Engineering of phenylalanine dehydrogenase from Thermoactinomyces intermedius for the production of a novel homoglutamate.

The dramatic increase in healthcare costs has become a significant burden to this era. Many patients are unable to access medication because of the high price of drugs. Genetic engineering has made advances to increase the yield, titer, and productivity in the bio-based production of chemicals, materials of interest, and identification of innovative targets for drug discovery. Currently, the production of homoglutamate (α-Aminoadipic acid) involves petrochemical routes that are costly with low yield and often not suitable for industrial production. Here, we established the development of NADH-dependent homoglutamate by engineering NADH-dependent phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius, which provides a novel tool for in-vivo metabolic engineering and in-vitro catalysis. Based on computational insight into the structure, we proposed the site-specific directed mutagenesis of the two important residues of PDH through docking simulations by AutoDock Vina which elucidated the binding mode of PDH with α-Ketoadipic acid and ligands. Our results demonstrated that the catalytic efficiency Km/Kcat of the final mutant Ala135Arg showed a 3-fold increase amination activity towards the ketoadipic acid as compared to the other mutant Gly114Arg, a double mutant Gly114Arg/Ala135Arg, and wild type TiPDH. Furthermore, we have introduced formate dehydrogenase as a cofactor regenerative system in this study which further made this study economically viable. Our study unfolds the possibility of biosynthesis of other non-proteinogenic amino acids that might be valuable pharmaceutical intermediaries.

Spatially resolved isotope tracing reveals tissue metabolic activity.

Isotope tracing has helped to determine the metabolic activities of organs. Methods to probe metabolic heterogeneity within organs are less developed. We couple stable-isotope-labeled nutrient infusion to matrix-assisted laser desorption ionization imaging mass spectrometry (iso-imaging) to quantitate metabolic activity in mammalian tissues in a spatially resolved manner. In the kidney, we visualize gluconeogenic flux and glycolytic flux in the cortex and medulla, respectively. Tricarboxylic acid cycle substrate usage differs across kidney regions; glutamine and citrate are used preferentially in the cortex and fatty acids are used in the medulla. In the brain, we observe spatial gradations in carbon inputs to the tricarboxylic acid cycle and glutamate under a ketogenic diet. In a carbohydrate-rich diet, glucose predominates throughout but in a ketogenic diet, 3-hydroxybutyrate contributes most strongly in the hippocampus and least in the midbrain. Brain nitrogen sources also vary spatially; branched-chain amino acids contribute most in the midbrain, whereas ammonia contributes in the thalamus. Thus, iso-imaging can reveal the spatial organization of metabolic activity.

A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis.

Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite ('counter-enzymes') rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations.

Natural Product Isoliquiritigenin Activates GABAB Receptors to Decrease Voltage-Gate Ca2+ Channels and Glutamate Release in Rat Cerebrocortical Nerve Terminals.

Reduction in glutamate release is a key mechanism for neuroprotection and we investigated the effect of isoliquiritigenin (ISL), an active ingredient of Glycyrrhiza with neuroprotective activities, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). ISL produced a concentration-dependent inhibition of glutamate release and reduced the intraterminal [Ca2+] increase. The inhibition of glutamate release by ISL was prevented after removing extracellular Ca2+ or blocking P/Q-type Ca2+ channels. This inhibition was mediated through the γ-aminobutyric acid type B (GABAB) receptors because ISL was unable to inhibit glutamate release in the presence of baclofen (an GABAB agonist) or CGP3548 (an GABAB antagonist) and docking data revealed that ISL interacted with GABAB receptors. Furthermore, the ISL inhibition of glutamate release was abolished through the inhibition of Gi/o-mediated responses or Gßγ subunits, but not by 8-bromoadenosine 3',5'-cyclic monophosphate or adenylate cyclase inhibition. The ISL inhibition of glutamate release was also abolished through the inhibition of protein kinase C (PKC), and ISL decreased the phosphorylation of PKC. Thus, we inferred that ISL, through GABAB receptor activation and Gßγ-coupled inhibition of P/Q-type Ca2+ channels, suppressed the PKC phosphorylation to cause a decrease in evoked glutamate release at rat cerebrocortical nerve terminals.

Genome editing of Corynebacterium glutamicum mediated with Cpf1 plus Ku/LigD.

OBJECTIVES: Corynebacterium glutamicum (C. glutamicum) has been harnessed for multi-million-ton scale production of glutamate and lysine. To further increase its amino acid production for fermentation industry, there is an acute need to develop next-generation genome manipulation tool for its metabolic engineering. All reported methods for genome editing triggered with CRISPR-Cas are based on the homologous recombination. While, it requires the generation of DNA repair template, which is a bottle-neck for its extensive application. RESULTS: In this study, we developed a method for gene knockout in C. glutamicum via CRISPR-Cpf1-coupled non-homologous end-joining (CC-NHEJ). Specifically, CRISPR-Cpf1 introduced double-strand breaks in the genome of C. glutamicum, which was further repaired by ectopically expressed two NHEJ key proteins (Mycobacterium tuberculosis Ku and ligase D). We provide the proof of concept, for CC-NHEJ, by the successful knockout of the crtYf/e gene in C. glutamicum with the efficiency of 22.00 ± 5.56%, or something like that. CONCLUSION: The present study reported a novel genome manipulation method for C. glutamicum.

The Function of Sialidase Revealed by Sialidase Activity Imaging Probe.

Sialidase cleaves sialic acid residues from glycans such as glycoproteins and glycolipids. In the brain, desorption of the sialic acid by sialidase is essential for synaptic plasticity, learning and memory and synaptic transmission. BTP3-Neu5Ac has been developed for sensitive imaging of sialidase enzyme activity in mammalian tissues. Sialidase activity in the rat hippocampus detected with BTP3-Neu5Ac increases rapidly by neuronal depolarization. It is presumed that an increased sialidase activity in conjunction with neural excitation is involved in the formation of the neural circuit for memory. Since sialidase inhibits the exocytosis of the excitatory neurotransmitter glutamate, the increased sialidase activity by neural excitation might play a role in the negative feedback mechanism against the glutamate release. Mammalian tissues other than the brain have also been stained with BTP3-Neu5Ac. On the basis of information on the sialidase activity imaging in the pancreas, it was found that sialidase inhibitor can be used as an anti-diabetic drug that can avoid hypoglycemia, a serious side effect of insulin secretagogues. In this review, we discuss the role of sialidase in the brain as well as in the pancreas and skin, as revealed by using a sialidase activity imaging probe. We also present the detection of influenza virus with BTP3-Neu5Ac and modification of BTP3-Neu5Ac.

Selection of Lactococcus lactis HY7803 for Glutamic Acid Production Based on Comparative Genomic Analysis.

Comparative genomic analysis was performed on eight species of lactic acid bacteria (LAB)-Lactococcus (L.) lactis, Lactobacillus (Lb.) plantarum, Lb. casei, Lb. brevis, Leuconostoc (Leu.) mesenteroides, Lb. fermentum, Lb. buchneri, and Lb. curvatus-to assess their glutamic acid production pathways. Glutamic acid is important for umami taste in foods. The only genes for glutamic acid production identified in the eight LAB were for conversion from glutamine in L. lactis and Leu. mesenteroides, and from glucose via citrate in L. lactis. Thus, L. lactis was considered to be potentially the best of the species for glutamic acid production. By biochemical analyses, L. lactis HY7803 was selected for glutamic acid production from among 17 L. lactis strains. Strain HY7803 produced 83.16 pmol/µl glutamic acid from glucose, and exogenous supplementation of citrate increased this to 108.42 pmol/µl. Including glutamic acid, strain HY7803 produced more of 10 free amino acids than L. lactis reference strains IL1403 and ATCC 7962 in the presence of exogenous citrate. The differences in the amino acid profiles of the strains were illuminated by principal component analysis. Our results indicate that L. lactis HY7803 may be a good starter strain for glutamic acid production.

Autophagy sustains glutamate and aspartate synthesis in Saccharomyces cerevisiae during nitrogen starvation.

Autophagy catabolizes cellular constituents to promote survival during nutrient deprivation. Yet, a metabolic comprehension of this recycling operation, despite its crucial importance, remains incomplete. Here, we uncover a specific metabolic function of autophagy that exquisitely adjusts cellular metabolism according to nitrogen availability in the budding yeast Saccharomyces cerevisiae. Autophagy enables metabolic plasticity to promote glutamate and aspartate synthesis, which empowers nitrogen-starved cells to replenish their nitrogen currency and sustain macromolecule synthesis. Our findings provide critical insights into the metabolic basis by which autophagy recycles cellular components and may also have important implications in understanding the role of autophagy in diseases such as cancer.

Postnatal Development of Glutamate and GABA Transcript Expression in Monkey Visual, Parietal, and Prefrontal Cortices.

Visuospatial working memory (vsWM) requires information transfer among multiple cortical regions, from primary visual (V1) to prefrontal (PFC) cortices. This information is conveyed via layer 3 glutamatergic neurons whose activity is regulated by gamma-aminobutyric acid (GABA)ergic interneurons. In layer 3 of adult human neocortex, molecular markers of glutamate neurotransmission were lowest in V1 and highest in PFC, whereas GABA markers had the reverse pattern. Here, we asked if these opposite V1-visual association cortex (V2)-posterior parietal cortex (PPC)-PFC gradients across the vsWM network are present in layer 3 of monkey neocortex, when they are established during postnatal development, and if they are specific to this layer. We quantified transcript levels of glutamate and GABA markers in layers 3 and 6 of four vsWM cortical regions in a postnatal developmental series of 30 macaque monkeys. In adult monkeys, glutamate transcript levels in layer 3 increased across V1-V2-PPC-PFC regions, whereas GABA transcripts showed the opposite V1-V2-PPC-PFC gradient. Glutamate transcripts established adult-like expression patterns earlier during postnatal development than GABA transcripts. These V1-V2-PPC-PFC gradients and developmental patterns were less evident in layer 6. These findings demonstrate that expression of glutamate and GABA transcripts differs across cortical regions and layers during postnatal development, revealing potential molecular substrates for vsWM functional maturation.

Role of GlnR in Controlling Expression of Nitrogen Metabolism Genes in Listeria monocytogenes.

Listeria monocytogenes is a fastidious bacterial pathogen that can utilize only a limited number of nitrogen sources for growth. Both glutamine and ammonium are common nitrogen sources used in listerial defined growth media, but little is known about the regulation of their uptake or utilization. The functional role of L. monocytogenes GlnR, the transcriptional regulator of nitrogen metabolism genes in low-G+C Gram-positive bacteria, was determined using transcriptome sequencing and real-time reverse transcription-PCR experiments. The GlnR regulon included transcriptional units involved in ammonium transport (amtB glnK) and biosynthesis of glutamine (glnRA) and glutamate (gdhA) from ammonium. As in other bacteria, GlnR proved to be an autoregulatory repressor of the glnRA operon. Unexpectedly, GlnR was most active during growth with ammonium as the nitrogen source and less active in the glutamine medium, apparently because listerial cells perceive growth with glutamine as a nitrogen-limiting condition. Therefore, paradoxically, expression of the glnA gene, encoding glutamine synthetase, was highest in the glutamine medium. For the amtB glnK operon, GlnR served as both a negative regulator in the presence of ammonium and a positive regulator in the glutamine medium. The gdhA gene was subject to a third mode of regulation that apparently required an elevated level of GlnR for repression. Finally, activity of glutamate dehydrogenase encoded by the gdhA gene appeared to correlate inversely with expression of gltAB, the operon that encodes the other major glutamate-synthesizing enzyme, glutamate synthase. Both gdhA and amtB were also regulated, in a negative manner, by the global transcriptional regulator CodY.IMPORTANCEL. monocytogenes is a widespread foodborne pathogen. Nitrogen-containing compounds, such as the glutamate-containing tripeptide, glutathione, and glutamine, have been shown to be important for expression of L. monocytogenes virulence genes. In this work, we showed that a transcriptional regulator, GlnR, controls expression of critical listerial genes of nitrogen metabolism that are involved in ammonium uptake and biosynthesis of glutamine and glutamate. A different mode of GlnR-mediated regulation was found for each of these three pathways.

Isolation, Structure Determination, and Synthesis of Cyclic Tetraglutamic Acids from Box Jellyfish Species Alatina alata and Chironex yamaguchii.

Cubozoan nematocyst venoms contain known cytolytic and hemolytic proteins, but small molecule components have not been previously reported from cubozoan venom. We screened nematocyst extracts of Alatina alata and Chironex yamaguchii by LC-MS for the presence of small molecule metabolites. Three isomeric compounds, cnidarins 4A (1), 4B (2), and 4C (3), were isolated from venom extracts and characterized by NMR and MS, which revealed their planar structure as cyclic γ-linked tetraglutamic acids. The full configurational assignments were established by syntheses of all six possible stereoisomers, comparison of spectral data and optical rotations, and stereochemical analysis of derivatized degradation products. Compounds 1-3 were subsequently detected by LC-MS in tissues of eight other cnidarian species. The most abundant of these compounds, cnidarin 4A (1), showed no mammalian cell toxicity or hemolytic activity, which may suggest a role for these cyclic tetraglutamates in nematocyst discharge.

Corynebacterium suranareeae sp. nov., a glutamate producing bacterium isolated from soil and its complete genome-based analysis.

Strain N24T was isolated from soil contaminated with starling's feces collected from Roi-Et province, Thailand. Cells of N24T were Gram-stain-positive rods, aerobic and non-spore-forming. N24T was positive for catalase, urease, citrate utilization, nitrate reduction and Methyl Red (MR) test but negative for oxidase, casein, gelatin liquefaction, tyrosine, Voges-Proskauer (VP) reaction and starch hydrolysis. Meso-diaminopimelic acid, rhamnose, ribose, arabinose and galactose were detected in its whole-cell hydrolysates. The results of the 16S rRNA gene sequence analysis indicated that N24T represented a member of the genus Corynebacterium. N24T was closely related to Corynebacterium glutamicum ATCC 13032T, with 99.0 % 16S rRNA gene sequence similarity. According to results obtained using in silico DNA-DNA hybridization approaches, N24T showed highest DNA-DNA relatedness (27.6 %) and average nucleotide identity (84.1 %) to Corynebacterium glutamicum ATCC 13032T. The DNA G+C content of N24T was 51.8 mol% (genome based). The major cellular fatty acids of N24T were C16 : 0, and C18 : 1ω9c. N24T had the nine isoprenes unit, MK-9(H2) as the predominant menaquinone. The predominant polar lipids were phosphatidylglycerol, phosphatidylinositol and diphosphatidylglycerol. Mycolic acids were also present. According to the complete genome sequence data, strain N24T and C. glutamicum ATCC 13032T are close phylogenetic neighbours, but have different genome characteristics. On the basis of the results of the genotypic and genomic studies and phenotypic characteristics including chemotaxonomy, strain N24T should be classified as representing a novel species of the genus Corynebacterium, for which the name Corynebacterium suranareeae sp. nov. is proposed. The type strain is N24T (TBRC 5845T=NBRC 113465T).

Improving synthetic methylotrophy via dynamic formaldehyde regulation of pentose phosphate pathway genes and redox perturbation.

Escherichia coli is an ideal choice for constructing synthetic methylotrophs capable of utilizing the non-native substrate methanol as a carbon and energy source. All current E. coli-based synthetic methylotrophs require co-substrates. They display variable levels of methanol-carbon incorporation due to a lack of native regulatory control of biosynthetic pathways, as E. coli does not recognize methanol as a proper substrate despite its ability to catabolize it. Here, using the E. coli formaldehyde-inducible promoter Pfrm, we implement dynamic expression control of select pentose-phosphate genes in response to the formaldehyde produced upon methanol oxidation. Genes under Pfrm control exhibited 8- to 30-fold transcriptional upregulation during growth on methanol. Formaldehyde-induced episomal expression of the B. methanolicus rpe and tkt genes involved in the regeneration of ribulose 5-phosphate required for formaldehyde fixation led to significantly improved methanol assimilation into intracellular metabolites, including a 2-fold increase of 13C-methanol into glutamate. Using a simple strategy for redox perturbation by deleting the E. coli NAD-dependent malate dehydrogenase gene maldh, we demonstrate 5-fold improved biomass formation of cells growing on methanol in the presence of a small concentration of yeast extract. Further improvements in methanol utilization are achieved via adaptive laboratory evolution and heterologous rpe and tkt expression. A short-term in vivo13C-methanol labeling assay was used to determine methanol assimilation activity for Δmaldh strains, and demonstrated dramatically higher labeling in intracellular metabolites, including a 6-fold and 1.8-fold increase in glycine labeling for the rpe/tkt and evolved strains, respectively. The combination of formaldehyde-controlled pentose phosphate pathway expression and redox perturbation with the maldh knock-out greatly improved both growth benefit with methanol and methanol carbon incorporation into intracellular metabolites.

Effects of proteases on L-glutamic acid fermentation.

In the process of L-glutamic acid fermentation, there are proteins that cannot be decomposed and utilized by bacteria and that secreted by bacteria at the same time, which cause problems such as increased foam production in the fermentation broth that lowers the dissolved oxygen, which makes the total fermentation efficiency low. Therefore, these proteins can be decomposed by adding proteases in the fermentation broth, and it is found that the best results are obtained by adding 0.5 g/L of trypsin. Proteins can be used by bacteria after being decomposed as well. The final L-glutamic acid production in our research was 177.0 g/L, which is 14.9% more than the control fermentation (154.0 g/L). Similarly, the glucose conversion rate was 68.3%, which is an increase of 4.0% as compared to the control fermentation (65.6%).

NMDA receptor modulation of glutamate release in activated neutrophils.

BACKGROUND: Neutrophil depletion improves neurologic outcomes in experimental sepsis/brain injury. We hypothesized that neutrophils may exacerbate neuronal injury through the release of neurotoxic quantities of the neurotransmitter glutamate. METHODS: Real-time glutamate release by primary human neutrophils was determined using enzymatic biosensors. Bacterial and direct protein-kinase C (Phorbol 12-myristate 13-acetate; PMA) activation of neutrophils in human whole blood, isolated neutrophils or human cell lines were compared in the presence/absence of N-Methyl-d-aspartic acid receptor (NMDAR) antagonists. Bacterial and direct activation of neutrophils from wild-type and transgenic murine neutrophils deficient in NMDAR-scaffolding proteins were compared using flow cytometry (phagocytosis, reactive oxygen species (ROS) generation) and real-time respirometry (oxygen consumption). FINDINGS: Both glutamate and the NMDAR co-agonist d-serine are rapidly released by neutrophils in response to bacterial and PMA-induced activation. Pharmacological NMDAR blockade reduced both the autocrine release of glutamate, d-serine and the respiratory burst by activated primary human neutrophils. A highly specific small-molecule inhibitor ZL006 that limits NMDAR-mediated neuronal injury also reduced ROS by activated neutrophils in a murine model of peritonitis, via uncoupling of the NMDAR GluN2B subunit from its' scaffolding protein, postsynaptic density protein-95 (PSD-95). Genetic ablation of PSD-95 reduced ROS production by activated murine neutrophils. Pharmacological blockade of the NMDAR GluN2B subunit reduced primary human neutrophil activation induced by Pseudomonas fluorescens, a glutamate-secreting Gram-negative bacillus closely related to pathogens that cause hospital-acquired infections. INTERPRETATION: These data suggest that release of glutamate by activated neutrophils augments ROS production in an autocrine manner via actions on NMDAR expressed by these cells. FUND: GLA: Academy Medical Sciences/Health Foundation Clinician Scientist. AVG is a Wellcome Trust Senior Research Fellow.

Interactions of Glutamatergic Neurotransmission and Brain-Derived Neurotrophic Factor in the Regulation of Behaviors after Nicotine Administration.

Nicotine causes tobacco dependence, which may result in fatal respiratory diseases. The striatum is a key structure of forebrain basal nuclei associated with nicotine dependence. In the striatum, glutamate release is increased when α7 nicotinic acetylcholine receptors expressed in the glutamatergic terminals are exposed to nicotine, and over-stimulates glutamate receptors in gamma amino-butyric acid (GABA)ergic neurons. These receptor over-stimulations in turn potentiate GABAergic outputs to forebrain basal nuclei and contribute to the increase in psychomotor behaviors associated with nicotine dependence. In parallel with glutamate increases, nicotine exposure elevates brain-derived neurotrophic factor (BDNF) release through anterograde and retrograde targeting of the synapses of glutamatergic terminals and GABAergic neurons. This article reviews nicotine-exposure induced elevations of glutamatergic neurotransmission, the bidirectional targeting of BDNF in the striatum, and the potential regulatory role played by BDNF in behavioral responses to nicotine exposure.

Metabolic engineering of Corynebacterium glutamicum by synthetic small regulatory RNAs.

Corynebacterium glutamicum is an important platform strain that is wildly used in industrial production of amino acids and various other biochemicals. However, due to good genomic stability, C. glutamicum is more difficult to engineer than genetically tractable hosts. Herein, a synthetic small regulatory RNA (sRNA)-based gene knockdown strategy was developed for C. glutamicum. The RNA chaperone Hfq from Escherichia coli and a rationally designed sRNA consisting of the E. coli MicC scaffold and a target binding site were proven to be indispensable for repressing green fluorescent protein expression in C. glutamicum. The synthetic sRNA system was applied to improve glutamate production through knockdown of pyk, ldhA, and odhA, resulting almost a threefold increase in glutamate titer and yield. Gene transcription and enzyme activity were down-regulated by up to 80%. The synthetic sRNA system developed holds promise to accelerate C. glutamicum metabolic engineering for producing valuable chemicals and fuels.

Methionine coordinates a hierarchically organized anabolic program enabling proliferation.

Methionine availability during overall amino acid limitation metabolically reprograms cells to support proliferation, the underlying basis for which remains unclear. Here we construct the organization of this methionine-mediated anabolic program using yeast. Combining comparative transcriptome analysis and biochemical and metabolic flux-based approaches, we discover that methionine rewires overall metabolic outputs by increasing the activity of a key regulatory node. This comprises the pentose phosphate pathway (PPP) coupled with reductive biosynthesis, the glutamate dehydrogenase (GDH)-dependent synthesis of glutamate/glutamine, and pyridoxal-5-phosphate (PLP)-dependent transamination capacity. This PPP-GDH-PLP node provides the required cofactors and/or substrates for subsequent rate-limiting reactions in the synthesis of amino acids and therefore nucleotides. These rate-limiting steps in amino acid biosynthesis are also induced in a methionine-dependent manner. This thereby results in a biochemical cascade establishing a hierarchically organized anabolic program. For this methionine-mediated anabolic program to be sustained, cells co-opt a "starvation stress response" regulator, Gcn4p. Collectively, our data suggest a hierarchical metabolic framework explaining how methionine mediates an anabolic switch.

Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma.

IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas.

Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production.

Methanol is a promising feedstock for bioproduction of fuels and chemicals, thus massive efforts have been devoted to engineering non-native methylotrophic platform microorganisms to utilize methanol. Herein, we rationally designed and experimentally engineered the industrial workhorse Corynebacterium glutamicum to serve as a methanol-dependent synthetic methylotroph. The cell growth of the methanol-dependent strain relies on co-utilization of methanol and xylose, and most notably methanol is an indispensable carbon source. Due to the methanol-dependent characteristic, adaptive laboratory evolution was successfully applied to improving methanol utilization. The evolved mutant showed a 20-fold increase in cell growth on methanol-xylose minimal medium and utilized methanol and xylose with a high mole ratio of 3.83:1. 13C-labeling experiments demonstrated that the carbon derived from methanol was assimilated into intracellular building blocks, high-energy carriers, cofactors, and biomass (up to 63% 13C-labeling). By inhibiting cell wall biosynthesis, methanol-dependent glutamate production was also achieved, demonstrating the potential application in bioconversion of methanol into useful chemicals. Genetic mutations detected in the evolved strains indicate the importance of intracellular NAD+/NADH ratio, substrate uptake, and methanol tolerance on methanol utilization. This study reports significant improvement in the area of developing fully synthetic methylotrophs.