Guido Hennig
email: ghennig@cebitec.uni-bielefeld.de

Carsten Haupka
email: chaupka@cebitec.uni-bielefeld.de

Luciana F Brito
email: luciana.f.d.brito@ntnu.no

Christian Rückert
email: christian.rueckert@cebitec.uni-bielefeld.de

Edern Cahoreau
email: cahoreau@insa-toulouse.fr

Stephanie Heux
email: heux@insa-toulouse.fr

F Volker

Wendisch

Stéphanie Heux

Volker F Wendisch
email: volker.wendisch@uni-bielefeld.de

Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway

Keywords: synthetic methylotrophy, methanol, ribulose monophosphate pathway, adaptive laboratory evolution, isotopic labeling, metabolic engineering

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

Introduction

Long-term transition from fossil fuels to renewable energy is inevitable and poses one of the major challenges for humanity in the 21st century. Methanol is a promising alternative renewable energy source with a price of 359 USD per metric ton (April 2020, Methanex, https://www.methanex.com/ourbusiness/pricing) and the feasibility of a "methanol economy" has been proposed before [START_REF] Olah | Towards oil independence through renewable methanol chemistry[END_REF]. Methanol can be synthesized from gasified biomass wastes or environmentally captured CO 2 -electrochemically or by hydrogenation. Therefore, pure H 2 is required which can be gained from electrolysis of water, powered by solar, wind or thermal energy. [START_REF] Aresta | Utilisation of CO 2 as a chemical feedstock: Opportunities and challenges[END_REF]. Moreover, the conversion of CO 2 to methanol by solar energy and the promising field of light-driven C 1 chemistry in general are expected to replace conventional thermal catalytic processes in the chemical industry and it is estimated that 40-70 Mt of methanol will be produced from CO 2 and H 2 by 2050 [START_REF] Chen | From Solar Energy to Fuels: Recent Advances in Light-Driven C 1 Chemistry[END_REF].

Methylotrophic bacteria on plant surfaces function as a sink for volatile organic compounds such as methanol, the most abundant organic gas in the atmosphere besides methane and, thus, improve the overall air quality and minimize global warming [START_REF] Kumar | Methylotrophic bacteria in sustainable agriculture[END_REF]. Additionally, they are an important group of bioinoculants which are applied in sustainable agriculture to potentially substitute for chemical fertilizers in order to provide nitrogen and phosphorus to plants without depletion of soil quality [START_REF] Kumar | Methylotrophic bacteria in sustainable agriculture[END_REF].

Natural methylotrophy in bacteria has been studied extensively in the past decades to reveal three distinct pathways for biomass-related C 1 utilization: the ribulose monophosphate pathway (RuMP) found e.g., in the Gram-positive, thermophilic Bacillus methanolicus [START_REF] Müller | Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity production from methanol[END_REF], the serine cycle/ethyl-malonyl-CoA-pathway found e.g., in the Gram-negative, mesophilic Methylobacterium extorquens [START_REF] Ochsner | Methylobacterium extorquens: Methylotrophy and biotechnological applications[END_REF] and the ribulose bisphosphate pathway (RuBP) found e.g., in Gram-negative, autotrophic Paracoccus denitrificans [START_REF] Erb | Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: The ethylmalonyl-CoA pathway[END_REF]. The RuMP pathway was shown to be the most efficient pathway in terms of energy, as it yields one molecule of nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) for each pyruvate produced, whereas the serine cycle and RuBP require energy investments for carbon fixation and utilization [START_REF] Anthony | Methanol oxidation and growth yields in methylotrophic bacteria: A review[END_REF][START_REF] Cotton | Renewable methanol and formate as microbial feedstocks[END_REF].

Methanol is an attractive carbon source for biotechnological application and natural methylotrophs have successfully been used to produce valuable products, such as amino acids or fine chemicals [START_REF] Pfeifenschneider | Methanol as carbon substrate in the bio-economy: Metabolic engineering of aerobic methylotrophic bacteria for production of value-added chemicals[END_REF]. Selected strains of B. methanolicus secreted up to 70 g/L of l-glutamate or 35 g/L of l-lysine in high-temperature fed-batch cultivations solely from methanol and the l-lysine/l-glutamate derived products cadaverine and gamma-aminobutyric acid have since been added to extend the product spectrum [START_REF] Brautaset | Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for L-lysine production from methanol at 50[END_REF][START_REF] Drejer | Methanol-based acetoin production by genetically engineered Bacillus methanolicus[END_REF][START_REF] Irla | Methanol-based γ-aminobutyric acid (GABA) production by genetically engineered Bacillus methanolicus strains[END_REF][START_REF] Naerdal | Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains[END_REF].

Although there have been recent advances in the development of tools for system engineering of B. methanolicus [START_REF] Carnicer | Quantitative metabolomics of the thermophilic methylotroph Bacillus methanolicus[END_REF][START_REF] Naerdal | L-lysine production by Bacillus methanolicus: Genome-based mutational analysis and L-lysine secretion engineering[END_REF][START_REF] Schultenkämper | Establishment and application of CRISPR interference to affect sporulation, hydrogen peroxide detoxification, and mannitol catabolism in the methylotrophic thermophile Bacillus methanolicus[END_REF], the genetic toolbox for these methylotrophic bacteria is still limited and metabolic engineering to optimize carbon fluxes for increased product titers or yields remains a challenge to this day [START_REF] Irla | Genome-based genetic tool development for Bacillus methanolicus: Theta-and rolling circle-replicating plasmids for inducible gene expression and application to methanol-based cadaverine production[END_REF]. Thus, the demand to transfer methylotrophy to mesophilic bacteria with well-established engineering toolset has been high in recent years [START_REF] Zhang | Current advance in bioconversion of methanol to chemicals[END_REF]. First attempts to engineer Escherichia coli and Corynebacterium glutamicum towards synthetic methylotrophy revealed functional operation of RuMP pathway enzymes but remained unsuccessful in establishing methanol utilization for biomass formation [START_REF] Leßmeier | Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate[END_REF][START_REF] Müller | Engineering Escherichia coli for methanol conversion[END_REF][START_REF] Witthoff | Metabolic engineering of Corynebacterium glutamicum for methanol metabolism[END_REF]. Recent approaches succeeded in re-routing the central carbon metabolism of non-methylotroph bacteria to create a methanol-dependency through metabolic cut-offs that required the co-utilization of methanol with gluconate or xylose through RuMP pathway reactions for partial complementation, which was subsequently enhanced through adaptive laboratory evolution (ALE) [START_REF] Chen | Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production[END_REF][START_REF] Meyer | Methanol-essential growth of Escherichia coli[END_REF][START_REF] Tuyishime | Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production[END_REF]. Moreover, a linear synthetic pathway named reductive glycine pathway was engineered in E. coli which enabled growth solely on C 1 compounds [START_REF] Kim | Growth of E. coli on formate and methanol via the reductive glycine pathway[END_REF].

Here, we engineered a methanol-dependency in C. glutamicum in two distinct approaches, deleting either the rpi gene, encoding ribose-5-phosphate isomerase or rpe, encoding ribulose-5-phosphate epimerase in the pentose-phosphate pathway. Hereby, we prevented the full metabolization of ribose, xylose or gluconate, forcing the co-utilization of methanol through RuMP pathway reactions for biomass formation. Methanol-dependent growth of the rpe-deletion strain was further improved until full complementation and 300% increased growth rate by ALE and mutations beneficial for methanol co-utilization were examined.

Results and Discussion

Methanol-Dependent Complementation of a Ribose 5-Phosphate Isomerase Mutant

Since methanol is a convenient alternative carbon source to sugar substrates that are commonly used in biotechnological processes and metabolic engineering of native methylotrophs is still limited, recent efforts focused on establishing synthetic methylotrophy bacteria to produce high-value chemicals from methanol [START_REF] Leßmeier | Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate[END_REF][START_REF] Chen | Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production[END_REF][START_REF] Meyer | Methanol-essential growth of Escherichia coli[END_REF][START_REF] Tuyishime | Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production[END_REF]. Since methanol is more reduced than glucose, it supports production of reduced compounds. Although omics data of native methylotrophs have been generated in the past decade [START_REF] Ochsner | Methylobacterium extorquens: Methylotrophy and biotechnological applications[END_REF][START_REF] Carnicer | Quantitative metabolomics of the thermophilic methylotroph Bacillus methanolicus[END_REF][START_REF] Naerdal | L-lysine production by Bacillus methanolicus: Genome-based mutational analysis and L-lysine secretion engineering[END_REF][START_REF] Irla | Complete genome sequence of Bacillus methanolicus MGA3, a thermotolerant amino acid producing methylotroph[END_REF], natural methanol utilization has not yet been fully understood, which makes the transfer of methylotrophy to non-methylotrophs a demanding challenge [START_REF] Müller | Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity production from methanol[END_REF][START_REF] Zhang | Current advance in bioconversion of methanol to chemicals[END_REF]. Here, we developed methanol-dependent C. glutamicum strains through rpe and rpi deletions that functioned as metabolic cut-offs to force methanol-utilization for biomass formation (Figure 1A,B). A metabolic cut-off to isolate ribose 5-phosphate-dependent anabolism from the rest of the cellular metabolism was constructed by deletion of ribose 5-phosphate isomerase gene rpi. Growth of this pentose phosphate pathway mutant with xylose, which required transformation with the plasmid pEKEx3-xylAB, was expected only upon supplementation with ribose (Figure 1B). Indeed, strain ∆ald ∆fadH ∆rpi (pEKEx3-xylAB) (MDS1) grew poorly on lysogeny broth (LB) or brain heart infusion (BHI) plates, whereas BHI plates supplemented with 20 mM ribose, 20 mM xylose and 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) supported efficient growth (data not shown). In minimal media, strain MDS1 grew with a rate of 0.05 h -1 with a mixture of ribose and xylose, but it did neither grow with xylose nor ribose as sole carbon source (Figure 2A). The parental strain MDS0(pEKEx3-xylAB), used as positive control, grew with growth rates of 0.15 h -1 with ribose, 0.08 h -1 with xylose and 0.10 h -1 with the mixture of xylose and ribose (Figure 2A). Next, we genetically complemented C. glutamicum strain MDS1 by transformation with pVWEx1-rpi. After lag phase, the strain MDS1(pVWEx1-rpi) grew on ribose as sole carbon source, while the empty vector carrying the control strain MDS1(pVWEx1) did not (Figure 2B). Thus, loss of function of rpi caused the observed growth phenotype. In order to test if a functional RuMP cycle for formaldehyde assimilation can circumvent feeding ribose to the rpi deletion mutant (Figure 1B), strain MDS1(pVWEx1-mdh-hxlAB) for expression of the genes encoding methanol dehydrogenase from B. methanolicus and 3-hexulose 6-phosphate synthase HxlA and hexulose 6-phosphate isomerase HxlB from B. subtilis was constructed and evaluated for methanol-dependent growth (Figure 2C). No growth of this strain was observed with xylose as sole carbon source, however, upon addition of methanol the strain grew with a growth rate 0.03 h -1 from an OD 600 of 0.52 ± 0.07 to 1.39 ± 0.37. As an independent test, growth with gluconate as sole carbon source with/without addition of methanol was tested since ribulose 5-phosphate is generated during gluconate catabolism. Indeed, strain MDS1(pVWEx1-mdh-hxlAB) grew with a growth rate of 0.04 h -1 from an initial OD600 of 0.50 ± 0.07 to 1.63 ± 0.10. Although no ALE was conducted with strain MDS1, the specific growth rates with both co-substrates were on par with, but not faster than previously reported growth rates for methanol-dependent growth of evolved C. glutamicum strain MX-11 with xylose (0.03 h -1 ) [START_REF] Tuyishime | Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production[END_REF].

To exclude that rpi deletion mutants can only be selected for if compensatory mutations occurred elsewhere in the genome, we re-sequenced the genome of strains MDS0(pEKEx3-xylAB) (pVWEx1-mdh-hxlAB) and MDS1(pVWEx1-mdh-hxlAB) that only differ by the presence/absence of rpi. For strain MDS0(pEKEx3-xylAB)(pVWEx1-mdh-hxlAB), 79.25%, 14.23% and 7.17% of the processed reads could be mapped to the genome, plasmid pEKEx3-xylAB and plasmid pVWEx1-mdh-hxlAB, respectively. For the rpi deletion mutant MDS1(pVWEx1-mdh-hxlAB), 79.81%, 13.43% and 6.33% of the processed reads were mapped to the genome, plasmid pEKEx3-xylAB and plasmid pVWEx1-mdh-hxlAB, respectively. As expected, reads mapping to the deleted genes ald (cg3096) and fadH (cg0387) were not found for both strains. As compared to the reference genome sequence, in both strains psp1 (cg2069) coding for a putative secreted protein of the prophage CGP3 was only partly covered and seven non-silent single-nucleotide polymorphisms (SNPs) were found (Table 1). Three occurred in the remainders of the deleted genes ald and fadH which were left to ensure in frame-deletions and prevent polar effects. The gene functions of loci cg0822 and cg1245 are yet unknown and additional SNPs were found in wzz and hrtA (cg0414, cg2204) encoding for proteins involved in cell surface polysaccharide biosynthesis, chain length determination and an ABC-type transport system and ATPase component [START_REF] Frunzke | Control of heme homeostasis in Corynebacterium glutamicum by the two-component system HrrSA[END_REF]. As expected, none of the SNPs matched the ones generated after ALE of a C. glutamicum strain with a similar background [START_REF] Tuyishime | Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production[END_REF]. Importantly, while sequenced reads were mapped to the rpi locus (cg2658) for strain MDS0(pEKEx3-xylAB)(pVWEx1-mdh-hxlAB), with a mean feature coverage of 12, no reads were mapped to this gene in strain MDS1(pVWEx1-mdh-hxlAB). Thus, the presence/absence of rpi was the only difference between the strains. Table 1. Single-nucleotide polymorphisms (SNPs) determined by whole-genome sequencing. Non-silent SNPs and mutations found in the strains ∆ald ∆fadH and ∆ald ∆fadH ∆rpi, as compared to the wildtype C. glutamicum ATCC13032, or strains MDS2T8 and MDS2T14 in comparison to their parental strain MDS2, including the corresponding loci, gene names, products and the resulting amino acid substitutions. To determine if methanol was assimilated via the RuMP pathway in the C. glutamicum strain MDS1(pVWEx1-mdh-hxlAB), the fate of 13 C-methanol was followed in the non-native product cadaverine. As reported previously, cadaverine production was enabled by overexpression of endogenous, feedback resistant aspartokinase (lysC T311I ) and heterologous expression of lysine decarboxylase (ldcC) from E. coli (using plasmid pEC-XT99A-lysC fbr -ldcC [START_REF] Leßmeier | Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate[END_REF]. Non-labeled gluconate was chosen as secondary substrate to 13 C-methanol to provide additional NADPH, benefitting production of cadaverine precursor L-lysine [START_REF] Leßmeier | Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate[END_REF]. In the chosen 13 C-labeling condition (20 mM gluconate and 500 mM 13 C-methanol), labeling at the C1/C5 or C2/C4 positions of the cadaverine molecule indicates utilization of methanol via RuMP pathway reactions, whereas C3 labeled cadaverine results from anaplerotic carboxylation reactions with 13 CO 2 that arose from oxidation of 13 C-methanol to 13 CO 2 (Figure 3A) [START_REF] Leßmeier | Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate[END_REF]. The 13 C-labeling pattern in 1.5 mM cadaverine that were produced after 48 h by MDS1(pVWEx1-mdh-hxlAB)(pEC-XT99A-lysC fbr -ldcC) besides production of 1.5 mM L-lysine indicated that 30 ± 1.0% of the cadaverine molecules originated from functional operation of the RuMP pathway, 14 ± 2.1% originated from 13 CO 2 and 56 ± 3.1% were unlabeled (Figure 3C). This indicates a 50% increased RuMP functionality over strain MDS0(pVWEx1-mdh-hxlAB)(pEC-XT99A-lysC fbr -ldcC) of which 20 ± 1.0% of the produced cadaverine was labeled at the C1/C5 or C2/C4 positions (Figure 3B) [START_REF] Leßmeier | Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate[END_REF] and is comparable to 24% methanol assimilation into hexose 6-phosphate achieved by engineered and evolved E. coli strains [START_REF] Meyer | Methanol-essential growth of Escherichia coli[END_REF]. (pEC-XT99A-lysC fbr -ldcC), respectively in shake flasks containing M9 minimal medium with 20 mM gluconate, 0.5 g/L yeast extract and 500 mM 13 C-methanol for 72 h. The pie charts show mean 13 C enrichment in cadaverine determined at carbon positions C1/C5 and C2/C4 for assimilation of carbon via RuMP pathway reactions (green) and C3 for assimilation of 13 CO 2 that has arisen from dissimilation of 13 C-methanol (blue).

Locus

Adaptive Laboratory Evolution for Accelerated Methanol-Dependent Complementation of a Ribulose 5-Phosphate Epimerase Mutant

To reduce the number of plasmids to one, we first constructed a new strain containing only one plasmid. Instead of rpi, the ribulose 5-phosphate epimerase gene rpe was deleted in MDS0 to isolate ribose-5-phosphate dependent anabolism from the rest of cellular metabolism. As a result, only one plasmid (i.e., pEKEx3-mdh-hxlAB) was required since in this metabolic setting C. glutamicum naturally catabolizes ribose. Methanol-dependency of the new C. glutamicum strain MDS2(pEKEx3-mdh-hxlAB) was examined in minimal medium with 20 mM ribose as co-substrate. No growth with ribose as sole carbon source was observed for 72 h. By contrast, with methanol plus ribose, strain MDS2(pEKEx3-mdh-hxlAB) formed biomass (∆OD of 1.32 ± 0.03) with specific growth rate of 0.03 ± 0.01 h -1 (Figure 4A). Thus, methanol dependent growth of two-vector strain MDS1(pVWEx1-mdh-hxlAB) with methanol plus xylose and one-vector strain MDS2(pEKEx3-mdh-hxlAB) with methanol plus ribose were comparable. C. glutamicum strain MDS2(pEKEx3-mdh-hxlAB) was subjected to ALE by iterative transfers of cells grown in minimal medium with ribose and methanol. After the first eight transfers, strain MDS2T8 was conserved for whole-genome sequencing and used to determine to what extent 13 C-methanol is assimilated via the RuMP pathway.

Carbon labeling of cadaverine from 13 C-methanol was investigated to determine if methanol is assimilated via the RuMP pathway in C. glutamicum strain MDS2T8 transformed with pEC-XT99A-lysC fbr -ldcC. The 13 C-labelling pattern of 1.6 mM cadaverine besides 1.6 mM L-lysine produced by MDS2T8(pEC-XT99A-lysC fbr -ldcC) indicated that 43% of the cadaverine molecules originated from operation of the RuMP pathway, 16% originated from 13 CO 2 and 41% were unlabeled (Figure 3D). Thus, the strain selected by ALE showed a higher proportion of cadaverine molecules originating from a functional RuMP pathway as compared to the non-evolved strain MDS1(pEKEx3-xylAB)(pVWEx1-mdh-hxlAB)(pEC-XT99A-lysC fbr -ldcC), whereas the dissimilatory pathway activity of both strains was similar (16% vs. 14%).

Furthermore, transfers of C. glutamicum strain MDS2T8 were performed from minimal medium to minimal medium with 0.5 g/L of yeast extract. After six further transfers, C. glutamicum strain MDS2T14 was selected as it reached a final ∆OD of 3.95 ± 0.37 in minimal medium with ribose and methanol with a specific growth rate of 0.10 ± 0.01 h -1 , which is comparable to the wild type (WT) grown with 20 mM ribose (∆OD of 3.90 ± 0.29) (Figure 4A). Therefore, MDS2T14 was 3-times as fast as strain C. glutamicum MX-11 which was cultivated with xylose as co-substrate [START_REF] Tuyishime | Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production[END_REF] and exceeded the growth rate of evolved methanol-essential growth with E. coli co-utilizing gluconate (0.081 ± 0.002 h -1 ) [START_REF] Meyer | Methanol-essential growth of Escherichia coli[END_REF]. Notably, strain MDS2T14 did not require yeast extract for methanol-dependent growth. Taken together, ALE allowed the selection of a mutant strain of MDS2 named MDS2T14 that showed full methanol-dependent complementation of the rpe deletion.

Genome Sequencing of ALE Strains Revealed Candidate Mutations that May Accelerate Methanol-Dependent Biomass Formation

The genome sequences of ALE strains MDS2T8 and MDS2T14 were determined and compared to that of the parental strain MDS2 to identify candidate mutations that may be causal for accelerated methanol-dependent growth. As compared to the parental C. glutamicum strain MDS2, strain MDS2T8 possessed three mutations: non-silent SNPs in the genes metK (S-adenosylmethionine synthetase) and res (site-specific recombinase) resulting in amino acid exchanges S288N and R91H, respectively, and a 15 bp deletion disrupting the coding sequence (CDS) of an uncharacterized gene (cg3104) which is annotated as ATPase involved in DNA repair. The latter shares structural motifs typically found in proteins with DNA exo-and endonuclease activity, such as the well-characterized SbcCD protein in E. coli [START_REF] Connelly | Purification, and Characterization of the SbcCD protein from Escherichia coli[END_REF] that was revealed to be capable of cleaving secondary hairpin structures during plasmid replication [START_REF] Pan | The roles of mutS, sbcCD and recA in the propagation of TGG repeats in Escherichia coli[END_REF]. The unique mutation found in strain MDS2T14 was a 1.4 kb transposon insertion near the deleted rpe gene which shares a leaderless promoter with the riboflavin synthesis genes ribG and ribC, encoding putative bifunctional riboflavin specific deaminase/reductase and putative riboflavin synthase, respectively (Figure 5A) [START_REF] Kalinowski | The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins[END_REF].

Identification of Mutations Causal for Methanol-Essential Growth

First, we analyzed the effect that deletion of rpe has on riboflavin biosynthesis. C. glutamicum MDS2(pEKEx3-mdh-hxlAB) and the evolved strains MDS2T8 and MDS2T14 were cultivated on glucose as sole carbon source with or without riboflavin supplementation to find out whether the transposon integration in MDS2T14 influenced growth without riboflavin. Growth of strains MDS2 and MDS2T8 on glucose minimal medium could be enhanced by riboflavin addition (Figure 5B) as it was previously reported in studies investigating riboflavin transport in a riboflavin deficient C. glutamicum strain [START_REF] Takemoto | The physiological role of riboflavin transporter and involvement of FMN-riboswitch in its gene expression in Corynebacterium glutamicum[END_REF]. By contrast, without added riboflavin strain MDS2T14 grew as fast as or faster than MDS2 and MDS2T8 with added riboflavin (Figure 5B). Thus, the growth advantage of strain MDS2T14 is riboflavin-dependent and likely due to the transposon integration which provided an additional potential -10 promoter region (TATTT) upstream of ribG and an alternative ribosome binding motif (AAGGG) 7 bp upstream of the ribG start-codon [START_REF] Pfeifer-Sancar | Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique[END_REF][START_REF] Vogl | Characterization of riboflavin (vitamin B2) transport proteins from Bacillus subtilis and Corynebacterium glutamicum[END_REF]. Deletion of cg3104 in parental strain MDS2 did not increase biomass formation (∆OD: 1.74 ± 0.08) as compared to MDS2 (∆OD: 2.08 ± 0.41) (Figure 4A). Based on its supposed function as putative ATPase involved in DNA repair, the plasmid copy number of plasmid pEKEx3-mdh-hxlAB was determined by qPCR (Figure 4C). Strains MDS2T14 and MDS2 ∆cg3104 showed approximately 40-60% increased relative plasmid copy number (1.85 ± 0.21 and 2.17 ± 0.05, respectively) as compared to that of the parental strain MDS2 (1.33 ± 0.02). Because of the elevated copy numbers, higher enzyme activities of the heterologously expressed RuMP genes were expected. A coupled assay of HxlA and HxlB revealed 60% higher enzyme activities (p < 0.05) of MDS2T14 (268 ± 21 mU/mg) and MDS2 ∆cg3104 (268 ± 26 mU/mg) compared to MDS2 (169 ± 22 mU/mg) (Figure 4B), which is beneficial for formaldehyde fixation. Improved channeling of formaldehyde into central metabolites has been shown to shift the equilibrium of Mdh towards the methanol-oxidation reaction in E. coli, for an overall increased utilization of methanol [START_REF] Price | Scaffoldless engineered enzyme assembly for enhanced methanol utilization[END_REF]. Efficient methanol-oxidation and formaldehyde channeling has been argued to be one of the major bottlenecks for synthetic methylotrophy [START_REF] Meyer | Methanol-essential growth of Escherichia coli[END_REF][START_REF] Woolston | Improving formaldehyde consumption drives methanol assimilation in engineered E. coli[END_REF], which is further reinforced by our finding that increased HxlAB activity is an essential factor for improved growth of strain MDS2T14.

Another key factor for improved methanol-dependent growth of MDS2T14 is an amino acid exchange from serine to asparagine found at position 288 in MetK (Table 1) which is next to the methionine binding K289. MetK catalyzes the formation of S-adenosylmethionine (SAM) from methionine and ATP (Figure 5C) [START_REF] Sekowska | Sulfur Metabolism in Escherichia coli and Related Bacteria: Facts and Fiction[END_REF]. Both E. coli and C. glutamicum only harbor one copy of the essential metK gene [START_REF] Großmann | Rapid cloning of metK encoding methionine adenosyltransferase from Corynebacterium glutamicum by screening a genomic library on a high density colony-array[END_REF]. When combining deletion of cg3104 with introduction of MetK amino acid exchange S288N, growth with ribose and methanol was improved as strain C. glutamicum MDS2 ∆cg3104 metK_S288N grew to a ∆OD of 4.20 ± 0.47, reaching comparable levels of biomass formation as the evolved strain MDS2T14 (3.95 ± 0.37) and C. glutamicum WT with 20 mM ribose (3.90 ± 0.29) (Figure 4A). Introduction of the MetK amino acid exchange S288N alone had no positive effect on biomass formation (∆OD: 2.20 ± 0.03). However, reverting the amino acid exchange in MetK in the evolved strain MDS2T14 from S288N to N288S, i.e., to the WT sequence, reduced growth on ribose and methanol considerably (∆OD of 1.11 ± 0.02) (Figure 4A). Taken together, the MetK amino acid exchange S288N combined with deletion of cg3104 and supplementation with riboflavin was sufficient to explain improved growth of the evolved strain MDS2T14 in minimal medium with ribose and methanol.

The antimicrobial methionine analogue O-methyl-homoserine [START_REF] Roblin | Studies in Chemotherapy. VIII. Methionine and Purine Antagonists and their Relation to the Sulfonamides[END_REF] was shown to be a naturally occurring side-product of O-acetyl-homoserine sulfhydrolase (MetY) of Corynebacterium acetophilum in the presence of homoserine and methanol since the latter is an analogue of methanethiol, the precursor of methionine (Figure 5C) [START_REF] Murooka | O-alkylhomoserine synthesis catalyzed by O-acetylhomoserine sulfhydrylase in microorganisms[END_REF]. The accumulation of growth-inhibiting O-methyl-homoserine in presence of methanol has been argued to be connected to methanol tolerance before, when MetY was found to be mutated leading to amino acid substitution A165T in a methanol tolerant C. glutamicum strain [START_REF] Leßmeier | Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase[END_REF]. Furthermore, metY was among the mutated genes of strain C. glutamicum MX-11 that exhibited improved methanol-dependent growth with xylose [START_REF] Tuyishime | Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production[END_REF]. During the writing process of this manuscript, a follow-up study from the same group appeared, further elucidating this mutation (G419D) [START_REF] Wang | Adaptive laboratory evolution enhances methanol tolerance and conversion in engineered Corynebacterium glutamicum[END_REF]. It was shown to increase the tolerance for methanol, which was confirmed by knock-down and knock-out experiments. Here, we observed elevated methanol-dependent growth with ribose and riboflavin after metY deletion in strain MDS2 ∆cg3104 (Figure 5D), which was similar to strain MDS2 ∆cg3104 metK_S288N, indicating that both encoded enzymes, MetK and MetY, presumably act on each other when methanol is present and significantly improve methanol-dependent biomass formation of C. glutamicum.

Materials and Methods

Microorganisms and Cultivation Conditions

E. coli DH5α strain was used as a host for cloning [START_REF] Hanahan | Techniques for Transformation of E. coli. DNA Cloning: A Practical Approach[END_REF], S17-1 for transconjugation [START_REF] Schäfer | Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: Selection of defined deletions in the chromosome of Corynebacterium glutamicum[END_REF]. Both strains were grown in lysogeny broth (LB) at 37 • C and supplemented with antibiotics (50 µg mL -1 kanamycin, 100 µg mL -1 spectinomycin, 10 µg mL -1 tetracycline) when appropriate. C. glutamicum ATCC13032 and derived mutants were cultivated in brain heart infusion (BHI), or CGXII minimal medium [START_REF] Eggeling | Experiments[END_REF] supplemented with 25 µg mL -1 kanamycin, 100 µg mL -1 spectinomycin, 5 µg mL -1 tetracycline, and 1 mM IPTG when appropriate. All strains and plasmids are listed in Tables 2 and3. For growth experiments, C. glutamicum overnight cultures in 50 mL BHI were harvested, washed twice in CGXII medium, inoculated to an OD 600 of 0.5 in 10 mL CgXII and supplemented with different carbon source combinations of methanol (500 mM) with gluconate, ribose or xylose (20 mM each) and 20 µM riboflavin for ALE experiments at 30 • C and 120 rpm. Growth was monitored in 100 mL shake flasks, by determination of the OD 600 with V-1200 Spectrophotometer (VWR, Radnor, PA, USA) and with a 48-well flower plate in the Biolector microfermentation system (m2p-labs, Baesweiler, Germany), respectively.

ALE Experiments

ALE of C. glutamicum strain MDS2 (methanol-dependent strain) was carried out in two phases: In phase 1 [MDS2 -MDS2T8] triplicates were cultivated in CGXII minimal medium with 20 mM ribose and 500 mM methanol with intermediate inoculations of LB overnight cultures and vice versa from the highest grown culture. In phase 2 [MDS2T8-MDS2T14], transfers to LB were omitted and minimal medium was supplemented with 0.5 g/L yeast extract, 20 mM ribose and 500 mM methanol.

Molecular Biology Methods

Genomic DNA of C. glutamicum was isolated as described previously [START_REF] Eikmanns | Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase[END_REF]. Classical methods including plasmid isolation, molecular cloning and transformation of E. coli by heat shock and of C. glutamicum by electroporation were performed as described before [START_REF] Eggeling | Experiments[END_REF]. DNA sequences were amplified with the ALLin HiFi DNA Polymerase (HighQu, Kraichtal, Germany) using plasmid or genomic DNA as template. The oligonucleotides used in this study are listed in Table S1. The gene rpi was amplified from C. glutamicum and assembled into PstI-and BamHI-digested pVWEx1 by Gibson Assembly using the respective primers. Likewise, the genes mdh, hxlA and hxlB were amplified as a single operon from the plasmid pEKEx3-mdh-hxlAB [START_REF] Leßmeier | Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate[END_REF] and cloned into pVWEx1 and pEC-XT99A linearized with BamHI. The constructed plasmids and empty vectors were transferred into C. glutamicum by transformation. For deletion, plasmid pK19mobsacB [START_REF] Schäfer | Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: Selection of defined deletions in the chromosome of Corynebacterium glutamicum[END_REF] digested with EcoRI and BamHI was assembled with amplified DNA fragments flanking the genes rpe and cg3104 by Gibson Assembly and transferred into E. coli S17-1 to follow a protocol for gene deletion routinely applied [START_REF] Eggeling | Experiments[END_REF].

Coupled in vitro Activity of HxlA and HxlB

C. glutamicum cells were grown overnight in BHI medium containing 1 mM IPTG and appropriate antibiotics, harvested by centrifugation (5 min, 4000 rpm), washed in 50 mM phosphate buffer and subsequently disrupted by sonication with an amplitude of 50% and a duty circle of 0.5 for 9 min. Afterwards, total protein concentrations were determined by the Bradford method with bovine serum albumin as standard. Activities of HxlA and HxlB in crude extracts were determined in a coupled assay, derived from a previously described method [START_REF] Leßmeier | Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate[END_REF]. A total volume of 1 mL was used for measurements, containing 25 mM phosphate buffer pH 7.4, 5 mM MgCl 2 , 5 mM ribose-5-phosphate, 0.5 mM NAD + , 2 U phosphoriboisomerase from spinach (Sigma), 2 U phosphoglucoisomerase from yeast (Sigma), 2 U Glucose-6-P dehydrogenase from Leuconostoc mesenteroides (Sigma) and 50 µL crude extract. An addition of 5 mM formaldehyde started the reaction and NADH formation was continuously measured at 340 nm and 30 • C for 6 min.

Whole-Genome Sequencing

Whole-genome sequencing was performed with isolated genomic DNA from C. glutamicum strains (PRJNA603493). DNA library preparation, trimming and mapping of the reads and visualization was performed as described previously [START_REF] López | Characterization of D-Arabitol as newly discovered carbon source of Bacillus methanolicus[END_REF]. For SNP detection in all CDS's of C. glutamicum, the built-in tool from ReadXplorer was used. Minimal scores for base quality, average base quality and average mapping quality were set to 20 and reads with a coverage between 5 and 10 were kept if the corresponding frequency was 100%. If the coverage was above 10, the minimum frequency was 90% instead. Additionally, genomic DNA of C. glutamicum ∆ald ∆fadH ∆rpe strains MDS2, MDS2T8 and MDS2T14 was sequenced using Nanopore MinION sequencing technology (Oxford Nanopore Technologies Oxford, UK) as described previously [START_REF] Kibret | Streptomyces spp. from Ethiopia producing antimicrobial compounds: Characterization via bioassays, genome analyses, and mass spectrometry[END_REF]. After assembly, 11 contigs for MDS2, 6 contigs for MDS2T8 and 2 contigs for MDS2T14 were revealed. Assembled genomes of MDS2T8 and MDS2T14 were aligned with the MDS2 genome sequence using SnapGene software v.4.3 (GSL Biotech, Chicago, USA) to visualize SNPs and insertions or deletions. Variant calling was performed manually to identify SNPs in the MDS2T8 and MDS2T14 genomes, after alignment to the MDS2 parental strain genome reference.

Quantitative PCR

Quantitative PCR (qPCR) was performed in 96-well plates, sealed with transparent adhesive cover, in a CFX Connect TM Real-Time PCR Detection System (Biorad, Hercules, USA). A total volume of 20 µL contained 10 µL SensiFAST TM SYBR No-ROX Kit from Bioline (Heidelberg, Germany), 100 nmol/L forward/reverse primer and 4 µL of diluted template DNA. Genomic template DNA, also containing plasmid DNA, was isolated as described before. Fragments of approximately 250 bp were amplified, targeting gntK encoding gluconate kinase on the chromosome and oriV Cg on the pEKEx3 plasmid. Serial dilutions of DNA from 100 to 0.1 ng/µL were used to construct relative standard curves for chromosomal and plasmid targets. Plasmid and chromosome-specific amplicons were additionally detected in separate reactions and technical triplicates at 10 ng/µL to calculate plasmid copy numbers.

Quantification of 13 C-Enrichment

Cultures of C. glutamicum MDS1 and MDS2T8 (pEC-XT99A-lysC fbr -ldcC), grown overnight in BHI medium, containing 1 mM IPTG and antibiotics were used to inoculate modified M9 medium with 20 mM gluconate and 500 mM 13 C-methanol to a starting OD 600 of 0.5. Modified M9 medium contained 7.5 g/L Na 2 HPO 4 , 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 0.25 g/L MgSO 4 7 H 2 O, 0.003 g/L CaCl 2 , 0.5 g/L NH 4 Cl and the trace elements as described for CGXII medium [START_REF] Eggeling | Experiments[END_REF]. Samples of 1 mL were taken after 24, 48 and 72 h of cultivation and subsequently centrifuged for 10 min at 10,000 rpm. Supernatants were frozen at -20 • C for analysis of isotopologues as previously described [START_REF] Leßmeier | Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate[END_REF].

Conclusions

By engineering two alternative metabolic cut-offs in the central carbon metabolism of C. glutamicum to reinforce methanol utilization through RuMP pathway reactions, we achieved methanol-dependent biomass formation on three different co-substrates (ribose, xylose and gluconate). Incorporation of methanol-carbon and functionality of the introduced RuMP pathway was confirmed by following the fate of 13 C-methanol in cadaverine. Growth rates and methanol-dependent biomass formation were improved through ALE. Analysis of mutations identified by genome sequencing of evolved strains revealed three mutations causing the observed metabolic changes concerning improved methanol utilization through an increased plasmid copy number, resistance against methionine-analogue toxicity due to modified metK sequence, which was also achieved by metY deletion and enhanced riboflavin supply because of alternative promoter and RBS motifs. The findings reported here will serve as a basis for future engineering approaches towards synthetic methylotrophy in C. glutamicum and lead to a better understanding of necessary requirements for synthetic methylotrophy in general to unleash the full potential of methanol as biotechnological carbon source for the sustainable production of valuable chemical compounds in the future.

Figure 1 .

 1 Figure 1. The ribulose monophosphate pathway (RuMP) implemented in C. glutamicum. (A) ∆rpe and (B) ∆rpi concepts for methanol-dependent complementation of two metabolic cut-offs of the pentose phosphate pathway in C. glutamicum, respectively. Substrates in grey boxes: MeOH, methanol; metabolites in black boxes: E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; FA, formaldehyde; GAP, glyceraldehyde 3-phosphate; Hu6P, hexulose 6-phosphate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; Xul, xylulose; Xu5P, xylulose 5-phosphate; interconnected pathways, violet boxes: PPP, pentose phosphate pathway; native or homologous overexpression of genes in orange circles: rpe, ribulose 5-phosphate epimerase; rpi, ribose 5-phosphate isomerase; tal, transaldolase; tkt, transketolase; xylA, xylose isomerase; xylB, xylulokinase; heterologous overexpression of xylA gene (xylose isomerase) from X. campestris in green circle; heterologous overexpression of RuMP pathway genes from B. subtilis in blue circles: hxlA, 3-hexulose 6-phosphate synthase; hxlB, 6-phospho 3-hexulose isomerase; heterologous overexpression of mdh gene (methanol dehydrogenase) from B. methanolicus in pink circle; red arrows, knocked out reactions; green arrows, complementing reactions.

Figure 2 .

 2 Figure 2. Loss and gain of Rpi Function. C. glutamicum strains cultivated in BioLector with 1 mL CGXII medium including carbon sources and 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 30 • C. (A) Mutants ∆ald ∆fadH (MDS0) (circles) and ∆ald ∆fadH ∆rpi (pEKEx3-xylAB) (MDS1) (triangles) comprising plasmid pEKEx3-xylAB were supplemented with 20 mM ribose (red), xylose (blue) and both (brown). (B) MDS0 (circles), MDS1(pVWEx1) (rectangles) and MDS1(pVWEx1-rpi) (diamonds) were supplemented with 20 mM ribose (red). (C) MDS1(pVWEx1-mdh-hxlAB) cultivated in 100 mL shakings flasks with 10 mL CGXII medium including carbon sources, 0.1 g L -1 yeast extract and 1 mM IPTG at 30 • C. Supplementation with either 20 mM xylose (blue) or gluconate (black) with and without 500 mM methanol (full, empty). Error bars indicate standard deviations of biological triplicates.

Figure 3 .

 3 Figure 3. Labeling of cadaverine from 13 C-methanol by C. glutamicum strains MDS1 and MDS2T8. (A) Possible 13 C-isotopologue distributions in the central carbon pathway. (B) Cadaverine was produced by the methanol-independent strain C. glutamicum MDS0 (pEKEx3-mdh-hxlAB) (pVWEx1-lysC fbr -ldcC) [20] (C) MDS1 (pVWEx1-mdh-hxlAB) (pEC-XT99A-lysC fbr -ldcC) and (D) MDS2T8(pEC-XT99A-lysC fbr -ldcC), respectively in shake flasks containing M9 minimal medium with 20 mM gluconate, 0.5 g/L yeast extract and 500 mM 13 C-methanol for 72 h. The pie charts show mean13 C enrichment in cadaverine determined at carbon positions C1/C5 and C2/C4 for assimilation of carbon via RuMP pathway reactions (green) and C3 for assimilation of13 CO 2 that has arisen from dissimilation of13 C-methanol (blue).

Figure 4 .

 4 Figure 4. Biomass formation with methanol as co-substrate (A) and relative copy numbers of plasmid pEKEx3-mdh-hxlAB (B) and HxlAB activities (C) in strain C. glutamicum MDS2 and its derivatives. (A) C. glutamicum wildtype (WT) and MDS2 were cultivated in CGXII medium with 20 mM ribose for 48 h and the maximal ∆OD was determined. C. glutamicum ∆ald ∆fadH ∆rpe (pEKEx3-mdh-hxlAB) (= MDS2), and derivatives were cultivated in CGXII medium containing 20 mM ribose and 500 mM methanol for 48 h to determine the maximum ∆OD and investigate the influence of mutations found in strain MDS2T14, obtained by ALE. Blue bars indicate addition of 20 µM riboflavin. (B) Quantitative PCR (qPCR) was performed on isolated genomic and plasmid DNA from overnight cultures of MDS2, MDS2T14 and MDS2 ∆cg3104. Relative plasmid copy numbers (PCN) were calculated using serial dilutions from 100 to 0.1 ng/µL as standard curve for the chromosomal (gntK) and plasmid (oriV Cg ) targets. Error bars depict standard deviations of technical triplicates. (C) Coupled HxlAB enzyme assays were performed with crude extracts of MDS2, MDS2T14 and MDS2 ∆cg3104.

Figure 5 .

 5 Figure 5. The influence of ALE mutations on methanol-dependent growth. (A) The insertion at the rpe locus in MDS2T14 is depicted. (B) C. glutamicum MDS2 (circles), MDS2T8 (triangles), MDS2T14 (squares) were cultivated using the BioLector with 1 mL filling volume of CGXII minimal medium with 20 mM glucose as sole carbon source (open symbols) or supplemented with 20 µM riboflavin (filled symbols). Backscatter was detected in BioLector with a gain of 20. Error bars depict standard deviations from biological triplicates. (C) The effect of ∆metY and metK S288N on methanol-essential growth and S-adenosylmethionine (SAM) synthesis is shown. (D) C. glutamicum strain MDS2 ∆cg3104 ∆metY was cultivated using the BioLector with 1 mL filling volume of CGXII minimal medium with 20 mM ribose and 20 µM riboflavin (empty symbols) and additional 500 mM methanol (filled symbols). Backscatter was detected in BioLector with a gain of 20. Error bars depict standard deviations from biological triplicates.

Table 2 .

 2 Bacterial strains used in this study.

	Strain	Relevant Characteristics	Reference
	E. coli
	DH5α	∆lacU169 (ϕ80lacZ ∆M15), supE44, hsdR17, recA1, endA1, gyrA96, thi-1, relA1	[43]
	S17-1	recA, pro, hsdR, RP4-2Tc::Mu Km::Tn7 integrated into the chromosome	[46]
		C. glutamicum
	WT	ATCC13032	ATCC
	MDS0	ATCC13032 ∆ald ∆fadH	[41]
	MDS1	ATCC13032 ∆ald ∆fadH ∆rpi (pEKEx3-xylAB)	This study
	MDS2	ATCC13032 ∆ald ∆fadH ∆rpe (pEKEx3-mdh-hxlAB)	This study
	MDS2T8	strain evolved from MDS2; after 8 transfers	This study
	MDS2T14	strain evolved from MDS2; after 14 transfers	This study
	MDS2 ∆cg3104	MDS2 with deletion ∆cg3104	This study
	MDS2 ∆cg3104 ∆metY	MDS2 ∆cg3104 with deletion ∆metY	This study
	MDS2 metK_S288N	MDS2 with SNP in metK resulting in amino acid exchange S288N This study
	MDS2 ∆cg3104 metK_S288N	MDS2 with deletion ∆cg3104 and SNP in metK, resulting in amino acid exchange S288N	This study
	MDS2T14 metK_N288S	MDS2T14 with reversion of metK to the WT sequence	This study

Table 3 .

 3 Plasmids used in this study.

	Plasmid	Relevant Characteristics	Reference
	pEC-XT99A	Tet R , C. glutamicum/E. coli shuttle vector (P trc lacI q , pGA1, oriV E.c. )	[47]
	pEC-XT99A-mdh-hxlAB	pEC-XT99A expressing mdh from B. methanolicus and hxlA and hxlB from B. subtilis	This study
	pEC-XT99A-lysC fbr -ldcC	pEC-XT99A expressing feedback-resistant lysC from C. glutamicum and ldcC from E. coli	This study
	pEKEx3	Spec R , C. glutamicum/E. coli shuttle vector (P tac lacI q pBL1, oriV Ec )	[48]
	pEKEx3-mdh-hxlAB	pEKEx3, expressing mdh from B. methanolicus and hxlA and hxlB from B. subtilis	[20]
	pEKEx3-xylAB	pEKEx3, expressing xylA from X. campestris and xylB from C. glutamicum	[49]
	pK19mobsacB	Kan R , mobilizable E. coli vector mutagenesis (oriV, sacB)	[44]
	pK19mobsacB-∆rpi	pK19mobsacB with a deletion construct rpi	This study
	pK19mobsacB-∆rpe	pK19mobsacB with a deletion construct rpe	This study
	pK19mobsacB-∆cg3104	pK19mobsacB with a deletion construct cg3104	This study
	pK19mobsacB-metK_S288N	pK19mobsacB with an amino acid exchange metK_S288N from C. glutamicum	This study
	pK19mobsacB-metK_N288S	pK19mobsacB with WT metK for reversion of amino acid exchange S288N in MDS2T14	This study
	pVWEx1	Kan R , C. glutamicum/E. coli shuttle vector (P tac , lacI q )	[50]
	pVWEx1-mdh-hxlAB	pVWEx1 expressing mdh from B. methanolicus and hxlA and hxlB from B. subtilis	This study
	pVWEx1-rpi	pVWEx1 expressing rpi from C. glutamicum	This study

Int. J. Mol. Sci. 2020, 21, 3617; doi:10.3390/ijms21103617 www.mdpi.com/journal/ijms

Acknowledgments:

The authors thank Lennart Leßmeier for providing the plasmid pK19mobsacB-∆rpe. The authors thank MetaToul-MetaboHUB (Metabolomics & Fluxomics Facitilies, Toulouse, France, www.metatoul. fr, ANR-11-INBS-0010, www.metabohub.fr) and its staff members for technical support and access to NMR facilities.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/10/ 3617/s1. The oligonucleotides used as primers are shown in Table S1.

Conflicts of Interest:

The authors declare no conflict of interest.


TOTH, STEPHEN A. Mettray. A History of France's Most Venerated Carceral Institution. Cornell University Press, Ithaca (NY) [etc.] 2019. xii, 263 pp. Ill. $43.95. (E-book: $21.99). This monograph by Stephen A. Toth, Associate Professor of Modern European History at Arizona State University, is dedicated to the history of the Mettray penal and agricultural colony. Founded in 1840 by Frédéric-Auguste Demetz, this institution aimed to socially rehabilitate young delinquents by subjecting them to agricultural work in order to promote their moral regeneration. An ancestor to the bagnes d'enfants that the journalist Henri Danjou denounced in 1932, the colony of Mettray has become famous thanks in particular to one of its most famous residents, Jean Genet, who wrote about it in his book Miracle de la Rose, and to the chapter devoted to Mettray by Michel Foucault in his book Surveiller et punir. As Toth notes in the introduction to his book, according to Foucault Mettray constitutes the most successful disciplinary model and the culmination of the modern prison system following its birth at the end of the eighteenth century. While opening up an extremely stimulating field of research for historians, Foucault also bequeathed to them a conceptual "toolbox" that Toth questions and uses by proposing a history of power (paying particular attention to the resistance it provokes) and a history of juvenile bodies subjected to the injunctive canons of the masculinity of an era. To do this, Toth immerses his reader in a micro-history based on a rich bibliography and, above all, by an exhaustive examination of the archives of the penal colony of Mettray kept in the departmental archives of Indre-et -Loire.

During the first half of the nineteenth century, philanthropists, penal reformers, and magistrates became interested in the question of juvenile delinquency and its causes ("Origins", chapter 1). Coming from the lower classes, the young Gavroche who walked the Parisian sidewalks were considered victims of a failed education provided by families too poor to support themselves. To remedy this situation, the lawyer Frédéric-Auguste Demetz envisaged a system of care for juvenile delinquents intended both to keep them out of prison, where they were subject to the deleterious influence of adults, and to keep them away from the corruption of major urban centres (particularly Paris). Drawing inspiration from the models he discovered during study trips to the United States and Europe, Demetz developed a penal colony project whose young inmates were subjected to a system of moral reform centred on a "substitute family". Under the leadership of the chef de famille, they were subjected to rural work. This was supposed to promote their regeneration by bringing them closer to nature and allow them to acquire agricultural training to repopulate the countryside (p. 32).

Established on lands donated by Viscount Hermann de Brétignières de Courteilles (who co-directed the colony with Demetz until his death in 1852), the colony of Mettray was built in 1839 and received its first residents the following year. The minors detained there could include those found guilty but acquitted for having acted "without proper judgment" (sans discernement) in the commission of a crime (article 66 of the penal code); those sentenced to between six months and two years in prison; and those subject to "paternal correction", that is to say imprisoned at the request of their fathers (they were imprisoned in Mettray in a maison paternelle, the subject of chapter 5). The colony comprised a central chapel flanked by ten pavilions and a disciplinary quarter. This architectural layout translated into stone the importance of religion, which remained, along with work, the cornerstone of the moral re-education intended in Mettray.

Even if the buildings were not enclosed by a wall, the disciplinary regime applied to the colony of Mettray rendered this institution a real penitentiary, the subject of the second chapter of the book ("Regime"). The children were divided into "families", each headed by a family head (chef de famille), who applied a military-type discipline that the inmates were required to obey without fail. Chefs de famille were foremen (contremaîtres), who were trained in Mettray and aided in their duties by an "elder brother" (frère aîné) chosen from among those with a record of good behaviour.

An inmate's day began at five a.m. and finished at nine p.m. Its course was immutable and included eight hours of manual work, one and a half hours of instruction, and two hours and forty-five minutes devoted to meals and recreation. It was only during these times of recreation that the inmates were allowed to speak. Silence was mandatory during all other activities. During the weekends, the inmates were occupied mainly with gymnastic exercises, and during a Sunday "parade" they were required to participate in a military review. These various activities were supposed to instil in them values of virility and masculinity intended to transform them into "citizen-soldiers" (p. 59). Those who did not comply with this harsh discipline faced a range of punishments. Despite this threat, many inmates resisted the institution and its relentless settlement, the subject of the third chapter ("Resistance"). The author lists the various types of refusal by the inmates, ranging from homosexual practices, tobacco use, tattooing, or attempted escapes.

Over the years, the disciplinary system at Mettray increasingly became the subject of sharp criticism, in particular from the press. And, from the 1880s, even some of the deputies were shocked by the level of violence at the colony ("Discord", chapter 4). This criticism and three scandals (in 1887, in 1909, and during the interwar period) led to the institution being discredited. This had an important impact on French society and led to its being closed on 5 November 1937 ("Denouement", chapter 6). As Toth shows, what began as a resolutely utopian project that emerged from an optimistic representation of juvenile delinquents by reformers in the first half of the nineteenth century was marked by a slow drift towards a strictly authoritarian and punitive model.

Jean-Lucien Sanchez 1

Ministère de la Justice Direction de l'administration pénitentiaire 13 place Vendôme, 75001 Paris, France E-mail: jean-lucien.sanchez@justice.gouv.fr

The author thanks Chris Gordon for his help in proofreading this review.