensitive to neomycin were known as Pop-In strains.

Figure 30 General overview of Pop-In and Pop-Out technique

Colonies which were grown only on neomycin-LB medium plates were treated as positive clones and were later verified through PCR using primer pair forward 1 and reverse 2 for the excision of the K7 cassette. Strains with no K7 cassette were preserved in glycerol stock at -80°C and were known as Pop-Out strains.

Insertion

All mutants were transformed with plasmid pBG400 for the integration of sfp gene at the amyE locus. The transformation protocol has been described in the next section. After transformation, the cells were spreaded on LB-Chloramphenicol plates at 37˚C overnight.

Each clone obtained were analyzed through replica plate method. LB-Chloramphenicol and LB-Spectinomycin were the chosen selective media. At an event of double cross-over integration, the clones will be resistant only to LB-Chloramphenicol and sensitive to LB-Spectinomycin. The positive clones were also analyzed through PCR using primer pair (sfp_fw and sfp_rev); the primer sequence is shown in Table 7. Integrated sfp gene in the strains resulted in positive haemolytic activity and negative amylase activity due to disruption of amyE locus.

In order to evaluate the role of promoter sequence on surfactin production, the native promoter of B. subtilis strain was replaced by a constitutive promoter P repU derived from S. aureus. The constitutive promoter B. subtilis strains obtained through transformation of the wildtype and derivative strains with pBG401. Upon transformation, they were lawned on LB-Erythromycin plates and incubated at 37˚C overnight. Clones obtained were analyzed through PCR using primer pair (P repU _fw and P repU _rev); the primer sequences are shown in Table 7.

Table 7 Primers used for insertion mutant construction 8 Transformation

E.coli

High efficiency E. coli JM109 strains were used for the amplification of plasmids.

Transformation was performed as described in pGEM ® -T and pGEM ® JM109 cells (50 μL) were transferred to a 15 ml falcon tube in which overnight ligation mixture was added. The tube were flicked gently and placed on ice for 20 min, and then heat-shock was applied to the cells by immersing the tube in water bath at 42°C for 90 secs. The tubes were returned immediately to ice for 2 minutes and after that 900 μL LB medium was added. The entire mixture was incubated at 37˚C with shaking for 1.5 hours.

After incubation, the entire mixture was spread on selective medium based on plasmid, with which it was transformed.

-TEasy vector systems technical manual from QIAGEN.

B. subtilis

Both B. subtilis 168 and BSB1 strains are naturally transformable strains. Strain(s) are streaked out on LB-plates (with/without antibiotic based on the strain) overnight at 37˚C.

A colony was inoculated in 2 ml of completed MC (APPENDIX II). The mixture was incubated at 37°C with shaking for 4-5 hours (or more until the culture is turbid). Then, 400 μL of cells were mixed with 1 μg of DNA (gDNA/plasmid) and it was incubated again at 37°C with shaking for 2 hours. After incubation, the entire mixture was spread on LBantibiotic plates based on the recipient resistance along with control cells without transformation in different plates.

Surfactin analysis

All mutants obtained were analyzed for surfactin production in order to determine the impact of gene for surfactin overproduction in comparison to control. The sfp gene required for lipopeptide production was inserted in all mutants prior to surfactin analysis.

This gene can promote surfactin production by converting the Apo-synthetase to holosynthetase.

Culture conditions

Firstly, single colony of mutant strain(s) was inoculated in LB-antibiotic medium. It was incubated at 37°C with shaking conditions. Next, required volume of culture was centrifuged at 8,000 rpm for 10 min. The cell pellets were washed twice to remove residual medium. Then, it was inoculated in the experimental media at 37°C with 160 rpm overnight to acclimatize the nutrient environment. Finally, the overnight culture was centrifuged at 8,000 rpm for 10 min and the cells were inoculated in the experimental medium. In all experiments, the inoculation OD 600 was maintained 0.2 and carried out at 37°C with variable shaking conditions. The surfactin analysis was carried out in both Conventional Erlenmeyer culture flask of 500 ml and 1000 ml with 10% filling ratio were used to carry out the shake flask experiment, as described previously (Coutte et al., 2010).

with a shaking diameter of 3 mm along with humidity control. A real-time monitoring of growth of the mutants was carried out using BioLection V.2.4.1.0 software. Experiments were carried out in triplicate and two biological replicates were performed. Results are expressed as mean value with standard deviation

In both conditions, at every time point, sample was transferred into 1.5 ml sterile eppendorf tubes. The samples were centrifuged at 8,000 rpm for 10 mins, the supernatant

CONFIDENTIAL DOCUMENT

were transferred into new sterile 1.5 ml eppendorf tubes for surfactin analysis and the sampling of the same time-point was carried out in triplicate.

Surfactin analysis by RP-HPLC

Surfactin was analyzed using reverse phase C18 HPLC (600 s, Waters, USA) equipped

LC-MS-MS analysis of surfactin isomer

Samples were analyzed by reverse phase UPLC-MS (UPLC, Waters, Acquity class H) coupled with a single quadrupole MS (SQDetector, Waters, Acquity) on an Acquity UPLC BEH C18 (Waters) 2.1 × 50 mm, 1.7 μm column. A method based on acetonitrile gradients that allowed the simultaneous detection of all three LP families. Elution was started at 30% acetonitrile (flow rate of 0.60 ml min -1 ). After 2.43 min, the percentage of acetonitrile was brought up to 95% and held until 5.2 min. Then, the column was stabilized at an acetonitrile percentage of 30% for 1.7 min. Compounds were identified on the basis of their retention times compared with authentic standards (98% purity, Lipofabrik, Villeneuve d'Ascq, France) and the masses detected in the SQDetector. Ionization and source conditions were set as follows: source temperature, 130°C; desolvation temperature, 400°C; nitrogen flow, 1000 l h -1 ; cone voltage, 120 V. This technique was carried out in Gembloux Agro-Bio Tech, University of Liege , Belgium.

At each time point, 2 ml of sample were filtered through 0.45 µm pore size filter (Sarstedt AG, Nürnberg, Germany) to obtain cell-free medium for the analysis of extracellular metabolome. All samples were stored at -20˚C prior to analysis.

During the analysis, 400 µl of sample was transferred to 5 mm glass tube (length 7 inch;

NORELL ST-500, NORELL, USA) and the sample was buffered to pH 7.0 by adding 200 µl of 0.2 mol/l sodium hydrogen phosphate buffer solution which was prepared with 50% D 2 O (Euriso-Top, St-Aubin Cedex, France), this solution provides a NMR-lock signal. In addition, the buffer solution also contains 1 mol/l TSP (3-trimethylsilyl-[2, 2, 3, 3-D4]-1propionic acid) (Sigma-Aldrich, USA) as an internal standard.

The analysis was carried out using 1 H-NMR and the samples were measured at 600.27

MHz and a temperature of 310K. The spectrum was obtained utilizing Bruker AVANCE-II 600 NMR spectrometer and was controlled by TOPSPIN 2.1 software (Bruker Biospin GmbH, Germany). Identification and quantification of NMR spectra was carried out using AMIX-Viewer v3.9.11 software (Bruker Biospin GmbH, Germany). Both standard compounds (Sigma-Aldrich, USA) and Human Metabolome Database (HMDB) was applied for the identification of spectra while the quantification was carried out by exploiting the signal area of Quant Ref signal at 15 ppm (Kohlstedt et al., 2014b). This technique was carried out in Ernst-Moritz-Arndt-University Greifswald, Institute of Biochemistry, Germany.

Is intracellular pool of leucine a limiting factor for surfactin biosynthesis?

B. subtilis BBG111 is a mutant of B. subtilis 168 able to produce two types of lipopeptides i.e. surfactin and fengycin. Coutte et al. previously showed that the deletion of pps gene which is responsible for the biosynthesis of fengycin leads to an increase of surfactin biosynthesis (Coutte et al., 2010). One of the hypotheses suggested by the authors to explain this result was a better availability of precursors required for surfactin biosynthesis as both the lipopeptides share several precursors. In addition, the replacement of the native promoter (P srfA ) of surfactin operon with a constitutive promoter (P repU ) did not lead to a high enhancement of surfactin production. Hence, it was again speculated that there might be limitation of precursor molecules, which are required for biosynthesis of surfactin.

Surfactin molecule contains seven amino amino acids within its peptide moeity. Out of the seven, five positions are occupied by branched chain amino acids i.e. valine and leucine. It was thus hypothesized that an increase in the intracellular pool of leucine pool will have a positive impact on the surfactin production as the molecule contains four molecules of leucine (Figure 33).

CONFIDENTIAL DOCUMENT

In order to investigate the hypothesis that an increase in the intracellular pool of leucine can lead to an increase in the surfactin production, the strain B. subtilis BBG111 (Coutte et al., 2010) was cultivated in the presence of L-leucine. Two different cultures were carried out in Erlenmeyer flask at 37˚C and an agitation of 160 rpm (materials and method:

Surfactin analysis). The first culture medium used was Landy [START_REF] Landy | Bacillomycin; an antibiotic from Bacillus subtilis active against pathogenic fungi[END_REF] with ammonium sulphate (instead of glutamic acid) and the second chosen nutrient environment was Landy medium with ammonium sulphate and L-leucine (instead of glutamic acid). The Landy medium with ammonium sulphate instead of glutamic acid will be called modified Landy medium momentarily.

Samples were withdrawn after 6 and 24 hours of incubation. Biomass and surfactin analysis were carried out (materials and method: Surfactin analysis). Cultures were performed in triplicate and results are expressed as mean value with standard deviation in Figure 34. When the culture was grown in modified Landy medium with only ammonium sulphate as the nitrogen source, specific surfactin production was 100 mg/g of biomass dryweight (DW) and 600 mg/g of DW at 6 h and 24 h of culture. However, upon addition of Lleucine (2.5 g/l) and (NH 4 ) 2 SO 4 at a concentration of 2.3 g/l, the specific surfactin production was enhanced 3-folds at 6 h and 24 h, respectively in comparison to the former condition. The maximum specific surfactin production of 1800 mg/g of DW was obtained in Landy medium supplemented with 2.3 g/l (NH 4 ) 2 SO 4 and 2.5 g/l of L-leucine for 24 h cultivation (Figure 33). Thus, it can be inferred from the results that the hypothesis raised at the beginning of the study was found to be true as it highlights the impact of leucine feeding on surfactin biosynthesis.

Hence, leucine as a monomer of surfactin forms an essential component for the enhancement of its production. The leucine intracellular pool results from leucine transporter efficiency on one side and from the anabolic pathway on the other side.

The metabolic flux analysis is a central method of cell engineering to increase the productivity of host microorganisms such as bacteria, yeast and fungi. In this area, many fundamental studies have been conducted on the pathway regulations and metabolic flux analysis [START_REF] Otero | Industrial systems biology[END_REF]. The use of bioinformatics in recent years helped to develop tools for reconstruction of metabolic pathways and genome scale metabolic modelling in order to overexpress metabolites, but most of them are only applied to primary metabolites and not on secondary metabolites [START_REF] Almaas | Global organization of metabolic fluxes in the bacterium Escherichia coli[END_REF][START_REF] Radeck | The Bacillus BioBrick Box : generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis The Bacillus BioBrick Box : generation and evaluation of essential genetic building blocks for standardized work with B[END_REF]) [START_REF] Goelzer | Reconstruction and analysis of the genetic and metabolic regulatory networks of the central metabolism of Bacillus subtilis[END_REF]) [START_REF] Sohn | Genome-scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production[END_REF]. In this work, the main emphasis is an overproduction of secondary metabolites, i.e. the surfactin biosynthesized by NRPS. To overcome the surfactin precursor limitation, here for the first time an original and fully integrated approach of synthetic biology to overproduce surfactin was developed (in collaboration with the Biocomputing team of CRIStAL laboratory of Lille).

How to enhance leucine production within the intracellular pool ? 2.1 Formalization of the model of BCAA metabolic pathway

Branched chain amino acids (BCAA) include isoleucine, valine and leucine. These amino acids are regulated in a similar way. A metabolic network for the production of these amino acids is presented in Figure 34.

CONFIDENTIAL DOCUMENT

The Figure 34 describes the metabolic transformations of Thr, Pyr and Akb into Ile, Val, and Leu, concomitantly with its regulation. This model mainly follows the abstraction level chosen by the previous work in [START_REF] Goelzer | Reconstruction and analysis of the genetic and metabolic regulatory networks of the central metabolism of Bacillus subtilis[END_REF] [START_REF] Mäder | SubtiWiki--a comprehensive community resource for the model organism Bacillus subtilis[END_REF], but the regulation is expressed more cleanly within this formal modeling language.

As the precise kinetics is unknown, the complex nature of the regulation of the inhibitors and the activators in the reaction were extracted based on comprehensive literature study and assumption. Although few detailed information regarding all inhibitors and activators were not available but the reaction network was formalized using this partial knowledge. The information regarding the metabolites, proteins and actors of this model and all the reactions have been provided APPENDIX III.

In order to group several proteins that participate in the same reaction, artificial species were introduced that were called protein clusters, and were represented as for instance . This protein cluster presents all the proteins generated by the bkL operon and in a similar fashion the proteins LeuB, LeuC and LeuD as well as IlvB and IlvH are represented in clusters LeuBCD and IlvBH, respectively.

Split-points were used to link different copies of the same species. For instance, one copy of Leu, which was drawn as was linked together with the original over a split point. Logically, there is no difference between a species and its copies; it just allows nicer graphs with fewer intersections of edges. Edge-clusters were used to represent several groups with the same sources or targets (for instance, the activator edges from BkL and YwaA to r 35 , r 32 , and r 29 are clustered).

Production of Isoleucine, Valine, and Leucine

For clarity, the intermediates that produce the outflows of the pathway, i.e. Ket a for Ile in r 26 , Ket b for Val in r 36 , and Ket c for Leu in r 45 were considered. All three reactions must be activated by YwaA/YbgE, that is either by protein YwaA (r 6 ) or protein YbgE (r 5 ), which are exchangeable catalysts following [START_REF] Thomaides | Essential bacterial functions encoded by gene pairs[END_REF]. There are also inverse reactions r 29 , r 32 , r 35 , but with [START_REF] Mäder | SubtiWiki--a comprehensive community resource for the model organism Bacillus subtilis[END_REF]. The genes of the proteins IlvBH, IlvC, LeuA, and LeuBCD are co-located in the same operon (ilv-leu), so they are under the dependence of the same single promoter P Ilv-Leu [START_REF] Tojo | Elaborate transcription regulation of the Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids through global regulators of CcpA, CodY and TnrA[END_REF]. This specific regulation is described below.

Regulation

The expression of the different enzymes involved in the production of branched chain amino acids (explained above) are denoted by the following reactions: r 22 for IlvA; r 25 for IlvD; r 23 for IlvBH; r 24 for IlvC; r 38 for LeuBCD; r 37 for LeuA; r 11 for BkL; r 10 for Bcd; r 47 for YwaA and r 46 for YbgE. All these expression reactions are controlled by a complex regulation [START_REF] Brinsmade | Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools[END_REF]) [START_REF] Molle | Additional targets of the Bacillus subtilis global regulator codY identified by chromatin immunoprecipitation and genome-wide transcript analysis[END_REF]) (Mäder et al., 2004b).

The proteins CcpA, CodY, TnrA, and BkdR have regulatory functions and are expressed by the reactions r 14 , r 15 , r 16 and r 12 , r 13 , respectively. The expression reactions of these regulators can be modulated by different factors from outside the context, which are not modeled here. GTP comes from the context and can be degraded through the reaction r 17 .

This metabolite increases the affinity of CodY for its binding sites, as well as Ile and Val [START_REF] Shivers | Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched-chain amino acids[END_REF]) [START_REF] Tojo | Elaborate transcription regulation of the Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids through global regulators of CcpA, CodY and TnrA[END_REF] [START_REF] Handke | Interaction of Bacillus subtilis CodY with GTP[END_REF] [START_REF] Molle | Additional targets of the Bacillus subtilis global regulator codY identified by chromatin immunoprecipitation and genome-wide transcript analysis[END_REF]) (Mäder et al., 2004b).

Expression of IlvA, IlvD, and YbgE are dependent on their own promoters and their expressions are down-regulated by CodY at the transcriptional level [START_REF] Molle | Additional targets of the Bacillus subtilis global regulator codY identified by chromatin immunoprecipitation and genome-wide transcript analysis[END_REF]) (Mäder et al., 2004b), which is represented by reactions r 22 , r 25 , and r 46 , respectively. [START_REF] Hatfield | Threonine deaminase from Bacillus subtilis. II. The steady state kinetic properties[END_REF] it was further showed that IlvA is deactivated by both Ile and Val (reactions r 20 , r 21 ). The transcription of all proteins IlvC, IlvBH, LeuA, and

CONFIDENTIAL DOCUMENT

LeuBCD starts through the activation of their promoter P Ilv-Leu , which controls the reactions r 23 , r 24 , r 37 and r 38 , respectively (Mäder et al., 2004b) [START_REF] Brinsmade | Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools[END_REF]. Its regulation is regulated by the reaction r 2 and is dependent on several mechanisms, which are described before. In addition to what has been described above, the down-regulation of the promoter P Ilv-Leu by Leu is also contributed by a ribosome-mediated attenuation mechanism Tbox (r 40 ) [START_REF] Brinsmade | Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools[END_REF] [START_REF] Grandoni | Transcriptional regulation of the ilv-leu operon of Bacillus subtilis[END_REF] and the deactivation of LeuA by Leu, which is represented by the reaction r 39 . Proteins BkL and Bcd lie under the action of two common but independent promoters (Prm b and Prm c ), whose activity level is represented by actor OP BkL-Bcd . Among them one is constitutive (no regulation, represented by the reaction r 4 ), and the other is positively impacted by BkdR and downregulated by both TnrA and CodY, as reflected by reaction r 3 (Michel Debarbouille et al., 1999). Although not regulated on the transcriptional level, protein BkdR is activated by Ile and Val, which is modeled by introducing the two BkdR producing reactions r 12 and r 13 .

Proteins YbgE (expressed by the reaction r 46 ) is down regulated by CodY. However, no regulation is known for protein YwaA, which is expressed by the reaction r 47 .

Knockout prediction

The knockout prediction algorithm was applied to the reaction network (Figure 34). The integrated approach was focused on a modelling language for reaction networks with partial information and on a new knockout prediction algorithm based on abstract interpretation that applies to all models written in this language [START_REF] Niehren | Predicting Changes of Reaction Networks with Partial Kinetic Information[END_REF].

There were two types of prediction provided, firstly, regarding the flux of various species or metabolites (s) and secondly, regarding single knock-out of genes. Both predictions were made based on increasing the production of leucine (y Leu = ). In this context, x s = denotes a decrease in the flux of species or metabolite (but not to zero) and y s = denotes an increase in the outflux of species or metabolite (but not from zero).

CONFIDENTIAL DOCUMENT

In all the flux solutions (Table 8) it was observed that x Glu = y oxoGlu = y Leu = , so it means that any increase of y Leu requires increasing the influx of Glu and outflux of OxoGlu.

The prediction based on flux does not require any reaction knockout within this context (Figure 34). In addition to these context changes, few others were also predicted which include y Acyl-CoA = , i.e. the outflux of Acyl-CoA is decreased, or else that x Pyr = , so that the influx of Pyr is increased. Both possibilities are plausible, since for overproducing Leu, either the production of Leu by r 45 must be increased, and this requires more Pyr, or else the outflux of Leu into the fatty acid pathway (via reactions r 35 and r 34 ) must be decreased, and this imposes a higher outflux of Acyl-CoA.

All solutions corresponding to single knockouts have been provided in Table 9. This can be done automatically by the tools developed by the Biocomputing team. In addition, an informal explanation of the positive effect of these single knockouts with respect to leucine overproduction has also been provided (Table 9).

CONFIDENTIAL DOCUMENT

Table 9 Single knockout predictions for leucine overproduction These effects can be seen in the model rather easily. It turns out, however that knockout of reactions r 14 and r 46 do not have a clear positive effect. The only effect of a knockout of reaction r 46 is the deactivation of r 45 producing Leu, which is unwanted. The knockout of reaction r 14 has a negative effect on the activity of P Ilv-Leu . This is not very plausible for leucine overproduction: it could still be argued that a decrease of the activity of P Ilv-Leu will decrease the speed of r 27 which in turn will increase the speed of r 30 , but at the same time, it also has a negative effect on the activation of r 30 , which counter balances this positive effect on the Leu production. Moreover, it has been demonstrated previously that a knockout of ccpA causes a decrease of ilv-leu expression [START_REF] Brinsmade | Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools[END_REF].

This new knockout prediction algorithm was applied for the overproduction of intracellular leucine production, which in turn might increase the production of surfactin in different mutants of B. subtilis 168 or B. subtilis BSB1 strain.

Table 11 Deletion mutants derived from B. subtilis BSB1 strain based on prediction results of the branched chain amino acid reaction network Thus, the markerless mutants developed along with BBG 111 and BBG 258 were treated as control strain for B. subtilis 168 and B. subtilis BSB1 strain for growth and surfactin analysis, respectively. The knockout strategy used provided complete deletion of the gene of interest. The markerless technique adopted here was found to be an efficient tool to carry out multiple deletion in one strain without antibiotic limitation.

According to the prediction results (Table 9) based on the model, various genes were deleted. Firstly, the CodY binding to its high affinity site on the P Ilv-Leu promoter (r 1 ) and secondly, the codY expression (r 15 ), the strains BBG251 and BBG254 were constructed in B. subtilis 168 strain while only the latter was constructed in B. subtilis BSB1 strain (BBG260). TnrA also negatively regulates the ilv-leu expression under nitrogen-limited conditions, the knockout predictions giving as a result the deletion of the TnrA binding on the P Ilv-Leu promoter (r 7 ) and the expression of tnrA (r 16 ). To achieve the same effect, the expression of tnrA (r 16 ) was completely suppressed in the mutant strain BBG256 and BBG262. Both the expression of codY (r 15 ) and tnrA (r 16 ) were suppressed simultaneously in one mutant strain BBG265. Leucine intracellular concentration can inhibit the expression of ilv-leu operon via Tbox attenuator. In the mutant strain BBG252, Tbox was suppressed (r 40 ), as recommended by the predictions. Lastly, prediction on leucine degradation was avoided by the knockout of the gene bcd (r 10 ), giving the strains BBG253 and BBG259 as well as deletion of lpdV (r 11 ), a gene within the bkL operon producing strains BBG255 and BBG261. The strains developed through this strategy were used for

91

CONFIDENTIAL DOCUMENT surfactin analysis. It was observed in Figure 34, that both TnrA and CodY binds to the upstream region of ilv-leu operon and repress its activity and this deletion was carried out to evaluate the role of double mutants in surfactin through this network so that the results can be used for the refinment of the model.

Growth and surfactin analysis

The mutants obtained with both B. subtilis 168 and B. subtilis BSB1 along with control strains were cultivated in high-throughput system of fermentation Biolector ® material and method-Surfactin analysis (Mp2-labs GmbH; Baesweiler; Germany) available on REALCAT platform of University Lille 1.

Cultures were performed in Landy medium with ammonium sulphate at 37˚C with shaking frequency of 1100 rpm. During the analysis of B .subtilis 168 and its derivative strain, the medium was supplemented with tryptophan at 16 mg/L. Samples were withdrawn when the cells were in exponential phase (about 6 h of culture) as described in (

). Indeed, at this time the ilvB operon [START_REF] Nicolas | Condition-Dependent Transcriptome Architecture in Bacillus subtilis[END_REF] is highly expressed and surfactin measurement was used as an indirect indicator to evaluate the impact of these genetic modification on the intracellular pool of leucine.

Growth analysis

Growth kinetics study of all the mutant strains were carried out during the exponential growth phase. No significant differences were observed for the growth rate of the two control strains (BBG111 and BBG258). The specific growth rate was found to be 0.47 ±0.06 h -1 and 0.42±0.01 h -1

Table 12 for BBG111 and BBG258, respectively. The specific growth rates of various mutants were different from the control strains BBG111 and BBG258 as shown in .

CONFIDENTIAL DOCUMENT

Table 12 Specific growth rate of all mutant strains derived from BBG111 and BBG258 in Landy medium with ammonium sulphate Most of the regulatory genes involved in the branched chain amino acid metabolic pathway are pleiotropic regulators in B. subtilis, so the effect on the growth of the strains in which these genes were deleted has also been studied. Growth analysis of the different mutant strains shows that the deletions of tnrA and Tbox have no significant negative impact on the specific growth rate of the strains. These data suggest that no significant influence was contributed by these regulators on the growth of the mutants. The disruption of high affinity CodY binding site and the codY deletion has no negative effect on the growth of the strains. Regarding the gene involved in the branched chain amino acid degradation, bcd and lpdV which code for branched chain amino acid dehydrogenase enzymes (Debarbouille et al., 1999) showed no growth defect too. Deletion may lead to a disturbance in the synthesis of branched-chain fatty acids, which plays an important role in maintaining the fluidity of the membrane lipids (Kaneda, 1991) but this was not observed

CONFIDENTIAL DOCUMENT

during growth analysis. All the obtained mutants were viable because these genes are not listed as essential for the cell [START_REF] Commichau | Essential genes in Bacillus subtilis: a reevaluation after ten years[END_REF].

Surfactin analysis

BBG111 and its derivative mutant strains shown in Table 10 (page no. 89) were evaluated for surfactin production. It can be observed from Figure 36, that all the mutant strains produced surfactin with varied concentrations. At 6 hours, the production of surfactin (mg/l) by the mutant strains was higher in comparison to control strain (BBG111). The maximum surfactin production of 324±9 mg/l was obtained with BBG256 (BS168, sfp + BBG258 was developed through the insertion of sfp gene in B. subtilis BSB1 strain. This strain (treated as control strain) along with its derivative mutant strains shown in , ΔtnrA). Although the figure does not represent the normalization concentration with dry weight of specific strain, this representation this beeen provided in the later section.

Table 11 were evaluated for surfactin production. All the mutant strains produced different concentration of surfactin, which can be seen in Figure 37. In comparison to control (BBG258) all the mutant strains produced higher surfactin at 6 hours except BBG261. BBG259 (BSB1, sfp + Thus, it can be deduced from ,Δbcd) produced maximum surfactin with a concentration of 1079±21 mg/l. The normalized representation of surfactin production has been provided in the later section.

CONFIDENTIAL DOCUMENT

Figure 36 and Figure 37 that sfp was properly integrated into the strains, leading to surfactin producing strain and the deletion has a role in the enhancement of surfactin production through an increase in leucine intracellular pool.

The surfactin concentration (mg/l) produced was normalized by the dryweight (g/l) of each strain at 6 hours to determine the specific surfactin yield by each mutant strain. The specific surfactin yield of BBG111 was found to be 194±32 mg of surfactin per g of dry weight (DW) of cell biomass, whereas for BBG251, BBG252, BBG253, BBG254, BBG255 and BBG256 was found to be 310±16, 366±5, 634±36, 1300±243,580±58, 818±59 mg of surfactin per g of DW of cell biomass (Figure 38). The maximum surfactin

CONFIDENTIAL DOCUMENT

yield was obtained BBG254 (BS168, sfp + , ΔcodY), which is 7-fold higher in comparison to the control (BBG111).

Figure 38 Specific surfactin yield (mg/g DW) of BBG111 and its derivative strains in Landy medium with ammonium sulphate. Results are expressed as mean value with standard deviation and statistical analysis of these data was made using one-way ANOVA with Tukey's multiple comparisons test.

In Figure 36, BBG256 (BS168, sfp +

, ΔtnrA) produced 323.9±9.44 mg/l, that was maximum in comparison to other mutant strains but upon normalization the surfactin yield is found to be 4 times higher than control. It can be observed ( ) that the strains BBG254, BBG256, BBG255 and BBG253 have significant impact on surfactin yield in comparison to control.

When similar experiment was performed with BBG258 and its derivative strains, the specific surfactin yield of BBG258 was found to be 252±24 mg of surfactin per g of cell DW. The surfactin yield for BBG259, BBG260, BBG261, BBG262 and BBG265 was found to be 1158±48, 1277±10,148±4, 1076±23 and 973±11 mg of surfactin per g of cell dry weight, respectively (Figure 39). The specific surfactin yield of BBG260 (BSB1, sfp + , ΔcodY) was found to be 5 times more than the control strain (BBG258) and a minimum d

CONFIDENTIAL DOCUMENT

surfactin yield of 3.8 times in comparison to control was found with BBG265 (BSB1, sfp + Figure 37 , ΔcodY, ΔtnrA). In , the strain BBG259 produced the maximum surfactin concentration but upon normalization the specific yield was found to be 4.6 times higher than the control.

Figure 39

Specific surfactin yield (mg/g DW) of BBG258 and its derivative strains in Landy medium with ammonium sulphate. Results are expressed as mean value with standard deviation and statistical analysis of these data was made using one-way ANOVA with Tukey's multiple comparisons test.

It can be observed (Figure 39) that the strains BBG260, BBG259, BBG262 and BBG265 have significant impact on enhancement of surfactin yield in comparison to control.

Thus, the mutant strains constructed with both B. subtilis 168 and B. subtilis BSB1 strains according to prediction results yielded in an increase in specific surfactin production from 1.6 to 7-folds more in comparison to the respective control strains (BBG111 and BBG258). It was suggested that the strains B. subtilis 168 and B. subtilis BSB1 are similar and the only difference is that the former is tryptophan auxotroph and the latter is tryptophan prototroph. Genetic studies revealed that there is no difference in the genome level but the analysis of specific surfactin yield of strains with bcd and lpdV deletion indicates that there might be some difference in the proteome level.

glutamine synthetase and glutamate synthase, respectively. B. subtilis requires NrgA for the transport of ammonium and NrgB is essential for the expression of nrgAB operon during ammonium assimilation [START_REF] Detsch | Ammonium utilization in Bacillus subtilis: Transport and regulatory functions of NrgA and NrgB[END_REF]. TnrA can upregulate the expression of nrgAB [START_REF] Wray | Bacillus subtilis CodY operators contain overlapping CodY binding sites[END_REF]. So it can be hypothesized that in BBG254 and BBG260 the production was due to high ilv-leu expression, which is supported by proper transport of ammonium ions for nitrogen metabolism required for surfactin synthesis. In BBG256 and BBG262, the production is lower due to lack of ammonium transport but there is still production due to relieve of repression of TnrA on P Ilv-Leu . But the specific production with BBG265 (ΔcodY, ΔtnrA) was lower than codY single mutants due to lack of nitrogen availability and almost equal to tnrA single mutants due to removal of repression on ilv-leu promoter. The result of the double knockout mutant suggests that the prediction algorithm is limited to the branched chain amino acid residue metabolism, which can predict for single knockout but do not ensure the enhancement for double knockout mutants. This result can also be explained by the fact that the version of prediction algorithm used here can predict multiple knockout without antagonistic effect but cannot accurately predict knockout having synergistic effect.

Intracellular concentration of leucine also downregulates the ilv-leu operon using a feedback loop through an attenuator [START_REF] Grandoni | Transcriptional regulation of the ilv-leu operon of Bacillus subtilis[END_REF]. This 80-bp leucine-dependent stem loop structure (Tbox) has been identified by the predictions as a target to be deleted (r 40 ). The effect of this deletion on specific surfactin production by the BBG252 strain was very low in the Landy medium supplemented with (NH 4 ) 2 SO 4 (1.5-fold increase) in comparison to BBG111. Due to absence of leucine in the extracellular medium the regulation does not occur in the Landy medium containing only (NH 4 ) 2 SO 4 as nitrogen source. Brinsmade et al. found that the copies of ilv-leu transcript were 19 times higher in Tbox mutant (SRB87) in comparison to the wild type strain, when leucine was present in the medium [START_REF] Brinsmade | Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools[END_REF].

The predictions obtained from the network (Figure 34) were related to the genes involved in the leucine degradation: ywaA via r 47 , bkL via r 11 and bcd via r 10 , and the mechanism that activates the expression of the two latter genes, i.e. expression of BkdR via Ile and Val activation via r 12 and r 13 and its binding on the promoter via r 3 or the constitutive expression of the promoter by r 4 . All these knockout predictions have the same effect of interrupting the two stages of reactions that allow the degradation of branched chain amino acids to make fatty acid precursors. To validate the prediction, bcd and lpdV were deleted 101 CONFIDENTIAL DOCUMENT

FACTORS INFLUENCING SURFACTIN PRODUCTION

Surfactin production depends on various factors which include nutrient composition, aeration and genotype of the strain used for production as discussed before in the literature reveiew section (Role of various factors-page no. 45). In this chapter, different experiments have been performed to study the impact of surfactin production in B. subtilis 168 and B. subtilis BSB1 strains.

Role of nutrient composition

In order to study the impact of nutrient composition, the strains (Table 10 page no. 89)

were cultivated in high-throughput system of fermentation Biolector ® Growth kinetics study of all the mutant strains were carried out during the exponential growth phase. The specific growth rate of BBG111 was found to be 0.47 ±0.06 h . Cultures were analysed in TSS medium supplemented with 16 amino acids with shaking frequency of 1100 rpm. Both media were supplemented with tryptophan at 16 mg/L. Samples were withdrawn when the cells were in exponential phase (about 6 h of culture).

-1 and 0.55±0.05 h -1 in Landy medium and TSS medium, respectively. The specific growth rates for BBG251, BBG252, BBG253, BBG254, BBG255 and BBG256 in Landy medium were found to be 0.58 ±0.06 h -1 , 0.55 ±0.02 h -1 , 0.38 ±0.08 h -1 , 0.39 ±0.03 h -1 , 0.54 ±0.04 and 0.59 ±0.01 h -1 , respectively. Whereas in TSS medium, the specific growth rates for BBG251, BBG252, BBG253, BBG254, BBG255 and BBG256 were found to be 0.51 ±0.01 h -1 , 0.51 ±0.02 h -1 , 0.39 ±0.05 h -1 , 0.48 ±0.03 h -1 , 0.58 ±0.07 and 0.47 ±0.12 h -1

Table 13 , respectively ( ). The specific growth rate of BBG254 increased in Landy medium in comparison to the specific growth rate in TSS medium whereas for the other strains the medium composition did not affect the growth rate.

CONFIDENTIAL DOCUMENT

Table 13 Specific growth rate of BBG111 and its derivative strains in Landy medium with ammonium sulphate and TSS medium supplemented with 16 amino acids

The decline in specific growth rate of BBG254 in TSS medium might be due to decrease in guanine nucleotide synthesis and CodY is guanine nucleotide sensor (Ratnayake- [START_REF] Ratnayake-Lecamwasam | Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels[END_REF]. So, in the absence of CodY in minimal media the level guanine nucleotide affects the growth rate of this mutant strain.

All the strains were evaluated for surfactin production in TSS medium. It can be observed from Figure 40, that all the strains produced surfactin with varied concentrations. At 6 hours, the production of surfactin (mg/l) by the mutant strains was higher except for BBG256 in comparison to control strain (BBG111). , ΔcodY), which is 21-fold higher in comparison to control, BBG111. In , BBG251 produced the maximum in comparison to other mutant strains but upon normalization the surfactin yield is found to be 9 times higher than control. The strain BBG256 showed the minimum enhancement in comparison to control. Through statistical analysis, it is determined that BBG254, BBG252, BBG251 and BBG253 have significantly enhanced the surfactin yield in comparison to BBG111. The low yield with BBG256 (BS168, sfp + It was reported that in the presence of amino acids in the medium CodY can repress srfA operon [START_REF] Serror | CodY is required for nutritional repression of Bacillus subtilis genetic competence[END_REF], which can justify the decline in surfactin yield in all mutants except BBG251 and BBG254. Only one site (site II) for CodY binding was deleted in BBG251 but the P Ilv-Leu promoter contains four binding sites for CodY [START_REF] Shivers | Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched-chain amino acids[END_REF], in the promoter-proximal CodY-I+II (nt -84/-32) site that is a Comparative analysis of the specific surfactin yield,revealed that the yield obtained with BBG251 and BBG254 was 2.3-fold and 2.4-fold higher in Landy medium with ammonium sulphate in comparison to TSS supplemented with 16 amino acids (Figure 42). So, it can be suggested that besides CodY regulation, nutrient composition also plays an important role in enhancing the specific surfactin yield. This impact may be directly related to glucose concentration as its concentration is 5 times more in former medium in comparison to latter medium.

CONFIDENTIAL DOCUMENT

The regulation of the ilv-leu operon is dependent on the availability and nature of carbon and nitrogen sources in the medium, since these are involved in various global regulators i.e. CcpA (carbon), TnrA (nitrogen) and CodY (amino acids and GTP) and other regulators, which depends upon the influence of these global regulators and change in concentration of various intracellular metabolites [START_REF] Brinsmade | Hierarchical expression of genes controlled by the Bacillus subtilis global regulatory protein CodY[END_REF].Therefore, the

CONFIDENTIAL DOCUMENT

results also depend on the medium that is chosen. To analyze the specific surfactin production the strains were cultivated in two different media, which contain a different composition in carbon and nitrogen sources. Indeed, TSS medium with 16 amino acid is low concentrated in glucose (5 g/l), but very rich in organic nitrogen (4.5 g/l), unlike the Landy medium supplemented with (NH 4 ) 2 SO 4 is very rich in glucose (20 g/l) and mainly contains inorganic nitrogen (3.6 g/l) and a small amount of organic nitrogen (1 g/l yeast extract). As shown in Figure 42, the differences in medium composition lead to differences in specific surfactin yield. The production is increased for the mutants in glucose rich medium (Landy). Brinsmade et al found that ilvB transcript level was enhanced 80 times, 28 times and 19 times, when codY, codY binding site II and Leucine T-box deletion strain were grown in TSS medium supplemented with 16 amino acids [START_REF] Brinsmade | Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools[END_REF].

These findings confirm the prediction made without knockout where an increase in pyruvate input leads to an increase of leucine production. Pyruvate is a direct result of glycolysis, an increase in glucose concentration in the medium may result in an increase of pyruvate intracellular concentration. Otherwise, the difference in nitrogen supply reveals a very different behaviour of the mutants.

CONFIDENTIAL DOCUMENT

Impact of oxygen transfer

Surfactin production highly depends on aeration and agitation. Surfactants at high concentration can affect oxygen transfer rate during fermentation [START_REF] Hbid | Influence of the production of two lipopeptides, iturin a and surfactin s1, on oxygen transfer during Bacillus subtilis fermentation[END_REF] and it was shown by Fahim et al., (2012) that an increase in volumetric oxygen transfer coefficicent (k L a) can lead to an enhancement of surfactin production. However in this study whether impact of k L a on strains after genetic modification remains directly proportional was analyzed. In this study, two strains BBG258 (BSB1, sfp + ) and BBG260 (BSB1, sfp +

The volumetric oxygen transfer co-efficicent in Biolector The specific growth rate of both strains were calculated in diffrent filling ratio in shake flask condition and in Biolector ® Table 14 have been shown in .

Table 14 Specific growth rate of BBG258 and BBG260 in different aeration conditions

The specific growth rate of both strains varied with different filling ratio conditions. The growth rate increased in both strains with the decrease in V L . This was well deduced from equation 1, according to which k L a will increase with decrease in V L in shake flask

CONFIDENTIAL DOCUMENT

condition. The different conditions did not affect the specfic growth rate of BBG260 in different conditions in comparison to BBG258 due to deletion of codY. The maximum specific surfactin yield obtained among two time points was plotted against different filling ratios. The maximum value was considered to minimize the effect of genetic modification on surfactin production. The maximum specfic surfactin yield for BBG258 was found to be 495±70, 421±23 and 234±12 mg of surfactin per g DW of cell biomass whereas for BBG260 it was found to be 911±4, 1475±196 and 1041±48 mg of surfactin per g DW of cell biomass at filling volume 0.05, 0.1 and 0.2, respectively. It can be deduced that with BBG258 the yield declines with the increase in filling volume. The relationship between filling ratio and yield is not linear in the case of BBG260 as the yield was maximum with 0.1 but it declines with 0.2 and again increased drastically with 0.3. It can be inferred that the volumetric oxygen transfer coefficicent might have a role on the yield of BBG260 as the geometry of the flask and the well of biolector was not same. The maximum specific surfactin yield for BBG258 was found to be 309±48, 268±75, 421±23 and 495±70 mg of surfactin per g DW of cell biomass whereas for BBG260 it was found to be 1041±48, 1277±10, 1475±196 and 911±4 mg of surfactin per g DW of cell biomass at k L a values of 199, 265, 355 and 632 h -1 Figure 43 respectively ( ).

It can be inferred that with increase in k L a, the yield also increased, but until certain limit of k L a. It was found for BBG258 at a very high k L a value of 632 h -1 , the yield did not increase. While with BBG260 at 632 h -1 k L a, have negative influence on the surfactin yield. The biomass of both the strains (BBG258 and BBG260) are same at k L a values 355 and 632 h -1 So, the maximum surfactin yield, was obtained with filling volume of 0.1 and at 250 rpm with this filling volume the volumetric oxygen transfer coefficient (k L a) will have positive impact on surfactin yield so these parameters were used for all the experiment in shake flask condition.

, so there might be another factor regulating the speicific surfactin yield of BBG260.

CONFIDENTIAL DOCUMENT

Figure 43 The role of volumetric oxygen transfer co-efficicent (k L a) in determining the maximum specfic surfactin yield of BBG258 and BBG260 Results are obtained in shake flask and in Biolector ® at 37 0 C and using Landy medium

The volumetric oxygen transfer coefficient is the key parameter controlling surfactin production and the selectivity of the parameters determining the aeration. Therefore, to scale-up fermentation process from flasks to large-scale fermentors, this parameter should be considered. Surfactin yield is clearly favoured at good oxygenation of the cells but there is an upper limit uptil which k L a has linear relationship with specific surfactin yield after that the relationship depends on the genetic construction of the strain.

Other factors

Besides glucose concentration, as shown in previous section that concentration of other nutrients can also have an impact on surfactin concentration. It was found that among the other nutrients (Mg 2+ , K + , Mn 2+ and Fe 2+ ), the concentration of Mg 2+

The strains ( influences the surfactin production the most [START_REF] Wei | Using Taguchi experimental design methods to optimize trace element composition for enhanced surfactin production by Bacillus subtilis ATCC 21332[END_REF].

Table 15) were grown in Landy medium with phosphate buffer (APPENDIX II) and at 6 h and 10 h the samples were analyzed for growth and surfactin analysis. The spcecific growth rate was measured during the exponential growth phase.

Table 15 BBG258 and its derivative strains used in this study

The specific growth rate of BBG258, BBG259, BBG260, BBG261 and BBG262 in this medium was found to be 0.37±0.01, 0.492±0.007, 0.481±0.011, 0.38±0.008 and 0.598±0.022, respectively (Table 16).

Table 16 Specific growth rate of BBG258 and its derivative strains in different media

CONFIDENTIAL DOCUMENT

Comparative growth analysis revealed that the specific growth rate of BBG258 and BBG259 was reduced in this medium in comparison to its growth rate in Landy medium with MOPS, but contrary results were obtained with BBG262. The growth rate of the other mutants did not decrease in this medium in comparison to their growth rate in Landy-MOPS medium. Thus, it can be inferred that the decline in magnesium concentration might have affected the growth rate of BBG258 and BBG259.

The surfactin concentration (mg/l) for BBG258, BBG259, BBG260, BBG261 and BBG262 was found to be 68±5, 392±35, 1243±153, 109±31 and 542±91 at 6 hour ; at 10 hour the surfactin concentration was found to be 221±83, 611±115, 2289±318, 252±21

and 1248±177 and at 24 hour the surfactin concentration was found to be 694±93, 736±103, 2911±141, 627±74 and 1639±358 respectively (Figure 44). The maximum concentration was produced by BBG260 (BSB1, sfp + ,∆codY) and it was found to be 18 times; 10 times and 4 times more at 6 hours; 10 hours and 24 hours, respectively in comparison to BBG258. Statistical analysis revealed that BBG260 at both 6, 10 and 24 hours and BBG262 at 24 hour had a significant impact on the enhancement of specific surfactin yield in comparison to BBG258.

It can be inferred from surfactin analysis, although Sfp protein needs magnesium ion as a cofactor [START_REF] Wei | Using Taguchi experimental design methods to optimize trace element composition for enhanced surfactin production by Bacillus subtilis ATCC 21332[END_REF], magnesium ion did not affect the production nor the yield of surfactin with BBG260 in comparison to BBG258, whereas the yield of BBG259 and BBG262 was affected as the end of 10 hours the yield was less or almost equal to one of BBG258 (Figure 37) (Figure 39).

The native promoter of srfA operon was exchanged with a constituitive promoter and affect of this new promoter was studied in this medium. The study was carried out to analyze whether the new promoter can enhance further the production of BBG260 in this new medium.

To study this objective two new strains (Table 17) were constructed with transformation of BBG258 and BBG260 with pBG401 (Figure 28) to replace the native promoter (P srfA ) by a constituitive promoter (P repU ). This constituitive promoter regulates the replication gene repU in S. aureus (Coutte et al., 2010).

Table 17 Genotype of BBG276 and BBG278 strain with a constituitive promoter

The specific growth rate and surfactin production was analyzed for these two strains in Landy-phosphate buffer medium. The specific growth rate was measured during the exponential growth phase and surfactin concentration was measured at 6 hours and 10 hours, respectively. ). The specfic growth rate of these new mutants did not vary in comparison to its parental strain. The replacement of the promoter did not affect the growth rate of the strain.

CONFIDENTIAL DOCUMENT

The surfactin concentration (mg/l) for BBG276 and BBG278 was found to be 116±33 and 731±139 at 6 hours, whereas at 10 hours the surfactin concentration was found to be 245±111 and 1435±179, respectively (Figure 46). The surfactin production of BBG278 was enhanced 6 times at both time points in comparison to control.

CONFIDENTIAL DOCUMENT

The surfactin concentration was normalized with dry weight of the strains at each time point and the specfic surfactin yield was determined at each time point. This value was represented in mg of surfactin per g dry weight of cell biomass. The specific surfactin yield of BBG276 and BBG278 was found to be 369±101 and 878±79 at 6 hours whereas at 10 hours the surfactin concentration was found to be 521±142 and 1613±150 respectively (Figure 47). The surfactin yield of BBG278 in comparison to control was enhanced 2 times and 3 times at both 6 hours and 10 hours respectively.

Major conclusions can be drawn from the above results. The promoter replacement did not have a great impact on the surfactin production. As BBG258 and BBG260 harboring the surfactin native promoter enhanced surfactin yield 1.4 times and 1.6 times, in comparison to its counterpart BBG276 and BBG278 having constitutive promoter at 6 hour (Figure 45). But at 10 hours the surfactin yield of BBG258 and BBG260 was found to be almost equal to BBG276 and BBG278, although the constitutive promoter (P repU ) gave 3 fold and 15 fold higher production of iturin A (K Tsuge et al., 2001) and mycosubtilin [START_REF] Leclère | Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism's antagonistic and biocontrol activities[END_REF] in comparison to corresponding strains with native promoter.

CONFIDENTIAL DOCUMENT

Publication and Valorization of these results

ORAL COMMUNICATION

Dhali.D , Coutte.F , Niehren.J , Versari.C, John.M, Bidenko.V, Auger.S, Jacques.P.

Metabolic engineering for the overproduction of surfactin: an integrative approach,

International

ROLE OF PYRUVATE FLUX

Besides single knockout prediction from the reaction network (Figure 34), the prediction based on flux was also provided (Table 8). The first solution of this prediction suggested that an increase in pyruvate flux can lead to an enhancement of leucine. In the glycolytic pathway (Figure 15), there are various genes which are essential for the growth of B. subtilis [START_REF] Commichau | Essential genes in Bacillus subtilis: a reevaluation after ten years[END_REF]. Besides these genes four genes were selected within the glycolytic pathway to analyze their role in surfactin production. All the partners in the European project also agreed to work on similar set of genes to have common strains for future experiments.

Mutant constructed

The glycolytic pathway mutants were constructed in B. subtilis BSB1 strain by the group of Prof. J Stülke (University of Göttingen, Germany) (Table 19).

These genes include pgcA: responsible for the interconversion of glucose-6-phosphate to α-glucose-1-phosphate; pfkA: catalyzes the conversion of fructose-6-phosphate to fructose-1, 6-bisphosphate; pyk: converts phosphoenol pyruvate to pyruvate and pgi: is an isomerase responsible for reversible reaction between glucose-6-phosphate and fructose-6phosphate.

Table 19 Deletion mutants obtained with Bacillus subtilis BSB1 strain with control based on glycolytic pathway

CONFIDENTIAL DOCUMENT

These mutants lack sfp gene and were thus transformed with pBG400 for the insertion of sfp gene to analyze the impact of the individual genes on surfactin production. The mutants were renamed after sfp gene insertion as shown in Table 20.

Table 20 Integration of sfp gene in the glycolytic mutants obtained with Bacillus subtilis BSB1 strain

Growth and surfactin analysis

The glycolytic mutants obtained with B. subtilis BSB1 strain shown in Table 14 were cultivated in high-throughput system of fermentation Biolector ® . Cultures were performed in both classical Landy medium and Amber medium at 37°C with shaking frequency of 1100 rpm. During the exponential phase, the glycolytic genes are highly expressed in order to correlate their influence on surfactin production the samples were withdrawn at 6 h and 8 h of culture.

Growth analysis

Growth kinetics study of the mutant strains were carried out during the exponential growth phase in classical Landy medium and Amber medium using the BioLection V.2.4.1.0 software (m2p-labs GmbH, Germany). The detailed composition of the media used has been provided in APPENDIX II. In both media, besides glucose, another carbon source has been used, as the mutants could not grow properly if only glucose is present. Classical Landy medium contains glucose and glutamic acid whereas AMBER medium contains glucose, L-malate and glutamic acid. Previous studies have revealed that malate is co-

CONFIDENTIAL DOCUMENT

metabolized with glucose, and malate metabolism is not prone to glucose dependent carbon catabolite repression [START_REF] Buescher | Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism[END_REF].

When BBG258 was grown in classical Landy medium, the specific growth rate was found to be 0.489±0.002 h -1 . But there was a decline in the specific growth rate of the mutants and it was found to be 0.232±0.014; 0.406±0.017; 0.394±0.006 and 0.335±0.023 h -1

When BBG258 was grown in Amber medium, the specific growth rate was found to be 0.355±0.002 h for BBG266; BBG267; BBG268 and BBG269. It was found that there was a significant decline in specific growth rate of BBG266 where the gene pfkA was deleted.

-1 . But there was a decline in the specific growth rate of the mutants and it was found to be 0.312±0.013; 0.394±0.013; 0.319±0.003 and 0.37±0.002 h -1

An overview of specific growth rate of BBG258 and its derivatives in different media have been provided in for BBG266; BBG267; BBG268 and BBG269.

Table 21. The deletion of the respective genes produces viable cells as the genes were not essential for the viability of the cells [START_REF] Commichau | Essential genes in Bacillus subtilis: a reevaluation after ten years[END_REF].

Table 21 Specific growth rate of glycolytic mutant strains and BBG258 in Landy medium and AMBER medium These genes are involved in the regulation of the glycolytic pathway and determine the flux towards pyruvate. Beside their role in the glycolytic pathway they can indirectly regulate various other pathways in B. subtilis. Growth analysis of BBG258 showed that the specific growth rate of this strain is better in Landy medium in comparison to AMBER medium. The reason for such a behaviour may be due to fact that glucose concentration (20 g/l) in Landy medium is 5 times more than glucose concentration (4 g/l) in Amber medium irrespective to the presence of other carbon sources and may be also due to presence of yeast extract in Landy medium.

The gene pfkA is responsible for the synthesis of fructose-1, 6-bisphosphate. There was a marked reduction is specific growth rate of this mutant in Landy medium than in Amber medium with respect to their control. In both media, the central pathway for the utilization is blocked due to deletion of respective genes but in Amber due to presence of malate which is responsible for gluconeogenesis, it can counter-balance the loss of glucose. The disruption of pgi did not affect the specific growth rate of the strain in both media as metabolic flux can flow through alternative pathway (pentose phosphate pathway) for the synthesis of respective metabolites (Figure 52). The deletion of pgcA also has no effect on the specific growth rate of the strain in both media as it is responsible for the conversion of glucose-1-phosphate to glucose-6-phosphate and did not affect the metabolic flow in the central pathway. In Landy medium, the strain with deletion of pykA has reduced specific growth rate but in AMBER medium it has no effect.

Surfactin analysis

The impact of gene deletion on surfactin production was analyzed. The surfactin concentration at each time point (6 h and 8 h) was determined through RP-HPLC.

It can be observed from Figure 48, that all the mutant strains produced surfactin with varied concentration in classical Landy medium. The control strain (BBG258) produced 746±29 mg/l and 1082±50 mg/l of surfactin at 6 hours and 8 hours, respectively. At both time points the concentration of surfactin (mg/l) by the glycolytic mutant strains was less in comparison to BBG258. The maximum surfactin production among the mutants was obtained with BBG268 (BSB1, sfp + , ΔpgcA) at both time points with a concentration of 483±7 mg/l and 1018±78 mg/l. The figure does not represent the normalization concentration with dry weight of specific strain; this representation has been provided in the later section. Thus, it can be deduced from Figure 48 and Figure 49 that modifying glycolytic pathway has a definitive effect on the surfactin production. It can be observed that the surfactin production of BBG258 in classical Landy medium was more than in AMBER medium, it is due to variation in glucose concentration. But the mutant strain BBG266 (BSB1, sfp +

CONFIDENTIAL DOCUMENT

, ΔpfkA) which is the least producer among the strains in both media was found to be better producer in AMBER medium than in classical Landy medium due to the presence of malate in the nutrient composition in the former medium which led to gluconeogenesis resulting in synthesis of few metabolites which could not be synthesized by glycolysis due to deletion of respective gene ( ).

The surfactin production was normalized with dry weight (DW) of each strain. The normalized surfactin production was renamed as surfactin yield (mg of surfactin per gm DW). The specific surfactin yield of BBG258 was found to be 868±78 and 1054±7 mg of surfactin per gm of dry weight (DW) of cell biomass at 6 hours and 8 hours, respectively in classical Landy medium. For BBG266, BBG267, BBG268, and BBG269 specific surfactin yield was found to be 264±94; 817±76; 966±15; 523±54 mg of surfactin per gm of DW of cell biomass at 6 hours while at 8 hours the specific surfactin yield was 375±15; 744±23; 1150±51; 684±22 for the respective strains (Figure 50). The minimum surfactin yield was obtained with BBG266 (BSB1, sfp +

CONFIDENTIAL DOCUMENT

CONFIDENTIAL DOCUMENT

When similar experiment was performed in AMBER medium with BBG258 and its derivative strains, the specific surfactin yield of BBG258 was found to be 760±34 and 941±31 mg of surfactin per gm of cell DW at 6 hour and 8 hour of incubation. The surfactin yield for BBG266, BBG267, BBG268 and BBG269 was found to be 341±79; 399±72; 711±18 and 369±75 mg of surfactin per gm of cell dry weight respectively at 6 hour while at 8 hour the specific surfactin yield was found to be 325±26; 421±12; 913±36 and 606±24 mg of surfactin per g DW for the respective strains (Figure 51).

The surfactin yield obtained with the strain BBG266 (BSB1, sfp + , ΔpfkA) was lowest among the glycolytic mutants. The specific surfactin yield of BBG266 was found to be 2 times and 3 times lower than the control strain, BBG258 at 6 hour and 8 hour, respectively.

The surfactin yield of strain BBG268 (BSB1, sfp +

, ΔpgcA) was equivalent to BBG258 at both time points. In , the surfactin concentration obtained with BBG268 was lower than BBG258 in both time points. This observation was due to the fact that the growth of BBG268 was lower than BBG258 in both the time points, which reflected in equal yield of surfactin by both strains.

CONFIDENTIAL DOCUMENT

Statististical analysis of Figure 50 and Figure 51, revealed that at 6 hour there is a significant decline in surfactin yield with BBG266 and BBG269 in Landy medium but in AMBER medium there is a significant decrease in yield with the strains BBG266, BBG267 and BBG269, respectively in comparison to BBG258. At 8 hour, there is a significant decrease with BBG266, BBG267 and BBG269, respectively, in both the condition in comparison to BBG258.

Thus, the glycolytic mutants have negative effect on surfactin production in both the media. Although the yield of surfactin was obtained more in classical Landy medium in comparison to AMBER medium with all mutant strains along with control. This observation reflected the fact that high glucose concentration can lead to high surfactin production, in this study which was present in classical Landy medium.

Carbon metabolism and surfactin production

Glycolytic pathway is responsible for the uptake of carbon source and is simultaneously distributed to various pathways. An important starting point for metabolic engineering is to determine the flow of carbon flux towards the metabolic link of various pathways.

Pyruvate forms this linkage, as carbon flux enters glycolytic pathways and is distributed to various pathways i.e. production of lactate; acetate; acetoin; ethanol; 2, 3-butandiol;

branched chain amino acids etc [START_REF] Romero | Metabolic engineering CONFIDENTIAL DOCUMENT of Bacillus subtilis for ethanol production: Lactate dehydrogenase plays a key role in fermentative metabolism[END_REF]. Different studies have been carried

out to determine the role of glycolytic pathway in the production of various secondary metabolites. In E. coli, the production of lycopene was limited by the concentration of glyceraldehyde-3-phosphate. This problem was overcome by reducing the metabolic flux to pyruvate by the deletion of pykA, responsible for the conversion of phosphoenol pyruvate to pyruvate [START_REF] Farmer | Precursor balancing for metabolic engineering of lycopene production in Escherichia coli[END_REF]. Similar study was performed with

Streptomyces clavuligerus to enhance the biosynthesis of clavulanic acid. Here, two genes gap1 and gap2 were deleted to increase the pool of glyceraldehyde-3-phosphate, which is one of the precursors for the production clavulanic acid [START_REF] Olano | Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering[END_REF]. Thus, the primary metabolism forms a determining factor for the supply of precursors required for production of secondary metabolites. Glucose is a good source of carbon as it can support growth as well as other physiological processes i.e. surfactin production. The regulation of surfactin production is very complex. The synthetase requires various monomers for the synthesis of this non-ribosomal lipopeptide. Previous studies suggested that surfactin

CONFIDENTIAL DOCUMENT

production depends on cell density and carbon concentration. Until now no data have been presented between surfactin production and central carbon metabolism. There are various genes within central carbon metabolism pathway which are essential for the growth of B.

subtilis. These genes include gapA; pgm and eno, which are responsible for the conversion of glyceraldehyde-3-phosphate to 1,3-bisphospahte glycerate; 3-phospho-glycerate; 2phospho-glycerate to phosphosphoenol pyruvate (Commichau and Stülke, 2008) (Figure 52). production. The production analysis showed that effect of nutrient composition can also have additional or counter balancing effect on the glycolytic mutants.

Based on the nutrient composition, the growth as well as the surfactin yield of BBG258 (control strain) was better in classical Landy medium in comparison to AMBER medium in all time points (6 hour and 8 hour). But the glycolytic mutants showed variation in specific growth rate in Landy medium but the differences in AMBER medium was closely clustered. The specific surfactin yield of these mutants in Landy medium was better than in AMBER medium at both time points reflecting that high flux of glucose is essential for the higher production of precursors required for surfactin biosynthesis.

Upon uptake of glucose by PtsGHI, glucose is phosphorylated to glucose-6-phosphate, although the biosynthesis of this molecule can also take place through the conversion of glucose-1-phosphate by pgcA. This gene does not lie within the central metabolic pathway and the substrate for this gene is provided by other carbon sources i.e. starch and sucrose (Figure 52). This gene is responsible for the conversion of glucose-1-phosphate to UDPglucose, which is essential for the synthesis of lipoteichoic acid, major and minor teichoic acid. These components are the constituents for the cell envelope biosynthetic process.

This gene produces a nucleotide sugar, which is a substrate for the eps pathway for the production of exopolysaccharide in B. subtilis and surfactin also plays a vital role in the arrangement of aerial structures on biofilm of B. subtilis [START_REF] Vlamakis | Sticking together: building a biofilm the Bacillus subtilis way[END_REF]. But no relation has been established between this gene and surfactin biosynthesis. The results (Figure 50 and Figure 51) showed that the gene cannot lower the flux for the synthesis of precursors required for the synthesis of surfactin.

The gene pgi is responsible for a reversible reaction between glucose-6-phosphate and fructose-6-phosphate. Presence of glucose can increase the expression of gap gene and pgk operon but the expression decreased in the absence of glucose. But this glucose dependent activity was not observed for genes pgi, pfk, fbaA and pykA [START_REF] Tobisch | Role of CcpA in regulation of the central pathways of carbon catabolism in Bacillus subtilis[END_REF]. It was reported that a mutant of pgi have poor growth in medium where carbon source is solely glucose as the flux is directed towards the pentose phosphate pathway [START_REF] Lin | Selection of a mutant of Bacillus subtilis deficient in glucose-6-phosphate dehydrogenase and phosphoglucoisomerase[END_REF]) (Figure 52). Similar behavior was observed in this study that when BBG267 (BSB1, sfp + : cm r , ∆pgi:kan r ) was grown in classical Landy medium, there was a slight decrease in specific growth rate but no effect was observed when it was grown in AMBER medium.

The reason for such a behavior is due to the fact that classical Landy has only glucose but AMBER media contains both glucose and malate. This gene is constitutively expressed

CONFIDENTIAL DOCUMENT

and an increase in intracellular concentration of this protein was observed upon salt stress (Kohlstedt et al., 2014a). But the salt stress condition was not imposed in this study. Thus, it can be inferred that an alteration of surfactin yield was observed with pgi mutant in comparison to control is solely due to decline in metabolic flux and less synthesis of precursors required for the biosynthesis of surfactin.

The conversion of fructose-6-phosphate to fructose-1, 6-bisphosphate is facilitated by pfkA whereas fbp is responsible for the reverse reaction. The former reaction is ATP-dependent that represents the initial step dedicated to glycolysis. Upon deletion of pfkA the doubling time was increased in comparison to control in both minimal and rich media (Muñoz-Márquez and Ponce-Rivas, 2010). This result is coherent with the observation in this study, as in both classical Landy and Amber media the strain BBG266 (BSB1, sfp + : cm r , ∆pfkA: kan r

) showed growth defects. The expression of this gene is induced by the presence of glucose thus regulating the direction of flux (Holger Ludwig et al., 2001).

Regulation of gene expression is controlled by messenger RNA turnover mechanism in bacteria. This mechanism is mediated by the role of various RNA binding proteins, noncoding RNAs and action of various ribonucleases (RNases) [START_REF] Newman | Dissection of the network of interactions that links RNA processing with glycolysis in the bacillus subtilis degradosome[END_REF].

The glycolytic enzymes enolase and phosphofructokinase (PfkA) along with four proteins (RNase Y, PNPase, RNase J1 and RNase J2) and RNA helicase CshA are involved in the metabolism of RNA and are known as degradasome [START_REF] Lehnik-Habrink | The RNA degradosome in Bacillus subtilis: Identification of csha as the major RNA helicase in the multiprotein complex[END_REF]. This association forms a link between glycolysis and RNA degradation. The degradasome proteins are found in abundance in B. subtilis and since PfkA is a part of the degradasome, it has multiple functions [START_REF] Cascante-Estepa | Localization of Components of the RNA-Degrading Machine in Bacillus subtilis[END_REF]. The glycolytic enzymes enolase and phosphofructokinase forms a complex known as glycosome, which may be involved in the increase of flux through glycolytic pathway [START_REF] Newman | Dissection of the network of interactions that links RNA processing with glycolysis in the bacillus subtilis degradosome[END_REF]. Deletion of pfkA can have affect on the levels of phosphorylated compounds in the cell and thus can influence the pyruvate pool (Muñoz-Márquez and Ponce-Rivas, 2010). Fructose-1, 6bisphosphate which is a product of PfkA can contribute to the activity CcpA and CcpA acts as an activator for the ilv-leu operon by binding to its cre-site [START_REF] Sonenshein | Control of key metabolic intersections in Bacillus subtilis[END_REF] ( ). So, in the absence of PfkA, there might be an alteration in the expression of ilv-leu operon due to the inactivation of CcpA and expression of this operon is essential for the biosynthesis of leucine, which is one of the most abundant precursor of surfactin within the peptidic moiety. Therefore, the deletion of pfkA has a dual repression affect on the surfactin production, firstly, lowering the flux towards pyruvate which is precursor for the synthesis of branched chain amino acids and secondly, through inactivation of CcpA.

Thus, it can be concluded that pfkA is essential for achieving both maximal growth rates and surfactin production.

The glycolytic enzymes Pfk and PykA play a crucial role in determining the carbon flux in the glycolytic pathway (Holger Ludwig et al., 2001). The gene pykA flanks to pfkA gene and both are expressed constitutively in B. subtilis [START_REF] Muñoz-Márquez | Effect of pfkA chromosomal interruption 167 CONFIDENTIAL DOCUMENT on growth, sporulation, and production of organic acids in bacillus subtilis[END_REF]. This gene is responsible for the synthesis of pyruvate, which is the final step of glycolysis and pyruvate forms the principal metabolite linking several important pathways (Figure 44). Any of these pathways can be associated to cell cycle processes, therefore cells might use pyruvate as a fundamental metabolite in processes that regulates cell cycle and division with growth. Disruption of pyruvate kinase (pyk) can hinder the normal role of FtsZ protein and its association into Z ring, which can affect normal cell growth [START_REF] Monahan | Coordinating bacterial cell division with nutrient availability: A role for glycolysis[END_REF]. It was also hypothesized that in the absence of pyk, it can result in the accumulation of phosphoenol pyruvate (PEP), which can lead to lowering of growth rate [START_REF] Fry | Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant[END_REF]. But in both cases, addition of pyruvate exogenously re-established the normal division in a pyk mutant strain. So it can be inferred that lowering of specific growth rate of BBG269 (BSB1, sfp + : cm r , ∆pyk: kan r ) observed in classical Landy medium was due to one of the reasons or the accumulation of PEP interacts with FtsZ protein and repressing its activity. But this lowering of specific growth rate was not observed in AMBER medium due to the presence of malate in the nutrient composition and malate can be converted to pyruvate by the action of three enzymes, MaeA, MalS and MleA [START_REF] Meyer | Malate metabolism in Bacillus subtilis: Distinct roles for three classes of malate-oxidizing enzymes[END_REF]. It was found that the glucose consumption rate was delayed in pyk mutant. Transcript level analysis of several genes showed that there was an upregulation of genes related to non-PTS glucose transporters and down regulation of genes related biosynthesis of metabolites, which use pyruvate as precursor. There was an increase in transcript level of genes responsible for the conversion of malate to pyruvate to compensate the pyruvate limitation. It was also found that the transcript level of genes in the pentose phosphate pathway and gene for the conversion of glutamate to α-ketoglutarate was increased too [START_REF] Cabrera-Valladares | Physiologic consequences of glucose transport and phosphoenolpyruvate node modifications in bacillus subtilis 168[END_REF]. In a pyk mutant due to accumulation of PEP, there was an increase in the production of various aromatic compounds i.e.

shikimic acid, dehydroshikimic acid etc [START_REF] Licona-Cassani | Inactivation of pyruvate kinase or the phosphoenolpyruvate: Sugar phosphotransferase system increases shikimic and dehydroshikimic acid yields from glucose in Bacillus subtilis[END_REF]. Thus, it can be concluded for these studies that the decline in surfactin production of BBG269 (BSB1, sfp + : cm r , ∆pyk: kan r ) was majorly due to pyruvate limitation. But still gradual increase in

CONFIDENTIAL DOCUMENT

yield was observed in both classical Landy and AMBER medium at 6 hours and 8 hours was due to re-routing of flux through pentose-phosphate to pyruvate as well as synthesis of pyruvate through TCA cycle metabolites i.e. malate and α-ketoglutarate.

The above results proved that the prediction based on pyruvate influx is true and an increase in pyruvate flux can indirectly lead to an enhancment in surfactin production.

IMPACT OF DELETION ON SURFACTIN VARIANTS

Previously, all the studies were linked to surfactin's quantitative analysis but in this section the main focus is on the impact of deletion mutant on surfactin isoform or qualitative analysis of surfactin. Surfactin is a heptapeptide linked to a β-hydoxy group of fatty acid chain forming a cyclic lactone ring structure. The peptidic moiety is arranged in a cyclic sequence of Glu-Leu-Leu-Val-Asp-Leu-Leu with chirality LLDLLDL. The β-OH fatty acid length varies from 12 to 16 carbon atoms. Studies have revealed that the position 2, 4 and 7 of the peptidic moiety are generally substituted, out of which position 4 has the greatest substrate variation. The D-amino acids present in position 3 and 6 are highly conserved within the peptidic moiety (Table 3 page no.35). The lipidic tail of surfactin also exhibits heterogeneity. The β-OH fatty acid can be iso, anteiso C 13 ; iso, normal C 14 and iso, anteiso C 15 (Peypoux et al., 1999a). The chain lengths C 14 and C 15 are usually predominant (Jacques, 2011).

This structure is mainly responsible for various activities of this molecule. In addition to the main surfactin variant, the producing strain also produces different variant of surfactin but the concentration of this variant differs based on the genetic make-up of the strain.

This variant production mostly depends on the amino acid present in the culture media or within the intracellular pool.

The bkd operon (ptb-bcd-buk-lpdV-bkdAA-bkdAB-bkdB) of B. subtilis encodes for enzymes responsible for the degradation of branched chain amino acids (valine, leucine and isoleucine) [START_REF] Nickel | Cold induction of the Bacillus subtilis bkd operon is mediated by increased mRNA stability[END_REF]. The promoter of the operon is present up-stream of ptb and the terminator is downstream of bkdB, thus encompassing the entire operon.

Moreover, it was speculated that another promoter might be present within the coding region of buk. The gene bkdR is essential for the expression of bkd operon. The genes lpdV, bkdAA, bkdAB and bkdB encode E3, E1α, E1β and E2 proteins which are functional components of branched chain α-keto dehydrogenase (M Debarbouille et al., 1999).

Isoform analysis

Isoform analysis was carried out using LC-MS-MS with the 6 hour sample of each mutant (BBG258, BBG260, BBG261 and BBG262) as it has been described in materials and

Extracellular metabolite analysis

Extracellular metabolites of the strains BBG258 and BBG261 were analyzed using nuclear magnetic resonance ( 1Samples were collected just after inoculation; this represents the concentration of the metabolites at 0 hour. The results of the extracellular depicts outflow of metabolite indicated by increase in concentration at a particular time into the extracellular medium whereas decrease in concentration at particular time indicates utilization or that metabolite was not produced.

H-NMR) spectroscopy at 310 K. Both standard compounds and Human Metabolome Database (HMDB) was applied for the identification of spectra while the quantification was carried out by exploiting the signal area of Quant Ref signal at 15 ppm (material and method: section 12). The strains were incubated in Landy medium with ammonium sulphate, buffered with phosphate, and the sampling was done at every 2 hour intervals upto 10 hours.

As shown in Figure 54, the metabolic flux from pyruvate, flows to various pathways producing numerous metabolites, which are efflux into the extracellular medium. Pyruvate was also released into the medium and afterwards it was consumed for the production of various metabolites. The strain BBG258 efflux pyruvate with the concentration reaching upto 0.13±0.009 mM at the end of 4 hours, whereas the maximum concentration of pyruvate in the medium by BBG261 was reached after 4 hours with a concentration 0.06±0.007 mM. After 4 hours and 6 hours, the strains BBG258 and BBG261, respectively starts to consume pyruvate.

The release of high concentration of pyruvate by BBG258 into extracellular medium also indicates that this metabolite was produced in higher concentrations and may be directed into various pathways or released in higher concentration in comparison to BBG261.

After 6 hours, the strain BBG258 starts to produce lactate while the strain BBG261 did not directs the metabolic flux towards the synthesis of lactate indicated by almost constant concentration of lactate in the extracellular medium.

Both strains start to consume isoleucine in a similar pattern and after 5 hours and 6 hours, isoleucine is released into the medium by the strain BBG258 and BBG261 but in a concentration ranging from 0.01 to 0.03 mM.

CONFIDENTIAL DOCUMENT

Acetate was the main overflow metabolite produced by both strains. The maximum concentration produced by BBG258 and BBG261 was 17±3.25 mM and 15±3.5 mM after 8 hours, respectively. The metabolic flux from pyruvate is directed towards 2-acetolatate and from there, it is simultaneously converted to valine, leucine or acetoin. The concentration of acetolactate changes dynamically in the extracellular medium which indicates that it is produced and consumed for the synthesis of other metabolites. Similar pattern of production and consumption was followed by both strains and changes are in the order of 10 -3 mM.

CONFIDENTIAL DOCUMENT

Acetoin was synthesized from 2-acetolatate. The maximum amount of flux was directed towards acetoin synthesis by BBG258 in comparison to BBG261. The maximum concentration in the medium reaching up to 14±1.6 mM and 5±0.7 mM for BBG258 and BBG261, respectively after 10 hours. It can be inferred that the metabolic flux in BBG261 was directed towards fatty acid synthesis and less towards acetoin synthesis in comparison to BBG258. Acetoin is converted to acetyl-coA, which is then used for the fatty acid biosynthesis. This hypothesis was also validated through the synthesis of 2, 3 -butandiol.

The strain BBG258 produces 2, 3 -butandiol at a concentration of 11±2.2 mM while a mere concentration of 0.7±0.14 mM was produced by BBG261 at the end of 10 hours. The metabolic flux from 2-acetolatate is also directed towards the synthesis of valine and leucine.

Leucine is converted by a reversible reaction to 2-oxo-isocaproic acid by either of these genes bcd, ywaA and ybgE. Then the gene cluster bkdAA and bkdAB is responsible for the conversion of 2-oxo-isocaproic acid to 3-Methyl 1-hydroxybutyl and is simultaneously converted to 3-Methyl butanoyl-dihydrlipoamide-E by the same set of genes. This metabolite is converted to isovalerate and dihydrlipoamide-E by bkdB. The former is used for the synthesis of branched chain fatty acids (isoC15:0 or isoC17:0), while the latter is converted to lipoamide by lpdV and this is used as a substrate by the enzyme encoded by bkdAA and bkdAB for the synthesis of 3-Methyl butanoyl-dihydrlipoamide-E. The gene lpdV encoding for the enzyme E3 lipoamide dehydrogenase was deleted in the strain BBG261, the gene is shown in red (Figure 54).

As shown in Figure 54, leucine was released into the medium after 4 hours and 6 hours by BBG258 and BBG261, respectively. But the efflux concentration differs as maximum concentration in the medium reached by BBG258 was 0.6±0.08 mM and for BBG261 it reached 0.18±0.04 mM. The concentration of 2-oxo-isocaproic acid changed in both directions for BBG261 during the course of experiment, as it was released and consumed, while BBG258 releases this metabolite into the environment after 4 hours. Thus, it can be hypothesized from the results that the flux in BBG258 was mostly directed towards the production of overflow metabolites i.e. lactate, acetate, acetoin and 2, 3butandiol and the branched chain acids (valine and leucine) was consumed in the initial stage and released into the environment in the later stage. But in the case of BBG261, the overflow metabolites were synthesized in less concentration except acetate in comparison to BBG258, as well as leucine and valine were not released into the medium. As a result, higher concentration of precursors are directed towards branched chain fatty acid synthesis from valine or leucine or towards conventinal fatty acid synthesis from acetoin.

The strains were also analyzed for the metabolism of valine and leucine; it was observed that due the deletion of lpdV, the recycling of dihydrolipoamide-E was inhibited leading to lower production of 2-Methyl propanoyl-dihydrlipoamide-E and 3-Methyl butanoyldihydrlipoamide-E with BBG261 in comparison to BBG258. This was well co-related by lower production or utilization of isobutyrate and isovalerate. This can also be speculated that these products are utilized by BBG261 for the production of branched chain fatty acids leading to production of isoforms of surfactin. The promoter of the bkd operon was reported to be induced by isoleucine and valine (M Debarbouille et al., 1999). The expression of this operon was reduced when the transporters responsible for the import of these two amino acids were deleted. It was observed that beside these two amino acids, leucine can also induce weaker activation of this promoter. The β-galactosidase activity of ptb-lacZ was observed 23 times and 17 times less with presence of leucine in comparison to isoleucine and valine respectively. Using ptb-lacZ fusion, it was observed that the promoter activity depends on the presence of amino acids in the following order isoleucine>valine>leucine [START_REF] Belitsky | Intermediate Levels of Bacillus subtilis CodY Activity Are Required for Derepression of the Branched-Chain Amino Acid Permease, BraB[END_REF].

Presence of valine serves as a precursor for the production of surfactin variants with even isoβ-hydroxy fatty acids whereas presence of isoleucine and leucine enhanced the production of surfactin variants with odd anteiso-and isoβ-hydroxy fatty acid, respectively. In the absence of any amino acids, the presence o f β-hydroxy fatty acid in surfactin isoform was found to be C 15 >C 14 >C 13 >C 16 (J. F. Liu et al., 2012). Similar results

CONFIDENTIAL DOCUMENT

were observed with BBG258 with no genetic alteration resulting in higher production of C 15 followed by C 14 and C 13 , respectively.

It was found that both [Leu7] -and [Val7] -gave rise to C 14 surfactin variant although in the former variant, the iso C 14 was mixed with small amount anteiso form depicting that leucine can also give rise to a mixture of iso C 14 [START_REF] Kowall | Separation and Characterization of Surfactin Isoforms Produced by Bacillus subtilis OKB 105[END_REF]. The concentration of leucine and isobutyrate present in extracellular medium at 6 hours was 0.18±0.02 mM and 0.15±0.009 mM for BBG258 whereas for BBG261 the concentration of the former and latter was 0.06±0.03 mM and 0.018±0.002 mM for leucine and isobutyrate at 6 hr, respectively (Figure 55 and Figure 56). Thus, it can be hypothesized from isoform analysis and surfactin analysis that in BBG261, deletion of lpdV directed the degradation of valine towards the synthesis of iso C 14 . This was well supported by the concentration of leucine and isobutyrate in the extracellular medium and high percentage of C 14 through LC-MS analysis.

The production of this isoform in high concentrations can be used for industrial application, specifically as biosurfactant. Indeed, the spreading activity of the biosurfactant produced by B. mojavensis was enhanced when percentage of C 14 was more than C 15 as even-numbered fatty acid provide an optimum hydrophilic-lipophilic balance, which is an essential criterion for surface activity [START_REF] Youssef | Importance of 3-Hydroxy Fatty Acid Composition of Lipopeptides for Biosurfactant Activity Importance of 3-Hydroxy Fatty Acid Composition of Lipopeptides for Biosurfactant[END_REF].

Publication and Valorization of these results

ARTICLE

Dhali, D., Coutte, F., Arias, A.A., Auger, S., Bidnenko, V., Chataignė, G., Lalk, M., Niehren, J., Sousa, J., Versari, C., Jacques, P. (2017). Genetic engineering of the branched fatty acid metabolic pathway of Bacillus subtilis for the overproduction of surfactin C 14 isoform. Biotechnology journal, (submitted) .

Table 22 The mutants obtained with the deletion of yvbW along with the replacement of native promoter with constitutive promoter in B. subtilis strains

The plasmid pBG401 harbours the P repU promoter sequence cloned between the homologous region of the native P srfA in B. subtilis 168 (Figure 28). The strains BBG258 and BBG257 were transformed with this plasmid through natural transformation technique and selected on LB-erythromycin. The integration of P repU promoter was verified through PCR. The genotype of the obtained mutants has been shown in Table 22.

Growth and surfactin analysis with native promoter and constitutive promoter

The mutants obtained with B. subtilis BSB1 (BBG257) along with control strain (BBG258) was cultivated in Landy medium with ammonium sulphate and phosphate buffer condition (APPENDIX II).

The experiment was performed in a shake flask at 37°C and 250 rpm. Suicidal flask strategy was used i.e. one flask for each time point (6 h, 10 h and 24 h). Experiments were carried out in triplicate and two biological replicates were performed. Results are expressed as mean value with standard deviation.

Similar strategy was used for the analysis of growth and surfactin production for the strains BBG275 and BBG276. But with these strains, the samples were collected only at 6 h and 10 h.

Strains with native promoter (P srfA )

The growth of the strains (BBG257 and BBG258) was analysed by measuring the optical density at 600 nm at each time point. Overview of the specific growth rate of all the strains 142 CONFIDENTIAL DOCUMENT is provided in Table 22. Growth kinetics study of all the strains was carried out during the exponential growth phase.

The specific growth rate of BBG258 in Landy medium with MOPS as buffering agent was found to be 0.42±0.01 (Table 16) but the specific growth of BBG258 was lowered in this condition and was found to be 0.37±0.01. The reason for such a behaviour is due to lower concentration of magnesium ions in this media in comparison to Landy medium with MOPS. Moreover this decline in concentration did not affect the growth rate of BBG257 in comparison to BBG258 in Landy medium with MOPS.

Table 23 Specific growth rate of BBG258 and BBG257 in modified Landy medium buffered with phosphate buffer Since most of the genes that regulates the branched chain amino acid metabolic pathway in B. subtilis are pleiotropic in nature, the effect of deletion on the growth of the strains was studied. Growth analysis revealed that the deletion did not affect the growth rate of the mutant strain.

Surfactin production was analyzed with the strains in this media condition at different time intervals. Both strains produced surfactin but with variable concentrations (Figure 57). The surfactin production for BBG258 was found to be 68±5 mg/l, 220±83 mg/l and 694±93

mg/l at 6 h, 10 h and 24 h, respectively, while the surfactin production for BBG257 was found to be 632±89 mg/l, 2453±254 mg/l and 4838±114 mg/l at 6 h, 10 h and 24 h, respectively. The maximum surfactin production was found at 24 hour for both strains. At this time point, BBG257 produced 7 times more than BBG258. strain. The structural organization of native promoter revealed the presence of dyad symmetries upstream of ribosomal binding site of srfA operon, these symmetries are site for ComA binding (Willenbacher et al., 2016). This might be the reason for the stronger expression of P srfA in relation to constitutive promoters (P repU and P veg ) as ComA positively regulates srfA expression through its phosphorylated product ( ).

Secondly, the gene yvbW is an interesting candidate for the overproduction of surfactin.

The strain BBG257 (BSB1, sfp +

The T-box anti-termination mechanism is highly sensitive and allows the bacteria to maintain steady level of aminoacyl tRNA [START_REF] Merino | Transcription attenuation: a highly conserved regulatory strategy used by bacteria[END_REF]. subtilis has leucine specific T-box located upstream of the gene. This gene belongs to APC family of transporters [START_REF] Wels | An in silico analysis of T-box regulated genes and T-box evolution in prokaryotes , with emphasis on prediction of substrate specificity of transporters 16[END_REF]. Under both high and limiting leucine condition, there is expression of yvbW but the level is higher in former condition and lower in later condition [START_REF] Rollins | Induced Transcriptional Expression of Bacillus subtilis Amino Acid Permease yvbW in Response to Leucine Limitation[END_REF].

, ∆yvbW) gave the highest yield of surfactin of 5.7 g per g of cell biomass, among the mutant strains screened in this study as well as all the strains reported in literature, in shake flask condition and without foam fractionation. It was found that this strain was able to convert 37% carbon of 20 g/l glucose to the carbon required for surfactin biosynthesis in shake flask condition. So, it can be hypothesized that upon deletion of this gene the export of leucine was inhibited upto certain extent and more leucine molecules are available as precursors for the surfactin synthetase for production of surfactin.

Publication and Valorization of these results

PATENT

Dhali, D., Coutte, F., Jacques P. Biosurfactant of B. subtilis : overproducing strains and genetic engineering methods to obtain (to be submitted)

CONCLUSION

The study started with a hypothesis that an increase in surfactin production can be obtained with an enhancement of precursor concentration. The reason behind this speculation was that previous studies revealed that the monoproducer lipopeptide strain as well as replacing the promoter did not enhance the surfactin yield. The peptidic moiety of surfactin contains four residues of leucine, so the strain was cultivated in the presence/absence of leucine (Figure 61-1). It was observed that the strain upon leucine feeding produced maximum 1.8 g of surfactin per g of cell biomass. This yield was 3 times higher than the control condition where there was no leucine. This result proved that leucine is one of the precursors required for increasing the production of surfactin or has an indirect role through the regulation of its biosynthesis. The next step was to have the intensive knowledge regarding the metabolism of leucine in B. subtilis. But the entire knowledge regarding the regulation and metabolism is not fully studied until now. Therefore, an integrated approach was required. A complete model of one of the most complex and regulated metabolic pathway of B. subtilis, i.e. the branched chain amino acid metabolic pathway was developed in collaboration with the Biocomputing team of CRIStAL laboratory Lille. This network constitutes the regulation and metabolism of the branched chain amino acids (isoleucine, valine and leucine) (Figure 61-2).This model was established using computational language. Based on this metabolic network, 12 plausible single knockout predictions were returned as well as 2 predictions regarding flux of metabolites in order to improve leucine intracellular pool. Out of 12 single knockout predictions, 6 single knockouts was carried out in B. subtilis 168 strain and 4 similar knockouts were carried out again in B. subtilis BSB1 strain. In Landy medium, with ammonium sulphate (Figure 61-2), the maximum specific surfactin yield was obtained with codY deletion was 1.3 g and 1.27 with the strains BBG254 and BBG260, respectively.

The deletion of codY has the maximum significant impact on surfactin yield as the yield was enhanced 7-fold and 5-fold in comparison to their respective control strains. This was coherent with the metabolic network information as from the metabolic network it was observed that CodY can repress leucine synthesis through various repression mechanism.

The specific yield of TnrA mutants (BBG256 and BBG262) derived from B. subtilis 168 and B. subtilis BSB1 was found to be significant in comparison to control but the yield was lower in comparison to respective codY mutant strains. But the yield obtained with the

CONFIDENTIAL DOCUMENT

double mutant of codY and TnrA (BBG265) was lower in comparison to codY and TnrA single knock-out strains. The reason for such observation was due to the fact that TnrA acts as an activator for the transport of ammonium. There was a significant impact on enhancement of surfactin production due to deletion of bcd gene as this gene is responsible for the degradation of leucine into its specific fatty acid. These set (Figure 61-2) of results validated the prediction provided from the metabolic network and that there is a definitive role of leucine in enhancing surfactin production. Besides low glucose concentration, the presence of amino acids (valine and isoleucine)

activates the CodY repression on ilv-leu and srfA operon. This repression was reflected on low surfactin yield in all mutants, except for the mutants with codY and codY site II deletion. The volumetric oxygen transfer coefficient is a key parameter for surfactin production and the selectivity of the parameters determining the aeration (Figure 61-3).

Therefore, to scale-up the fermentation process from flasks to large-scale fermentors, this parameter should be considered. Surfactin yield is clearly favoured at good oxygenation of the cells (199 h -1 >k L a <400 h -1 ). Thus, there is an upper limit until which k L a has a linear relationship with specific surfactin yield, after that the relatioship depends on the genetic construction of the strain.This parameter also depends on the genotype of the strain, as in this study it was shown that the maximum surfactin yield of BBG258 did not decrease at high k L a value but the yield of BBG260 was affected when this value was enhanced beyond 400 h -1

. The promoter replacement did not have great impact on the surfactin yield ( -3). The surfactin yield obtained with the strains harboring native promoter was higher in comparison to the strains having constitutive promoter at 6 hours. But at 10 hours, the surfactin yield from strains with either promoter was found to be equal.

Impact on pyruvate flux was analyzed with deletion of genes in the glycolytic pathway (Figure 61-4). All the mutants except ΔpgcA showed significant decrease in specific surfactin yield in comparison to control strain (BBG258). The maximum deterioration in surfactin yield was obtained with pfkA deletion mutant as the product of this gene can increase pyruvate flux but also can activate CcpA protein, which is essential for ilv-leu operon to inhibit binding of CodY. It was also observed that in the presence of glutamic acid in Landy medium, the specific surfactin yield of BBG258 was enhanced in comparison to Landy medium with ammonium sulphate.

Besides quantitative analysis of surfactin, qualitative analysis was also carried out (Figure 61-5). It showed that the lpdV mutant resulted in greater production of C 14 -isoform in comparison to other mutant. This could be due to the fact that the flux was more directed towards the production of branched chain fatty acids or normal fatty acid synthesis. This hypothesis was verified through the analysis of extracellular metabolites. The decline in concentration of acetoin, isovalerate and isobutyrate in comparison to control strain supports the fact that the flux was directed towards fatty acid synthesis.

A positive influence of the knockout of a protein with a hypothetical function as leucine permease was verified . A specfic yield of 5.7 g per g of DW was observed, which is reported to be the highest in shake flask condition without foam fractionation.

  to Dr. Joachim Niehren and Dr. Cristian Versari for the development of the metabolic network and providing interesting prediction which really helped me a lot. I am thankful to Prof. Pascal Dhulster for allowing me to access the laboratory and research facilities. My work and stay at INRA was meaningful due to the assistance of Dr. Sandrine Auger and Dr. Vladimir Bidenko. I am really grateful to Dr. Vladimir Bidenko for providing lots of in-depth knowledge of Pop in -Pop out technique as well as helping in getting my first meaningful achievement. I am really thankful to him as he drove me to the lab even on Saturdays and Sundays so I can work and get the results required. I am thankful to the members in Gembloux for LC-MS analysis. I am thankful to Prof. Michael Lalk for allowing me to work in his laboratory and accepting to review my thesis. I am also thankful to Prof. Manuel Canovas for accepting to review my thesis. And, I would like to express gratitude to Dr. Valerie Leclere and Dr.

  really helped me during my stay in Greifswald and helped me a lot to perform and learn the technique to analyze extracellular metabolites. Paola also helped with my work during my stay in INRA. I am thankful to Hannah for assisting me in the last phase of my experiments.

6 Figure 3 Figure 5 Figure 9

 6359 Figure 2 Overview of regulatory pathways affecting competence development as well as positive and negative regulation of comK and related genes are indicated by arrows and Tbars (Hamoen et al., 2003) ..................................................................................................... 6 Figure 3 Amino acid recognition and activation by ATP hydrolysis by Adenylation (A) domain (Sieber and Marahiel, 2005) ................................................................................... 11 Figure Role of sfp gene for the attachment of prosthetic arm (Sieber and Marahiel, 2005) ............................................................................................................................................. 12 Figure 5 Activated amino-acyl adenylate forming a covalent bond with the free thiol of PCP domain (Sieber and Marahiel, 2005) ........................................................................... 13 Figure Peptide bond formation between acceptor (a) substrate and donor (d) substrate is facilitated by C-domain (Sieber and Marahiel, 2005) ......................................................... 14 Figure Role of TE domain for peptide release (Sieber and Marahiel, 2005) ................... 15 Figure Role of epimerization (E) domain during elongation (Sieber and Marahiel, 2005) ............................................................................................................................................. 16 Figure 9 Reaction involving N-methyltransferase (Mt) domain, the yellow box indicates the modification (Marahiel, 2009) ....................................................................................... 17

Figure 10

 10 Figure 10 Reaction involving Formylation (F) domain, the yellow box indicates the modification (Marahiel, 2009) ............................................................................................. 18 Figure 11 Reaction involving oxidation (Ox) and reduction(R) domains, the yellow box indicates the modification (Marahiel, 2009) ....................................................................... 18 Figure Assembly and mode of synthesis of linear NRPSs(Mootz et al., 2002) ............ 19

  Figure Role of glycolytic pathway and production of various metabolites as a result of this pathway (Tojo et al., 2010) ........................................................................................... 22 Figure 16 Complex regulation of branched chain amino acid pathway (Brinsmade et al., 2010) .................................................................................................................................... 25 Figure 17 Role of various genes for the production of isoleucine, leucine and valine (Belitsky, 2015) ................................................................................................................... 26 Figure The role of various enzymes in the synthesis of branched chain fatty acids ...... 27

Figure 46

 46 Figure 46 Surfactin production (mg/l) by BBG276 and BBG278 in Landy medium with phosphate buffer at 37 0 C in shake flask condition with 250 rpm. Results are expressed as mean value with standard deviation .................................................................................. 114 Figure 47 Specific surfactin yield obtained with BBG276 and BBG278 in Landy medium with phosphate buffer at 37 0 C in shake flask condition with 250 rpm. Results are expressed as mean value with standard deviation .............................................................................. 115 Figure 48 Surfactin production (mg/l) of BBG258 and its derivative strains in classical Landy medium at 6 hour and 8 hour, respectively in Biolector ® at 37˚C with shaking frequency 1100 rpm. Results are expressed as mean value with standard deviation ........ 121

  genes responsible in B. subtilis .............................................................................................. 8

  .................................................................................................................................... 28 Table 3 Various isoforms of surfactin based on its peptidic moiety along with surfactin (S) native structure .................................................................................................................... 35 Table 4 Different plasmids and their genotype used in this study ....................................... 64 Table 5 Different E.coli strains and their genotype used in this study ................................ 64 Table 6 Primers used for deletion mutant construction ....................................................... 70 Table 7 Primers used for insertion mutant construction ...................................................... 73 Table 8 Solutions related to flux where no knockouts are required .................................... 86 Table 9 Single knockout predictions for leucine overproduction ........................................ 87 Table 10 Deletion mutants derived from B. subtilis 168 strain based on prediction results of the branched chain amino acid reaction network ............................................................ 89 Table 11 Deletion mutants derived from B. subtilis BSB1 strain based on prediction results of the branched chain amino acid reaction network ............................................................ 90

Figure 1 Figure 1 C

 11 Figure 1 Distribution of essential genes in B. subtilis 168 genome

Figure 2

 2 Figure 2 Overview of regulatory pathways affecting competence development as well as positive and negative regulation of comK and related genes are indicated by arrows and Tbars[START_REF] Hamoen | Controlling competence in Bacillus subtilis: shared use of regulators[END_REF]

Figure 3

 3 Figure 3 Amino acid recognition and activation by ATP hydrolysis by Adenylation (A)

Figure 6

 6 Figure 6 Peptide bond formation between acceptor (a) substrate and donor (d) substrate is catalyzed by C-domain[START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF]

  i) Transferring the final peptide to the active serine residue of the terminal PCP domain of the NRPS assembly (with the formation of oxoester linkage from thioester linkage); ii) producing peptidyl-acyl-enzyme intermediate (acyl-O-TE) following dissociation by an internal nucleophilic reaction (intramolecular region and stereoselective macrocyclization)

Figure 8

 8 Figure 8 Role of epimerization (E) domain during elongation[START_REF] Sieber | Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics[END_REF]

Figure 9

 9 Figure 9 Reaction involving N-methyltransferase (Mt) domain, the yellow box indicates the modification (Marahiel, 2009)

Figure 10

 10 Figure 10 Reaction involving Formylation (F) domain, the yellow box indicates the modification (Marahiel, 2009)

Figure 11

 11 Figure 11 Reaction involving oxidation (Ox) and reduction(R) domains, the yellow box indicates the modification (Marahiel, 2009)

Figure 14

 14 Figure 14Assembly and mode of synthesis of nonlinear NRPSs[START_REF] Mootz | An improvement of surfactin production by Bacillus subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor[END_REF]

Figure 15

 15 Figure 15Role of glycolytic pathway and production of various metabolites as a result of this pathway[START_REF] Tojo | Heavy involvement of stringent transcription control depending on the adenine or guanine species of the transcription initiation site in glucose and pyruvate metabolism in Bacillus subtilis[END_REF]

Figure 17 .

 17 Figure17

Figure 17

 17 Figure 17Role of various genes for the production of isoleucine, leucine and valine(Belitsky, 2015)

Figure 18

 18 Figure 18

Figure 19

 19 Figure 19 Branched chain amino acid transporters A) YvbW (both import and export of leucine); B) BcaP, BrnQ and BraB (import of atleast one BCAA) ; C) AzlCD export of

Figure 20

 20 Figure 20Primary structure of surfactin, where n represents the chain length of fatty acids(Seydlová and Svobodová, 2008)

Figure 21

 21 Figure 21 Operon responsible for the biosynthesis of surfactin, A: denoting the domains present in each sub-unit, B: indicates the amino acid incorporated by each subunit in the final peptide moiety(Jacques,2011)

Figure 23 ,

 23 Figure 23, bacteria produce various quorum sensing molecules (PhrC, PhrF, PhrG, PhrH and ComX). Accumulation of these molecules triggers a cascade of reaction which regulates the expression of srfA. These molecules also help the bacteria to communicate among themselves. Primary regulation is contributed by ComX, which is secreted out of the cell by ComQ. At a particular concentration of ComX, ComP can

Figure 23

 23 Figure23An overview of regulatory pathways affecting surfactin operon expression(Jacques, 2011)

  4 ] or[Ile 4 ] variant surfactin, whereas the presence of L-valine or L-isoleucine can lead to 40-45% production of[Val 7 ]

  e. the elution time of surfactin variant should be [Leu7] < [Val7] < [Ileu7]. They can also differentiate based on their fatty-acid chain as the column contains a modified C 18 silice, the elution time of the fraction with longer fatty acid chain is longer in comparison to the fraction with shorter fatty acid chain.

K

  ; V L is the filling volume ratio; d oThe volumetric oxygen transfer co-efficicent of the Biolector was calculated according to the equation, is shaking diameter; d is the maximum inner diameter of flask(Fahim et al., 2012a). The agitation speed , shaking diameter and inner diameter of the flask was kept constant during the entire study with values 250 rpm, 5 cm and 12 cm, respectively. K L a = OTR max / Lo 2 oxygen transfer capacity; oxygen solubility (Lo 2 = 9.89*10 -4 mol/L/bar at 37˚C) and partial pressure of oxygen in gas phase (p G =0.2095 bar)[START_REF] Bu | The Baffled Microtiter Plate : Increased Oxygen Transfer and Improved Online Monitoring in Small Scale Fermentations[END_REF])(Gnaiger, 2001). The working volume used in Biolector was 1ml with total well volume 3.6 ml.High shaking rate in flask condition is beneficial for the production of lipopeptides by B.

Liu

  et al. (2012) carried out a feeding experiment with different amino acids supplied to the media and the surfactin variants were analyzed using B. subtilis TD7 strain. Addition of amino acids not only affects the amino acids moiety in the peptide ring but also affects the hydroxyl fatty acid moiety. It was found that addition of L-valine to the culture can lead to the production of[Val 7

  Bacillus subtilis ATCC 168 and Bacillus subtilis BSB1

Figure 24

 24 Figure 24Development of master strain B. subtilis BSB1N[START_REF] Tanaka | Building the repertoire of dispensable chromosome regions in Bacillus subtilis entails major refinement of cognate largescale metabolic model[END_REF]

Figure 26 A

 26 Figure 26 A) Plasmid K7010; B) upp-phleo-cI cassette used for markerless deletion

  of B. subtilis strains was performed using the Wizard® Genomic DNA Purification Kit (Promega Corp., Madison, WI, USA). The extraction procedure comprises four steps; the first step is the lysis of cells using lysozyme and the RNA is digested by RNase. The cellular proteins are then removed by a salt precipitation step, while leaving the high molecular weight genomic DNA in solution. Finally, the genomic DNA is concentrated and desalted by isopropanol precipitation and dissolved in a TE buffer. The concentration and purity (A 260 /A 280 ) were measured using NanoDrop (Thermo Scientific NanoDrop Lite Spectrophotometer, USA).

Figure

  Figure 29 Various identifiers in the reaction network

  Figure 31 (Mp2-labs GmbH; Baesweiler; Germany) is a high-throughput fermentation system ( A) available on REALCAT platform of the University of Lille-Sciences and Technologies. Cultures were carried out in 48 wells flower plate (Figure 31B)

  with a C18 column (250 mm x 3.0 mm, Grace, USA). It was quantified using surfactin standard with 98% purity (Lipofabrik, Villeneuve d'Ascq, France). Surfactin was eluted during 20 min under an isocratic condition (80:20) of ACN/TFA (0.1% TFA in 100% ACN) and H2O/TFA (0.1% TFA in 100% ACN) at a flow rate of 0.6 ml/min. The chromatogram at 214 nm was extracted and the peaks were selected on the basis of certain curve of secondary derivative at 214 nm with Savitzky-Golay smoothing of 15. The surfactin concentration was normalized each time with the dry weight of the strain at that particular time interval. Results are expressed as mean values with standard deviation and the statistical analysis of the data has been made using one-way ANNOVA with Tukey's multiple comparison test (p-value <0.0001).

Figure 32

 32 Figure 32 General surfactin structure: It consists of four leucine molecules among seven amino seven amino acids present within the peptidic moeity, the position of the leucine molecules are encircled

Figure 33

 33 Figure 33 Leucine feeding : Impact on specfic surfactin yield with B. subtilis BBG111 in two different media condition with and without the presence of leucine in the culture media at 37˚C in shake flask condition with 160 rpm. Results are expressed as mean value with standard deviation.

Figure 34

 34 Figure 34 Branched chain amino acid reaction network. The concrete syntax of our reaction networks is based on XML, from which the graphs are computed. The XML representation is also the input for the prediction algorithm.

  different activators, the proteins Bcd and YwaA. The intermediates Ket a , Ket b , and Ket c can be transformed alternatively into Acyl-CoA by reactions r 28 , r 31 , and r 34 , which can then flow out into the context for fatty acid biosynthesis. This is regulated by protein BkL. The creation of Ile, Val, and Leu from Ket a , Ket b and Ket c is always coupled with a parallel transformation of Glu into OxoGlu, and the inverse decompositions are coupled with the inverse transformation of OxoGlu into Glu. The required Glu for the production of Ile, Val, and Leu, can be input from the glutamate biosynthesis cycle of the context (derived from OxoGlu) produced OxoGlu can flow out from this pathway to the context (as for example used in the TCA cycle). The intermediates are produced as follows: Ket a is produced from Akb by r 27 , Ket b from Pyr by r 30 , and Ket c from Ket b by r 33 . Akb can be produced from Thr by reaction r 41 , which in turn can also be input from the threonine biosynthesis pathway of the context (which is linked to the glycolysis pathway via the aspartate biosynthesis). Pyr can be input from the glycolysis pathway of the context. IlvA activates the conversion of Thr into Akb. IlvBH (a cluster of IlvB and IlvH), IlvC and IlvD activate the transformation of Akb to Ket a . The proteins LeuA and LeuBCD (a cluster of LeuA, LeuB, and LeuC) perform a sequence of reactions justifying the transformation of Ket b to Ket c

Figure 36

 36 Figure 36 Surfactin production (mg/l) of BBG111 and its derivative strains (B. subtilis 168) in Landy medium with ammonium sulphate at 37˚C in Biolector with shaking frequency of 1100 rpm. Results are expressed as mean value with standard deviation.

Figure 37

 37 Figure 37 Surfactin production (mg/l) of BBG258 and its derivative strains (B. subtilis BSB1) in Landy medium with ammonium sulphate at 37˚C in Biolector with shaking frequency of 1100 rpm. Results are expressed as mean value with standard deviation.

Figure 40

 40 Figure 40 Surfactin production (mg/l) of BBG111 and its derivative strains in TSS medium supplemented with 16 amino acids at 37˚C in Biolector ®

Figure

  Figure41).

Figure 41

 41 Figure 41 Specific surfactin yield (mg/g DW) in TSS medium supplemented with 16 amino acids with BBG111 and its derivative strains. Results are expressed as mean value with standard deviation and statistical analysis of these data was made using one-way ANOVA with Tukey's multiple comparisons test.

,

  ΔtnrA) is due to dual repression, firstly CodY repressing both ilv-leu and srfA operon and secondly in the absence of ammomium is affected. 105 CONFIDENTIAL DOCUMENT high-affinity binding site of CodY, while the promoter-distal CodY-III (nt-154/-107) and CodY-IV (185/-168) sites are low-affinity ones can confirm the yield difference between BBG251 and BBG254.

Figure 42

 42 Figure 42 Comparative analysis on the basis of specific surfactin yield of BBG251 and BBG254 in Landy medium with ammonium sulphate and TSS medium supplemented with 16 amino acids. Results are expressed as mean value with standard deviation.

,

  ∆codY) were grown in Landy medium supplemented with ammonium sulphate. The strains were grown in 1000 ml flask with different filling ratio, V L (0.05, 0.1 and 0.2) and after each time point (6 h and 10 h) the growth and surfactin production were analyzed. Suicidal flask strategy i.e. each flask was dedicated to each time point so that there is no alteration of volume after each time point. Experiments were carried out in triplicate and two biological replicates were performed. Results are expressed as mean value with standard deviation. ® Oxygenation and in shaking flask condition with each filling ratio was calculated with the equation 1 and 2 ( page no. 48)

Figure 44

 44 Figure 44 Surfactin production (mg/l) by BBG258 and its derivative strains in Landy medium with phosphate buffer at 37˚C in shake flask condition with 250 rpm. Results are expressed as mean value with standard deviation.

Figure 46

 46 Figure 46 Surfactin production (mg/l) by BBG276 and BBG278 in Landy medium with phosphate buffer at 37˚C in shake flask condition with 250 rpm. Results are expressed as mean value with standard deviation.

Figure 47

 47 Figure 47 Specific surfactin yield obtained with BBG276 and BBG278 in Landy medium with phosphate buffer at 37˚C in shake flask condition with 250 rpm. Results are expressed as mean value with standard deviation.

Figure 48 Figure 49

 4849 Figure 48 Surfactin production (mg/l) of BBG258 and its derivative strains in classical Landy medium at 6 hours and 8 hours, respectively in Biolector ® at 37˚C with shaking frequency 1100 rpm. Results are expressed as mean value with standard deviation.

Figure 50

 50 Figure 50 Specific surfactin yield obtained with BBG258 and its derivative strains in classical Landy medium at 6 hour and 8 hour respectively. Results are expressed as mean value with standard deviation and statistical analysis of these data was made using oneway ANOVA with Tukey's multiple comparisons test.

Figure 48 ,

 48 Figure48, ΔpfkA), which is 3 fold lower in both time points in comparison to control, BBG258. Although in , the strain BBG268 (BSB1, sfp +

Figure 39 and

 39 Figure 39 and Figure 50. The surfactin yield was increased 3.5 folds in Landy medium with glutamic acid in comparison to Landy medium with ammonium sulphate.

Figure 51

 51 Figure 51Specific surfactin yield obtained with BBG258 and its derivative strains in AMBER medium at 6 hours and 8 hours, respectively. Results are expressed as mean value with standard deviation and statistical analysis of these data was made using oneway ANOVA with Tukey's multiple comparisons test.

Figure 52

 52 Figure 52 Regulation of glycolytic pathway and genes marked in red were deleted to analyze their roles in surfactin production

Figure 54

 54 Figure 54 Time-resolved analysis of overflow metabolites and its associated metabolic pathways. Arrows indicate direct conversion, whereas metabolites in boxes indicate the intermediates. Results are expressed as mean value with standard deviation.

Figure 55

 55 Figure 55 Extracellular concentration of leucine and its degradation products for BBG258 and BBG261, Dashed lines indicated conversion to respective fatty acid. Gene in red deleted. Results are expressed as mean value with standard deviation.

Figure 56

 56 Figure 56 Extracellular concentration of valine and its degradation product for BBG258 and BBG261, Dashed lines indicates conversion to respective fatty acid. Gene in red deleted

Figure 57

 57 Figure 57 Surfactin production (mg/l) by BBG258 and BBG257 in Landy medium with phosphate buffer at 37˚C with 250 rpm in shake flask condition. Results are expressed as mean value with standard deviation.

Figure 58

 58 Figure 58 Specific surfactin yield obtained with BBG258 and BBG257 in Landy medium with phosphate buffer. Results are expressed as mean value with standard deviation.

  The T-box controls genes that are involved in amino acid biosynthesis and transport. Most of the genes involved in the transport of amino acid are regulated by T-box mechanism. The gene yvbW found in B.

Figure 61

 61 Figure 61 Overview of approaches to determine the role of precursors for the enhancement of surfactin in B. subtilis

pBC16FIGUREFIGUREFIGUREFIGURE

  FIGURE Plasmid pBC16 (sfp) used for the construction of pBG400

Table 12

 12 Specific growth rate of all mutant strains derived from BBG111 and BBG258 in Landy medium with ammonium sulphate ........................................................................... 92Table 13 Specific growth rate of BBG111 and its derivative strains in Landy medium with ammonium sulphate and TSS medium supplemented with 16 amino acids ..................... 102 Table 14 Specific growth rate of BBG258 and BBG260 in different aeration conditions 107

Table 15

 15 BBG258 and its derivative strains used in this study ........................................ 110Table 16 Specific growth rate of BBG258 and its derivative strains in different media .. 110

Table Genotype of

 Genotype BBG276 and BBG278 strain with a constituitive promoter .......... 113 Table Specfic growth rate of BBG276 and BBG278 in Landy-phosphate buffer medium ........................................................................................................................................... 114 Table Deletion mutants obtained with Bacillus subtilis BSB1 strain with control based on glycolytic pathway ........................................................................................................ 117 Table Integration of sfp gene in the glycolytic mutants obtained with Bacillus subtilis BSB1 strain ........................................................................................................................ 118 Table Specific growth rate of glycolytic mutant strains and BBG258 in Landy medium and Amber medium ........................................................................................................... 119 Table 22 The mutants obtained with the deletion of yvbW along with the replacement of native promoter with constitutive promoter in B. subtilis strains ...................................... 141 Table 23 Specific growth rate of BBG258 and BBG257 in modified Landy medium buffered with phosphate buffer .......................................................................................... 142 Table 24 Specific growth rate of BBG258 and BBG257 in modified Landy medium buffered with phosphate buffer .......................................................................................... 144

	Methods :	LIST OF ABBREVIATIONS
	PCR :	Polymerase chain reation
	Microorganisms : RP-HPLC : Reverse phase -high performance liquid chromatography
	B. subtilis : Bacillus subtilis NMR: Nuclear magnetic resonance spectroscopy
	E. coli: Units :	Escherichia coli
	S. aureus : OD 600 :	Staphylococcus aureus Optical density at 600 nm
	Chemicals: h :	hour
	EDTA : min :	Ethylenediametetraacetic acid minute
	MOPS : g:	3-Morpholinopropane-1-sulfonic acid gram
	X-Gal: mg :	5-bromo-4-chloro-3-indolyl ß-galactopyranoside milligram
	Metabolites: cm :	centimetre
	Glc : l:		Glucose litre
	BCAA: ml :	Branched chain amino acid millilitre
	Ile : μl :		Isoleucine microlitre
	Val : M :		Valine mole/l
	Leu : mM :	Leucine millimole/l
	Thr : EXTRAS:	Threonine
	Acyl-CoA: NRPS :	Acyl Coenzyme A Non-ribosomal peptide synthesis
	Akb : DW:	L-2-amino-acetoacetate Dry weight
	Glu:		Glutamate
	GTP :	Guanosine triphosphate
	Ket a	:	2-keto-3-methylvalerate
	Ket	b	:	2-keto-isovalerate
	Ket	c :	2-keto-isocaproate
	OxoGlu:	Oxoglutarate
	Pyr:		Pyruvate
			1	CONFIDENTIAL DOCUMENT

  To enhance the biotechnological applications, large scale genome minimization studies are being carried out, strain

(MBG874) lacks 874kb (20.7%) of the B. subtilis genome and B. subtilis Δ6 lacks 376 genes at seven different locations in reference to B. subtilis 168

  Coutte et al. (2010) constructed a mutant strain in which the native promoter was replaced with constitutive promoter P repU (BBG 113), a promoter which regulates the replication gene repU in S. aureus as well as a mutant strain with same promoter construction but the ppsA gene was disrupted (BBG 131). Both mutant strains were obtained in B. subtilis 168 background and the control strain was named as BBG 111.

To restore lipopeptide production, sfp gene from B. subtilis ATCC21332 was inserted in order to convert the Apo-synthetase to Holo-synthetase. The promoter substitution increased the surfactin production five times during the early stage of growth. Continuing the incubation period, it was found an enhancement of 1.3 times of surfactin production for BBG 113, and an increase of 1.2 times with BBG 131 in comparison to BBG 111 at 24 hours in Landy medium. Consequently the haemolytic activity was enhanced; the maximum halo diameter was obtained with BBG 131 after 3 days of incubation. Another constitutive promoter P veg which is endogenous promoter of B. subtilis regulating vegetative gene veg

Table 8

 8 Solutions related to flux where no knockouts are required

Table 18

 18 Specfic growth rate of BBG276 and BBG278 in Landy-phosphate buffer medium The specific growth rate of BBG276 and BBG278 was found to be 0.397±0.002 and 0.472±0.016 h -1 Table18(

engineering of the branched chain amino-acid biosynthetic pathway for the overproduction of surfactin by Bacillus subtilis : Application of biocomputing predictions, 10th

  EuropeanSymposium on Biochemical Engineering Science, Lille, France, 8-10 September 2014.

	Conference on Metabolic Engineering in Bacteria, Amsterdam, The
	Netherlands, 16-17 April 2015.
	POSTER COMMUNICATION
	Coutte, F., John, M., Dhali, D., Béchet, M., Niehren J., Jacques P. Genetic

ACKNOWLEDGMENTS

LIST OF FIGURES

CONFIDENTIAL DOCUMENT

Plasmid with constitutive promoter P repU -ermC construct

A customized plasmid containing erythromycin (ermC) resistance gene sequence (APPENDIX I) and P repU gene sequence was synthesized and inserted into MCS of pUC57 (APPENDIX I). The plasmid was transformed into competent JM109 E. coli cells for its amplification. Subsequently, the plasmid was digested with AccI (Xmil I) and PmlI (Eco721). The desired fragment (ermC-prepU) was purified by agarose gel electrophoresis and extracted from the gel. The pBG133 plasmid was digested with AccI and PmlI in order to excise the P repU -neo fragment. The fragments were separated by agarose gel electrophoresis and extracted. The former fragment was then ligated with the digested pBG133 fragment in order to obtain a pBG401 (Figure 28).

Figure 28

The integration plasmid pBG401 used for the integration of P repU -ermC gene

Genotype of plasmids and E.coli strains

The genotype of the plasmids used here are provided in Table 4 and the strain containing these plasmid are listed in Table 5.

Table 4 Different plasmids and their genotype used in this study

Table 5 Different E.coli strains and their genotype used in this study

CONFIDENTIAL DOCUMENT

Mutant constructed

Previous section provided various single knockout candidates (Table 9) by abstract interpretation for the overproduction of leucine based on the reaction network (Figure 34).

Based on the prediction, selected few genes which regulated the expression of ilv-leu operon were deleted in order to validate the prediction results and evaluate its effect on surfactin production.

The mutants were constructed both with B. subtilis 168 and B. subtilis BSB1, in order to check the efficacy of the prediction results based on B. subtilis strains.

The deletion was carried out in the model organism B. subtilis BSB1 strain using PopIn -PopOut technique (Figure 30). This technique produced entire removal of the gene, which was crucial to study their impact on intracellular leucine pool and eventually on surfactin production. After deletion, the gene of interest was knockout entirely, which can be verified through PCR verification using forward 1 and reverse 2 ). The band size obtained was 1982 bps, 3075 bps, 1953 bps and 1819 bps for the deletion of bcd (lane 1), codY (lane 2), lpdV (lane 3) and tnrA (lane 4) genes, respectively (Figure 35). The mutants with desired gene deletion were selected on LB-agar containing phleomycin (4 µg/ml). Two other strains SRB94 and SRB87 were obtained from Sonenshein Lab [START_REF] Brinsmade | Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools[END_REF]. SRB94 was generated through site directed mutagenesis in the region II of CodY binding site at ilv-leu promoter, while SRB87 is a mutant strain devoid of stem-loop terminator structure responsible for leucine-dependent transcriptional regulation; sfp gene was inserted in the former and latter strain to generate BBG 251 and BBG 252. The mutants with B. subtilis 168 as background strains are provided in Table 10.

CONFIDENTIAL DOCUMENT

B. subtilis BSB1 strain is also devoid of sfp gene, which is essential for the conversion of apo-synthetase to holo-synthetase and results in the production of lipopeptides. This strain was transformed with pBG400 using natural transformation method for the insertion of sfp gene in the amyE locus and positive mutants were selected as described in material and methods. The sfp gene was inserted in all the markerless mutant strains obtained through PopIn -PopOut technique. Double knockout strains were also obtained through the transformation of genomic DNA of PopIn mutant strain into PopOut mutant strain and simultaneously selected on selective LB-agar plates. The mutant strains were verified through PCR. The mutants with B. subtilis BSB1 as background strain are provided in Table 11.

CONFIDENTIAL DOCUMENT

The expression of ilv-leu operon is regulated by CodY in the presence of branched chain amino acid and is known to respond to high content of GTP present in exponentially growing phase [START_REF] Ratnayake-Lecamwasam | Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels[END_REF]. In the Landy medium supplemented with (NH 4 ) 2 SO 4 , there was an increase of seven times and five times in the specific surfactin production with BBG254 and BBG260 (codY deleted; r 15 ), which reached an exceptional value of 1.3 g and 1.27 g of surfactin produced per gram of dried biomass.

This provides an evidence for higher expression of ilv-leu in the mutant strains, thus, results in an increase of intracellular pool of leucine.

The production of the strain BBG251 (BS CodY deleted; r 1 ) was not significantly different in this condition compared to the control strain. In this case, it can be supposed that CodY remains active and was able to repress the ilv-leu operon via its other binding sites. Indeed, the P Ilv-Leu promoter contains four binding sites for CodY, in the promoter-proximal CodY-I+II (nt -84/-32) site that is a high-affinity binding site of CodY, while the promoter-distal

CodY-III (nt-154/-107) and CodY-IV (185/-168) sites are low-affinity ones [START_REF] Shivers | Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched-chain amino acids[END_REF]. This shows that modification of CodY regulation in the cell led to an enhancement of specific surfactin production by increasing the production of leucine via the overexpression of ilv-leu operon. This observation has been perfectly predicted (Table 9) in the model (Figure 34) that CodY has an impact on the promoter P Ilv-Leu through the reaction r 15. The P Ilv-Leu is also subject to a negative regulation by TnrA through its binding to the specific sites. This stimulates DNA binding inhibiting transcription initiation and repressing the ilv-leu expression [START_REF] Fujita | CcpA-mediated catabolite activation of the Bacillus subtilis ilv-leu operon and its negation by either CodY-or TnrAmediated negative regulation[END_REF]. To avoid this regulation, the prediction suggested deletion of input of TnrA (r 16 or to suppress its binding on the P Ilv-Leu : r 7 ). This repression was disrupted by the deletion of tnrA in the strain BBG256 and BBG262. In Landy medium supplemented with (NH 4 ) 2 SO 4 , where the concentration of organic nitrogen was lower, a 4 times increase in specific surfactin production was observed with both strains. These results are quite logical since the impact of this regulator is bestowed only in organic nitrogen limited conditions [START_REF] Fisher | Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence![END_REF].

When both the regulatory genes tnrA and codY were deleted in the strain BBG265, an enhancement of only 3.8 times in specific surfactin production was observed. The production was lower than the one obtained after single knockout of codY and almost equal to single knockout of tnrA, respectively. Glutamate is the best source of nitrogen utilized by B. subtilis. Moreover in the absence of glutamate, ammonium is utilized as a nitrogen source. This is then used for the synthesis of glutamine and glutamate by

CONFIDENTIAL DOCUMENT

seperately in the strains BBG253 and BBG259; BBG255 and BBG261, respectively. Here, ywaA was not chosen because it is also involved in leucine production through the reaction r 45 . However, YwaA action in this step might be able to be offset by YbgE as these two enzymes are exchangeable and have the same function [START_REF] Thomaides | Essential bacterial functions encoded by gene pairs[END_REF]. In Landy medium supplemented with (NH 4 ) 2 SO 4 , the effect of knockout on bcd led to an enhancement of three times and four times in specific surfactin production, respectively.

But the deletion had no impact in case of BBG261 whereas an enhancment of three times was observed with BBG255.

A complete model of the most complex and regulated metabolic pathway of B. subtilis, i.e. the branched chain amino acid metabolic pathway has been developed.

When applied to the model of BCAA metabolic pathway (Figure 34), the prediction algorithm returned 12 plausible single knockout predictions. From these 12 predictions, five mutants were constructed in B. subtilis 168 and three similar mutants were constructed in B. subtilis BSB1 strain to study the consistency of the prediction. Since, the peptide moiety of surfactin contains four leucine residues; the surfactin concentration was measured as an indicator to detect the intracellular level of leucine. All these results discussed above confirm the efficiency and the relevancy of the modelling language and the abstract interpretation algorithm developed for gene knockout prediction. Indeed in the tested medium conditions, all the predicted knockouts led to an increase in surfactin production. In summary, this work opens numerous perspectives, firstly, to improve the models and the algorithm and secondly, to apply this original approach to more complex secondary metabolites biosynthesis or more complex metabolic pathways such as may be found in eukaryotic organisms.

Publication and Valorization of these results

ARTICLE

CONFIDENTIAL DOCUMENT

The surfactin concentration was normalized with dry weight of the strains at each time point and the specific surfactin yield was determined at each time point. This value was represented in mg of surfactin per g dry weight of cell biomass.

The specific surfactin yield (mg of surfactin/g DW) for BBG258 , BBG259, BBG260, BBG261 and BBG262 was found to be 257±11, 414±55, 1483±233, 368±96 and 276±32 at 6 hours; at 10 hours the surfactin yield was found to be 526±80, 360±69, 1556±123, 782±51 and 560±70 and 24 hours the surfactin yield was found to be 1480±195, 809±114, 3138±112, 981±112 and 2526±388, respectively (Figure 45). The maximum yield was obtained with BBG260 (BSB1, sfp + ,∆codY) and the yield was enhanced 6 times, 3 times and 2 times at 6 hours, 10 hours and 24 hours in comparison to control (BBG258).

CONFIDENTIAL DOCUMENT

methods ( LC-MS-MS analysis of surfactin isomer, page no. 76). This time point was selected as at this time the ilvB operon [START_REF] Nicolas | Condition-Dependent Transcriptome Architecture in Bacillus subtilis[END_REF]) is highly expressed.

The variation in concentration of fatty acid chain length for two strains (BBG258 and BBG261) at 6 hours is shown in Figure 53.The percentage of C 14 was 32.2 and 67.2 for BBG258 and BBG261, respectively, whereas the percentage of C 15 was 56.9 and 24.9 for BBG258 and BBG261, respectively. The quantity of C 14 isoform was twice more in BBG261 in comparison to BBG258. The two other mutants produced C14 in less concentration in comparison to BBG261(APPENDIX III). Thus a mutant strain was successfully developed which is a high producer of C 14 isoform. The role of overflow metabolites in the production of this isoform has been verified through extracellular metabolite analysis.

CONFIDENTIAL DOCUMENT

PERMEASE

Previously, in the section about transporters it was studied that B. subtilis contains three different types of transporters. Firstly, a set of transporter proteins function as importer of branched chain amino acids (valine, isoleucine and leucine). This set of proteins includes BcaP, BrnQ and BraB. The second set proteins contains that are involved in the export of branched chain amino acids, these include AzlC and AzlD (Figure 19). Lastly, it was proposed that a particular protein YvbW is a leucine specific permease [START_REF] Wels | An in silico analysis of T-box regulated genes and T-box evolution in prokaryotes , with emphasis on prediction of substrate specificity of transporters 16[END_REF].

Moreover, it was recently hypothesized that YvbW might be a candidate for the transport of leucine specifically as it was observed that a quadruple mutant strain (braB brnQ bcaP leuB) was able to grow in Leu (40 µg/ml) containing minimal medium (Boris R Belitsky, 2015). As this gene was not included in the metabolic network (Figure 34), so this gene was not predicted among the single knockout prediction (Table 9). It can be hypothesized that this gene can have a role in increasing the intracellular pool of leucine. The surfactin production was studied both in the presence of native and constitutive promoter controlling srfA operon.

Mutant construction

The gene yvbW was deleted in the model organism B.subtilis BSB1 strain using Pop In -Pop Out technique (Figure 30). The genotype of the mutant strains are provided in Table 22.

CONFIDENTIAL DOCUMENT

The surfactin concentration was normalized with the cell biomass dry weight (DW) of each strain at each time point to obtain the specific surfactin yield which is represented by mg of surfactin per gm of dry weight. The specific surfactin yield obtained with BBG258 was 257±11; 639±203; 1480±195 mg of surfactin per gm of DW whereas with BBG257 it was obtained 839±126; 2324±174; 5696±842 mg of surfactin per gm of DW at 6 h , 10 h and 24 h, respectively (Figure 58). At 24 hours, the specific surfactin yield was found to be 4 folds higher with BBG257 in comparison to BBG258.

Strains with constitutive promoter (P repU )

The growth of the strains (BBG275 and BBG276) was analysed by measuring the optical density at 600 nm at each time point. Overview of the specific growth rate of all the strains is provided in Table 24. Growth kinetics study of all the strains was carried out during the exponential growth phase.

Table 24 Specific growth rate of BBG258 and BBG257 in modified Landy medium buffered with phosphate buffer

The specific growth rate of BBG258 and BBG257 in this medium was found to be 0.37±0.01 and 0.501±0.018 (Table 24) and the specific growth rate of BBG276 and BBG275 was found to be 0.397±0.002 and 0.501±0.007, respectively. The specific growth rate of the strains did not change significantly, when the native surfactin promoter (P srfA ) was exchanged with a constitutive one (P repU ), controlling the expression of surfactin operon (srfA).

Surfactin production was analyzed with the strains in this media condition at 6 hours and 10 hours. Both strains produced surfactin but with diverse concentrations (Figure 59). The surfactin production for BBG276 was found to be 134±18 mg/l and 293±103 mg/l at 6 h

CONFIDENTIAL DOCUMENT

and 10 h, respectively. While the surfactin production for BBG275 was found to be 1048±47 mg/l and 1511±93 mg/l at 6 h and 10 h, respectively. The maximum surfactin production was found at 10 hours for both strains. At this time point, BBG276 produced 5 times surfactin more than BBG275. The surfactin concentration was normalized with the cell biomass dry weight of (DW) of each strain at each time point to obtain the specific surfactin yield in mg of surfactin per g of dry weight (Figure 60). The specific surfactin yield obtained with BBG276 was 369±101; 521±142 mg of surfactin per g of DW whereas with BBG275 it was obtained 1076±142; 1143±66 mg of surfactin per g of DW at 6 h and 10 h respectively. At 10 hours the specific surfactin yield of BBG275 was twice higher than that of BBG276. Two major conclusions can be drawn from the above results; Firstly, the surfactin native promoter (P srfA ) of B. subtilis is a highly efficient promoter for the production of surfactin in comparison to the constitutive P repU promoter. As BBG257 harboring the surfactin native promoter enhanced surfactin yield twice in comparison to its counterpart BBG275, having constitutive promoter at 10 hours. On the basis of native promoter controlling srfA expression, it was proved that P srfA of B. subtilis 168 was stronger than P srfA of B. subtilis ATCC6633 [START_REF] Duitman | Novel methods for genetic transformation of natural Bacillus subtilis isolates used to study the regulation of the mycosubtilin and surfactin synthetases[END_REF]. But in comparison to an other study, utilizing P repU promoter for surfactin production (F. Coutte et al., 2010), the specific surfactin yield with BBG113 (BS168, sfp + , P repU -srfA) was 880 mg of surfactin per g of DW at 24 hour, which is 1.3 times less than the yield obtained with BBG275 at 10 hour although the former study was carried out in Landy MOPS medium with glutamic acid and 130 rpm, while the latter was carried out in Landy with ammonium sulphate with phosphate buffer at 250 rpm.

CONFIDENTIAL DOCUMENT

Although the constitutive promoter (P repU ) gave 3-folds and 15-folds higher production of iturin A (K Tsuge et al., 2001) and mycosubtilin [START_REF] Leclère | Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism's antagonistic and biocontrol activities[END_REF] in comparison to corresponding strain with native promoter. Another constitutive promoter P veg was inserted into B. subtilis 3A38 to enhance the production of surfactin but a maximum production of

CHAPTER 4: CONCLUSION AND PERSPECTIVES

CONFIDENTIAL DOCUMENT

The deletion of this mutant directed 37% of the carbon in glucose towards carbon synthesis of surfactin molecule. But when the promoter was replaced with a constituitive one, there was only 2-fold enhancement in comparison to its counterpart and yield obtained was 1.14 g per g DW. The yield obtained with native promoter with the deletion of yvbW was 5-times more in comparison to strain with constituitive promoter with same gene deletion. This difference in yield might be due to the presence of dyad symmetries in the upstream region of native promoter for ComA binding.

Thus, the precursor limitation can be overcome with this integrative approach and can lead to high production of surfactin, which is an essential criteria for its proper industrial application.

PERSPECTIVES

1. Development of a double mutant (ΔcodY, ΔyvbW) and analyze the yield of this mutant.

2. To analyze extracellular and intracellular metabolites for the overproducing mutant strains in the definitive conditions.

3. Extension of the modeling to all other metabolism, in order to have an integrative approach on the entire metabolism.

4. To study whether there might be a role of any other amino acid or fatty acid as a limiting factor for the production of surfactin.