Gregory Guirimand

Natalja Kulagina

Nicolas Papon

Tomohisa Hasunuma
email: hasunuma@port.kobe-u.ac.jp

Vincent Courdavault
email: vincent.courdavault@univ-tours.fr

Innovative Tools and Strategies for Optimizing Yeast Cell Factories

Keywords: Metabolic engineering, genome editing, cis/trans regulators, metabolic fluxes, subcellular compartmentation, adaptive laboratory evolution

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Towards the ideal yeast cell factory: from rational design to adaptive laboratory evolution Medicinal natural products (NPs) (see Glossary) suffer from recurrent shortages, mainly due to the overexploitation of their sources (https://www.nytimes.com/2019/10/14/health/cancerdrug-shortage.html) [START_REF] Ehrenworth | Accelerating the semisynthesis of alkaloid-based drugs through metabolic engineering[END_REF][START_REF] Courdavault | Towards the Microbial Production of Plant-Derived Anticancer Drugs[END_REF][3]. While the well-known and long-established uses of synthetic chemistry (SC) allow producing many NPs and their derivatives, at large scales, some compounds such as plant-derived NPs (monoterpene indole alkaloids or MIAs, benzylisoquinoline alkaloids or BIAs, etc.) and mammal derived NPs (such as hormones and neurotransmitters) are too complex to be synthesized by SC alone at an industrial scale because of asymmetric carbons, long multi-step biosynthetic pathways, and other factors [3][START_REF] Nielsen | Engineering Cellular Metabolism[END_REF][START_REF] Lian | Recent advances in metabolic engineering of Saccharomyces cerevisiae: New tools and their applications[END_REF][START_REF] Chen | Advanced Strategies for Production of Natural Products in Yeast[END_REF]. Although recent progress now allows very complex NPs such as vitamin B12 [START_REF] Eschenmoser | Natural product synthesis and vitamin B12[END_REF] or strictosidine [START_REF] Sakamoto | Total Syntheses of (-)-Strictosidine and Related Indole Alkaloid Glycosides[END_REF] to be produced synthetically, chemical synthesis is not always the most suitable approach in terms of ecological impact and cost efficiency. Therefore, alternative strategies have been considered to produce these NPs in greater quantities, such as using metabolic engineering (ME), which aims to rewire cellular metabolism, has empowered and accelerated the development of yeast cell factories (YCFs) by enhancing the TRY of the bioproduced target compounds, broadening the spectrum of these compounds (e.g., taxol, opioids), increasing the range of substrates (e.g., xylose, arabinose), and enhancing strain physiological properties (e.g., global fitness, stress tolerance) [START_REF] Courdavault | Towards the Microbial Production of Plant-Derived Anticancer Drugs[END_REF][3][START_REF] Nielsen | Engineering Cellular Metabolism[END_REF][START_REF] Lian | Recent advances in metabolic engineering of Saccharomyces cerevisiae: New tools and their applications[END_REF][START_REF] Chen | Advanced Strategies for Production of Natural Products in Yeast[END_REF]. In particular, baker's yeast (Saccharomyces cerevisiae), as well as some non-conventional yeasts with specific features of interest, such as high secretion ability or lipophilic compounds accumulation (including Pichia pastoris and Yarrowia lipolytica [START_REF] Raschmanová | Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects[END_REF][START_REF] Larroude | Synthetic biology tools for engineering Yarrowia lipolytica[END_REF]), generally regarded as safe (GRAS) and robust microorganisms, have their genome entirely known and easy to manipulate. In recent decades, remarkable progress has been made in the field of molecular and cell biology of yeast, due to the rapid development of genome sequencing, cell engineering and synthetic biology (SB) [3][START_REF] Nielsen | Engineering Cellular Metabolism[END_REF][START_REF] Lian | Recent advances in metabolic engineering of Saccharomyces cerevisiae: New tools and their applications[END_REF][START_REF] Chen | Advanced Strategies for Production of Natural Products in Yeast[END_REF]. However, many limiting factors remain that make genome manipulation and the construction of YCFs inefficient and time-consuming [START_REF] Lian | Recent advances in metabolic engineering of Saccharomyces cerevisiae: New tools and their applications[END_REF][START_REF] Chen | Advanced Strategies for Production of Natural Products in Yeast[END_REF]. Indeed, creating YCFs commonly implies the integration of complex biosynthetic pathways, which requires the highfidelity assembly of long DNA fragments and sophisticated genome editing tools [START_REF] Raschmanová | Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects[END_REF][START_REF] Choi | Systems Metabolic Engineering Strategies: Integrating Systems and Synthetic Biology with Metabolic Engineering[END_REF][START_REF] Zhang | Recent Advances of CRISPR/Cas9-Based Genetic Engineering and Transcriptional Regulation in Industrial Biology[END_REF].

Novel strategies and approaches are continuously developing to facilitate multiplex markerfree genomic integration, DNA assembly and transformation efficiency [4-6, 20, 22].

Additionally, integrating heterologous pathways in yeast generally involves fine-tuning gene expression, which has been limited by the availability of characterized native gene regulatory elements, because they are lengthy and unable to cover the required range of the expression strength [START_REF] Partow | Characterisation of different promoters for designing a new expression vector in Saccharomyces cerevisiae[END_REF][START_REF] Sun | Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae[END_REF][START_REF] Peng | Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: A comparison of yeast promoter activities[END_REF]. Multiple approaches aiming at optimizing and extending transcription regulation [START_REF] Blazeck | Promoter engineering: Recent advances in controlling transcription at the most fundamental level[END_REF][START_REF] Redden | The synthetic biology toolbox for tuning gene expression in yeast[END_REF] are still constantly evolving to further broaden the assortment of regulatory elements and overcome their limitations. Moreover, the overexpression of heterologous biosynthetic pathways is frequently responsible for massive stress affecting global cell fitness, which includes the accumulation of toxic intermediates, carbon source competition, and a loss of reducing power (i.e. oxidative stress, and/or unbalanced/competitive use of cofactors) [START_REF] Lian | Recent advances in metabolic engineering of Saccharomyces cerevisiae: New tools and their applications[END_REF][START_REF] Chen | Advanced Strategies for Production of Natural Products in Yeast[END_REF][START_REF] Rahmat | Yeast metabolic engineering for the production of pharmaceutically important secondary metabolites[END_REF]. In particular, developing an industrial bioprocess based on YCFs compatible with commercial purposes, with a maximal product TRY and optimal metabolic fluxes of the multiple integrated genes, demands precise control and balance and remains a key goal for already-established recombinant strains [START_REF] Eriksen | Protein design for pathway engineering[END_REF][START_REF] Martin | Synthetic Metabolism: Engineering Biology at the Protein and Pathway Scales[END_REF].

Furthermore, enzyme and substrate differential subcellular localization [START_REF] Zhou | Harnessing Yeast Peroxisomes for Biosynthesis of Fatty-Acid-Derived Biofuels and Chemicals with Relieved Side-Pathway Competition[END_REF][START_REF] Inokuma | Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains[END_REF][START_REF] Pompon | Nanotechnology for Synthetic Biology: Crossroads Throughout Spatial Confinement[END_REF], enzymatic promiscuity, and pathway lateral branches generally decrease substrate availability and metabolic fluxes by hijacking biosynthetic intermediates [START_REF] Scalcinati | Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fedbatch mode[END_REF][START_REF] Tippmann | Affibody scaffolds improve sesquiterpene production in saccharomyces cerevisiae[END_REF]. As such, formal strategies are also required to redesign metabolic pathways in yeast. This comprehensive review provides an integrative and clear view of the most recent and innovative molecular tools and approaches dedicated to the implementation and improvement of YCFs for the bioproduction of valuable molecules. It will especially emphasize specific challenges associated with already existing methods, and discuss successful examples that highlight the latest tools and advances, and future perspectives.

Genome editing tools

In yeast genome editing, homologous recombination and cre/lox-mediated integration have largely been replaced by the CRISPR/Cas9 system, which is still continuously improving to facilitate and accelerate DNA assembly and genome manipulation [START_REF] Raschmanová | Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects[END_REF][START_REF] Zhang | Recent Advances of CRISPR/Cas9-Based Genetic Engineering and Transcriptional Regulation in Industrial Biology[END_REF]. Particularly laborious was the assembly of large template DNA (multiple expression cassettes), which is now generally realized in vivo via overlapping single-stranded oligonucleotides and homologous recombination (HR), the technique described as DNA assembler [START_REF] Shao | DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways[END_REF]. It bypasses multistep and multi plasmid cloning and was successfully employed in an assembly of up to eight-gene biosynthetic pathways in S. cerevisiae with the efficiency being inversely correlated to the number of genes [START_REF] Shao | DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways[END_REF]. Another limiting factor consisted of multiplex genomic integration, which requires multiple gRNAs. Recently, several novel approaches were developed, aimed to perform multisite targeting and exploiting the simultaneous expression of numerous gRNA using Cas9-expressing yeast strains or all-in-one plasmids (Fig. 2A). For instance, in the CasEMBLR method (Cas9-facilitated multi loci integration of assembled DNA parts into S. cerevisiae chromosomes, in combination with DNA assembler techniques), DNA fragments are amplified by PCR to contain homologous overhangs for further in vivo assembly, and marker-free integration was reported to be successful into up to five sites with 50-100% efficiency, which implies both gRNAs-expressing plasmid and Cas9-expressing yeast strain [START_REF] Jakočiunas | CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae[END_REF][START_REF] Jakočiunas | Assembly and Multiplex Genome Integration of Metabolc Pathways in Yeast Using CasEMBLR[END_REF]. All-in-one plasmids, such as gRNAs-Cas9 co-expressing vectors with an inducible promoter for Cas9 (also available in constitutive version), enable dissociating cloning from genome editing by yeast pre-transformation with all CRISPR/Cas9 components prior to the introduction of template DNA [START_REF] Degreif | Preloading budding yeast with all-in-one CRISPR/Cas9 vectors for easy and high-efficient genome editing[END_REF]. Time-effectively, the all-in-one plasmids contain a universal gRNA expression cassette, and the introduction of an appropriate gRNA targeting sequence upon plasmid recircularization takes place in vivo in yeast via HR [START_REF] Degreif | Preloading budding yeast with all-in-one CRISPR/Cas9 vectors for easy and high-efficient genome editing[END_REF]. However, the conventional expression and delivery of multiple gRNAs are delicate, either an individual expression cassette is required for each gRNA, or a common transcript is generated and further cleaved via various strategies into individual gRNAs [START_REF] Stovicek | CRISPR/Cas system for yeast genome engineering: advances and applications[END_REF]. Both approaches are limited by the number of gRNAs expressed in the system and the processing efficiency. Recently, alternative strategies were reported. In the GTR-CRISPR (Fig. 2B) (tandem gRNA-tRNA array for CRISPR/Cas9) the use of a gRNA-tRNA array within the Cas9-expressing plasmid, and exploiting the endogenous tRNA-processing system, which precisely cleaves the precursor of tRNA and enables the release of single gRNAs from the common gRNA-tRNA transcript, resulted in the simultaneous disruption of 8 genes in S. cerevisiae with 87% efficiency [START_REF] Zhang | A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae[END_REF].

Moreover,

Lightning GTR-CRISPR, an E.coli-free Golden Gate cloning system [START_REF] Engler | A one pot, one step, precision cloning method with high throughput capability[END_REF] compatible version can be employed (the Golden Gate reaction is directly transformed into yeast), which was shown to result in 96% and 60% efficiency of correct editing, illustrated through the disruption of four and six genes, respectively [START_REF] Zhang | A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae[END_REF]. Alternatively, a plasmid-free gRNA delivery method gRNA-Transient Expression System (TES) was described, where gRNA-encoding PCR fragments, composed of the promoter, guiding sequence and gRNA scaffold (two gRNAs are required to cleave both sides of the targeted region), are transformed together with template DNA into Cas9-expressing S. cerevisiae strain, which resulted in the substitution of different sized regions of S. cerevisiae chromosome 4 by template DNA with 67-100% efficiency [START_REF] Easmin | gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing[END_REF].

In parallel, the CRISPR/Cas9 system has been explored to enhance the efficiency of its components (Fig. 2B). In this context Cpf1 (CRISPR from Prevotella and Francisella 1), a family of class 2/type V CRISPR bacterial endonucleases was found to display several advantages compared to Cas9. For instance, Cpf1 generates sticky ends instead of blunt ends upon DNA cleavage [START_REF] Easmin | gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing[END_REF], which potentially facilitates template DNA integration. Moreover, Cfp1 possesses both RNAase and DNAse activities and does not need RNAse III, which results in the requirement of only crispr RNA (crRNA) instead of a longer complex of transactivating crispr RNA (tracrRNA) and crRNA [START_REF] Zetsche | Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System[END_REF][START_REF] Safari | CRISPR Cpf1 proteins: Structure, function and implications for genome editing[END_REF]. Thus, S. cerevisiae strain producing patchoulol was generated using self-cloning Cpf1-crRNA co-expressing plasmid, where singleplex and triplex genomic integrations of in vivo assembled template DNA were achieved with 80% and 32% of efficiency, respectively [START_REF] Li | Self-cloning CRISPR/Cpf1 facilitated genome editing in saccharomyces cerevisiae[END_REF].

In addition, dCas9-mediated Target-AID (Activation Induced cytidine Deaminase from vertebrate), a synthetic hybrid complex that performs highly efficient C to G and C to T mutations, was successfully used in high throughput loss of function screens [START_REF] Després | Double selection enhances the efficiency of target-AID and Cas9-based genome editing in yeast. G3 Genes, Genomes[END_REF], which is an appealing application to screen for yeast de novo features. On the other hand, the σ sequences, which are a family of repetitive DNA sequences (at least 100 copies) in the S.

cerevisiae genome [START_REF] Cameron | Evidence for transposition of dispersed repetitive DNA families in yeast[END_REF], were used together with the CRISPR/Cas9 system in a novel method of genome shuffling [START_REF] Mitsui | Improved Stress Tolerance of Saccharomyces cerevisiae by CRISPR-Cas-Mediated Genome Evolution[END_REF]. Cas9, guided by the gRNA targeting the σ sequences, cleaves DNA at multiple sites, thus promoting endogenous DNA repair and mutagenesis [START_REF] Pâques | Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae[END_REF], which can lead to the improvement of yeast characteristics when cultivated under specific conditions (e.g. thermotolerant S. cerevisiae obtained under high-temperature conditions [START_REF] Mitsui | Improved Stress Tolerance of Saccharomyces cerevisiae by CRISPR-Cas-Mediated Genome Evolution[END_REF]). These approaches demonstrate the application of the CRISPR/Cas9 system beyond gene integration, notably useful in adaptive laboratory evolution (ALE), which will be presented in the last section of this review.

Transcriptional ("cis") and post-transcriptional ("trans") regulator toolboxes

Alteration and regulation of gene expression at the transcriptional level via promoter and terminator elements have been unceasingly considered as a powerful approach. Previously characterized long native elements were initially employed (S. cerevisiae pTEF1, pGAL1-10, tCYC1, tADH1, etc) [START_REF] Partow | Characterisation of different promoters for designing a new expression vector in Saccharomyces cerevisiae[END_REF][START_REF] Sun | Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae[END_REF][START_REF] Peng | Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: A comparison of yeast promoter activities[END_REF], which, however, pointed out an extensive necessity of development of novel synthetic units, more compact and functionally diversified. Indeed, the integration of complex multigenic heterologous pathways in yeast mobilizes a promoter and a terminator for each gene, which considerably extends template DNA length, increases the risk of self-recombination, as well as generally demands optimization of gene expression. To address these issues, several regulatory sequence libraries were recently constructed and investigated (Fig. 3A). For instance, a library of short 69-bp semi-synthetic promoters, covering an 8.0-fold expression range, was generated from the determined minimal length of S. cerevisiae native pTEF1 [START_REF] Liu | Engineering yeast artificial core promoter with designated base motifs[END_REF] (Fig. 3A1). Moreover, the use of upstream activating sequences (UAS) expanded the library to reach a 20-fold expression range and a maximal length of 130 bp. Importantly, some of the pTEF1 variants demonstrated expression strength comparable to native pTEF1 and pPGK1. Another library of Y. lipolytica semi-synthetic promoters was generated, also based on the promoter core region between the TATA-box and the transcriptional starting site (TSS) upstream the 5' untranslated region (5'UTR) sequence [START_REF] Liu | Engineering yeast artificial core promoter with designated base motifs[END_REF] (Fig. 3A2). Artificial sequences of 30 bp in length were designed (various combinations of T-rich and G/C-rich fragments) and substituted in Y. lipolytica strong native pEXP1 and pGDP to assess expression levels of crtY enzyme and, consequently, conversion of lycopene to βcarotene. The results demonstrated a population of novel Y. lipolytica promoters showing an up to a 5.5-fold increase in lycopene conversion. On the other hand, the impact of 10-bp terminator linker 1 (the sequence between the efficiency element and the positioning element) on gene expression was investigated via synthetic terminator library in S. cerevisiae [START_REF] Wang | Yeast Synthetic Terminators: Fine Regulation of Strength through Linker Sequences[END_REF] (Fig. 3A3). A 6.0-fold expression range was achieved and it was demonstrated that sequences with low GC content and enriched in T were conferring higher levels of expression.

In addition to constitutive regulation of gene expression, metabolic biosensors play a central role in metabolic rewiring and the optimization of productivity in YCFs. For instance,

Metabolite responsive allosteric transcription factors (aTFs or MRTFs) from bacteria

interact with their corresponding metabolites prior to acting as repressors or activators of transcription [START_REF] Wan | Engineering metabolite-responsive transcriptional factors to sense small molecules in eukaryotes: Current state and perspectives[END_REF]. To alter transcription, MRTFs bind promoter-specific sequences and, therefore, restrict or facilitate the access to RNA polymerase [START_REF] Wan | Engineering metabolite-responsive transcriptional factors to sense small molecules in eukaryotes: Current state and perspectives[END_REF]. Thus, MRTFs enable dynamic control of heterologous gene expression and, consequently, regulate metabolic activity. However, the engineering of MRTFs in eukaryotic cells remains more than challenging. Recently, a novel strategy has been reported and aimed to generate user-defined biosensors via evolution-guided toggled selection (directed biosensor evolution and library construction, followed by the selection of mutants via alternating conditions according to the set criteria), based on the mutagenesis of aTF effector binding domain (EBD) and

Fluorescence-Activated Cell Sorting (FACS)-mediated selection [START_REF] Snoek | Evolution-guided engineering of small-molecule biosensors[END_REF] (Fig. 3B1). Thus, a variety of de novo features were identified, such as inversion of function and change of specificity, as well as modification of dynamic and operational range. Another approach focused on the optimization of biosensor reporter promoters in a binding site-dependent manner [START_REF] Ambri | High-resolution scanning of optimal biosensor reporter promoters in yeast[END_REF] (Fig. 3B2). The constructed synthetic promoter libraries (from native pTEF1 and truncated pCYC1) covered all the possible positions for introducing aTFs binding site within promoter core region, which resulted in the identification of function-related positions for most of the screened repressor or activator type aTFs [START_REF] Ambri | High-resolution scanning of optimal biosensor reporter promoters in yeast[END_REF]. The complementary aspect of gene expression regulation via promoters is related to the carbon source of yeast metabolism. Given that multiple commonly used native promoters are associated with glycolytic genes and are less effective under glucose starvation [START_REF] Peng | Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: A comparison of yeast promoter activities[END_REF], novel promoters, activated upon diauxic shift, are required to maintain constitutive gene expression (or to disassociate growth from production if required). Recently, carbon source response elements (CSRE) of gluconeogenic promoters were introduced upstream UAS of S. cerevisiae pTDH3, substituting the binding site of glycolytic genes transcriptional activator Gcr1 and surrounding neutral sequence [START_REF] Rajkumar | Engineered Reversal of Function in Glycolytic Yeast Promoters[END_REF] (Fig. 3C). This approach demonstrated strong induction of reporter Yellow Fluorescent Protein (YFP) in the low-glucose medium instead of a glucose-rich medium, which was validated in a vanillin-β-glucoside-producing yeast strain. Indeed, when glycolytic promoters were employed, the production of vanillin-β-glucoside took place mostly during the ethanol phase leading to the accumulation of the cytotoxic intermediate protocatechuic acid (PCA). The use of generated gluconeogenic promoters to control the expression of enzymes converting PCA to vanillin-βglucoside showed an enhanced yeast growth, lower accumulation of PCA, and conserved vanillin-β-glucoside bioconversion [START_REF] Rajkumar | Engineered Reversal of Function in Glycolytic Yeast Promoters[END_REF]. Taken together, these advances are broadening the collection of available gene regulation elements and deliver compelling guidance to combinatorial approaches for metabolic rewiring in YCF.

Rewiring of primary metabolism

The strategies to enhance TRY imply the optimization of host primary metabolism, which includes the modification of native metabolic fluxes and coupling/decoupling growth with production. Sufficient availability of the NADPH cofactor, required by numerous enzymes, in some cases (excluding, for instance, hydrocortisone biosynthesis [START_REF] Szczebara | Total biosynthesis of hydrocortisone from a simple carbon source in yeast[END_REF]) stands as a limiting factor (e.g. S. cerevisiae 3-hydroxy-3-methylglutaryl-CoA reductase Hmg1, S. cerevisiae squalene synthase Erg9 etc) [START_REF] Kim | Rerouting of NADPH synthetic pathways for increased protopanaxadiol production in Saccharomyces cerevisiae[END_REF][START_REF] Paramasivan | Regeneration of NADPH Coupled with HMG-CoA Reductase Activity Increases Squalene Synthesis in Saccharomyces cerevisiae[END_REF], given that glucose is dispatched in both glycolysis and pentose phosphate pathway (PPP) upon consumption [START_REF] Stincone | The return of metabolism: biochemistry and physiology of the pentose phosphate pathway[END_REF]. Several successful approaches have been implemented to enhance NADPH (re)generation (Fig. 4A) and, therefore, heterologous/homologous production. For instance, a number of S. cerevisiae genes involved in NADPH synthesis were deleted or overexpressed to assess the effect on NADPH concentration in the cytosol and heterologous protopanaxadiol (PPD) production from endogenous squalene [START_REF] Kim | Rerouting of NADPH synthetic pathways for increased protopanaxadiol production in Saccharomyces cerevisiae[END_REF]. In the best performing PPD-producing S. cerevisiae strain, the deletion of NADH-generating ALD2 and overexpression of NADPH-generating ALD6 aldehyde dehydrogenases-encoding genes, involved in ethanol metabolism, resulted in a 1.3-fold increase of NADPH cytosolic concentration and a 4.5-fold increase in PPD production [START_REF] Kim | Rerouting of NADPH synthetic pathways for increased protopanaxadiol production in Saccharomyces cerevisiae[END_REF].

Likewise, the positive effect of the overexpression of the Ald6-encoding gene was demonstrated in recombinant noscapine-producing S. cerevisiae [START_REF] Tomàs-Gamisans | Redox Engineering by Ectopic Overexpression of NADH Kinase[END_REF]. The overexpression of S. cerevisiae full-length mitochondrial or truncated cytoplasmic NADH kinase Pos5, which catalyzes NADPH-generating reactions, led to the 9.0-fold and 7.0-fold enhancement of homologous squalene production respectively [START_REF] Paramasivan | Regeneration of NADPH Coupled with HMG-CoA Reductase Activity Increases Squalene Synthesis in Saccharomyces cerevisiae[END_REF]. Similarly, the overexpression of truncated cytoplasmic Pos5 in antibody fragment-expressing P. pastoris was shown to significantly increase the production of recombinant protein [START_REF] Tomàs-Gamisans | Redox Engineering by Ectopic Overexpression of NADH Kinase[END_REF].

Another important cofactor in cellular metabolism is S-adenosylmethionine (SAM), which is a donor of the methyl group upon methylation catalyzed by SAM-dependent methyltransferases (Mtases) [START_REF] Struck | S-Adenosyl-Methionine-Dependent Methyltransferases: Highly Versatile Enzymes in Biocatalysis, Biosynthesis and Other Biotechnological Applications[END_REF]. Methylation, being required by a wide range of cellular processes, is used in biotechnological approaches for the heterologous production of valuable methylated compounds [START_REF] Schönherr | Profound methyl effects in drug discovery and a call for new C-H methylation reactions[END_REF]. Engineering of SAM-dependent Mtases has been challenging, mostly due to the lack of efficiency and consistency. Recently, methylation was coupled to growth by combining enhanced Mtase activity and cysteine biosynthesis [START_REF] Luo | Coupling S-adenosylmethionine-dependent methylation to growth: Design and uses[END_REF] (Fig. 4B), which was achieved via Mtase adaptive laboratory evolution in vivo and deletion of several S. cerevisiae genes. On the other hand, certain enzymes require specific chemical elements for their formation and activity such as the bacterial xylonate dehydratase (XylD, involved in xylose metabolism) that contains iron-sulfur (Fe-S) cluster [START_REF] Bamba | Production of 1,2,4-butanetriol from xylose by Saccharomyces cerevisiae through Fe metabolic engineering[END_REF]. Although S. cerevisiae Fe metabolism was not reported to be limiting for bovine Fe-S adrenodoxin and hydrocortisone biosynthesis [START_REF] Szczebara | Total biosynthesis of hydrocortisone from a simple carbon source in yeast[END_REF], the folding and the activity of several bacterial Fe-S enzymes were reported to be insufficient in yeast [START_REF] Carlsen | Heterologous expression and characterization of bacterial 2-Cmethyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae[END_REF][START_REF] Partow | Reconstruction and Evaluation of the Synthetic Bacterial MEP Pathway in Saccharomyces cerevisiae[END_REF][START_REF] Benisch | The bacterial Entner-Doudoroff pathway does not replace glycolysis in Saccharomyces cerevisiae due to the lack of activity of iron-sulfur cluster enzyme 6-phosphogluconate dehydratase[END_REF], still being a key concern in the functional expression of bacterial Fe-S proteins. Recently, Fe metabolism of yeast cytosolic Fe-S cluster machinery was modified to enhance Fe uptake, XylD generation, and 1,2,4-butanetriol production [START_REF] Bamba | Production of 1,2,4-butanetriol from xylose by Saccharomyces cerevisiae through Fe metabolic engineering[END_REF] (Fig. 4C). In the best performing recombinant S. cerevisiae strain, the overexpression of truncated Tyw1 protein (tTyw1), unable to bind and sequestrate Fe-S clusters, and the deletion of one of the components of negative regulation of Fe uptake, resulted in 6.0-fold and 1.4-fold higher XylD activity and 1,2,4-butanetriol production, respectively [START_REF] Bamba | Production of 1,2,4-butanetriol from xylose by Saccharomyces cerevisiae through Fe metabolic engineering[END_REF].

Spatial reconfiguration strategies

The development of valuable compound-producing YCFs usually involves complex enzymatic pathways, requiring specific physicochemical conditions, substrates and cofactors, potentially affected by adverse side reactions or toxic intermediates [START_REF] Chen | Advanced Strategies for Production of Natural Products in Yeast[END_REF][START_REF] Choi | Systems Metabolic Engineering Strategies: Integrating Systems and Synthetic Biology with Metabolic Engineering[END_REF]. To meet these challenges, several approaches based on the spatial rearrangement of the desired pathway(s) have emerged, from the metabolic engineering of organelles (MEO) to the construction of artificial metabolons [START_REF] Zhou | Harnessing Yeast Peroxisomes for Biosynthesis of Fatty-Acid-Derived Biofuels and Chemicals with Relieved Side-Pathway Competition[END_REF][START_REF] Inokuma | Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains[END_REF][START_REF] Pompon | Nanotechnology for Synthetic Biology: Crossroads Throughout Spatial Confinement[END_REF]. Recently, successful artificial compartmentalization of the triterpene biosynthetic pathway in the peroxisome of S. cerevisiae allowed producing a high concentration of squalene (Fig. 5A) revealing this organelle as a promising site for the biosynthesis and storage of terpene compounds [START_REF] Liu | The yeast peroxisome: A dynamic storage depot and subcellular factory for squalene overproduction[END_REF]. In particular, the rapid and highly efficient protein import machinery of peroxisomes, along with their high plasticity (number and size dynamically adjusted according to the physiological state of the cells), combined to a channeling effect insured by the close vicinity of overexpressed heterologous biosynthetic enzymes, make this subcellular compartment an ideal target for improvement of YCFs.

Previously, the successful bioproduction of hydrocortisone in yeast elegantly showed the flexibility of yeast in accommodating the relocalization of membrane-bound enzymes to a different subcellular compartment, without affecting the final titer of hydrocortisone produced [START_REF] Szczebara | Total biosynthesis of hydrocortisone from a simple carbon source in yeast[END_REF][START_REF] Kelly | Rewiring yeast for drug synthesis[END_REF]. More recently, the successful artificial compartmentalization of isoprenoid biosynthesis into the mitochondria was achieved (Fig. 5B), with an enhanced supply of acetyl-CoA and tricarboxylic acid cycle intermediates [START_REF] Yee | Engineered mitochondrial production of monoterpenes in Saccharomyces cerevisiae[END_REF]. However, as mitochondrion is gathering crowds of essential proteins involved in respiration, the compartmentalization of heterologous biosynthetic pathways in this organelle might be, in some cases, responsible for metabolic stresses. In MEO, important physiological parameters such as the size and the biogenesis of organelles can increase the physical space available for enzyme encapsulation and storage of metabolites [START_REF] Orrenius | Phenobarbital-induced synthesis of the microsomal drugmetabolizing enzyme system and its relationship to the proliferation of endoplasmic membranes[END_REF][START_REF] Kanai | Quantitative analysis of smooth and rough endoplasmic reticulum proliferation in differentiating hepatocytes of midpostnatal mice treated with phenobarbital[END_REF][START_REF] Kim | Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway[END_REF][START_REF] Ma | Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene[END_REF]. For example, increasing the size of lipid droplets, by modulating triacylglycerol metabolism, allowed a 1.25-fold increase in lycopene bio-production in a recombinant strain of S. cerevisiae, correlated to the improved storage capacity of hydrophilic lycopene [START_REF] Ma | Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene[END_REF]. Similarly, the overexpression of a key ER size regulatory factor gene, INO2, allowed a significant increase of the surface of the ER [START_REF] Kim | Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway[END_REF], associated with a drastic augmentation of production of P450s [START_REF] Orrenius | Phenobarbital-induced synthesis of the microsomal drugmetabolizing enzyme system and its relationship to the proliferation of endoplasmic membranes[END_REF][START_REF] Kanai | Quantitative analysis of smooth and rough endoplasmic reticulum proliferation in differentiating hepatocytes of midpostnatal mice treated with phenobarbital[END_REF][START_REF] Kim | Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway[END_REF], and lead to an 8-fold increase in PPD bioproduction, along with a 7.1-fold increase of protein secretion, emphasizing the crucial role of ER in protein synthesis and folding, to circumvent potential metabolic constraints [START_REF] Kim | Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway[END_REF][START_REF] Silva | Engineering the early secretory pathway for increased protein secretion in Saccharomyces cerevisiae[END_REF].

In parallel to MEO, another approach has emerged allowing the display of enzymes directly at the surface of the yeast cells (Fig. 5C) [START_REF] Inokuma | Whole Cell Biocatalysts Using Enzymes Displayed on Yeast Cell Surface[END_REF]. The cell surface engineering (CSE) approach enables the generation of the whole cell catalysts to achieve the hydrolysis of numerous substrates, including lignocellulosic biomass, consecutively converted by fermentation into a variety of valuable compounds such as ethanol and xylitol [START_REF] Inokuma | Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains[END_REF][START_REF] Inokuma | Whole Cell Biocatalysts Using Enzymes Displayed on Yeast Cell Surface[END_REF][START_REF] Guirimand | Cell-surface display technology and metabolic engineering of: Saccharomyces cerevisiae for enhancing xylitol production from woody biomass[END_REF][START_REF] Anandharaj | Constructing a yeast to express the largest cellulosome complex on the cell surface[END_REF]. In brief, CSE consists of targeting heterologous enzymes to the cell wall, through the secretion pathway, to expose their catalytic sites toward the extracellular environment. The enzymes can be displayed either individually or gathered in association with a protein-based scaffold to form the cellulosome, a multi-enzymatic complex structure capable of accommodating up to 63 enzymes for the largest one reported to date [START_REF] Inokuma | Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains[END_REF][START_REF] Anandharaj | Constructing a yeast to express the largest cellulosome complex on the cell surface[END_REF]. One of the main challenges in CSE resides in controlling the efficiency of the display, as well as the anchorage position of the target protein within the cell wall. In the case of the bio-production of xylitol from lignocellulose by CSE, the uptake of xylose across the membrane of the cells constitutes a critical point, as the bioconversion reaction requires NADH and cannot be achieved at the surface of the cells [START_REF] Guirimand | Cell-surface display technology and metabolic engineering of: Saccharomyces cerevisiae for enhancing xylitol production from woody biomass[END_REF][START_REF] Nijland | Engineering of Pentose Transport in Saccharomyces cerevisiae for Biotechnological Applications[END_REF].

Interestingly, the co-expression of a homologous maltose transporter (ScMal11) along with a beta-glucosidase (AaBgl) degrading cello-oligosaccharides (e.g. cellobiose) on the cell surface of S. cerevisiae allowed to enhancing xylitol production from the co-utilization of cellobiose/xylose contained in lignocellulose [START_REF] Guirimand | Combined Cell Surface Display of β-d-Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic B[END_REF].

To prevent the accumulation of toxic/unstable intermediates as well as the loss of the desired intermediates, another interesting option has emerged based on the spatial (re)arrangement of enzymes into artificial metabolon, ensuring substrate channeling for a continuous metabolic flux within the YCF [START_REF] Pompon | Nanotechnology for Synthetic Biology: Crossroads Throughout Spatial Confinement[END_REF][START_REF] Rabeharindranto | Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae[END_REF]. Two main approaches enable constructing such artificial metabolons: one consisting of a direct fusion of the enzymes to each other (protein linker), and one relying on the interaction of enzymes with protein (or nucleic acid)-based scaffolds (Fig. 5D). While protein fusion is considered as the easiest way to enhance substrate channeling, this approach may alter the structure of the enzymes and is always restricted to a very limited (two to three) number of enzymes. Nevertheless, the over-expression of an engineered tridomain enzyme (CrtB, CrtI, CrtY) harboring the full β-carotene biosynthetic pathway lead to an improved bio-production of the pigment in S. cerevisiae highlighting the potential of this approach for the improvement of YCFs [START_REF] Rabeharindranto | Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae[END_REF]. On another hand, artificial metabolon using protein-based scaffold technologies allows the gathering of several enzymes in close vicinity through affinity binding, leading to significantly improved xylose utilization and resveratrol bioproduction in S. cerevisiae and to decrease the accumulation of by-product xylitol [START_REF] Thomik | An artificial transport metabolon facilitates improved substrate utilization in yeast[END_REF].

Adaptive laboratory evolution strategies

Besides pathway engineering and metabolic flux rewiring approaches, the development of robust YCFs is crucial for industrial application purposes, due to harsh culture conditions and frequent limiting toxicity of intermediates and products [START_REF] Sandberg | The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology[END_REF][START_REF] Kang | Linking genetic, metabolic, and phenotypic diversity among Saccharomyces cerevisiae strains using multi-omics associations[END_REF]. Adaptive laboratory evolution (ALE) relies on yeast adaptation capacity allowed by multiple DNA recombination events and high genomic plasticity, utterly difficult to achieve through the rational engineering approach alone (Fig. 6A) [START_REF] Sandberg | The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology[END_REF]. For instance, ALE led to improved xylose utilization ability in S. cerevisiae along with an enhanced isobutanol bioproduction due to point mutations in the CCR4 and TIF1

genes, and fine-tuning of gene expression in the evolved strain (Fig. 6B) [START_REF] Promdonkoy | Improvement in d-xylose utilization and isobutanol production in S. cerevisiae by adaptive laboratory evolution and rational engineering[END_REF]. Associated with high throughput screening methodologies [START_REF] Kang | Linking genetic, metabolic, and phenotypic diversity among Saccharomyces cerevisiae strains using multi-omics associations[END_REF][START_REF] Tian | A multiple-step strategy for screening Saccharomyces cerevisiae strains with improved acid tolerance and aroma profiles[END_REF], ALE constitutes a powerful approach allowing speeding up the conception of highly efficient recombinant YCFs (Fig. 6C-E). In particular, ALE allows overcoming numerous physiological limitations (i.e. thermotolerance, osmotic stress, low pH, toxicity, etc) [START_REF] Mitsui | Improved Stress Tolerance of Saccharomyces cerevisiae by CRISPR-Cas-Mediated Genome Evolution[END_REF][START_REF] Kang | Linking genetic, metabolic, and phenotypic diversity among Saccharomyces cerevisiae strains using multi-omics associations[END_REF][START_REF] Promdonkoy | Improvement in d-xylose utilization and isobutanol production in S. cerevisiae by adaptive laboratory evolution and rational engineering[END_REF][START_REF] Tian | A multiple-step strategy for screening Saccharomyces cerevisiae strains with improved acid tolerance and aroma profiles[END_REF][START_REF] Pereira | Adaptive laboratory evolution of tolerance to dicarboxylic acids in Saccharomyces cerevisiae[END_REF][START_REF] Randez-Gil | Myriocin-induced adaptive laboratory evolution of an industrial strain of Saccharomyces cerevisiae reveals its potential to remodel lipid composition and heat tolerance[END_REF][START_REF] Zhang | Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation[END_REF][START_REF] Xu | Evolutionary engineering in Saccharomyces cerevisiae reveals a TRK1-dependent potassium influx mechanism for propionic acid tolerance[END_REF][START_REF] Zhu | Enhancement of sphingolipid synthesis improves osmotic tolerance of saccharomyces cerevisiae[END_REF][START_REF] Caspeta | Engineering high-gravity fermentations for ethanol production at elevated temperature with Saccharomyces cerevisiae[END_REF] as well as to complement the rational engineering approaches presented in the above sections, and providing new targets for the next round of rational design [START_REF] Luo | Coupling S-adenosylmethionine-dependent methylation to growth: Design and uses[END_REF][START_REF] Sandberg | The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology[END_REF][START_REF] Kang | Linking genetic, metabolic, and phenotypic diversity among Saccharomyces cerevisiae strains using multi-omics associations[END_REF][START_REF] Promdonkoy | Improvement in d-xylose utilization and isobutanol production in S. cerevisiae by adaptive laboratory evolution and rational engineering[END_REF][START_REF] Tian | A multiple-step strategy for screening Saccharomyces cerevisiae strains with improved acid tolerance and aroma profiles[END_REF][START_REF] Caspeta | Engineering high-gravity fermentations for ethanol production at elevated temperature with Saccharomyces cerevisiae[END_REF][START_REF] Jakočiūnas | CasPER, a method for directed evolution in genomic contexts using mutagenesis and CRISPR/Cas9[END_REF][START_REF] Papapetridis | Laboratory evolution for forced glucose-xylose coconsumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae[END_REF][START_REF] Strucko | Laboratory evolution reveals regulatory and metabolic tradeoffs of glycerol utilization in Saccharomyces cerevisiae[END_REF][START_REF] Zhang | Adaptive Evolution Relieves Nitrogen Catabolite Repression and Decreases Urea Accumulation in Cultures of the Chinese Rice Wine Yeast Strain Saccharomyces cerevisiae XZ-11[END_REF][START_REF] Qi | Engineering microbial membranes to increase stress tolerance of industrial strains[END_REF][START_REF] Kawai | Identification of metabolic engineering targets for improving glycerol assimilation ability of Saccharomyces cerevisiae based on adaptive laboratory evolution and transcriptome analysis[END_REF][START_REF] Seppälä | Heterologous transporters from anaerobic fungi bolster fluoride tolerance in Saccharomyces cerevisiae[END_REF][100][101][102]. Noteworthily, the use of CRISPR/Cas9 system in combination with ALE approach enabled the generation of evolved YCF with improved thermotolerance, as mentioned in the "Genome editing" section of this review [START_REF] Mitsui | Improved Stress Tolerance of Saccharomyces cerevisiae by CRISPR-Cas-Mediated Genome Evolution[END_REF].

Other stricking studies, such as growth-coupling strategies associated to ALE, can be cited [START_REF] Luo | Coupling S-adenosylmethionine-dependent methylation to growth: Design and uses[END_REF]103]. In particular, when the production of the essential amino acid cysteine was tied to the activity of methyltransferases, ALE was used to select for both E. coli and S. cerevisiae strains with mutations leading to 2-fold increases in heterologous methyltransferase activity [START_REF] Luo | Coupling S-adenosylmethionine-dependent methylation to growth: Design and uses[END_REF]. In this study, adaptive mutations were forced to preferentially target the methyltransferase activity bottleneck which was limiting for growth-rate. This approach allowed to significantly improve activity of both N-and O-type methyltransferases, as illustrated in the "Rewiring of primary metabolism" section of this review.

In addition, ALE virtually allows to improve the "Design, Build, Test, Learn" (DBTL) cycle classically used in ME to generate YCFs (Fig. 6E), when performed in the continuity of the "Build" step for rescuing/optimizing a strain with decreased fitness [START_REF] Sandberg | The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology[END_REF]. Nevertheless, given that that evolution of the strains often drives the population towards a low-production highfitness phenotype, which constitutes an important limitation of ALE, an extensive effort is still required to overcome this problem [83-85, 103, 104]. In that sense, combining ALE with multiomic technologies is emerging as the most promising and efficient approach in YCFs engineering [START_REF] Mitsui | Improved Stress Tolerance of Saccharomyces cerevisiae by CRISPR-Cas-Mediated Genome Evolution[END_REF][START_REF] Luo | Coupling S-adenosylmethionine-dependent methylation to growth: Design and uses[END_REF][START_REF] Kang | Linking genetic, metabolic, and phenotypic diversity among Saccharomyces cerevisiae strains using multi-omics associations[END_REF][START_REF] Tian | A multiple-step strategy for screening Saccharomyces cerevisiae strains with improved acid tolerance and aroma profiles[END_REF][START_REF] Strucko | Laboratory evolution reveals regulatory and metabolic tradeoffs of glycerol utilization in Saccharomyces cerevisiae[END_REF]102,104,105].

Concluding remarks: Future Challenges and Directions

Using synthetic biology to implement YCFs is a promising approach to produce very complex compounds (including vinblastine and vincristine, among many others) that are almost impossible to produce by synthetic chemistry alone because of challenges like asymmetric carbons. Although several molecular tools (e.g., halogenases) have been characterized in marine bacteria and plants, reports on bioproduction of halogenated NPs in yeast is scarce Therefore, SC is still indispensable to generate valuable fluorinated or chlorinated NPs derivatives, usually presenting much higher pharmacodynamic characteristics, and a larger demand in drug industries. One example is dexamethasone, the fluorinated derivative of hydrocortisone, which is 40 times more potent than hydrocortisone and far more stable in vivo.

In that sense, a strong complementarity between SB and SC exists, while SB is a highly sustainable and ecological way to produce platform compounds, SC can be employed in downstream fine-tuning and upstream precursor-directed yeast-mediated biosynthesis (bioconversion, [110]). Despite some technical limitations, yeast cells represent a useful eukaryotic tool suitable for the bio-production of valuable and complex molecules. Meanwhile, constant progress in SB has accelerated the industrial applications of YCFs [START_REF] Ehrenworth | Accelerating the semisynthesis of alkaloid-based drugs through metabolic engineering[END_REF][START_REF] Courdavault | Towards the Microbial Production of Plant-Derived Anticancer Drugs[END_REF][3][START_REF] Nielsen | Engineering Cellular Metabolism[END_REF][START_REF] Lian | Recent advances in metabolic engineering of Saccharomyces cerevisiae: New tools and their applications[END_REF][START_REF] Chen | Advanced Strategies for Production of Natural Products in Yeast[END_REF]. Besides model yeasts such as S. cerevisiae, several non-conventional chassis present increasing interest in ME applications due to their unique features [START_REF] Raschmanová | Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects[END_REF][START_REF] Larroude | Synthetic biology tools for engineering Yarrowia lipolytica[END_REF]. While SC has made huge progress in recent years, including the recent syntheses of molecules as complex as Vitamin B12 [START_REF] Eschenmoser | Natural product synthesis and vitamin B12[END_REF] or strictosidine [START_REF] Sakamoto | Total Syntheses of (-)-Strictosidine and Related Indole Alkaloid Glycosides[END_REF], tailoring ideal YCFs that can produce large-scale NPs as well as their derivatives is essential to establish next-generation bio-foundries as cost-and timeefficient alternatives. Such progress in the field of SB will constitute an invaluable chance to further achieving a better and more sustainable future, in agreement with the Sustainable Development Goals (SDGs) of the United Nations (https://www.un.org/sustainabledevelopment/sustainable-development-goals/). The coming decade is undoubtedly going to be full of exciting improvements in the YCF technology, leading to the ultimate convergence of SC and SB: the production of a truly infinite palette of complex molecules. ("trans") regulation, rewiring of yeast primary metabolism and adaptive laboratory evolution (ALE), which will be discussed in this review. However, heterologous production also presents several limitations/challenges, which, in tandem with synthetic chemistry (purple circle and arrows), potentially can be resolved (e.g. substrate chemical synthesis, product modification), and represent promising prospects.

Figure360. An author presentation of Figure 1. constitutive or inducible promoter, according to user needs). This is achieved either in tandem with gRNA(s) [START_REF] Degreif | Preloading budding yeast with all-in-one CRISPR/Cas9 vectors for easy and high-efficient genome editing[END_REF], followed by the Cas9-gRNA(s)-expressing strain transformation with template DNA, or yeast pre-transformation with Cas9-expressing plasmid only, further followed by the transformation of Cas9-expressing strain with template DNA and gRNA(s) plasmid [START_REF] Jakočiunas | CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae[END_REF][START_REF] Jakočiunas | Assembly and Multiplex Genome Integration of Metabolc Pathways in Yeast Using CasEMBLR[END_REF]. A recent gRNA delivery method transient expression system (TES) was described, where the PCR fragments encoding the gRNAs (and containing the promoter, the guiding sequence and the gRNA scaffold) are transformed together with template DNA into Cas9-expressing yeast strain [START_REF] Easmin | gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing[END_REF]. The template DNA (multiple expression cassettes, composed of promoter and terminator for each gene of interest) is assembled in vivo via overlapping oligonucleotides (here represented as colored lines at the extremities of expression cassettes, black lines represent the so-called Arms -the sequences homologous to the specific yeast genomic regions) and homologous recombination (HR) [START_REF] Jakočiunas | CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae[END_REF][START_REF] Jakočiunas | Assembly and Multiplex Genome Integration of Metabolc Pathways in Yeast Using CasEMBLR[END_REF]. The schematic example of a single expression cassette is shown. (B) Advances in CRISPR/Cas9 system. The GTR-CRISPR (tandem gRNA-tRNA array for CRISPR/Cas9) is based on the use of a gRNA-tRNA array encoded in a plasmid with Cas9, and the native tRNA-processing system (the precursor of tRNA is cleaved, which releases the individual gRNAs from the common transcript) [START_REF] Zhang | A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae[END_REF]. Cpf1

(CRISPR from Prevotella and Francisella 1 endonuclease) demonstrates several advantages compared to Cas9 (generation of sticky ends, RNAse and DNAse activity, requirement of only crRNA) [START_REF] Safari | CRISPR Cpf1 proteins: Structure, function and implications for genome editing[END_REF]. dCas9-mediated Target-AID (Activation Induced cytidine Deaminase from vertebrate) is a synthetic hybrid complex, which performs C to G and C to T mutations with high accuracy [START_REF] Després | Double selection enhances the efficiency of target-AID and Cas9-based genome editing in yeast. G3 Genes, Genomes[END_REF]. The dashed line rectangle -variant selection, the purple background -control strain not expressing MRTF. The criteria for selection vary according to user needs [START_REF] Snoek | Evolution-guided engineering of small-molecule biosensors[END_REF]. 2) Activator-or repressor-type MRTF binding site (BS) were inserted in front of every nucleotide of native pTEF1 or truncated pCYC1 core regions to generate MRTF operator library [START_REF] Ambri | High-resolution scanning of optimal biosensor reporter promoters in yeast[END_REF]. (C) Construction of synthetic gluconeogenic promoters in yeast [START_REF] Rajkumar | Engineered Reversal of Function in Glycolytic Yeast Promoters[END_REF]. Native promoters were reshaped from glycolytic to gluconeogenic via substitution of the glycolytic genes transcriptional activator Gcr1 BS by carbon source response elements (CSRE) of native gluconeogenic promoters. sP -synthetic promoter, purple and black circles/curves -ethanol and glucose, respectively. Overlapping of curves -diauxic shift. engineering to enhance heterologous Fe-S enzyme activity (adapted from [START_REF] Bamba | Production of 1,2,4-butanetriol from xylose by Saccharomyces cerevisiae through Fe metabolic engineering[END_REF]). The assembly of Fe-S clusters is mediated by the assembly complex. The targeting complex guides Fe-S clusters to the corresponding apoprotein to form Fe-S proteins. Tyw1 binds Fe-S clusters to regulate Fe-S excess in the cell. Truncated Tyw1 (tTyw1) is not able to bind Fe-S clusters.

Aft1/2 TFs enhance Fe uptake in the case of Fe deficiency, which is inhibited by the inhibitory complex. Yap5 TF promotes Fe sequestration when in excess. All the represented complexes contain several components omitted here. In the best performing strain, tTyw1 was overexpressed and the inhibitory complex component Bol2 was deleted (Δbol2). The red cross represents the abolishment of inhibitory complex activity. (A) Artificial compartmentalization of the triterpene biosynthetic pathway in the peroxisome of S. cerevisiae, leading to a significant increase in squalene bio-production (adapted from [START_REF] Liu | The yeast peroxisome: A dynamic storage depot and subcellular factory for squalene overproduction[END_REF]).

(B) Artificial compartmentalization of the isoprenoid biosynthetic pathway in the mitochondria of S. cerevisiae, leading to a significant increase of geraniol bio-production (adapted from [START_REF] Yee | Engineered mitochondrial production of monoterpenes in Saccharomyces cerevisiae[END_REF]). (C) Cell surface engineering of S. cerevisiae for the efficient bio-production of ethanol and xylitol from lignocellulosic biomass (adapted from [START_REF] Guirimand | Cell-surface display technology and metabolic engineering of: Saccharomyces cerevisiae for enhancing xylitol production from woody biomass[END_REF][START_REF] Anandharaj | Constructing a yeast to express the largest cellulosome complex on the cell surface[END_REF][START_REF] Guirimand | Combined Cell Surface Display of β-d-Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic B[END_REF]). (D) Two different types of artificial metabolons in S. cerevisiae (adapted from [START_REF] Pompon | Nanotechnology for Synthetic Biology: Crossroads Throughout Spatial Confinement[END_REF]). Titer, rate, and yield (TRY): refers to production metrics of product concentration, the time required to generate it, and the final amount.

Yeast cell factories (YCFs): an approach in bio-engineering, which relies on metabolic engineering and employs yeast cells as a production unit.

[

  106-108]. This kind of subtle modification (such as chlorination or fluorination) remains a future challenge for engineering YCFs to generate new-to-nature compounds with high pharmaceutical interest (see Outstanding Questions) [1, 2, 106-109]. Recently, an indirect way to obtain such compounds was reported, consisting of incorporating precursor derivatives (in this case, halogenated tyrosine) to produce S-reticuline derivatives [60]. Another recent study elegantly showed the bioproduction of halogenated oxo-(2-aminophenyl) and quinoline scaffolds in S. cerevisiae, by overexpressing regiospecific L-tryptophan halogenases [109].

Figure 1 ,

 1 Figure 1, Key Figure: Comprehensive overview of cutting-edge molecular tools and strategies to implement the ideal yeast cell factory (YCF). This figure illustrates various synthetic biology tools that are employed for the YCF construction and optimization (grey arrows), such as advanced genome editing tools, transcriptional ("cis") and post-transcriptional

Figure 2 .

 2 Figure 2. Advanced genome editing tools in yeast (A) DNA assembly and CRISPR/Cas9mediated yeast transformations. Cas9 is expressed in yeast (plasmid-encoded under

Figure 3 . 2 )

 32 Figure 3. New tools for the regulation of gene expression in yeast. (A) Generation of synthetic gene regulatory element libraries. Promoter (P) core region between the TATA box and the transcriptional starting site (TSS) was mutated. 1) The determined minimal sequence of S. cerevisiae pTEF1 core region was mutated. The variants, with or without UASs, were screened by measuring yECitrine (yECit, yellow-green fluorescent protein) fluorescence [50].2) The artificial sequences enriched in T and/or G/C were substituted in Y. lipolytica pEXP1 and pGDP core regions. The screening -colony-color spectrum due to crtY activity (accumulation of carotene pigments)[START_REF] Liu | Engineering yeast artificial core promoter with designated base motifs[END_REF]. 3) In the S. cerevisiae terminator library terminator (T) linker 1 sequence between the efficiency element (EE) and the positioning element (PE) was randomized. The screens were done using green fluorescent protein (GFP) fluorescence as a reporter[START_REF] Wang | Yeast Synthetic Terminators: Fine Regulation of Strength through Linker Sequences[END_REF]. (B) Engineering of metabolite sensing and downstream regulation in S. cerevisiae. 1) The method enables the design of user-defined MRTFs by random mutagenesis of the MRTF effector binding domain (EBD) upstream DNA binding domain (DBD). The

Figure 4 .

 4 Figure 4. Engineering of primary metabolism in yeast. (A) Redox engineering. PPP stands for the pentose phosphate pathway. The best performing strains are illustrated. Green color indicates overexpression while velvet color -deletion. ALD6 was overexpressed [57, 60] and ALD2 deleted [57]. Full-length POS5/truncated tPOS5 were overexpressed [58], as well as POS5 [61]. NADPH availability is estimated by the activity of heterologous NADPH-dependent enzymes. (B) Coupling methylation to growth via Mtase selection (adapted from [64]). Black arrows represent the native SAM cycle. SAH stands for S-adenosylhomocysteine, velvet cross represents the inhibition of reaction by gene deletion, green arrows show heterologous reactions. The graphics demonstrate the growth (optical density OD) of yeast strains (the tested strain on the left and the control strain on the right) over time, in the presence (+X) or absence (-X) of the corresponding heterologous Mtase substrate. (C) Fe metabolic

Figure 5 .

 5 Figure 5. Strategies of spatial (re)configuration for the metabolic engineering of YCF.

Figure 6 .

 6 Figure6. The integrated approach of adaptive laboratory evolution (ALE) to improve YCF, (adapted from[START_REF] Sandberg | The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology[END_REF][START_REF] Promdonkoy | Improvement in d-xylose utilization and isobutanol production in S. cerevisiae by adaptive laboratory evolution and rational engineering[END_REF]).(A) Initial YCFs, obtained by rational ME steps, are cultured in desired growth conditions for an extended period, allowing natural selection to enrich for mutant strains with improved fitness. ALE then occurs via n repetitions of propagation of batch cultures, until obtaining the most efficiently evolved YCF (endpoint). (B) Enhancement of D-xylose utilization and isobutanol production by combining the rational DBTL approach and ALE. The ALE experiment was

Acknowledgments

The authors are truly grateful to the two reviewers who utterly improved the quality of this work by their valuable comments on this review. GG acknowledges the research fellowship of Le Studium-Institute for Advanced Studies, Loire Valley, Orléans, France. We acknowledge funding from the ARD2020 Biopharmaceutical Program of the Région Centre Val de Loire (BioPROPHARM, ETOPOCentre and CatharSIS projects), La Ligue Contre le Cancer and Le Studium (Consortium fellowship). The synthetic biology research of TH is supported by project P16009, Development of Production Techniques for Highly Functional Biomaterials Using Smart Cells of Plants and Other Organisms (Smart Cell Project), from the New Energy and Industrial Technology Development Organization (NEDO). TH also acknowledges support from JSPS KAKENHI (JP15H05557) and by the Advanced Low Carbon Technology Research and Development Program (ALCA, JPMJAL1306) from the Japan Science and Technology Agency (JST), the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. The authors apologize for other excellent studies that have not been cited here due to space limitations.

Glossary

Adaptive laboratory evolution (ALE): integrated approach relying on the capacity of microorganisms to evolve in response to specific cultivation conditions, under selection pressure, and along an extended period (hundreds to thousands of generations), to obtain improved strains (growth, tolerance, titer, etc).

Carbon source response elements (CSRE): short promoter sequences, identified as activating motifs of yeast gluconeogenic genes, and responsive to ethanol.

Cell surface engineering (CSE): recombinant protein expression and incorporation into the yeast cell wall, to generate a whole-cell catalyst capable to accommodate different enzymatic reactions directly on the extracellular interface.

Cellulosome: multi-enzyme complexes associated with the cell surface of cellulolytic microorganisms, mediating cell attachment to insoluble substrates for degradation into soluble products.

"Design, Build, Test, Learn" (DBTL) cycle: an integrated iterative approach for metabolic engineering of high-performance microbial biocatalysts and improvement of the commercially relevant metrics of titer, rate, and yield.

Genome editing: genetic modification using genome editing tools that result in DNA deletion, integration or substitution within a genome.

Global fitness: physiological state of living cells at a given time.

Metabolic biosensors: biomolecules able to detect/respond to specific metabolites within metabolic pathways.