Structural variations between small alarmone hydrolase dimers support different modes of regulation of the stringent response.

The bacterial stringent response involves wide-ranging metabolic reprogramming aimed at increasing long-term survivability during stress conditions. One of the hallmarks of the stringent response is the production of a set of modified nucleotides, known as alarmones, which affect a multitude of cellular pathways in diverse ways. Production and degradation of these molecules depend on the activity of enzymes from the RelA/SpoT homologous family, which come in both bifunctional (containing domains to both synthesize and hydrolyze alarmones) and monofunctional (consisting of only synthetase or hydrolase domain) variants, of which the structure, activity, and regulation of the bifunctional RelA/SpoT homologs have been studied most intensely. Despite playing an important role in guanosine nucleotide homeostasis in particular, mechanisms of regulation of the small alarmone hydrolases (SAHs) are still rather unclear. Here, we present crystal structures of SAH enzymes from Corynebacterium glutamicum (RelHCg) and Leptospira levettii (RelHLl) and show that while being highly similar, structural differences in substrate access and dimer conformations might be important for regulating their activity. We propose that a varied dimer form is a general property of the SAH family, based on current structural information as well as prediction models for this class of enzymes. Finally, subtle structural variations between monofunctional and bifunctional enzymes point to how these different classes of enzymes are regulated.

Effect of Clp protease from Corynebacterium glutamicum on heterologous protein expression.

The protease present in a host may reduce the yield and biological activity of heterologous proteins. In this study, we used protease overexpression and deletion strategies to examine the effect of the Clp protease system in Corynebacterium glutamicum on the recombinant protein and to produce a highly efficient heterologous protein expression host. In this study, we identified seven genes in the Clp protease family in Corynebacterium glutamicum ATCC 13032 through bioinformatics analysis, and studied their effects on the enhanced green fluorescent protein (EGFP) reporter protein. The fluorescence intensity of the knockout strain was significantly higher, and the effect of the clpS deletion strain was the most obvious. To verify the universal effect of the lack of clpS, the excellent industrial strain C. glutamicum 1.15647 was transformed to form recombinant 15647-ΔclpS. Based on the results, 15647-ΔclpS had a more significant effect on improving protein expression. Furthermore, recombinant human teriparatide (rhPTH) and variable domain of heavy chain of heavy-chain antibody (VHH) were selected to verify the universal applicability of the knockout strain for expressing heterologous proteins. Accordingly, we found that protease deficiency could increase the production of heterologous proteins. Finally, through a large-scale fermentation, the 15647-ΔclpS strain was used to produce VHH. Its yield was approximately 530 mg/L, which was 65% higher than that of WT-15647. In this study, a host that could effectively increase heterologous protein expression was successfully obtained.

Cg10062 Catalysis Forges a Link between Acetylenecarboxylic Acid and Bacterial Metabolism.

The reliance of biocatalysis on plant-derived carbon for the synthesis of fuels and chemicals places it in direct competition with food production for resources. A potential solution to this problem is development of a metabolic link between alternative carbon sources and bacterial metabolism. Acetylenecarboxylic acid, which can be synthesized from methane and carbon dioxide, could enable this connection. It was previously shown that the enzyme Cg10062 catalyzes hydration of acetylenecarboxylate to afford malonate semialdehyde. Subsequent hydration-dependent decarboxylation to form acetaldehyde (81%), which was also observed, limits its biocatalytic usefulness. Several Cg10062 variants including E114Q and E114D do not catalyze decarboxylation and provide malonate semialdehyde as the sole product, albeit with substantially reduced catalytic activity. To identify an efficient enzyme capable of catalyzing acetylenecarboxylate hydration without decarboxylation, we undertook a mechanistic investigation of Cg10062 using mutagenesis, kinetic characterization, and X-ray crystallography. Cg10062 is a member of the tautomerase superfamily of enzymes, characterized by their ß-α-ß protein fold and an N-terminal proline residue situated at the center of the enzyme active site. Along with Pro-1, five additional active site residues (His-28, Arg-70, Arg-73, Tyr-103, and Glu-114) are required for Cg10062 activity. Incubation of crystals of four catalytically slow variants of Cg10062 with acetylenecarboxylate resulted in atomic resolution structures of Pro-1 bound to a complete set of intermediates, fully elaborating the detailed mechanism of the enzyme and establishing the process to involve covalent catalysis. Further, the intermediate-bound E114D structure explains the mechanism governing decarboxylation suppression. Together, these studies provide the most detailed picture of the catalytic mechanism of a tautomerase enzyme to date.

Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids.

BACKGROUND: Branched chain amino acids (BCAAs) are widely applied in the food, pharmaceutical, and animal feed industries. Traditional chemical synthetic and enzymatic BCAAs production in vitro has been hampered by expensive raw materials, harsh reaction conditions, and environmental pollution. Microbial metabolic engineering has attracted considerable attention as an alternative method for BCAAs biosynthesis because it is environmentally friendly and delivers high yield. MAIN TEXT: Corynebacterium glutamicum (C. glutamicum) possesses clear genetic background and mature gene manipulation toolbox, and has been utilized as industrial host for producing BCAAs. Acetohydroxy acid synthase (AHAS) is a crucial enzyme in the BCAAs biosynthetic pathway of C. glutamicum, but feedback inhibition is a disadvantage. We therefore reviewed AHAS modifications that relieve feedback inhibition and then investigated the importance of AHAS modifications in regulating production ratios of three BCAAs. We have comprehensively summarized and discussed metabolic engineering strategies to promote BCAAs synthesis in C. glutamicum and offer solutions to the barriers associated with BCAAs biosynthesis. We also considered the future applications of strains that could produce abundant amounts of BCAAs. CONCLUSIONS: Branched chain amino acids have been synthesized by engineering the metabolism of C. glutamicum. Future investigations should focus on the feedback inhibition and/or transcription attenuation mechanisms of crucial enzymes. Enzymes with substrate specificity should be developed and applied to the production of individual BCAAs. The strategies used to construct strains producing BCAAs provide guidance for the biosynthesis of other high value-added compounds.

Enhancement of large ring cyclodextrin production using pretreated starch by glycogen debranching enzyme from Corynebacterium glutamicum.

Synthesis of large-ring cyclodextrins (LR-CDs) in any significant amount has been challenging. This study enhanced the LR-CDs production by Thermus filiformis amylomaltase (TfAM) enzyme by starch pretreatment using glycogen debranching enzyme from Corynebacterium glutamicum (CgGDE). CgGDE pretreated tapioca starch gave LR-CD conversion of 31.2 ± 2.2%, compared with LR-CDs produced from non-treated tapioca starch (16.0 ± 2.4%). CgGDE pretreatment enhanced amylose content by approximately 30%. Notably, a shorter incubation time of 1 h is sufficient for CgGDE starch pretreatment to produce high LR-CD yield, compared with 6 h required for the commercial isoamylase. High-Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD) and Gel Permeable Chromatography (GPC) revealed that CgGDE is more efficient than the commercial isoamylase in debranching tapioca starch and gave lower molecular weight products. In addition, lower amount of by-products (linear oligosaccharides) were detected in cyclization reaction when using CgGDE-pretreated starch. In conclusion, CgGDE is a highly effective enzyme to promote LR-CD synthesis from starch with a shorter incubation time than the commercial isoamylase.

Evaluation of bacterial hosts for conversion of lignin-derived p-coumaric acid to 4-vinylphenol.

Hydroxycinnamic acids such as p-coumaric acid (CA) are chemically linked to lignin in grassy biomass with fairly labile ester bonds and therefore represent a straightforward opportunity to extract and valorize lignin components. In this work, we investigated the enzymatic conversion of CA extracted from lignocellulose to 4-vinylphenol (4VP) by expressing a microbial phenolic acid decarboxylase in Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The performance of the recombinant strains was evaluated in response to the substrate concentration in rich medium or a lignin liquor and the addition of an organic overlay to perform a continuous product extraction in batch cultures. We found that using undecanol as an overlay enhanced the 4VP titers under high substrate concentrations, while extracting > 97% of the product from the aqueous phase. C. glutamicum showed the highest tolerance to CA and resulted in the accumulation of up to 187 g/L of 4VP from pure CA in the overlay with a 90% yield when using rich media, or 17 g/L of 4VP with a 73% yield from CA extracted from lignin. These results indicate that C. glutamicum is a suitable host for the high-level production of 4VP and that further bioprocess engineering strategies should be explored to optimize the production, extraction, and purification of 4VP from lignin with this organism.

Polar Growth in Corynebacterium glutamicum Has a Flexible Cell Wall Synthase Requirement.

Members of the Corynebacterineae suborder of bacteria, including major pathogens such as Mycobacterium tuberculosis, grow via the insertion of new cell wall peptidoglycan (PG) material at their poles. This mode of elongation differs from that used by Escherichia coli and other more well-studied model organisms that grow by inserting new PG at dispersed sites along their cell body. Dispersed cell elongation is known to strictly require the SEDS-type PG synthase called RodA, whereas the other major class of PG synthases called class A penicillin-binding proteins (aPBPs) are not required for this mode of growth. Instead, they are thought to be important for maintaining the integrity of the PG matrix in organisms growing by dispersed elongation. In contrast, based on prior genetic studies in M. tuberculosis and related members of the Corynebacterineae suborder, the aPBPs are widely believed to be essential for polar growth, with RodA being dispensable. However, polar growth has not been directly assessed in mycobacterial or corynebacterial mutants lacking aPBP-type PG synthases. We therefore investigated the relative roles of aPBPs and RodA in polar growth using Corynebacterium glutamicum as a model member of Corynebacterineae. Notably, we discovered that the aPBPs are dispensable for polar growth and that this growth mode can be mediated by either an aPBP-type or a SEDS-type enzyme functioning as the sole elongation PG synthase. Thus, our results reveal that the mechanism of polar elongation is fundamentally flexible and, unlike dispersed elongation, can be effectively mediated in C. glutamicum by either a SEDS-bPBP or an aPBP-type synthase. IMPORTANCE The Corynebacterineae suborder includes a number of major bacterial pathogens. These organisms grow by polar extension unlike most well-studied model bacteria, which grow by inserting wall material at dispersed sites along their length. A better understanding of polar growth promises to uncover new avenues for targeting mycobacterial and corynebacterial infections. Here, we investigated the roles of the different classes of cell wall synthases for polar growth using Corynebacterium glutamicum as a model. We discovered that the polar growth mechanism is surprisingly flexible in this organism and, unlike dispersed synthesis, can function using either of the two known types of cell wall synthase enzymes.

Electrocatalytic evidence of the diversity of the oxygen reaction in the bacterial bd oxidase from different organisms.

Cytochrome bd oxidase is a bacterial terminal oxygen reductase that was suggested to enable adaptation to different environments and to confer resistance to stress conditions. An electrocatalytic study of the cyt bd oxidases from Escherichia coli, Corynebacterium glutamicum and Geobacillus thermodenitrificans gives evidence for a different reactivity towards oxygen. An inversion of the redox potential values of the three hemes is found when comparing the enzymes from different bacteria. This inversion can be correlated with different protonated glutamic acids as evidenced by reaction induced FTIR spectroscopy. The influence of the microenvironment of the hemes on the reactivity towards oxygen is discussed.

Increasement of O-acetylhomoserine production in Escherichia coli by modification of glycerol-oxidative pathway coupled with optimization of fermentation.

OBJECTIVE: O-acetylhomoserine (OAH) is an important platform chemical to produce high-valuable chemicals. To improve the production of O-acetylhomoserine from glycerol, the glycerol-oxidative pathway was investigated and the optimization of fermentation with crude glycerol was carried out. RESULTS: The glycerol-uptake system and glycerol-oxidative pathway were modified and O-acetyltransferase from Corynebacterium glutamicum was introduced into the engineered strain to produce O-acetylhomoserine. It was found that overexpression of glycerol 3-phosphate dehydrogenase improved the OAH production to 6.79 and 4.21 g/L from pure and crude glycerol, respectively. And the higher OAH production depending on higher level of transcription of glpD. Two-step statistical approach was employed to optimize the fermentation conditions. The significant effects of glycerol, ammonium chloride and yeast extract were screened applying Plackett-Burman design and were optimized further by employing the Response Surface Methodology. Under optimized conditions, the OAH production was up to 9.42 and 7.01 g/L when pure and crude glycerol were used in shake flask cultivations, respectively. CONCLUSIONS: The enzymatic step catalyzing the oxidation of glycerol through GlpD was the key step for OAH production, which served the foundation for realization of a consistent OAH production from crude glycerol in the future.

Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.

Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1-7 nt at the 5' end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the - 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.

Enhancement of fatty acid biosynthesis by exogenous acetyl-CoA carboxylase and pantothenate kinase in Escherichia coli.

OBJECTIVES: To establish a technique for efficient fatty acid production through enhancement of coenzyme A (CoA) biosynthesis and malonyl-CoA supply by introducing exogenous pantothenate kinase (coaA) and acetyl-CoA carboxylase (acc) in Escherichia coli. RESULTS: The expression of acc, obtained from Corynebacterium glutamicum, accumulated 2.2-fold more fatty acids in E. coli. The addition of coaA from Pseudomonas putaida or fatty acid synthase (fasA) from C. glutamicum resulted in a 3.1- and 3.6-fold increase in fatty acid synthesis in E. coli cells, which expressed acc and coaA, or acc and fasA, respectively. The transformants, simultaneously possessing all three genes, produced 5.6-fold more fatty acids. The strain possessing acc, coaA, and fasA stored 691 mg/L of fatty acids, primarily as phospholipids, inside the inner membrane after 72-h cultivation. In addition, 19% of the total CoA pool was occupied by malonyl-CoA. CONCLUSIONS: Increased malonyl-CoA significantly contributed to fatty acid production, and the effect was boosted by the expanded total CoA pool. Manipulation of the intracellular CoA species is effective for fatty acid production in E. coli.

In vitro reconstitution and characterization of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum.

Pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) are critical enzymes in central carbon metabolism. In Corynebacterium glutamicum, an unusual hybrid complex consisting of CgE1p (thiamine diphosphate-dependent pyruvate dehydrogenase, AceE), CgE2 (dihydrolipoamide acetyltransferase, AceF), CgE3 (dihydrolipoamide dehydrogenase, Lpd), and CgE1o (thiamine diphosphate-dependent 2-oxoglutarate dehydrogenase, OdhA) has been suggested. Here, we elucidated that the PDH-ODH hybrid complex in C. glutamicum probably consists of six copies of CgE2 in its core, which is rather compact compared with PDH and ODH in other microorganisms that have twenty-four copies of E2. We found that CgE2 formed a stable complex with CgE3 (CgE2-E3 subcomplex) in vitro, hypothetically comprised of two CgE2 trimers and four CgE3 dimers. We also found that CgE1o exists mainly as a hexamer in solution and is ready to form an active ODH complex when mixed with the CgE2-E3 subcomplex. Our in vitro reconstituted system showed CgE1p- and CgE1o-dependent inhibition of ODH and PDH, respectively, actively supporting the formation of the hybrid complex, in which both CgE1p and CgE1o associate with a single CgE2-E3. In gel filtration chromatography, all the subunits of CgODH were eluted in the same fraction, whereas CgE1p was eluted separately from CgE2-E3, suggesting a weak association of CgE1p with CgE2 compared with that of CgE1o. This study revealed the unique molecular architecture of the hybrid complex from C. glutamicum and the compact-sized complex would provide an advantage to determine the whole structure of the unusual hybrid complex.

Efficient synthesis of γ-glutamyl compounds by co-expression of γ-glutamylmethylamide synthetase and polyphosphate kinase in engineered Escherichia coli.

γ-Glutamyl compounds have unveiled their importance as active substances or precursors of pharmaceuticals. In this research, an approach for enzymatic synthesis of γ-glutamyl compounds was developed using γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays and polyphosphate kinase (PPK) from Corynebacterium glutamicum. GMAS and PPK were co-recombined in pETDuet-1 plasmid and co-expressed in E. coli BL21 (DE3), and the enzymatic properties of GMAS and PPK were investigated, respectively. Under the catalysis of the co-expression system, L-theanine was synthesized with 89.8% conversion when the substrate molar ratio of sodium glutamate and ethylamine (1:1.4) and only 2 mM ATP were used. A total of 14 γ-glutamyl compounds were synthesized by this one-pot method and purified by cation exchange resin and isoelectric point crystallization with a yield range from 22.3 to 72.7%. This study provided an efficient approach for the synthesis of γ-glutamyl compounds by GMAS and PPK co-expression system.

[Expression, purification and characterization of catalase from Corynebacterium glutamicum].

Catalase catalyzes the decomposition of H2O2 to H2O and O2, and has a wide range of industrial applications. However, most catalases used in the textile and paper industries are often subjected to high-alkaline challenges which makes it necessary to develop alkaline catalase. In this study, a catalase from Corynebacterium glutamicum was expressed in Escherichia coli, and the expression conditions were optimized. The recombinant catalase was purified by Ni-chelating affinity chromatography, and the recombinant enzyme was characterized. The optimal conditions of producing the recombinant catalase were: an IPTG concentration of 0.2 mmol/L, a culturing temperature of 25 °C and a culturing time of 11 h. The purified catalase had a specific activity of 55 266 U/mg, and it had a high activity in the pH range of 4.0 to11.5, with the highest activity at pH 11.0. When treated in pH 11.0 for 3 h, the enzyme retained 93% of its activity, indicating that the enzyme was qualified with a favorable stability under high-alkaline condition. The recombinant catalase had maximal activity at 30 °C, and showed a satisfactory thermal stability at a range of 25 °C to 50 °C. The apparent Km and Vmax values of purified catalase were 25.89 mmol/L and 185.18 mmol/(minmg), respectively. Besides, different inhibitors, such as sodium dodecyl sulfate (SDS), urea, NaN2, ß-mercaptoethanol, and EDTA had different degrees of inhibition on enzyme activity. The catalase from C. glutamicum shows high catalytic efficiency and high alkaline stability, suggesting its potential utilization in industrial production.

Characterization of the Corynebacterium glutamicum dehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid.

The dehydroshikimate dehydratase (DSD) from Corynebacterium glutamicum encoded by the qsuB gene is related to the previously described QuiC1 protein (39.9% identity) from Pseudomonas putida. Both QuiC1 and QsuB are two-domain bacterial DSDs. The N-terminal domain provides dehydratase activity, while the C-terminal domain has sequence identity with 4-hydroxyphenylpyruvate dioxygenase. Here, the QsuB protein and its N-terminal domain (N-QsuB) were expressed in the T7 system, purified and characterized. QsuB was present mainly in octameric form (60%), while N-QsuB had a predominantly monomeric structure (80%) in aqueous buffer. Both proteins possessed DSD activity with one of the following cofactors (listed in the order of decreasing activity): Co2+, Mg2+, Mn2+. The Km and kcat values for the QsuB enzyme (Km ~ 1 mM, kcat ~ 61 s-1) were two and three times higher than those for N-QsuB. 3,4-DHBA inhibited QsuB (Ki ~ 0.38 mM, Ki' ~ 0.96 mM) and N-QsuB (Ki ~ 0.69 mM) enzymes via mixed and noncompetitive inhibition mechanism, respectively. E. coli MG1655ΔaroEPlac‒qsuB strain produced three times more 3,4-DHBA from glucose in test tube fermentation than the MG1655ΔaroEPlac‒n-qsuB strain. The C-terminal domain activity towards 3,4-DHBA was not established in vitro. This domain was proposed to promote protein oligomerization for maintaining structural stability of the enzyme. The dimer formation of QsuB protein was more predictable (ΔG = ‒15.8 kcal/mol) than the dimerization of its truncated version N-QsuB (ΔG = ‒0.4 kcal/mol).

Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered Corynebacterium glutamicum.

Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L-1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates C. glutamicum, changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L-1. Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers.

Role of the unique, non-essential phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) in Corynebacterium glutamicum.

Bacterial lipoproteins are secreted proteins that are post-translationally lipidated. Following synthesis, preprolipoproteins are transported through the cytoplasmic membrane via the Sec or Tat translocon. As they exit the transport machinery, they are recognized by a phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), which converts them to prolipoproteins by adding a diacylglyceryl group to the sulfhydryl side chain of the invariant Cys+1 residue. Lipoprotein signal peptidase (LspA or signal peptidase II) subsequently cleaves the signal peptide, liberating the α-amino group of Cys+1, which can eventually be further modified. Here, we identified the lgt and lspA genes from Corynebacterium glutamicum and found that they are unique but not essential. We found that Lgt is necessary for the acylation and membrane anchoring of two model lipoproteins expressed in this species: MusE, a C. glutamicum maltose-binding lipoprotein, and LppX, a Mycobacterium tuberculosis lipoprotein. However, Lgt is not required for these proteins' signal peptide cleavage, or for LppX glycosylation. Taken together, these data show that in C. glutamicum the association of some lipoproteins with membranes through the covalent attachment of a lipid moiety is not essential for further post-translational modification.

Enhanced succinic acid production by Mannheimia employing optimal malate dehydrogenase.

Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH (CgMDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH (MsMDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate (ki of 67.4 and 588.9 µM for MsMDH and CgMDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L-1 of SA with the maximum productivity of 21.3 g L-1 h-1, demonstrating the importance of enzyme optimization in strain development.

Requirement of de novo synthesis of pyruvate carboxylase in long-term succinic acid production in Corynebacterium glutamicum.

Protein turnover through de novo synthesis is critical for sustainable cellular functions. We previously found that glucose consumption rate in Corynebacterium glutamicum under anaerobic conditions increased at temperature higher than the upper limit of growth temperature. Here, we showed that production of lactic and succinic acids increased at higher temperature for long-term (48 h) anaerobic reaction in metabolically engineered strains. At 42 °C, beyond the upper limit of growth temperature range, biomass-specific lactic acid production rate was 8% higher than that at 30 °C, the optimal growth temperature. In contrast, biomass-specific succinic acid production rate was highest at 36 °C, 28% higher than that at 30 °C, although the production at 42 °C was still 23% higher than that at 30 °C. As enzymes are usually unstable at high temperatures, we investigated whether protein turnover of metabolic enzymes is required for the production of lactic and succinic acids under these conditions. Interestingly, when de novo protein synthesis was inhibited by addition of chloramphenicol, after 6 h, only succinic acid production was inhibited. Because glycolytic enzymes are involved in both lactic and succinic acids synthesis, enzymes in the anaplerotic pathway and the tricarboxylic acid (TCA) cycle leading to succinic acid synthesis were likely to be responsible for its decreased production. Among the five enzymes examined, the specific activity of only pyruvate carboxylase was drastically decreased after 48 h at 42 °C. Thus, the de novo synthesis of pyruvate carboxylase is required for long-term production of succinic acid. Graphical abstract KEY POINTS: • Long-term reaction for organic acids can be improved at temperature beyond ideal growth conditions. • De novo synthesis of pyruvate carboxylase is required for long-term succinic acid production.

Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps.

Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second rate-limiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.