Acetohydroxy Acid Isomeroreductase Research Articles

Highly efficient production of L-valine by multiplex metabolic engineering of Corynebacterium glutamicum

As a branched chain amino acid, L-valine is widely used in the medicine and feed sectors. In this study, a microbial cell factory for efficient production of L-valine was constructed by combining various metabolic engineering strategies. First, precursor supply for L-valine biosynthesis was enhanced by strengthening the glycolysis pathway and weakening the metabolic pathway of by-products. Subsequently, the key enzyme in the L-valine synthesis pathway, acetylhydroxylate synthase, was engineered by site-directed mutation to relieve the feedback inhibition of the engineered strain. Moreover, promoter engineering was used to optimize the gene expression level of key enzymes in L-valine biosynthetic pathway. Furthermore, cofactor engineering was adopted to change the cofactor preference of acetohydroxyacid isomeroreductase and branched-chain amino acid aminotransferase from NADPH to NADH. The engineered strain C. glutamicum K020 showed a significant increase in L-valine titer, yield and productivity in 5 L fed-batch bioreactor, up to 110 g/L, 0.51 g/g and 2.29 g/(L‧h), respectively.

High-level and -yield production of L-leucine in engineered Escherichia coli by multistep metabolic engineering

L-leucine is an essential amino acid widely used in food and pharmaceutical industries. However, the relatively low production efficiency limits its large-scale application. In this study, we rationally developed an efficient L-leucine-producing Escherichia coli strain. Initially, the L-leucine synthesis pathway was enhanced by overexpressing feedback-resistant 2-isopropylmalate synthase and acetohydroxy acid synthase both derived from Corynebacterium glutamicum, along with two other native enzymes. Next, the pyruvate and acetyl-CoA pools were enriched by deleting competitive pathways, employing the nonoxidative glycolysis pathway, and dynamically modulating the citrate synthase activity, which significantly promoted the L-leucine production and yield to 40.69 g/L and 0.30 g/g glucose, respectively. Then, the redox flux was improved by substituting the native NADPH-dependent acetohydroxy acid isomeroreductase, branched chain amino acid transaminase, and glutamate dehydrogenase with their NADH-dependent equivalents. Finally, L-leucine efflux was accelerated by precise overexpression of the exporter and deletion of the transporter. Under fed-batch conditions, the final strain LXH-21 produced 63.29 g/L of L-leucine, with a yield and productivity of 0.37 g/g glucose and 2.64 g/(L h), respectively. To our knowledge, this study achieved the highest production efficiency of L-leucine to date. The strategies presented here will be useful for engineering E. coli strains for producing L-leucine and related products on an industrial scale.

Molecular Characterization of Germin-like Protein Genes in Zea mays (ZmGLPs) Using Various In Silico Approaches.

Germin (GER) and germin-like proteins (GLPs) play an important role in various plant processes. Zea mays contains 26 germin-like protein genes (ZmGLPs) located on chromosomes 2, 4, and 10; most of which are functionally unexplored. The present study aimed to characterize all ZmGLPs using the latest computational tools. All of them were studied at a physicochemical, subcellular, structural, and functional level, and their expression was predicted in plant development, against biotic and abiotic stresses using various in silico approaches. Overall, ZmGLPs showed greater similarity in their physicochemical properties, domain architecture, and structure, mostly localized in the cytoplasmic or extracellular regions. Phylogenetically, they have a narrow genetic background with a recent history of gene duplication events on chromosome 4. Functional analysis revealed novel enzymatic activities of phosphoglycolate phosphatase, adenosylhomocysteinase, phosphoglycolate phosphatase-like, osmotin/thaumatin-like, and acetohydroxy acid isomeroreductase largely mediated by disulfide bonding. Expression analysis revealed their crucial role in the root, root tips, crown root, elongation and maturation zones, radicle, and cortex with the highest expression being observed during germination and at the maturity levels. Further, ZmGLPs showed strong expression against biotic (Aspergillus flavus, Colletotrichum graminicola, Cercospora zeina, Fusarium verticillioides, and Fusarium virguliforme) while limited expression was noted against abiotic stresses. Concisely, our results provide a platform for additional functional exploration of the ZmGLP genes against various environmental stresses.

Identification of novel smORFs and microprotein acting in response to rehydration of Nostoc flagelliforme.

Nostoc flagelliforme, a terrestrial cyanobacterium spread throughout arid and semi-arid areas, has been long known for its outstanding adaptability to extremely dry conditions. This microorganism is able to recover biological activities within hours after months of anhydrobiosis state, attracting investigation through proteomic analysis. Except for canonical proteome, microproteins encoded by small ORFs (smORFs) have recently been regarded as indispensable participants in metabolic processes. However, the involvement of smORFs in Nostoc flagelliforme remains unknown. Here we first constructed a smORF database in Nostoc flagelliforme using bioinformatic prediction, resulting in 6072 novel smORFs. Then LS-MS/MS analysis was applied to identify expression patterns of microproteins and seek smORFs and their encoded microprotein playing a role during rehydration. In total, 18 novel microproteins were mined based on a smORF searching strategy combined with three proteomic assays, of which five were annotated as ribosomal proteins, one as RNA polymerase subunit, and one as acetohydroxy acid isomeroreductase. We also suggested the possible functions of smORFs according to their expression pattern and discovered two neighboring and homologous smORFs. All these results will expand our knowledge of smORFs-encoded microproteins and their relation to the stress response of extremophilic microorganisms. This article is protected by copyright. All rights reserved.

Expressing 2-keto acid pathway enzymes significantly increases photosynthetic isobutanol production

BackgroundCyanobacteria, photosynthetic microorganisms, are promising green cell factories for chemical production, including biofuels. Isobutanol, a four-carbon alcohol, is considered as a superior candidate as a biofuel for its high energy density with suitable chemical and physical characteristics. The unicellular cyanobacterium Synechocystis PCC 6803 has been successfully engineered for photosynthetic isobutanol production from CO2 and solar energy in a direct process.ResultsHeterologous expression of α-ketoisovalerate decarboxylase (KivdS286T) is sufficient for isobutanol synthesis via the 2-keto acid pathway in Synechocystis. With additional expression of acetolactate synthase (AlsS), acetohydroxy-acid isomeroreductase (IlvC), dihydroxy-acid dehydratase (IlvD), and alcohol dehydrogenase (Slr1192OP), the Synechocystis strain HX42, with a functional 2-keto acid pathway, showed enhanced isobutanol production reaching 98 mg L−1 in short-term screening experiments. Through modulating kivdS286T copy numbers as well as the composition of the 5′-region, a final Synechocystis strain HX47 with three copies of kivdS286T showed a significantly improved isobutanol production of 144 mg L−1, an 177% increase compared to the previously reported best producing strain under identical conditions.ConclusionsThis work demonstrates the feasibility to express heterologous genes with a combination of self-replicating plasmid-based system and genome-based system in Synechocystis cells. Obtained isobutanol-producing Synechocystis strains form the base for further investigation of continuous, long-term-photosynthetic isobutanol production from solar energy and carbon dioxide.Graphic abstract

High-yield production of L-valine in engineered Escherichia coli by a novel two-stage fermentation

L-valine is an essential amino acid and an important amino acid in the food and feed industry. The relatively low titer and low fermentation yield currently limit the large-scale application of L-valine. Here, we constructed a chromosomally engineered Escherichia coli to efficiently produce L-valine. First, the synthetic pathway of L-valine was enhanced by heterologous introduction of a feedback-resistant acetolactate acid synthase from Bacillus subtilis and overexpression of other two enzymes in the L-valine synthetic pathway. For efficient efflux of L-valine, an exporter from Corynebacterium glutamicum was subsequently introduced. Next, the precursor pyruvate pool was increased by knockout of GTP pyrophosphokinase and introduction of a ppGpp 3′-pyrophosphohydrolase mutant to facilitate the glucose uptake process. Finally, in order to improve the redox cofactor balance, acetohydroxy acid isomeroreductase was replaced by a NADH-preferring mutant, and branched-chain amino acid aminotransferase was replaced by leucine dehydrogenase from Bacillus subtilis. Redox cofactor balance enabled the strain to synthesize L-valine under oxygen-limiting condition, significantly increasing the yield in the presence of glucose. Two-stage fed-batch fermentation of the final strain in a 5 L bioreactor produced 84 g/L L-valine with a yield and productivity of 0.41 g/g glucose and 2.33 g/L/h, respectively. To the best of our knowledge, this is the highest L-valine titer and yield ever reported in E. coli. The systems metabolic engineering strategy described here will be useful for future engineering of E. coli strains for the industrial production of L-valine and related products.

Isobutanol production in Corynebacterium glutamicum: Suppressed succinate by-production by pckA inactivation and enhanced productivity via the Entner–Doudoroff pathway

On the basis of our previous studies of microbial L-valine production under oxygen deprivation, we developed isobutanol-producing Corynebacterium glutamicum strains. The artificial isobutanol synthesis pathway was composed of the first three steps of the L-valine synthesis pathway; and the subsequent Ehrlich Pathway: pyruvate was converted to 2-ketoisovalerate in the former reactions; and the 2-keto acid was decarboxylated into isobutyraldehyde, and subsequently reduced into isobutanol in the latter reactions. Although there exists redox cofactor imbalance in the overall reactions, i.e., NADH is generated via glycolysis whereas NADPH is required to synthesize isobutanol, it was resolved by taking advantage of the NAD-preferring mutant acetohydroxy acid isomeroreductase encoded by ilvCTM and the NAD-specific alcohol dehydrogenase encoded by adhA. Each enzyme activity to synthesize isobutanol was finely tuned by using two kinds of lac promoter derivatives. Efficient suppression of succinate by-production and improvement of isobutanol yield resulted from inactivation of pckA, which encodes phosphoenolpyruvate carboxykinase, whereas glucose consumption and isobutanol production rates decreased because of the elevated intracellular NADH/NAD+ ratio. On the other hand, introduction of the exogenous Entner–Doudoroff pathway effectively enhanced glucose consumption and productivity. Overexpression of phosphoenolpyruvate:carbohydrate phosphotransferase system specific to glucose and deletion of ilvE, which encodes branched-chain amino acid transaminase, further suppressed by-products and improved isobutanol productivity. Finally, the produced isobutanol concentration reached 280 mM at a yield of 84% (mol/mol glucose) in 24 h.

Improvement of l-Leucine Production in Corynebacterium glutamicum by Altering the Redox Flux.

The production of l-leucine was improved by the disruption of ltbR encoding transcriptional regulator and overexpression of the key genes (leuAilvBNCE) of the l-leucine biosynthesis pathway in Corynebacterium glutamicum XQ-9. In order to improve l-leucine production, we rationally engineered C. glutamicum to enhance l-leucine production, by improving the redox flux. On the basis of this, we manipulated the redox state of the cells by mutating the coenzyme-binding domains of acetohydroxyacid isomeroreductase encoded by ilvC, inserting NAD-specific leucine dehydrogenase, encoded by leuDH from Lysinibacillus sphaericus, and glutamate dehydrogenase encoded by rocG from Bacillus subtilis, instead of endogenous branched-chain amino acid transaminase and glutamate dehydrogenase, respectively. The yield of l-leucine reached 22.62 ± 0.17 g·L−1 by strain ΔLtbR-acetohydroxyacid isomeroreductase (AHAIR)M/ABNCME, and the concentrations of the by-products (l-valine and l-alanine) increased, compared to the strain ΔLtbR/ABNCE. Strain ΔLtbR-AHAIRMLeuDH/ABNCMLDH accumulated 22.87±0.31 g·L−1 l-leucine, but showed a drastically low l-valine accumulation (from 8.06 ± 0.35 g·L−1 to 2.72 ± 0.11 g·L−1), in comparison to strain ΔLtbR-AHAIRM/ABNCME, which indicated that LeuDH has much specificity for l-leucine synthesis but not for l-valine synthesis. Subsequently, the resultant strain ΔLtbR-AHAIRMLeuDHRocG/ABNCMLDH accumulated 23.31 ± 0.24 g·L−1 l-leucine with a glucose conversion efficiency of 0.191 g·g−1.

Isobutanol and 2-ketoisovalerate production by Klebsiella pneumoniae via a native pathway

Isobutanol is a valuable chemical and is considered a new generation biofuel. Construction of isobutanol synthesis pathways in bacteria is a hot topic in isobutanol production. Here, we show that an isobutanol synthesis pathway exists naturally in Klebsiella pneumoniae; however, this pathway is dormant in the wild-type bacterium. K. pneumoniae is a 2,3-butanediol producer, and the synthesis pathways of 2,3-butanediol, valine and isobutanol all start from condensation of two pyruvate molecules to yield α-acetolactate. Inactivation of α-acetolactate decarboxylase (encoded by budA) resulted in α-acetolactate flowing into the valine pathway, which led to synthesis of isobutanol and 2-ketoisovalerate (a precursor of isobutanol). ldhA (lactate dehydrogenase) deletion further increased the isobutanol and 2-ketoisovalerate production. In the first step of this pathway, BudB (α-acetolactate synthase) was identified as responsible for most of the α-acetolactate synthesis. Complementation of ilvBN or ilvIH (isoenzymes of budB) both resulted in a remarkable increase in 2-ketoisovalerate production. Thus, α-acetolactate formation is the rate-limiting step of 2-ketoisovalerate production. ilvC (acetohydroxy acid isomeroreductase) and ilvD (dihydroxy acid dehydratase) were identified responsible for 2-ketoisovalerate synthesis from α-acetolactate. ipdC, which encodes an indole-3-pyruvate decarboxylase, was identified responsible for most of the isobutyraldehyde formation from 2-ketoisovalerate, and isobutanol production was increased 15.7 fold in the ipdC complementation strain, with a final titer of 2.45g/L. Isobutanol dehydrogenase activity is distributed across multiple alcohol dehydrogenase enzymes expressed by K. pneumoniae. BudC, DhaT, DhaD and YqhD all had isobutanol dehydrogenase activity in vitro. YqhD uses NADPH as the coenzyme, while the other dehydrogenases use NADH. However, inactivating one or two of these dehydrogenases had no effect on isobutanol production in vivo with isobutyraldehyde as the substrate. These results reveal a novel method for biological production of isobutanol and 2-ketoisovalerate.

Improvement of lincomycin production by mutant selection and metabolic regulation

ABSTRACTLincomycin is a lincosamide antibiotic produced by Streptomyces lincolnensis. Through mutagenesis by ethylmethansulfonate (EMS) and ultraviolet (UV) irradiation repeatedly, M2 was picked out in plate with glutamine and propylproline orderly. In 50-L stirred bioreactor, the production of lincomycin, fermented by M2, was increased to 8136 u/ml under the optimal condition as compared to original strain S. lincolnensis 07–5 (6634 u/ml). Two-dimensional gel electrophoresis (2-D GE) and mass spectrometry (MS)-shown LmbG, LmbI, and acetohydroxy acid isomeroreductase were remarkably synthesized in M2. The gene lmbG and lmbI are responsible for methylation in the lincomycin biosynthetic cluster, while acetohydroxy acid isomeroreductase contributes to stronger metabolic capability. Finally, we obtained a better strain for industrial production.

Effective trapping of fruit flies with cultures of metabolically modified acetic acid bacteria.

Acetoin in vinegar is an attractant to fruit flies when combined with acetic acid. To make vinegar more effective in attracting fruit flies with increased acetoin production, Komagataeibacter europaeus KGMA0119 was modified by specific gene disruption of the acetohydroxyacid isomeroreductase gene (ilvC). A previously constructed mutant lacking the putative ligand-sensing region in the leucine-responsive regulatory protein (KeLrp, encoded by Kelrp) was also used. The ilvC and Kelrp disruptants (KGMA5511 and KGMA7203, respectively) produced greater amounts of acetoin (KGMA5511, 0.11%; KGMA7203, 0.13%) than the wild-type strain KGMA0119 (0.069%). KGMA7203 produced a trace amount of isobutyric acid (0.007%), but the other strains did not. These strains produced approximately equal amounts of acetic acid (0.7%). The efficiency of fruit fly attraction was investigated with cultured Drosophila melanogaster. D. melanogaster flies (approximately 1,500) were released inside a cage (2.5 m by 2.5 m by 1.5 m) and were trapped with a device containing vinegar and a sticky sheet. The flies trapped on the sticky sheet were counted. The cell-free supernatant from KGMA7203 culture captured significantly more flies (19.36 to 36.96% of released flies) than did KGMA0119 (3.25 to 11.40%) and KGMA5511 (6.87 to 21.50%) cultures. Contrastingly, a 0.7% acetic acid solution containing acetoin (0.13%) and isobutyric acid (0.007%), which mimicked the KGMA7203 supernatant, captured significantly fewer flies (0.88 to 4.57%). Furthermore, the KGMA0119 supernatant with additional acetoin (0.13%) and isobutyric acid (0.007%) captured slightly more flies than the original KGMA0119 supernatant but fewer than the KGMA7203 supernatant, suggesting that the synergistic effects of acetic acid, acetoin, isobutyric acid, and unidentified metabolites achieved the efficient fly trapping of the KGMA7203 supernatant.

Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16.

2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by L-valine (IC50=1.2 mM), L-isoleucine (IC50=2.3 mM), and L-leucine (IC50=5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (KM=10.5 μM) and is highly selective towards 2-ketobutyrate (R=140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for value-added materials.

Metabolic engineering of Corynebacterium glutamicum for 2-ketoisocaproate production

2-Ketoisocaproate (KIC) is used as a therapeutic agent, and a KIC-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered Corynebacterium glutamicum for the production of KIC from glucose by deletion of ltbR and ilvE, encoding the transcriptional repressor LtbR and transaminase B, respectively, and additional overexpression of ilvBNCD, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. The KIC-producing strain was improved by deletion of the methylcitrate synthase genes and by decreasing citrate synthase activity by exchange of the native promoter of the citrate synthase gene. In shake-flask fermentations under L-leucine limitation, the newly constructed strain C. glutamicum VB (pJC4ilvBNCD) produced 31 ± 2 mM (4.0 ± 0.3 g l(-1)) KIC and showed a product yield of about 0.26 ± 0.02 mol per mole (0.19 ± 0.01 g per gram) of glucose. As by-product, the strain formed about 33 mM 2-ketoisovalerate, which is a precursor of KIC. KIC production was further improved by additional expression of an isopropylmalate synthase allele (leuA (EC-G462D)), encoding an enzyme resistant towards L-leucine inhibition, and by addition of acetate as additional substrate. With glucose and acetate, the newly constructed strain produced 71 ± 3.2 mM (9.2 ± 0.4 g l(-1)) KIC with a yield of 0.24 ± 0.01 mol C (KIC) per mole C (in both substrates) and with nearly no 2-ketoisovalerate by-product formation (<2 mM). Investigating the activities and regulation of the native isopropylmalate synthase and dehydratase of C. glutamicum, we observed competitive and noncompetitive inhibition, respectively, by KIC.

Strain optimization for efficient isobutanol production using Corynebacterium glutamicum under oxygen deprivation

Microbial production of isobutanol is made difficult by the chemical's high cell toxicity. Corynebacterium glutamicum, inherently one of the more isobutanol-tolerant industrial microorganisms, exhibits unprecedented productivity under oxygen deprivation, potentially allowing for high productivity of such toxic chemicals as isobutanol. Here, we show that development of C. glutamicum strains proficient in isobutanol production depends not only on modulating the activity of 2-keto acid decarboxylase (KDC) and isobutanol dehydrogenase (IBDH) and suppressing by-product formation, but also on optimizing the production process to eschew product inhibition. Isobutanol production under oxygen deprivation reached 343 mM (3.2% v/v) in strain IBU5 expressing kivd (encoding KDC) under the control of ldhA promoter and adhP (encoding IBDH from Escherichia coli MG1655) under the control of gapA promoter. This productivity is double the previously reported best productivity of 1.6% (v/v) and exceeds the 2.5% (v/v) limit beyond which cell growth becomes too severely suppressed. Irrespective, a cumulative 56.5% improvement on yield was possible with the combined effects of disruption of the ppc gene, encoding phosphoenolpyruvate carboxylase (PEPC), use of a NAD⁺-specific mutant acetohydroxyacid isomeroreductase (AHAIR), and overexpression of select glycolytic genes. Using oleyl alcohol to continuously extract the isobutanol from reaction mixture and tripling the cell concentration in the reaction mixture to 60 g dry cell/L stretched the yield to 78.1% and volumetric productivity to 981 mM (9.1% v/v).

Engineering of Corynebacterium glutamicum for High-Yieldl-Valine Production under Oxygen Deprivation Conditions

We previously demonstrated efficient L-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of l-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the L-valine yield. Eliminating these by-products therefore was deemed key to improving theL-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and L-valine production dropped considerably due to the severely elevated intracellular NADH/NAD(+) ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher L-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM L-valine at a yield of 88% mol mol of glucose(-1) after 24 h under oxygen deprivation, a vastly improved yield over our previous best.

Improvement of the Redox Balance Increasesl-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

Production of L-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the L-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall L-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of L-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for L-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD(+) ratio significantly decreased, and glucose consumption and L-valine production drastically improved. Moreover, L-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD(+) ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for L-valine production under oxygen deprivation conditions. The L-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM L-valine after 24 h with a yield of 0.63 mol mol of glucose(-1), and the L-valine productivity reached 1,940 mM after 48 h.

Corynebacterium glutamicum Tailored for Efficient Isobutanol Production

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (Y(P/S)) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the Y(P/S) to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y(P/S), indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH + H+ to NADPH + H+. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h⁻¹, and showed an overall Y(P/S) of about 0.48 mol per mol of glucose in the production phase.

Escherichia coli W as a new platform strain for the enhanced production of L‐Valine by systems metabolic engineering

A less frequently employed Escherichia coli strain W, yet possessing useful metabolic characteristics such as less acetic acid production and high L-valine tolerance, was metabolically engineered for the production of L-valine. The ilvA gene was deleted to make more pyruvate, a key precursor for L-valine, available for enhanced L-valine biosynthesis. The lacI gene was deleted to allow constitutive expression of genes under the tac or trc promoter. The ilvBN(mut) genes encoding feedback-resistant acetohydroxy acid synthase (AHAS) I and the L-valine biosynthetic ilvCED genes encoding acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and branched chain amino acid aminotransferase, respectively, were amplified by plasmid-based overexpression. The global regulator Lrp and L-valine exporter YgaZH were also amplified by plasmid-based overexpression. The engineered E. coli W (ΔlacI ΔilvA) strain overexpressing the ilvBN(mut) , ilvCED, ygaZH, and lrp genes was able to produce an impressively high concentration of 60.7 g/L L-valine by fed-batch culture in 29.5 h, resulting in a high volumetric productivity of 2.06 g/L/h. The most notable finding is that there was no other byproduct produced during L-valine production. The results obtained in this study suggest that E. coli W can be a good alternative to Corynebacterium glutamicum and E. coli K-12, which have so far been the most efficient L-valine producer. Furthermore, it is expected that various bioproducts including other amino acids might be more efficiently produced by this revisited platform strain of E. coli.

Metabolic Engineering of Corynebacterium glutamicum for 2-Ketoisovalerate Production

2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ± 28 mM (21.8 ± 3.2 g liter(-1)) 2-ketoisovalerate and showed a product yield of about 0.47 ± 0.05 mol per mol (0.3 ± 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ± 0.6 mM (0.53 ± 0.07 g liter(-1)) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate.

Factors enhancing l-valine production by the growth-limited l-isoleucine auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB ilvNM13 (pECKAilvBNC)

Cell growth limitation is known to be an important condition that enhances L: -valine synthesis in Corynebacterium glutamicum recombinant strains with L: -isoleucine auxotrophy. To identify whether it is the limited availability of L: -isoleucine itself or the L: -isoleucine limitation-induced rel-dependent ppGpp-mediated stringent response that is essential for the enhancement of L: -valine synthesis in growth-limited C. glutamicum cells, we deleted the rel gene, thereby constructing a relaxed (rel (-) ) C. glutamicum DeltailvA DeltapanB Deltarel ilvNM13 (pECKAilvBNC) strain. Variations in enzyme activity and L: -valine synthesis in rel (+) and rel (-) strains under conditions of L: -isoleucine excess and limitation were investigated. A sharp increase in acetohydroxy acid synthase (AHAS) activity, a slight increase in acetohydroxyacid isomeroreductase (AHAIR) activity, and a dramatic increase in L: -valine synthesis were observed in both rel (+) and rel (-) cells exposed to L: -isoleucine limitation. Although the positive effect of induction of the stringent response on AHAS and AHAIR upregulation in cells was not confirmed, we found the stringent response to be beneficial for maintaining increased AHAS, dihydroxyacid dehydratase, and transaminase B activity and L: -valine synthesis in cells during the stationary growth phase.