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Chitin is an abundant waste product from shrimp and mushroom industries and as such, an appropriate secondary feedstock for biotechnological processes. However, chitin is a crystalline substrate embedded in complex biological matrices, and, therefore, difficult to utilize, requiring an equally complex chitinolytic machinery. Following a bottom-up approach, we here describe the step-wise development of a mutualistic, non-competitive consortium in which a lysine-auxotrophic Escherichia coli substrate converter cleaves the chitin monomer N-acetylglucosamine (GlcNAc) into glucosamine (GlcN) and acetate, but uses only acetate while leaving GlcN for growth of the lysine-secreting Corynebacterium glutamicum producer strain. We first engineered the substrate converter strain for growth on acetate but not GlcN, and the producer strain for growth on GlcN but not acetate. Growth of the two strains in co-culture in the presence of a mixture of GlcN and acetate was stabilized through lysine cross-feeding. Addition of recombinant chitinase to cleave chitin into GlcNAc2, chitin deacetylase to convert GlcNAc2 into GlcN2 and acetate, and glucosaminidase to cleave GlcN2 into GlcN supported growth of the two strains in co-culture in the presence of colloidal chitin as sole carbon source. Substrate converter strains secreting a chitinase or a β-1,4-glucosaminidase degraded chitin to GlcNAc2 or GlcN2 to GlcN, respectively, but required glucose for growth. In contrast, by cleaving GlcNAc into GlcN and acetate, a chitin deacetylase-expressing substrate converter enabled growth of the producer strain in co-culture with GlcNAc as sole carbon source, providing proof-of-principle for a fully integrated co-culture for the biotechnological utilization of chitin. Graphical abstract Key Points• A bacterial consortium was developed to use chitin as feedstock for the bioeconomy.• Substrate converter and producer strain use different chitin hydrolysis products.• Substrate converter and producer strain are mutually dependent on each other.

The aromatic amino acid tryptophan serves as a precursor for many valuable compounds such as neuromodulators, indoleamines and indole alkaloids. In this work, tryptophan biosynthesis was extended by halogenation followed by decarboxylation to the respective tryptamines or cleavage to the respective indoles. Either the tryptophanase genes tnaAs from E. coli and Proteus vulgaris or the aromatic amino acid decarboxylase genes AADCs from Bacillus atrophaeus, Clostridium sporogenes, and Ruminococcus gnavus were expressed in C. glutamicum strains producing (halogenated) tryptophan. Regarding indoles, final titers of 16 mg L-1 7-Cl-indole and 23 mg L-1 7-Br-indole were attained. Tryptamine production led to a much higher titer of 2.26 g L-1 upon expression of AADC from B. atrophaeus. AADCs were shown to be active with halogenated tryptophan in vitro and in vivo and supported production of 0.36 g L-1 7-Br-tryptamine with a volumetric productivity of 8.3 mg L-1 h-1 in a fed-batch fermentation.

The aim of this work was to study the fermentative production of the N-methylated amino acid sarcosine by C. glutamicum. Characterization of the imine reductase DpkA from Pseudomonas putida revealed that it catalyses N-methylamination of glyoxylate to sarcosine. Heterologous expression of dpkA in a C. glutamicum strain engineered for glyoxylate overproduction enabled fermentative production of sarcosine from sugars and monomethylamine. Glucose-based fermentation reached sarcosine production titers of 2.4 ± 0.1 g L−1. Sarcosine production based on the second generation feedstocks xylose and arabinose led to higher product titers of 2.7 ± 0.1 g L−1 and 3.4 ± 0.3 g L−1, respectively, than glucose-based production. Optimization of production conditions with xylose and potassium acetate blends increased sarcosine titers to 8.7 ± 0.2 g L−1 with a yield of 0.25 g g−1. This is the first example in which a C. glutamicum process using lignocellulosic pentoses is superior to glucose-based production.

Coenzyme Q10 (CoQ10) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. For the microbial production, so far only bacteria have been used that naturally synthesize CoQ10 or a related CoQ species. Since the whole pathway involves many enzymatic steps and has not been fully elucidated yet, the set of genes required for transfer of CoQ10 synthesis to a bacterium not naturally synthesizing CoQ species remained unknown. Here, we established CoQ10 biosynthesis in the non-ubiquinone-containing Gram-positive Corynebacterium glutamicum by metabolic engineering. CoQ10 biosynthesis involves prenylation and, thus, requires farnesyl diphosphate as precursor. A carotenoid-deficient strain was engineered to synthesize an increased supply of the precursor molecule farnesyl diphosphate. Increased farnesyl diphosphate supply was demonstrated indirectly by increased conversion to amorpha-4,11-diene. To provide the first CoQ10 precursor decaprenyl diphosphate (DPP) from farnesyl diphosphate, DPP synthase gene ddsA from Paracoccus denitrificans was expressed. Improved supply of the second CoQ10 precursor, para-hydroxybenzoate (pHBA), resulted from metabolic engineering of the shikimate pathway. Prenylation of pHBA with DPP and subsequent decarboxylation, hydroxylation, and methylation reactions to yield CoQ10 was achieved by expression of ubi genes from Escherichia coli. CoQ10 biosynthesis was demonstrated in shake-flask cultivation and verified by liquid chromatography mass spectrometry analysis. To the best of our knowledge, this is the first report of CoQ10 production in a non-ubiquinone-containing bacterium.

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (~10 %) compared to glucose (~60 %). Consequently, the biosynthesis of NADPH demanding products, e.g. L-lysine, by C. glutamicum is largely decreased, when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and consequently NADPH availability. Here, we generated deletion strains either lacking in the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose specific PTS, as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased biomass formation, reduced side-product accumulation, and increased L-lysine production by 50 %.

N-alkylated amino acids are intermediates of natural biological pathways and can be found incorporated in peptides or have physiological roles in their free form. The N-ethylated amino acid l-theanine shows taste-enhancing properties and health benefits. It naturally occurs in green tea as major free amino acid. Isolation of l-theanine from Camilla sinensis shows low efficiency, and chemical synthesis results in a racemic mixture. Therefore, biochemical approaches for the production of l-theanine gain increasing interest. Here, we describe metabolic engineering of Pseudomonas putida KT2440 for the fermentative production of l-theanine from monoethylamine and carbon sources glucose, glycerol, or xylose using heterologous enzymes from Methylorubrum extorquens for l-theanine production and heterologous enzymes from Caulobacter crescentus for growth with xylose. l-Theanine (15.4 mM) accumulated in shake flasks with minimal medium containing monoethylamine and glucose, 15.2 mM with glycerol and 7 mM with xylose. Fed-batch bioreactor cultures yielded l-theanine titers of 10 g L-1 with glucose plus xylose, 17.2 g L-1 with glycerol, 4 g L-1 with xylose, and 21 g L-1 with xylose plus glycerol, respectively. To the best of our knowledge, this is the first l-theanine process using P. putida and the first compatible with the use of various alternative carbon sources.

The soil microbe Corynebacterium glutamicum is a leading workhorse in industrial biotechnology and has become famous for its power to synthetise amino acids and a range of bulk chemicals at high titre and yield. The product portfolio of the microbe is continuously expanding. Moreover, metabolically engineered strains of C. glutamicum produce more than 30 high value active ingredients, including signature molecules of raspberry, savoury, and orange flavours, sun blockers, anti-ageing sugars, and polymers for regenerative medicine. Herein, we highlight recent advances in engineering of the microbe into novel cell factories that overproduce these precious molecules from pioneering proofs-of-concept up to industrial productivity.

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (∼10%) compared to glucose (∼60%). Consequently, the biosynthesis of NADPH demanding products, e.g., L-lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose-specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and, consequently, NADPH availability. Here, we generated deletion strains lacking either the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose-specific PTS as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose-specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, and P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased ODs, reduced side-product accumulation, and increased L-lysine production by 50%.

l-2-hydroxyglutarate (l-2HG) is a trifunctional building block and highly attractive for the chemical and pharmaceutical industries. The natural l-lysine biosynthesis pathway of the amino acid producer Corynebacterium glutamicum was extended for the fermentative production of l-2HG. Since l-2HG is not native to the metabolism of C. glutamicum metabolic engineering of a genome-streamlined l-lysine overproducing strain was required to enable the conversion of l-lysine to l-2HG in a six-step synthetic pathway. To this end, l-lysine decarboxylase was cascaded with two transamination reactions, two NAD(P)-dependent oxidation reactions and the terminal 2-oxoglutarate-dependent glutarate hydroxylase. Of three sources for glutarate hydroxylase the metalloenzyme CsiD from Pseudomonas putida supported l-2HG production to the highest titers. Genetic experiments suggested a role of succinate exporter SucE for export of l-2HG and improving expression of its gene by chromosomal exchange of its native promoter improved l-2HG production. The availability of Fe2+ as cofactor of CsiD was identified as a major bottleneck in the conversion of glutarate to l-2HG. As consequence of strain engineering and media adaptation product titers of 34 ± 0 mM were obtained in a microcultivation system. The glucose-based process was stable in 2 L bioreactor cultivations and a l-2HG titer of 3.5 g L−1 was obtained at the higher of two tested aeration levels. Production of l-2HG from a sidestream of the starch industry as renewable substrate was demonstrated. To the best of our knowledge, this study is the first description of fermentative production of l-2HG, a monomeric precursor used in electrochromic polyamides, to cross-link polyamides or to increase their biodegradability.

Methanol is inexpensive, is easy to transport, and can be produced both from renewable and from fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as Bacillus methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories or to design methylotrophic capability in other strains already used by the industry., Bacillus methanolicus MGA3 is a thermotolerant and relatively fast-growing methylotroph able to secrete large quantities of glutamate and lysine. These natural characteristics make B. methanolicus a good candidate to become a new industrial chassis organism, especially in a methanol-based economy. Intriguingly, the only substrates known to support B. methanolicus growth as sole sources of carbon and energy are methanol, mannitol, and, to a lesser extent, glucose and arabitol. Because fluxomics provides the most direct readout of the cellular phenotype, we hypothesized that comparing methylotrophic and nonmethylotrophic metabolic states at the flux level would yield new insights into MGA3 metabolism. In this study, we designed and performed a 13C metabolic flux analysis (13C-MFA) of the facultative methylotroph B. methanolicus MGA3 growing on methanol, mannitol, and arabitol to compare the associated metabolic states. On methanol, results showed a greater flux in the ribulose monophosphate (RuMP) pathway than in the tricarboxylic acid (TCA) cycle, thus validating previous findings on the methylotrophy of B. methanolicus. New insights related to the utilization of cyclic RuMP versus linear dissimilation pathways and between the RuMP variants were generated. Importantly, we demonstrated that the linear detoxification pathways and the malic enzyme shared with the pentose phosphate pathway have an important role in cofactor regeneration. Finally, we identified, for the first time, the metabolic pathway used to assimilate arabitol. Overall, those data provide a better understanding of this strain under various environmental conditions. IMPORTANCE Methanol is inexpensive, is easy to transport, and can be produced both from renewable and from fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as Bacillus methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories or to design methylotrophic capability in other strains already used by the industry.

In bacterial system, direct conversion of xylose to xylonic acid is mediated through NAD-dependent xylose dehydrogenase (xylB) and xylonolactonase (xylC) genes. Heterologous expression of these genes from Caulobacter crescentus into recombinant Corynebacterium glutamicum ATCC 13032 and C. glutamicum ATCC 31831 (with an innate pentose transporter, araE) resulted in an efficient bioconversion process to produce xylonic acid from xylose. Process parameters including the design of production medium was optimized using a statistical tool, Response Surface Methodology (RSM). Maximum xylonic acid of 56.32 g/L from 60 g/L xylose, i.e. about 76.67% of the maximum theoretical yield was obtained after 120 h fermentation from pure xylose with recombinant C. glutamicum ATCC 31831 containing the plasmid pVWEx1 xylB. Under the same condition, the production with recombinant C. glutamicum ATCC 13032 (with pVWEx1 xylB) was 50.66 g/L, i.e. 69% of the theoretical yield. There was no significant improvement in production with the simultaneous expression of xylB and xylC genes together indicating xylose dehydrogenase activity as one of the rate limiting factor in the bioconversion. Finally, proof of concept experiment in utilizing biomass derived pentose sugar, xylose, for xylonic acid production was also carried out and obtained 42.94 g/L xylonic acid from 60 g/L xylose. These results promise a significant value addition for the future bio refinery programs.

N‐ functionalized amines play important roles in nature and occur, for example, in the antibiotic vancomycin, the immunosuppressant cyclosporine, the cytostatic actinomycin, the siderophore aerobactin, the cyanogenic glucoside linamarin, and the polyamine spermidine. In the pharmaceutical and fine‐chemical industries N‐ functionalized amines are used as building blocks for the preparation of bioactive molecules. Processes based on fermentation and on enzyme catalysis have been developed to provide sustainable manufacturing routes to N‐ alkylated, N‐ hydroxylated, N‐ acylated, or other N‐ functionalized amines including polyamines. Metabolic engineering for provision of precursor metabolites is combined with heterologous N‐ functionalizing enzymes such as imine or ketimine reductases, opine or amino acid dehydrogenases, N‐ hydroxylases, N‐ acyltransferase, or polyamine synthetases. Recent progress and applications of fermentative processes using metabolically engineered bacteria and yeasts along with the employed enzymes are reviewed and the perspectives on developing new fermentative processes based on insight from enzyme catalysis are discussed.

l-Carnitine is essential in the intermediary metabolism of eukaryotes and is involved in the β-oxidation of medium- and long-chain fatty acids; thus, it has applications for medicinal purposes and ...

Methanol is an attractive alternative non-food feedstock for industrial fermentations that can be used instead of sugar-based raw materials. Here, the thermophilic and methylotrophic bacterium Bacillus methanolicus MGA3 was metabolically engineered to produce the platform chemical (R)-acetoin from methanol at 50 °C. Three different heterologous alsSD/budAB operons, each encoding acetolactate synthase and acetolactate decarboxylase, were functionally expressed under control of an inducible promoter in B. methanolicus MGA3, resulting in up to 0.26 ± 0.04 g L−1 of (R)-acetoin titer in shake flask cultivations. To further improve acetoin production, a total of six different genes or operons were expressed in the acetoin producing strains to increase supply of the acetoin precursor pyruvate. In particular, expression of a gene coding for malic enzyme from Geobacillus stearothermophilus in combination with the isocitrate lyase gene from B. methanolicus MGA3 increased acetoin titers 1.6-fold up to 0.42 ± 0.01 g L−1 which corresponds to 0.07 g g−1 methanol. This resulted in an MGA3 strain overproducing 4 recombinant enzymes in total from two different plasmids with two distinct antibiotics resistance markers, demonstrating the increased complexity of metabolic engineering allowed by newly developed genetic tools for this organism. To our knowledge, this is the first demonstration of microbial production of acetoin from methanol.

In the biorefinery concept renewable feedstocks are converted to a multitude of value-added compounds irrespective of seasonal or other variations of the complex biomass substrates. Conceptionally, this can be realized by specialized single microbial strains or by co-culturing various strain combinations. In the latter approach strains for substrate conversion and for product formation can be combined. This study addressed the construction of binary microbial consortia based on starch- and sucrose-based production of l-lysine and derived value-added compounds. A commensalism-based synthetic consortium for l-lysine production from sucrose was developed combining an l-lysine auxotrophic, naturally sucrose-negative E. coli strain with a C. glutamicum strain able to produce l-lysine that secretes fructose when grown with sucrose due to deletion of the fructose importer gene ptsF. Mutualistic synthetic consortia with an l-lysine auxotrophic, α-amylase secreting E. coli strain and naturally amylase-negative C. glutamicum strains was implemented for production of valuable fine chemicals from starch.

Background Transaminases have become a key tool in biocatalysis to introduce the amine functionality into a range of molecules like prochiral α-ketoacids and ketones. However, due to the necessity of shifting the equilibrium towards the product side (depending on the amine donor) an efficient amination system may require three enzymes. So far, this well-established transformation has mainly been performed in vitro by assembling all biocatalysts individually, which comes along with elaborate and costly preparation steps. We present the design and characterization of a flexible approach enabling a quick set-up of single-cell biocatalysts producing the desired enzymes. By choosing an appropriate co-expression strategy, a modular system was obtained, allowing for flexible plug-and-play combination of enzymes chosen from the toolbox of available transaminases and/or recycling enzymes tailored for the desired application. Results By using a two-plasmid strategy for the recycling enzyme and the transaminase together with chromosomal integration of an amino acid dehydrogenase, two enzyme modules could individually be selected and combined with specifically tailored E. coli strains. Various plug-and-play combinations of the enzymes led to the construction of a series of single-cell catalysts suitable for the amination of various types of substrates. On the one hand the fermentative amination of α-ketoacids coupled both with metabolic and non-metabolic cofactor regeneration was studied, giving access to the corresponding α-amino acids in up to 96% conversion. On the other hand, biocatalysts were employed in a non-metabolic, “in vitro-type” asymmetric reductive amination of the prochiral ketone 4-phenyl-2-butanone, yielding the amine in good conversion (77%) and excellent stereoselectivity (ee = 98%). Conclusions The described modularized concept enables the construction of tailored single-cell catalysts which provide all required enzymes for asymmetric reductive amination in a flexible fashion, representing a more efficient approach for the production of chiral amines and amino acids. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0750-5) contains supplementary material, which is available to authorized users.

Bacteria are widely used as cell factories for production of enzymes and chemicals, mostly from sugars. Methylotrophic bacteria can utilize the one-carbon compound methanol as sole carbon source for growth, and metabolic engineering is being used to develop bioprocesses based on these organisms for conversion of methanol into value-added chemicals. Methylotrophic model strains include both Gram-positive and Gram-negative bacteria and in all cases methanol metabolism proceeds via the cell-toxic intermediate formaldehyde. Thus, understanding the genetics, biochemistry, and regulation of methylotrophic pathways is crucial for successful strain development and for their concomitant fermentations. Also, such basic knowledge has proven useful for design of strategies and approaches for the rational transfer of methylotrophy into non-methylotrophic bacteria. In the current review we highlight the best studied methylotrophic model organisms, with particular focus on the Gram-positive Bacillus methanolicus and the Gram-negative Methylobacterium extorquens, including their genetics and physiology. We present successful metabolic engineering examples leading to the construction of recombinant strains for the production of amino acids, platform chemicals, terpenoids and dicarboxylic acids derivatives from methanol as sole carbon source. © 2017 Society of Chemical Industry and John Wiley & Sons, Ltd

Gamma-aminobutyric acid (GABA) is a non-protein amino acid widespread in Nature. Among the various uses of GABA, its lactam form 2-pyrrolidone can be chemically converted to the biodegradable plastic polyamide-4. In metabolism, GABA can be synthesized either by decarboxylation of l-glutamate or by a pathway that starts with the transamination of putrescine. Fermentative production of GABA from glucose by recombinant Corynebacterium glutamicum has been described via both routes. Putrescine-based GABA production was characterized by accumulation of by-products such as N-acetyl-putrescine. Their formation was abolished by deletion of the spermi(di)ne N-acetyl-transferase gene snaA. To improve provision of l-glutamate as precursor 2-oxoglutarate dehydrogenase activity was reduced by changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and by maintaining the inhibitory protein OdhI in its inhibitory form by changing amino acid residue 15 from threonine to alanine. Putrescine-based GABA production by the strains described here led to GABA titers up to 63.2 g L-1 in fed-batch cultivation at maximum volumetric productivities up to 1.34 g L-1 h-1 , the highest volumetric productivity for fermentative GABA production reported to date. Moreover, GABA production from the carbon sources xylose, glucosamine, and N-acetyl-glucosamine that do not have competing uses in the food or feed industries was established. Biotechnol. Bioeng. 2017;114: 862-873. © 2016 Wiley Periodicals, Inc.

The construction of microbial platform organisms by means of genome reduction is an ongoing topic in biotechnology. In this study, we investigated whether the deletion of single or multiple gene clusters has a positive effect on the secretion of cutinase from Fusarium solani pisi in the industrial workhorse Corynebacterium glutamicum. A total of 22 genome‐reduced strain variants were compared applying two Sec signal peptides from Bacillus subtilis. High‐throughput phenotyping using robotics‐integrated microbioreactor technology with automated harvesting revealed distinct cutinase secretion performance for a specific combination of signal peptide and genomic deletions. Strikingly, bioreactor fed‐batch cultivations at controlled growth rates resulted in a complete reversal of the batch screening results, with the cutinase yield for strain GRS41_51_NprE dropping by ~ 25% compared to the reference strain. Thus, the choice of bioprocess conditions may turn a “high‐performance” strain from batch screening into a “low‐performance” strain in fed‐batch cultivation., Summary The construction of microbial platform organisms by means of genome reduction is an ongoing topic in biotechnology. In this study, we investigated whether the deletion of single or multiple gene clusters has a positive effect on the secretion of cutinase from Fusarium solani pisi in the industrial workhorse Corynebacterium glutamicum. A total of 22 genome‐reduced strain variants were compared applying two Sec signal peptides from Bacillus subtilis. High‐throughput phenotyping using robotics‐integrated microbioreactor technology with automated harvesting revealed distinct cutinase secretion performance for a specific combination of signal peptide and genomic deletions. The biomass‐specific cutinase yield for strain GRS41_51_NprE was increased by ~ 200%, although the growth rate was reduced by ~ 60%. Importantly, the causative deletions of genomic clusters cg2801‐cg2828 and rrnC‐cg3298 could not have been inferred a priori. Strikingly, bioreactor fed‐batch cultivations at controlled growth rates resulted in a complete reversal of the screening results, with the cutinase yield for strain GRS41_51_NprE dropping by ~ 25% compared to the reference strain. Thus, the choice of bioprocess conditions may turn a ‘high‐performance’ strain from batch screening into a ‘low‐performance’ strain in fed‐batch cultivation. In conclusion, future studies are needed in order to understand metabolic adaptations of C. glutamicum to both genomic deletions and different bioprocess conditions.

Bacillus methanolicus is a thermophilic, Gram-positive, rod-shaped bacterium. It is a facultative methylotroph which can use carbon and energy sources including mannitol and the one-carbon (C1) and non-food substrate methanol for growth and overproduction of amino acids, which makes it a promising candidate for biotechnological applications. Despite a growing tool box for gene cloning and expression, tools for targeted chromosomal gene knockouts and gene repression are still missing for this organism. Here, the CRISPRi-dCas9 technique for gene repression was established in B. methanolicus MGA3. Significantly reduced spore formation on the one hand and increased biofilm formation on the other hand could be demonstrated when the stage zero sporulation protein A gene spo0A was targeted. Furthermore, when the mannitol-1-phosphate 5-dehydrogenase gene mtlD was targeted by CRISPRi, mtlD RNA levels, and MtlD specific activities in crude extracts were decreased to about 50 % which resulted in reduced biomass formation from mannitol. As a third target, the catalase gene katA was chosen. Upon targeting katA by CRISPRi, catalase activity was decreased to about 25 % as shown in H2O2 drop assays and by determination of specific catalase activity in crude extracts. Our results support the predicted functions of Spo0A in sporulation and biofilm formation, of MtlD for mannitol catabolism, and of catalase in hydrogen peroxide dismutation. Thus, CRISPR interference as developed here serves as basis for the functional characterization of B. methanolicus physiology as well as for its application in biotechnology.

The aim of the present study is the development of a consolidated bioprocess for the production of lysine with recombinant Corynebacterium glutamicum DM1729 strains expressing endoglucanase and β-glucosidase genes. Here, the endoglucanase genes from Xanthomonas campestris XCC3521 and XCC2387 and betaglucosidase gene from Saccharophagus degradans Sde1394 were cloned in C. glutamicum DM1729 and expressed either extracellularly or on cell surface. The highest β-glucosidase activity of 9±0.5U/OD600 of 1 and endoglucanase activity of 5.5±0.8U was obtained in C. glutamicum DM 1729 (pVWEx1-TATXCC2387) (pEKEx3-PorC-Sde1394) when cellobiose (20g/L) alone or in combination with carboxymethyl cellulose (20g/L) was used as the carbon sources respectively. The overall efforts resulted in a lysine titre of 5.9±0.5mM. The ability of the constructs to utilize carboxymethyl cellulose and cellobiose for growth and amino acid production proves the concept of utilization of C. glutamicum as a biocatalyst in the lignocellulosic biorefinery.

The Gram-positive Corynebacterium glutamicum is widely used for fermentative production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose uptake and phosphorylation by C. glutamicum mainly occur by the phosphotransferase system (PTS) and to lesser extent by inositol permeases and glucokinases. Heterologous expression of the genes for the high-affinity glucose permease from Streptomyces coelicolor and Bacillus subtilis glucokinase fully compensated for the absence of the PTS in Δhpr strains. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and glucokinase from B. subtilis were overproduced with balanced translation initiation rates using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid or with pVWEx1-IolTBest, 18 to 20 % higher volumetric productivities and 70 to 72 % higher specific productivities as compared to the parental strain resulted. The non-proteinogenic amino acid L-pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics, or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the constructed L-lysine-producing strain, the L-lysine 6-dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were overexpressed as synthetic operon. This enabled C. glutamicum to produce L-PA with a yield of 0.09 ± 0.01 g g(-1) and a volumetric productivity of 0.04 ± 0.01 g L(-1) h(-1).To the best of our knowledge, this is the first fermentative process for the production of L-PA from glucose.

Corynebacterium glutamicum is an important organism for the industrial production of amino acids. Metabolic pathways in this organism are usually engineered by conventional methods such as homologous recombination, which depends on rare double-crossover events. To facilitate the mapping of gene expression levels to metabolic outputs, we applied CRISPR interference (CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in C. glutamicum. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of pgi and pck up to 98%, and of pyk up to 97%, resulting in titer enhancement ratios of l-lysine and l-glutamate production comparable to levels achieved by gene deletion. This approach for C. glutamicum metabolic engineering, which only requires 3 days, indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection.

The glutamate-derived bioproducts ornithine, citrulline, proline, putrescine, and arginine have applications in the food and feed, cosmetic, pharmaceutical, and chemical industries. Corynebacterium glutamicum is not only an excellent producer of glutamate but also of glutamate-derived products. Here, engineering targets beneficial for ornithine production were identified and the advantage of rationally constructing a platform strain for the production of the amino acids citrulline, proline, and arginine, and the diamine putrescine was demonstrated. Feedback alleviation of N-acetylglutamate kinase, tuning of the promoter of glutamate dehydrogenase gene gdh, lowering expression of phosphoglucoisomerase gene pgi, along with the introduction of a second copy of the arginine biosynthesis operon argCJB(A49V,M54V)D into the chromosome resulted in a C. glutamicum strain producing ornithine with a yield of 0.52 g ornithine per g glucose, an increase of 71% as compared to the parental ΔargFRG strain. Strains capable of producing 0.41 g citrulline per g glucose, 0.29 g proline per g glucose, 0.30 g arginine per g glucose, and 0.17 g putrescine per g glucose were derived from the ornithine-producing platform strain by plasmid-based overexpression of appropriate pathway modules with one to three genes.

Halogenated compounds, like 7-chloro- l -tryptophan, are important intermediates or components of bioactive substances relevant for the pharmaceutical, chemical and agrochemical industries. About 20% of all pharmaceutical small molecule drugs and around 30% of all active compounds in agrochemistry are halogenated. Chemical halogenation procedures usually are characterized by the use of hazardous or even highly toxic chemicals. Recently, a biocatalytic process for l -tryptophan halogenation at the gram-scale using FAD-dependent halogenase and NADH-dependent flavin reductase enzymes has been described. Many proteinogenic amino acids are produced by fermentation using Corynebacterium glutamicum. The fermentative production of l -glutamate and l -lysine, for example, is operated at the million-ton scale. However, fermentative production of halogenated amino acids has not yet been described. In this study, fermentative production of the halogenated amino acid 7-chloro- l -tryptophan from sugars, ammonium and chloride salts was achieved. This required metabolic engineering of an l -tryptophan producing C. glutamicum strain for expression of the genes coding for FAD-dependent halogenase RebH and NADH-dependent flavin reductase RebF from Lechevalieria aerocolonigenes. Chlorination of l -tryptophan to 7-chloro- l -tryptophan by recombinant C. glutamicum was improved by optimizing the RBS of rebH. Metabolic engineering enabled production of 7-chloro- l -tryptophan and l -tryptophan from the alternative carbon sources arabinose, glucosamine and xylose.

Corynebacterium glutamicum is a workhorse of industrial amino acid production employed for more than five decades for the million-ton-scale production of L-glutamate and L-lysine. This bacterium is pigmented due to the biosynthesis of the carotenoid decaprenoxanthin. Decaprenoxanthin is a carotenoid with 50 carbon atoms, and, thus, C. glutamicum belongs to the rare group of bacteria that produce long-chain C50 carotenoids. C50 carotenoids have been mainly isolated from extremely halophilic archaea (Kelly and Jensen, Acta Chem Scand 21:2578, 1967; Pfander, Pure Appl Chem 66:2369-2374, 1994) and from Gram-positive bacteria of the order Actinomycetales (Netzer et al., J Bacteriol 192:5688-5699, 2010). The characteristic yellow phenotype of C. glutamicum is due to the cyclic C50 carotenoid decaprenoxanthin and its glycosides. Decaprenoxanthin production has been improved by plasmid-borne overexpression of endogenous genes of carotenogenesis. Gene deletion resulted in the production of the C40 carotenoid lycopene, an intermediate of decaprenoxanthin biosynthesis. Heterologous gene expression was required to develop strains overproducing nonnative carotenoids and terpenes, such as astaxanthin (Henke et al., Mar Drugs 14:E124, 2016) and (+)-valencene (Frohwitter et al., J Biotechnol 191:205-213, 2014). Integration of additional copies of endogenous genes expressed from strong promoters improved isoprenoid biosynthesis. Here, we describe C. glutamicum strains, plasmids, and methods for overexpression of endogenous and heterologous genes, gene deletion, replacement, and genomic integration. Moreover, strain cultivation as well as extraction, identification, and quantitative determination of terpenes and carotenoids produced by C. glutamicum is detailed.

Corynebacterium glutamicum is used for production of the food and feed amino acids l-glutamate and l-lysine at the million-ton-scale. One feed formulation of l-lysine simply involves spray-drying of the fermentation broth, thus, including secreted l-lysine and C. glutamicum cells which are pigmented by the C50 carotenoid decaprenoxanthin. C. glutamicum has been engineered for overproduction of various compounds including carotenoids. In this study, C. glutamicum was engineered for coproduction of a secreted amino acid with a cell-bound carotenoid. Asa proof of principle, coproduction of l-glutamate with the industrially relevant astaxanthin was shown. This strategy was applied to engineer l-lysine overproducing strains for combined overproduction of secreted l-lysine with the cell-bound carotenoids decaprenoxanthin, lycopene, β-carotene, zeaxanthin, canthaxanthin and astaxanthin. By fed-batch fermentation 48g/Ll-lysine and 10mg/L astaxanthin were coproduced. Moreover, C. glutamicum was engineered for coproduction of l-lysine and β-carotene from xylose and arabinose as alternative feedstocks.

The Gram-positive Corynebacterium glutamicum co-metabolizes most carbon sources such as the phosphotransferase system (PTS) sugar glucose and the non-PTS sugar maltose. Maltose is taken up via the ABC-transporter MusEFGK2I, and is further metabolized to glucose phosphate by amylomaltase MalQ, maltodextrin phosphorylase MalP, glucokinase Glk and phosophoglucomutase Pgm. Surprisingly, growth of C. glutamicum strains lacking the general PTS components EI or HPr was strongly impaired on the non-PTS sugar maltose. Complementation experiments showed that a functional PTS phosphorelay is required for optimal growth of C. glutamicum on maltose, implying its involvement in the control of maltose metabolism and/or uptake. To identify the target of this PTS-dependent control, transport measurements with 14C-labelled maltose, Northern blot analyses and enzyme assays were performed. The activities of the maltose transporter and enzymes MalQ, Pgm and GlK were not decreased in PTS-deficient C. glutamicum strains, which was corroborated by comparable transcript amounts of musE, musK and musG, as well as of malQ, in C. glutamicum ΔptsH and WT. By contrast, MalP activity was significantly reduced and only residual amounts of malP transcripts were detected in C. glutamicum ΔptsH when compared to WT. Promoter activity assays with the malP promoter in C. glutamicum ΔptsH and WT confirmed that malP transcription is reduced in the PTS-deficient strain. Taken together, we show here for what is to the best of our knowledge the first time a regulatory function of the PTS in C. glutamicum and identify malP transcription as its target.

Methylotrophic bacteria utilize methanol and other reduced one-carbon compounds as their sole source of carbon and energy. For this purpose, these bacteria evolved a number of specialized enzymes and pathways. Here, we used a synthetic biology approach to select and introduce a set of "methylotrophy genes" into Escherichia coli based on in silico considerations and flux balance analysis to enable methanol dissimilation and assimilation. We determined that the most promising approach allowing the utilization of methanol was the implementation of NAD-dependent methanol dehydrogenase and the establishment of the ribulose monophosphate cycle by expressing the genes for hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi). To test for the best-performing enzymes in the heterologous host, a number of enzyme candidates from different donor organisms were selected and systematically analyzed for their in vitro and in vivo activities in E. coli. Among these, Mdh2, Hps and Phi originating from Bacillus methanolicus were found to be the most effective. Labeling experiments using (13)C methanol with E. coli producing these enzymes showed up to 40% incorporation of methanol into central metabolites. The presence of the endogenous glutathione-dependent formaldehyde oxidation pathway of E. coli did not adversely affect the methanol conversion rate. Taken together, the results of this study represent a major advancement towards establishing synthetic methylotrophs by gene transfer.

Here, a new metabolic pathway for the production of 5-aminovalerate (5AVA) from l-lysine via cadaverine as intermediate was established and this three-step-pathway comprises l-lysine decarboxylase (LdcC), putrescine transaminase (PatA) and γ-aminobutyraldehyde dehydrogenase (PatD). Since Corynebacterium glutamicum is used for industrial l-lysine production, the pathway was established in this bacterium. Upon expression of ldcC, patA and patD from Escherichia coli in C. glutamicum wild type, production 5AVA was achieved. Enzyme assays revealed that PatA and PatD also converted cadaverine to 5AVA. Eliminating the by-products cadaverine, N-acetylcadaverine and glutarate in a genome-streamlined l-lysine producing strain expressing ldcC, patA and patD improved 5AVA production to a titer of 5.1gL-1, a yield of 0.13gg-1 and a volumetric productivity of 0.12gL-1h-1. Moreover, 5AVA production from the alternative feedstocks starch, glucosamine, xylose and arabinose was established.

The biotechnological production of amino acids occurs at the million-ton scale and annually about 6milliontons of l-glutamate and l-lysine are produced by Escherichia coli and Corynebacterium glutamicum strains. l-glutamate and l-lysine production from starch hydrolysates and molasses is very efficient and access to alternative carbon sources and new products has been enabled by metabolic engineering. This review focusses on genetic and metabolic engineering of amino acid producing strains. In particular, rational approaches involving modulation of transcriptional regulators, regulons, and attenuators will be discussed. To address current limitations of metabolic engineering, this article gives insights on recent systems metabolic engineering approaches based on functional tools and method such as genome reduction, amino acid sensors based on transcriptional regulators and riboswitches, CRISPR interference, small regulatory RNAs, DNA scaffolding, and optogenetic control, and discusses future prospects.

It is long known that Kasugamycin inhibits translation of canonical transcripts containing a 5’-UTR with a Shine Dalgarno (SD) motif, but not that of leaderless transcripts. To gain a global overview of the influence of Kasugamycin on translation efficiencies, the changes of the translatome of Escherichia coli induced by a 10 minutes Kasugamycin treatment were quantified. The effect of Kasugamycin differed widely, 102 transcripts were at least twofold more sensitive to Kasugamycin than average, and 137 transcripts were at least twofold more resistant, and there was a more than 100-fold difference between the most resistant and the most sensitive transcript. The 5’-ends of 19 transcripts were determined from treated and untreated cultures, but Kasugamycin resistance did neither correlate with the presence or absence of a SD motif, nor with differences in 5’-UTR lengths or GC content. RNA Structure Logos were generated for the 102 Kasugamycin-sensitive and for the 137 resistant transcripts. For both groups a short Shine Dalgarno (SD) motif was retrieved, but no specific motifs associated with resistance or sensitivity could be found. Notably, this was also true for the region -3 to -1 upstream of the start codon and the presence of an extended SD motif, which had been proposed to result in Kasugamycin resistance. Comparison of the translatome results with the database RegulonDB showed that the transcript with the highest resistance was leaderless, but no further leaderless transcripts were among the resistant transcripts. Unexpectedly, it was found that translational coupling might be a novel feature that is associated with Kasugamycin resistance. Taken together, Kasugamycin has a profound effect on translational efficiencies of E. coli transcripts, but the mechanism of action is different than previously described.

The use of methanol as a carbon source for biotechnological processes has recently attracted great interest due to relatively low price, high abundance, high purity of methanol, and the fact that it is a non-food raw material. In this study, methanol-based production of γ-aminobutyric acid (GABA), which is a component of drugs and functional foods and is used as monomer for production of the biodegradable plastic polyamide 4, was established using recombinant Bacillus methanolicus strains. This was achieved by heterologous overexpression of glutamate decarboxylase genes from Sulfobacillus thermosulfidooxidans (gad St ) or Escherichia coli ( gadB ) in methylotrophic B. methanolicus MGA3. Strains expressing either gad St or gadB accumulated between 0.03 and 0.4 g/L of GABA in shake flask experiments. Initially, controlled methanol fed-batch fermentations yielded low GABA concentration (0.1 g/L). However, employing a two-phase production strategy with an initial high-cell-density fermentation phase for growth and l -glutamate accumulation followed by pH reduction from 6.5 to 4.6 after 27 h for enzymatic conversion of glutamate to GABA led to 90-fold increased GABA accumulation to a final titer of 9 g/L. To the best of our knowledge, this study represents the first demonstration of methanol-based production of GABA.

Corynebacterium glutamicum, a yellow-pigmented soil bacterium that synthesizes the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides, has been engineered for the production of various carotenoids. CrtE was assumed to be the major geranylgeranyl pyrophosphate (GGPP) synthase in carotenogenesis; however, deletion of crtE did not abrogate carotenoid synthesis. In silico analysis of the repertoire of prenyltransferases encoded by the C. glutamicum genome revealed two candidate GGPPS genes (idsA and ispB). The absence of pigmentation of an idsA deletion mutant and complementation experiments with a double deletion mutant lacking both idsA and crtE showed that IdsA is the major GGPPS of C. glutamicum and that crtE overexpression compensated for the lack of IdsA, whereas plasmid-borne overexpression of ispB did not. Purified His-tagged CrtE was active as a homodimer, whereas the active form of IdsA was homotetrameric. Both enzymes catalyzed prenyl transfer with isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesylphosphate (FPP) as substrates. IdsA showed the highest catalytic efficiency with dimethylallyl pyrophosphate and IPP, whereas the catalytic efficiency of CrtE was highest with geranyl pyrophosphate and IPP. Finally, application of prenyltransferase overexpression revealed that combined overexpression of idsA and the IPP isomerase gene idi in the absence of crtE led to the highest decaprenoxanthin titer reported to date.

The cyclic amino acid ectoine is a compatible solute serving as a protective substance against osmotic stress. Ectoine finds various applications due to its moisturizing effect. To avoid the disadvantages of the prevailing so-called "bacterial milking ectoine production process" caused by the high salt concentration, low salt fermentation strategies are sought after. As l-lysine and ectoine biosynthesis share l-aspartate-semialdehyde as common precursor, l-lysine producing strains can be converted to ectoine producing strains. Corynebacterium glutamicum, which is used for l-lysine production in the million-ton-scale, was engineered for ectoine production by heterologous expression of the ectoine biosynthesis operon ectABC from Chromohalobacter salexigens. Derepression of glucose metabolism by deletion of the regulatory gene sugR and avoiding l-lactate formation by deletion of the lactate dehydrogenase gene ldhA increased ectoine productivity. In bioreactor fed-batch cultivations an ectoine titer of 22gL-1 and a volumetric productivity of 0.32gL-1h-1 were obtained. The ectoine yield of 0.16gg-1, to the best of our knowledge, exceeded previously reported yields. Moreover, ectoine production from the alternative carbon sources glycerol, glucosamine, xylose, arabinose, and soluble starch was achieved.

The yellow-pigmented soil bacterium Corynebacterium glutamicum ATCC13032 is accumulating the cyclic C-50 carotenoid decaprenoxanthin and its glucosides. Carotenoid pathway engineering was previously shown to allow for efficient lycopene production. Here, engineering of C. glutamicum for production of endogenous decaprenoxanthin as well as of the heterologous C-50 carotenoids C.p.450 and sarcinaxanthin is described. Plasmid-borne overexpression of genes for lycopene cyclization and hydroxylation from C. glutamicum, Dietzia sp., and Micrococcus luteus, in a lycopene-producing platform strain constructed here, resulted in accumulation of these three C-50 carotenoids to concentrations of about 3-4 mg/g CDW. Chromosomal deletion of a putative carotenoid glycosyltransferase gene cg0730/crtX in these strains entailed production of non-glucosylated derivatives of decaprenoxanthin, C.p.450, and sarcinaxanthin, respectively. Upon introduction of glucosyltransferase genes from M. luteus, C. glutamicum, and Pantoea ananatis, these hydroxylated C-50 carotenoids were glucosylated. We here also demonstrate production of the C-40 carotenoids beta-carotene and zeaxanthin in recombinant C. glutamicum strains and co-expression of the P. ananatis crtX gene was used to obtain glucosylated zeaxanthin. Together, our results show that C. glutamicum is a potentially valuable host for production of a wide range of glucosylated C-40 and C-50 carotenoids.

Corynebacterium glutamicum metabolizes sialic acid (Neu5Ac) to fructose-6-phosphate (fructose-6P) via the consecutive activity of the sialic acid importer SiaEFGI, N -acetylneuraminic acid lyase (NanA), N -acetylmannosamine kinase (NanK), N -acetylmannosamine-6P epimerase (NanE), N -acetylglucosamine-6P deacetylase (NagA), and glucosamine-6P deaminase (NagB). Within the cluster of the three operons nagAB , nanAKE , and siaEFGI for Neu5Ac utilization a fourth operon is present, which comprises cg2936 , encoding a GntR-type transcriptional regulator, here named NanR. Microarray studies and reporter gene assays showed that nagAB , nanAKE , siaEFGI , and nanR are repressed in wild-type (WT) C. glutamicum but highly induced in a Δ nanR C. glutamicum mutant. Purified NanR was found to specifically bind to the nucleotide motifs A[AC]G[CT][AC]TGATGTC[AT][TG]ATGT[AC]TA located within the nagA-nanA and nanR-sialA intergenic regions. Binding of NanR to promoter regions was abolished in the presence of the Neu5Ac metabolism intermediates GlcNAc-6P and N -acetylmannosamine-6-phosphate (ManNAc-6P). We observed consecutive utilization of glucose and Neu5Ac as well as fructose and Neu5Ac by WT C. glutamicum , whereas the deletion mutant C. glutamicum Δ nanR simultaneously consumed these sugars. Increased reporter gene activities for nagAB , nanAKE , and nanR were observed in cultivations of WT C. glutamicum with Neu5Ac as the sole substrate compared to cultivations when fructose was present. Taken together, our findings show that Neu5Ac metabolism in C. glutamicum is subject to catabolite repression, which involves control by the repressor NanR. IMPORTANCE Neu5Ac utilization is currently regarded as a common trait of both pathogenic and commensal bacteria. Interestingly, the nonpathogenic soil bacterium C. glutamicum efficiently utilizes Neu5Ac as a substrate for growth. Expression of genes for Neu5Ac utilization in C. glutamicum is here shown to depend on the transcriptional regulator NanR, which is the first GntR-type regulator of Neu5Ac metabolism not to use Neu5Ac as effector but relies instead on the inducers GlcNAc-6P and ManNAc-6P. The identification of conserved NanR-binding sites in intergenic regions within the operons for Neu5Ac utilization in pathogenic Corynebacterium species indicates that the mechanism for the control of Neu5Ac catabolism in C. glutamicum by NanR as described in this work is probably conserved within this genus.

Xylitol is a non-fermentable sugar alcohol used as sweetener. Corynebacterium glutamicum ATCC13032 was metabolically engineered for xylitol production from the lignocellulosic pentose sugars xylose and arabinose. Direct conversion of xylose to xylitol was achieved through the heterologous expression of NAD(P)H-dependent xylose reductase (xr) gene from Rhodotorula mucilaginosa. Xylitol synthesis from arabinose was attained through polycistronic expression of l-arabinose isomerase (araA), d-psicose 3 epimerase (dpe) and l-xylulose reductase (lxr) genes from Escherichia coli, Agrobacterium tumefaciens and Mycobacterium smegmatis, respectively. Expression of xr and the synthetic araA-dpe-lxr operon under the control of IPTG-inducible Ptac promoter enabled production of xylitol from both xylose and arabinose in the mineral (CGXII) medium with glucose as carbon source. Additional expression of a pentose transporter (araTF) gene enhanced xylitol production by about four-fold compared to the parent strain. The constructed strain Cg-ax3 produced 6.7±0.4g/L of xylitol in batch fermentations and 31±0.5g/L of xylitol in fed-batch fermentations with a specific productivity of 0.28±0.05g/g cdw/h. The strain Cg-ax3 was also validated for xylitol production from pentose rich, acid pre-treated liquor of sorghum stover (SAPL) and the results were comparable in both SAPL (27±0.3g/L) and mineral medium (31±0.5g/L).

l-arginine is a semi-essential amino acid with application in cosmetic, pharmaceutical, and food industries. Metabolic engineering strategies have been applied for overproduction of l-arginine by Corynebacterium glutamicum. LysE was the only known l-arginine exporter of this bacterium. However, an l-arginine-producing strain carrying a deletion of lysE still accumulated about 10 mM l-arginine in the growth medium. Overexpression of the putative putrescine and cadaverine export permease gene cgmA was shown to compensate for the lack of lysE with regard to l-arginine export. Moreover, plasmid-borne overexpression of cgmA rescued the toxic effect caused by feeding of the dipeptide Arg-Ala to lysE-deficient C. glutamicum and argO-deficient Escherichia coli strains. Deletion of the repressor gene cgmR improved l-arginine titers by 5 %. Production of l-lysine and l-citrulline was not affected by cgmA overexpression. Taken together, CgmA may function as an export system not only for the diamine putrescine and cadaverine but also for l-arginine. The major export system for l-lysine and l-arginine LysE may also play a role in l-citrulline export since production of l-citrulline was reduced when lysE was deleted and improved by 45 % when lysE was overproduced.

Corynebacterium glutamicum is a Gram-positive nonpathogenic bacterium that is used for the biotechnological production of amino acids. Here, we investigated the transcriptional control of the malE gene encoding malic enzyme (MalE) in C. glutamicum ATCC 13032, which is known to involve the nitrogen regulator AmtR. Gel shift experiments using purified regulators RamA and RamB revealed binding of these regulators to the malE promoter. In DNA-affinity purification experiments a hitherto uncharacterized transcriptional regulator belonging to the MarR family was found to bind to malE promoter DNA and was designated as MalR. C. glutamicum cells overexpressing malR showed reduced MalE activities in LB medium or in minimal media with acetate, glucose, pyruvate or citrate. Deletion of malR positively affected MalE activities during growth in LB medium and minimal media with pyruvate, glucose or the TCA cycle dicarboxylates l-malate, succinate and fumarate. Transcriptional fusion analysis revealed elevated malE promoter activity in the malR deletion mutant during growth in pyruvate minimal medium suggesting that MalR acts as a repressor of malE. Purified MalR bound malE promoter DNA in gel shift experiments. Two MalR binding sites were identified in the malE promoter by mutational analysis. Thus, MalR contributes to the complex transcriptional control of malE which also involves RamA, RamB and AmtR.

Corynebacterium glutamicum possesses export systems for various amino acids including BrnFE, a two-component export system for L-methionine and the branched-chain amino acids L-valine, L-isoleucine and L-leucine. A gene for a putative transcriptional regulator of the Lrp family is transcribed divergently to the brnFE operon and is required for L-isoleucine export. By comparing global gene expression changes due to L-isoleucine addition we revealed increased brnFE expression in response to L-isoleucine in C. glutamicum wild type but not in an lrp deletion mutant. ChIP-to-chip analysis, band shift experiments and DNAse footprint analysis demonstrated that Lrp binds to the intergenic region between lrp and brnF. Expression analysis of transcriptional fusions with the lrp and brnFE promoters indicated that branched-chain amino acids and L-methionine when added to the growth medium stimulated brnFE expression in the order L-leucine > L-methionine > L-isoleucine > L-valine and that Lrp was required for activation of brnFE expression. Thus, regulation of brnFE by Lrp ensures that BrnFE is synthesized only if its substrate amino acids accumulate in cells which is commensurate with its role to counteract such situations of metabolic imbalance.

Gamma-aminobutyric acid (GABA), a non-protein amino acid widespread in nature, is a component of pharmaceuticals, foods, and the biodegradable plastic polyamide 4. Corynebacterium glutamicum shows great potential for the production of GABA from glucose. GABA added to the growth medium hardly affected growth of C. glutamicum, since a half-inhibitory concentration of 1.1 M GABA was determined. As alternative to GABA production by glutamate decarboxylation, a new route for the production of GABA via putrescine was established in C. glutamicum. A putrescine-producing recombinant C. glutamicum strain was converted into a GABA producing strain by heterologous expression of putrescine transaminase (PatA) and gamma-aminobutyraldehyde dehydrogenase (PatD) genes from Escherichia coli. The resultant strain produced 5.3 ± 0.1 g L-1 of GABA. GABA production was improved further by adjusting the concentration of nitrogen in the culture medium, by avoiding the formation of the by-product N-acetylputrescine and by deletion of the genes for GABA catabolism and GABA re-uptake. GABA accumulation by this strain was increased by 51 % to 8.0 ± 0.3 g L-1, and the volumetric productivity was increased to 0.31 g L-1 h-1; the highest volumetric productivity reported so far for fermentative production of GABA from glucose in shake flasks was achieved.

The pstSCAB operon of Corynebacterium glutamicum, which encodes a high affinity transport system for uptake of the phosphorus source inorganic phosphate, is induced upon phosphate starvation involving activation by the two-component regulatory system PhoS-PhoR. Partial phosphate starvation induction of the pstSCAB operon in a ΔphoRS mutant indicated the involvement of (an) additional transcriptional regulator(s). Here, GlxR, a global cAMP-dependent transcriptional regulator, was shown to bind to the pstS promoter -133 bp to -117 bp upstream of the transcriptional start site as shown by gel shift and mutation experiments. Transcriptional fusion analysis revealed that GlxR activates the pstSCAB operon under phosphate limiting conditions in a carbon source dependent manner. Commensurate with these findings, overexpression of glxR was shown to stimulate growth under phosphate limiting conditions with glucose, but not with acetate, as carbon source. Thus, in C. glutamicum pstSCAB expression is regulated in response to the availability of phosphorus and carbon sources.

Expression profiling of Corynebacterium glutamicum in comparison to a derivative deficient in the transcriptional regulator AtlR (previously known as SucR or MtlR) revealed eight genes showing more than 4-fold higher mRNA levels in the mutant. Four of these genes are located in the direct vicinity of the atlR gene, i.e., xylB, rbtT, mtlD, and sixA, annotated as encoding xylulokinase, the ribitol transporter, mannitol 2-dehydrogenase, and phosphohistidine phosphatase, respectively. Transcriptional analysis indicated that atlR and the four genes are organized as atlR-xylB and rbtT-mtlD-sixA operons. Growth experiments with C. glutamicum and C. glutamicum ΔatlR, ΔxylB, ΔrbtT, ΔmtlD, and ΔsixA derivatives with sugar alcohols revealed that (i) wild-type C. glutamicum grows on D-arabitol but not on other sugar alcohols, (ii) growth in the presence of D-arabitol allows subsequent growth on D-mannitol, (iii) D-arabitol is cometabolized with glucose and preferentially utilized over D-mannitol, (iv) RbtT and XylB are involved in D-arabitol but not in D-mannitol metabolism, (v) MtlD is required for D-arabitol and D-mannitol metabolism, and (vi) SixA is not required for growth on any of the substrates tested. Furthermore, we show that MtlD confers D-arabitol and D-mannitol dehydrogenase activities, that the levels of these and also xylulokinase activities are generally high in the C. glutamicum ΔatlR mutant, whereas in the parental strain, they were high when cells were grown in the presence of D-arabitol and very low when cells were grown in its absence. Our results show that the XylB, RbtT, and MtlD proteins allow the growth of C. glutamicum on D-arabitol and that D-arabitol metabolism is subject to arabitol-dependent derepression by AtlR.

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l -lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis , alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae , and expression of the pntAB transhydrogenase genes from Escherichia coli . The resulting strain produced isobutanol with a substrate-specific yield (Y P/S ) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum , and overexpression of the corresponding adhA gene increased the Y P/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y P/S , indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H + to NADPH+H + . In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum Δ aceE Δ pqo Δ ilvE Δ ldhA Δ mdh (pJC4 ilvBNCD-pntAB )(pBB1 kivd-adhA ), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h −1 , and showed an overall Y P/S of about 0.48 mol per mol of glucose in the production phase.

In bacteria, the uptake of dicarboxylates, such as the tricarboxylic acid (TCA) cycle intermediates succinate, fumarate, and l-malate, is mediated by transporters of different protein families. Whereas Dcu-type transporters facilitate dicarboxylate uptake under anaerobic conditions, the most common aerobic dicarboxylate transporters are members of the dicarboxylate amino acid-cation symporter (DAACS), divalent anion sodium symporter (DASS), tripartite ATP-independent periplasmic (TRAP), and CitMHS transporter families. DAACS transporters are responsible for C4-dicarboxylate uptake under aerobic conditions in various bacteria, e.g., DctA from Escherichia coli, Bacillus subtilis, or Rhizobium leguminosarum, and are involved in different physiological functions (2, 4, 27, 41). The first described member of the TRAP family is the C4-dicarboxylate transporter DctPQM from Rhodobacter capsulatus, which facilitates substrate uptake by the use of an extracytoplasmic solute receptor (8). An example of the DASS family, members of which occur in bacteria, as well in eukaryotes, is the well-characterized transporter SdcS from Staphylococcus aureus (13). Members of the CitHMS family import citrate in symport with the cation Mg2+ or Ca2+. Whereas E. coli possesses one DctA and four different Dcu carriers, no Dcu transporter-encoding genes were found in Corynebacterium glutamicum (16, 19), which is used for the industrial production of amino acids, such as glutamate (33) or l-lysine (39), and is capable of succinate and l-lactate production under oxygen deprivation conditions. A dctA gene was annotated (19); however, C. glutamicum is not able to utilize succinate, malate, or fumarate as a sole carbon source. The uptake systems CitH and TctCBA have been characterized recently as citrate uptake systems (3, 26). Interestingly, we and others have shown that C. glutamicum possesses a DASS family transporter (DccT) for uptake of the C4-dicarboxylates succinate, fumarate, and l-malate (36, 40). Spontaneous mutants showing fast growth in succinate or fumarate minimal medium were isolated and shown to possess promoter-up mutations in the dccT gene (40). In l-malate minimal medium, these spontaneous mutants showed relatively slow growth, and the affinity of DccT for succinate and fumarate was found to be 5- and 12-fold higher than for l-malate, respectively (40). These findings prompted us to search for other uptake systems for l-malate in C. glutamicum. Here, we describe the identification and characterization of the DAACS family protein DctA from C. glutamicum as a proton motive force-driven uptake system for C4-dicarboxylate intermediates of the TCA cycle. Additionally, we compare both uptake systems, DccT and DctA, from C. glutamicum.

Corynebacterium glutamicum accumulates up to 300 mM of inorganic polyphosphate (PolyP) in the cytosol or in granules. The gene products of cg0488 ( ppx1 ) and cg1115 ( ppx2 ) were shown to be active as exopolyphosphatases (PPX), as overexpression of either gene resulted in higher exopolyphosphatase activities in crude extracts and deletion of either gene with lower activities than those of the wild-type strain. PPX1 and PPX2 from C. glutamicum share only 25% identical amino acids and belong to different protein groups, which are distinct from enterobacterial, archaeal, and yeast exopolyphosphatases. In comparison to that in the wild type, more intracellular PolyP accumulated in the Δ ppx1 and Δ ppx2 deletion mutations but less when either ppx1 or ppx2 was overexpressed. When C. glutamicum was shifted from phosphate-rich to phosphate-limiting conditions, a growth advantage of the deletion mutants and a growth disadvantage of the overexpression strains compared to the wild type were observed. Growth experiments, exopolyphosphatase activities, and intracellular PolyP concentrations revealed PPX2 as being a major exopolyphosphatase from C. glutamicum . PPX2 His was purified to homogeneity and shown to be active as a monomer. The enzyme required Mg 2+ or Mn 2+ cations but was inhibited by millimolar concentrations of Mg 2+ , Mn 2+ , and Ca 2+ . PPX2 from C. glutamicum was active with short-chain polyphosphates, even accepting pyrophosphate, and was inhibited by nucleoside triphosphates.

The transcriptional regulator SugR from Corynebacterium glutamicum represses genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Growth experiments revealed that the overexpression of sugR not only perturbed the growth of C. glutamicum on the PTS sugars glucose, fructose, and sucrose but also led to a significant growth inhibition on ribose, which is not taken up via the PTS. Chromatin immunoprecipitation combined with DNA microarray analysis and gel retardation experiments were performed to identify further target genes of SugR. Gel retardation analysis confirmed that SugR bound to the promoter regions of genes of the glycolytic enzymes 6-phosphofructokinase ( pfkA ), fructose-1,6-bisphosphate aldolase ( fba ), enolase ( eno ), pyruvate kinase ( pyk ), and NAD-dependent l -lactate dehydrogenase ( ldhA ). The deletion of sugR resulted in increased mRNA levels of eno , pyk , and ldhA in acetate medium. Enzyme activity measurements revealed that SugR-mediated repression affects the activities of PfkA, Fba, and LdhA in vivo. As the deletion of sugR led to increased LdhA activity under aerobic and under oxygen deprivation conditions, l -lactate production by C. glutamicum was determined. The overexpression of sugR reduced l -lactate production by about 25%, and sugR deletion increased l -lactate formation under oxygen deprivation conditions by threefold. Thus, SugR functions as a global repressor of genes of the PTS, glycolysis, and fermentative l -lactate dehydrogenase in C. glutamicum .

Many bacteria can utilize C 4 -carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and l -malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and l -malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT , in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and l -malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na + symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, l -malate, and likely also oxaloacetate in a sodium-dependent manner.