Comprehensive analysis of genomic variation, pan-genome and biosynthetic potential of Corynebacterium glutamicum strains.

Corynebacterium glutamicum is a non-pathogenic species of the Corynebacteriaceae family. It has been broadly used in industrial biotechnology for the production of valuable products. Though it is widely accepted at the industrial level, knowledge about the genomic diversity of the strains is limited. Here, we investigated the comparative genomic features of the strains and pan-genomic characteristics. We also observed phylogenetic relationships among the strains based on average nucleotide identity (ANI). We found diversity between strains at the genomic and pan-genomic levels. Less than one-third of the C. glutamicum pan-genome consists of core genes and soft-core genes. Whereas, a large number of strain-specific genes covered about half of the total pan-genome. Besides, C. glutamicum pan-genome is open and expanding, which indicates the possible addition of new gene families to the pan-genome. We also investigated the distribution of biosynthetic gene clusters (BGCs) among the strains. We discovered slight variations of BGCs at the strain level. Several BGCs with the potential to express novel bioactive secondary metabolites have been identified. Therefore, by utilizing the characteristic advantages of C. glutamicum, different strains can be potential applicants for natural drug discovery.

Enhancing astaxanthin biosynthesis and pathway expansion towards glycosylated C40 carotenoids by Corynebacterium glutamicum.

Astaxanthin, a versatile C40 carotenoid prized for its applications in food, cosmetics, and health, is a bright red pigment with powerful antioxidant properties. To enhance astaxanthin production in Corynebacterium glutamicum, we employed rational pathway engineering strategies, focused on improving precursor availability and optimizing terminal oxy-functionalized C40 carotenoid biosynthesis. Our efforts resulted in an increased astaxanthin precursor supply with 1.5-fold higher ß-carotene production with strain BETA6 (18 mg g-1 CDW). Further advancements in astaxanthin production were made by fine-tuning the expression of the ß-carotene hydroxylase gene crtZ and ß-carotene ketolase gene crtW, yielding a nearly fivefold increase in astaxanthin (strain ASTA**), with astaxanthin constituting 72% of total carotenoids. ASTA** was successfully transferred to a 2 L fed-batch fermentation with an enhanced titer of 103 mg L-1 astaxanthin with a volumetric productivity of 1.5 mg L-1 h-1. Based on this strain a pathway expansion was achieved towards glycosylated C40 carotenoids under heterologous expression of the glycosyltransferase gene crtX. To the best of our knowledge, this is the first time astaxanthin-ß-D-diglucoside was produced with C. glutamicum achieving high titers of microbial C40 glucosides of 39 mg L-1. This study showcases the potential of pathway engineering to unlock novel C40 carotenoid variants for diverse industrial applications.

Enhancing Poly-γ-glutamic Acid Production in Bacillus tequilensis BL01 through a Multienzyme Assembly Strategy and Expression Features of Glutamate Synthesis from Corynebacterium glutamicum.

The enhancement of intracellular glutamate synthesis in glutamate-independent poly-γ-glutamic acid (γ-PGA)-producing strains is an essential strategy for improving γ-PGA production. Bacillus tequilensis BL01ΔpgdSΔggtΔsucAΔgudB:P43-ppc-pyk-gdhA for the efficient synthesis of γ-PGA was constructed through expression of glutamate synthesis features of Corynebacterium glutamicum, which increased the titer of γ-PGA by 2.18-fold (3.24 ± 0.22 g/L) compared to that of B. tequilensis BL01ΔpgdSΔggtΔsucAΔgudB (1.02 ± 0.11 g/L). To further improve the titer of γ-PGA and decrease the production of byproducts, three enzymes (Ppc, Pyk, and AceE) were assembled to a complex using SpyTag/Catcher pairs. The results showed that the γ-PGA titer of the assembled strain was 31.31% higher than that of the unassembled strain. To further reduce the production cost, 25.73 ± 0.69 g/L γ-PGA with a productivity of 0.48 g/L/h was obtained from cheap molasses. This work provides new metabolic engineering strategies to improve the production of γ-PGA in B. tequilensis BL01. Furthermore, the engineered strain has great potential for the industrial production of γ-PGA from molasses.

Chemical Proteomics Strategies for Analyzing Protein Lipidation Reveal the Bacterial O-Mycoloylome.

Protein lipidation dynamically controls protein localization and function within cellular membranes. A unique form of protein O-fatty acylation in Corynebacterium, termed protein O-mycoloylation, involves the attachment of mycolic acids─unusually large and hydrophobic fatty acids─to serine residues of proteins in these organisms' outer mycomembrane. However, as with other forms of protein lipidation, the scope and functional consequences of protein O-mycoloylation are challenging to investigate due to the inherent difficulties of enriching and analyzing lipidated peptides. To facilitate the analysis of protein lipidation and enable the comprehensive profiling and site mapping of protein O-mycoloylation, we developed a chemical proteomics strategy integrating metabolic labeling, click chemistry, cleavable linkers, and a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing LC separation and complementary fragmentation methods tailored to the analysis of lipophilic, MS-labile O-acylated peptides. Using these tools in the model organism Corynebacterium glutamicum, we identified approximately 30 candidate O-mycoloylated proteins, including porins, mycoloyltransferases, secreted hydrolases, and other proteins with cell envelope-related functions─consistent with a role for O-mycoloylation in targeting proteins to the mycomembrane. Site mapping revealed that many of the proteins contained multiple spatially proximal modification sites, which occurred predominantly at serine residues surrounded by conformationally flexible peptide motifs. Overall, this study (i) discloses the putative protein O-mycoloylome for the first time, (ii) yields new insights into the undercharacterized proteome of the mycomembrane, which is a hallmark of important pathogens (e.g., Corynebacterium diphtheriae, Mycobacterium tuberculosis), and (iii) provides generally applicable chemical strategies for the proteomic analysis of protein lipidation.

[Advances in fermentative production of L-tryptophan: a review].

L-tryptophan is an essential amino acid that is widely used in food, medicine and feed sectors. L-tryptophan can be produced through fermentation, and the main producing strains are engineered Escherichia coli and Corynebacterium glutamicum, which are constructed by rational design methods based on metabolic engineering and synthetic biology. However, due to the long metabolic pathway, complex and unclear regulatory mechanism for L-tryptophan production in microbial cells, the production efficiency and robustness of L-tryptophan producing strains are still low. In this connection, irrational design methods such as laboratory adaptive evolution, are often applied to improve the performance of L-tryptophan producing strains. This review summarizes the recent progress on biosynthesis metabolism of L-tryptophan and its regulation, the construction and optimization of L-tryptophan producing strains, and fermentative production of L-tryptophan, and prospects future development perspective. This review may facilitate research and development for fermentative production of L-tryptophan.

Efficient Fermentative Production of d-Alanine and Other d-Amino Acids by Metabolically Engineered Corynebacterium glutamicum.

d-Amino acids (d-AAs) have wide applications in industries such as pharmaceutical, food, and cosmetics due to their unique properties. Currently, the production of d-AAs has relied on chemical synthesis or enzyme catalysts, and it is challenging to produce d-AAs via direct fermentation from glucose. We observed that Corynebacterium glutamicum exhibits a remarkable tolerance to high concentrations of d-Ala, a crucial characteristic for establishing a successful fermentation process. By optimizing meso-diaminopilmelate dehydrogenases in different C. glutamicum strains and successively deleting l-Ala biosynthetic pathways, we developed an efficient d-Ala fermentation system. The d-Ala titer was enhanced through systems metabolic engineering, which involved strengthening glucose assimilation and pyruvate supply, reducing the formation of organic acid byproducts, and attenuating the TCA cycle. During fermentation in a 5-L bioreactor, a significant accumulation of l-Ala was observed in the broth, which was subsequently diminished by introducing an l-amino acid deaminase. Ultimately, the engineered strain DA-11 produced 85 g/L d-Ala with a yield of 0.30 g/g glucose, accompanied by an optical purity exceeding 99%. The fermentation platform has the potential to be extended for the synthesis of other d-AAs, as demonstrated by the production of d-Val and d-Glu.

RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria.

Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.

Metabolic engineering with adaptive laboratory evolution for phenylalanine production by Corynebacterium glutamicum.

The mutants resistant to a phenylalanine analog, 4-fluorophenylalanine (4FP), were obtained for metabolic engineering of Corynebacterium glutamicum for producing aromatic amino acids synthesized through the shikimate pathway by adaptive laboratory evolution. Culture experiments of the C. glutamicum strains which carry the mutations found in the open reading frame from the 4FP-resistant mutants revealed that the mutations in the open reading frames of aroG (NCgl2098), pheA (NCgl2799) and aroP (NCgl1062) encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate, prephenate dehydratase, and aromatic amino acid transporter are responsible for 4FP resistance and higher concentration of aromatic amino acids in their culture supernatants in the 4FP-resistant strains. It was expected that aroG and pheA mutations would release feedback inhibition of the enzymes involved in the shikimate pathway by phenylalanine and that aroP mutations would prevent intracellular uptake of aromatic amino acids. Therefore, we conducted metabolic engineering of the C. glutamicum wild-type strain for aromatic amino acid production and found that phenylalanine production at 6.11 ± 0.08 g L-1 was achieved by overexpressing the mutant pheA and aroG genes from the 4FP-resistant mutants and deleting aroP gene. This study demonstrates that adaptive laboratory evolution is an effective way to obtain useful mutant genes related to production of target material and to establish metabolic engineering strategies.

Rewiring Metabolic Flux in Corynebacterium glutamicum Using a CRISPR/dCpf1-Based Bifunctional Regulation System.

Corynebacterium glutamicum, a microorganism classified as generally recognized as safe for use in the industrial production of food raw materials and additives, has encountered challenges in achieving widespread adoption and popularization as microbial cell factories. These obstacles arise from the intricate nature of manipulating metabolic flux through conventional methods, such as gene knockout and enzyme overexpression. To address this challenge, we developed a CRISPR/dCpf1-based bifunctional regulation system to bidirectionally regulate the expression of multiple genes in C. glutamicum. Specifically, through fusing various transcription factors to the C-terminus of dCpf1, the resulting dCpf1-SoxS exhibited both CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) capabilities in C. glutamicum by altering the binding sites of crRNAs. The bifunctional regulation system was used to fine-tune metabolic flux from shikimic acid (SA) and l-serine biosynthesis, resulting in 27-fold and 10-fold increases in SA and l-serine production, respectively, compared to the original strain. These findings highlight the potential of the CRISPR/dCpf1-based bifunctional regulation system in effectively enhancing the yield of target products in C. glutamicum.

Corynebacterium glutamicum cell factory design for the efficient production of cis, cis-muconic acid.

Cis, cis-muconic acid (MA) is widely used as a key starting material in the synthesis of diverse polymers. The growing demand in these industries has led to an increased need for MA. Here, we constructed recombinant Corynebacterium glutamicum by systems metabolic engineering, which exhibit high efficiency in the production of MA. Firstly, the three major degradation pathways were disrupted in the MA production process. Subsequently, metabolic optimization strategies were predicted by computational design and the shikimate pathway was reconstructed, significantly enhancing its metabolic flux. Finally, through optimization and integration of key genes involved in MA production, the recombinant strain produced 88.2 g/L of MA with the yield of 0.30 mol/mol glucose in the 5 L bioreactor. This titer represents the highest reported titer achieved using glucose as the carbon source in current studies, and the yield is the highest reported for MA production from glucose in Corynebacterium glutamicum. Furthermore, to enable the utilization of more cost-effective glucose derived from corn straw hydrolysate, we subjected the strain to adaptive laboratory evolution in corn straw hydrolysate. Ultimately, we successfully achieved MA production in a high solid loading of corn straw hydrolysate (with the glucose concentration of 83.56 g/L), resulting in a titer of 19.9 g/L for MA, which is 4.1 times higher than that of the original strain. Additionally, the glucose yield was improved to 0.33 mol/mol. These provide possibilities for a greener and more sustainable production of MA.

A genome-reduced Corynebacterium glutamicum derivative discloses a hidden pathway relevant for 1,2-propanediol production.

BACKGROUND: 1,2-propanediol (1,2-PDO) is widely used in the cosmetic, food, and drug industries with a worldwide consumption of over 1.5 million metric tons per year. Although efforts have been made to engineer microbial hosts such as Corynebacterium glutamicum to produce 1,2-PDO from renewable resources, the performance of such strains is still improvable to be competitive with existing petrochemical production routes. RESULTS: In this study, we enabled 1,2-PDO production in the genome-reduced strain C. glutamicum PC2 by introducing previously described modifications. The resulting strain showed reduced product formation but secreted 50 ± 1 mM D-lactate as byproduct. C. glutamicum PC2 lacks the D-lactate dehydrogenase which pointed to a yet unknown pathway relevant for 1,2-PDO production. Further analysis indicated that in C. glutamicum methylglyoxal, the precursor for 1,2-PDO synthesis, is detoxified with the antioxidant native mycothiol (MSH) by a glyoxalase-like system to lactoylmycothiol and converted to D-lactate which is rerouted into the central carbon metabolism at the level of pyruvate. Metabolomics of cell extracts of the empty vector-carrying wildtype, a 1,2-PDO producer and its derivative with inactive D-lactate dehydrogenase identified major mass peaks characteristic for lactoylmycothiol and its precursors MSH and glucosaminyl-myo-inositol, whereas the respective mass peaks were absent in a production strain with inactivated MSH synthesis. Deletion of mshA, encoding MSH synthase, in the 1,2-PDO producing strain C. glutamicum ΔhdpAΔldh(pEKEx3-mgsA-yqhD-gldA) improved the product yield by 56% to 0.53 ± 0.01 mM1,2-PDO mMglucose-1 which is the highest value for C. glutamicum reported so far. CONCLUSIONS: Genome reduced-strains are a useful basis to unravel metabolic constraints for strain engineering and disclosed in this study the pathway to detoxify methylglyoxal which represents a precursor for 1,2-PDO production. Subsequent inactivation of the competing pathway significantly improved the 1,2-PDO yield.

Isopropanol production using engineered Corynebacterium glutamicum from waste rice straw biomass.

Isopropanol, a well-known biofuel, is a widely used precursor for chemical products that can replace nonrenewable petroleum energy. Here, engineered Corynebacterium glutamicum that can effectively utilize all xylose and glucose in agricultural waste rice straw to produce isopropanol was described. First, codon mutations were introduced into transporters and glycolytic-related genes to decrease the glucose preference of C. glutamicum. A more energetically favorable xylose oxidative pathway was constructed that replaced traditional xylose isomerization pathways, saving twice the number of enzymatic steps. A succinate auxiliary module was incorporated into the tricarboxylic acid cycle (TCA), connecting the xylose-utilized pathway with the isopropanol pathway to maximize xylose orientation towards the product. The final engineered strain successfully consumed 100 % of the xylose from NaOH-pretreated, enzyme-hydrolyzed rice straw and effectively synthesized 4.91 g/L isopropanol. This study showcases the successful conversion of agricultural waste into renewable energy, unveiling new possibilities for advancing biological fermentation technology.

Production of bilirubin via whole-cell transformation utilizing recombinant Corynebacterium glutamicum expressing a ß-glucuronidase from Staphylococcus sp. RLH1.

Bilirubin, a key active ingredient of bezoars with extensive clinical applications in China, is produced through a chemical process. However, this method suffers from inefficiency and adverse environmental impacts. To address this challenge, we present a novel and efficient approach for bilirubin production via whole-cell transformation. In this study, we employed Corynebacterium glutamicum ATCC13032 to express a ß-glucuronidase (StGUS), an enzyme from Staphylococcus sp. RLH1 that effectively hydrolyzes conjugated bilirubin to bilirubin. Following the optimization of the biotransformation conditions, a remarkable conversion rate of 79.7% in the generation of bilirubin was obtained at temperate 40 °C, pH 7.0, 1 mM Mg2+ and 6 mM antioxidant NaHSO3 after 12 h. These findings hold significant potential for establishing an industrially viable platform for large-scale bilirubin production.

Establishment of synthetic microbial consortia with Corynebacterium glutamicum and Pseudomonas putida: Design, construction, and application to production of γ-glutamylisopropylamide and l-theanine.

Microbial synthetic consortia are a promising alternative to classical monoculture for biotechnological applications and fermentative processes. Their versatile use offers advantages in the degradation of complex substrates, the allocation of the metabolic burden between individual partners, or the division of labour in energy utilisation, substrate supply or product formation. Here, stable synthetic consortia between the two industrially relevant production hosts, Pseudomonas putida KT2440 and Corynebacterium glutamicum ATCC13032, were established for the first time. By applying arginine auxotrophy/overproduction and/or formamidase-based utilisation of the rare nitrogen source formamide, different types of interaction were realised, such as commensal relationships (+/0 and 0/+) and mutualistic cross-feeding (+/+). These consortia did not only show stable growth but could also be used for fermentative production of the γ-glutamylated amines theanine and γ-glutamyl-isopropylamide (GIPA). The consortia produced up to 2.8 g L-1 of GIPA and up to 2.6 g L-1 of theanine, a taste-enhancing constituent of green tea leaves. Thus, the advantageous approach of using synthetic microbial consortia for fermentative production of value-added compounds was successfully demonstrated.

Metabolic engineering of Corynebacterium glutamicum for the production of anthranilate from glucose and xylose.

Anthranilate and its derivatives are important basic chemicals for the synthesis of polyurethanes as well as various dyes and food additives. Today, anthranilate is mainly chemically produced from petroleum-derived xylene, but this shikimate pathway intermediate could be also obtained biotechnologically. In this study, Corynebacterium glutamicum was engineered for the microbial production of anthranilate from a carbon source mixture of glucose and xylose. First, a feedback-resistant 3-deoxy-arabinoheptulosonate-7-phosphate synthase from Escherichia coli, catalysing the first step of the shikimate pathway, was functionally introduced into C. glutamicum to enable anthranilate production. Modulation of the translation efficiency of the genes for the shikimate kinase (aroK) and the anthranilate phosphoribosyltransferase (trpD) improved product formation. Deletion of two genes, one for a putative phosphatase (nagD) and one for a quinate/shikimate dehydrogenase (qsuD), abolished by-product formation of glycerol and quinate. However, the introduction of an engineered anthranilate synthase (TrpEG) unresponsive to feedback inhibition by tryptophan had the most pronounced effect on anthranilate production. Component I of this enzyme (TrpE) was engineered using a biosensor-based in vivo screening strategy for identifying variants with increased feedback resistance in a semi-rational library of TrpE muteins. The final strain accumulated up to 5.9 g/L (43 mM) anthranilate in a defined CGXII medium from a mixture of glucose and xylose in bioreactor cultivations. We believe that the constructed C. glutamicum variants are not only limited to anthranilate production but could also be suitable for the synthesis of other biotechnologically interesting shikimate pathway intermediates or any other aromatic compound derived thereof.

Development of Corynebacterium glutamicum as a monoterpene production platform.

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.

De novo biosynthesis of 2'-fucosyllactose by bioengineered Corynebacterium glutamicum.

2'-Fucosyllactose (2'-FL) which is well-known human milk oligosaccharide was biotechnologically synthesized using engineered Corynebacterium glutamicum, a GRAS microbial workhorse. By construction of the complete de novo pathway for GDP-L-fucose supply and heterologous expression of Escherichia coli lactose permease and Helicobacter pylori α-1,2-fucosyltransferase, bioengineered C. glutamicum BCGW_TL successfully biosynthesized 0.25 g L-1 2'-FL from glucose. The additional genetic perturbations including the expression of a putative 2'-FL exporter and disruption of the chromosomal pfkA gene allowed C. glutamicum BCGW_cTTLEΔP to produce 2.5 g L-1 2'-FL batchwise. Finally, optimized fed-batch cultivation of the BCGW_cTTLEΔP using glucose, fructose, and lactose resulted in 21.5 g L-1 2'-FL production with a productivity of 0.12 g L-1 •h, which were more than 3.3 times higher value relative to the batch culture of the BCGW_TL. Conclusively, it would be a groundwork to adopt C. glutamicum for biotechnological production of other food additives including human milk oligosaccharides.

Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization.

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

Combining protein and metabolic engineering strategies for high-level production of L-theanine in Corynebacterium glutamicum.

L-theanine is a natural non-protein amino acid with wide applications. Thus, a high yield of L-theanine production is required on an industrial scale. Herein, an efficient L-theanine-producing strain of Corynebacterium glutamicum was constructed by combining protein and metabolic engineering. Firstly, a γ-glutamylmethylamide synthetase from Paracoccus aminovorans (PaGMAS) was isolated and engineered by computer-aided design, the resulting mutant E179K/N105R improved L-theanine yield by 36.61 %. Subsequently, to increase carbon flux towards L-theanine production, the gene ggt which degrades L-theanine, the gene alaT which participated in L-alanine synthesis, and the gene NCgl1221 which encodes glutamate-exporting protein were deleted. Finally, ppk gene was overexpressed to enhance intracellular ATP production. The reprogramed strain produced 44.12 g/L L-theanine with a yield of 57.11 % and productivity of 1.16 g/L/h, which is the highest L-theanine titer reported by Corynebacterium glutamicum. This study provides an efficient and economical biosynthetic pathway for the industrial production of L-theanine.

Structural and Biochemical Analysis of 3-Dehydroquinate Dehydratase from Corynebacterium glutamicum.

Dehydroquinate dehydratase (DHQD) catalyzes the conversion of 3-dehydroquinic acid (DHQ) into 3-dehydroshikimic acid in the mid stage of the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids and folates. Here, we report two the crystal structures of type II DHQD (CgDHQD) derived from Corynebacterium glutamicum, which is a widely used industrial platform organism. We determined the structures for CgDHQDWT with the citrate at a resolution of 1.80Å and CgDHQDR19A with DHQ complexed forms at a resolution of 2.00 Å, respectively. The enzyme forms a homododecamer consisting of four trimers with three interfacial active sites. We identified the DHQ-binding site of CgDHQD and observed an unusual binding mode of citrate inhibitor in the site with a half-opened lid loop. A structural comparison of CgDHQD with a homolog derived from Streptomyces coelicolor revealed differences in the terminal regions, lid loop, and active site. Particularly, CgDHQD, including some Corynebacterium species, possesses a distinctive residue P105, which is not conserved in other DHQDs at the position near the 5-hydroxyl group of DHQ. Replacements of P105 with isoleucine and valine, conserved in other DHQDs, caused an approximately 70% decrease in the activity, but replacement of S103 with threonine (CgDHQDS103T) caused a 10% increase in the activity. Our biochemical studies revealed the importance of key residues and enzyme kinetics for wild type and CgDHQDS103T, explaining the effect of the variation. This structural and biochemical study provides valuable information for understanding the reaction efficiency that varies due to structural differences caused by the unique sequences of CgDHQD.