Metabolic engineering for improved production of ethanol by Corynebacterium glutamicum

Jojima, Toru; Noburyu, Ryoji; Sasaki, Miho; Tajima, Takahisa; Suda, Masako; Yukawa, Hideaki; Inui, Masayuki

doi:10.1007/s00253-014-6223-4

Cited by 72 publications

(45 citation statements)

References 30 publications

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“…Notably, in contrast to the ΔpfkB mutant that requires expensive fructose as a carbon source for cell growth to induce the enhanced productivity, the engineered strains that can use inexpensive glucose are cost-effective in commercial production. Although it has already been reported that overexpression of the glycolytic genes increases glucose consumption and productivity (11,15,49), we further demonstrate that overexpression of the PTS genes is also effective in C. glutamicum. These findings will be helpful in understanding the regulatory mechanisms of glycolytic flux and can be applicable to the improvement of microbial production of various kinds of useful chemicals.…”

Section: Resultsmentioning

confidence: 66%

“…Consequently, under anaerobic conditions, overexpression of GAPDH and perhaps of other glycolytic enzymes should be effective to enhance glycolytic flux. Indeed, we have demonstrated that glucose consumption and productivity were enhanced by overexpressing gapA (11), pyk, pfkA, pgi (49), and tpi (15) under oxygen deprivation, whereas gapA overexpression had no effect on the glucose consumption rate under aerobic conditions (11). In this study, overexpression of pgi, pfkA, tpi, gapA, and pgk increased oxygen-deprived glucose consumption and organic acid production as well (Fig.…”

Section: Resultsmentioning

confidence: 76%

“…Under these conditions, the glucose consumption rate becomes higher than that attained during aerobic cultivation (the Pasteur effect [8]), and improved product yields are additionally expected due to repression of carbon loss by cell growth and respiration. On the basis of these characteristics, we have demonstrated overproduction of organic acids (9,10), amino acids (11)(12)(13), and a biofuel (14,15) by engineered C. glutamicum R strains under oxygen deprivation.…”

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confidence: 99%

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Enhanced Glucose Consumption and Organic Acid Production by Engineered Corynebacterium glutamicum Based on Analysis of a pfkB1 Deletion Mutant

Hasegawa

Tanaka

Suda

et al. 2017

Appl Environ Microbiol

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In the analysis of a carbohydrate metabolite pathway, we found interesting phenotypes in a mutant strain of Corynebacterium glutamicum deficient in pfkB1, which encodes fructose-1-phosphate kinase. After being aerobically cultivated with fructose as a carbon source, this mutant consumed glucose and produced organic acid, predominantly L-lactate, at a level more than 2-fold higher than that of the wild-type grown with glucose under conditions of oxygen deprivation. This considerably higher fermentation capacity was unique for the combination of pfkB1 deletion and cultivation with fructose. In the metabolome and transcriptome analyses of this strain, marked intracellular accumulation of fructose-1-phosphate and significant upregulation of several genes related to the phosphoenolpyruvate:carbohydrate phosphotransferase system, glycolysis, and organic acid synthesis were identified. We then examined strains overexpressing several of the identified genes and demonstrated enhanced glucose consumption and organic acid production by these engineered strains, whose values were found to be comparable to those of the model pfkB1 deletion mutant grown with fructose. L-Lactate production by the ppc deletion mutant of the engineered strain was 2,390 mM (i.e., 215 g/liter) after 48 h under oxygen deprivation, which was a 2.7-fold increase over that of the wild-type strain with a deletion of ppc.IMPORTANCE Enhancement of glycolytic flux is important for improving microbiological production of chemicals, but overexpression of glycolytic enzymes has often resulted in little positive effect. That is presumably because the central carbon metabolism is under the complex and strict regulation not only transcriptionally but also posttranscriptionally, for example, by the ATP/ADP ratio. In contrast, we studied a mutant strain of Corynebacterium glutamicum that showed markedly enhanced glucose consumption and organic acid production and, based on the findings, identified several genes whose overexpression was effective in enhancing glycolytic flux under conditions of oxygen deprivation. These results will further understanding of the regulatory mechanisms of glycolytic flux and can be widely applied to the improvement of the microbial production of useful chemicals.KEYWORDS Corynebacterium glutamicum, fructose metabolism, fructose-1-phosphate kinase, organic acid production, overexpression of glycolytic genes, regulation of glycolytic flux C orynebacterium glutamicum has been used as a powerful workhorse in the industrial production of amino acids, such as L-glutamate and L-lysine (1, 2). This organism behaves rather similarly to an aerobe because little growth is identified anaerobically without an alternative electron acceptor, nitrate (3, 4). In contrast, under oxygen-deprived conditions, C. glutamicum metabolizes glucose to L-lactate, succinate,

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Section: Resultsmentioning

confidence: 66%

Section: Resultsmentioning

confidence: 76%

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confidence: 99%

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Enhanced Glucose Consumption and Organic Acid Production by Engineered Corynebacterium glutamicum Based on Analysis of a pfkB1 Deletion Mutant

Hasegawa

Tanaka

Suda

et al. 2017

Appl Environ Microbiol

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“…orynebacterium glutamicum represents one of the most important biotechnological platform organisms and massively contributes to the industrial production of amino acids (1)(2)(3)(4)(5), but it has also been engineered, for instance, for the production of lower alcohols (6)(7)(8)(9), organic acids (10-13), diamines (14,15), and carotenoids (16)(17)(18). However, currently applied expression setups show only moderate performance regarding both precise control and expression homogeneity.…”

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confidence: 99%

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

Binder

Frohwitter

Mahr

et al. 2016

Appl Environ Microbiol

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Precise control of microbial gene expression resulting in a defined, fast, and homogeneous response is of utmost importance for synthetic bio(techno)logical applications. However, even broadly applied biotechnological workhorses, such as Corynebacterium glutamicum, for which induction of recombinant gene expression commonly relies on the addition of appropriate inducer molecules, perform moderately in this respect. Light offers an alternative to accurately control gene expression, as it allows for simple triggering in a noninvasive fashion with unprecedented spatiotemporal resolution. Thus, optogenetic switches are promising tools to improve the controllability of existing gene expression systems. In this regard, photocaged inducers, whose activities are initially inhibited by light-removable protection groups, represent one of the most valuable photoswitches for microbial gene expression. Here, we report on the evaluation of photocaged isopropyl-␤-D-thiogalactopyranoside (IPTG) as a light-responsive control element for the frequently applied tac-based expression module in C. glutamicum. In contrast to conventional IPTG, the photocaged inducer mediates a tightly controlled, strong, and homogeneous expression response upon short exposure to UV-A light. To further demonstrate the unique potential of photocaged IPTG for the optimization of production processes in C. glutamicum, the optogenetic switch was finally used to improve biosynthesis of the growth-inhibiting sesquiterpene (؉)-valencene, a flavoring agent and aroma compound precursor in food industry. The variation in light intensity as well as the time point of light induction proved crucial for efficient production of this toxic compound. IMPORTANCEOptogenetic tools are light-responsive modules that allow for a simple triggering of cellular functions with unprecedented spatiotemporal resolution and in a noninvasive fashion. Specifically, light-controlled gene expression exhibits an enormous potential for various synthetic bio(techno)logical purposes. Before our study, poor inducibility, together with phenotypic heterogeneity, was reported for the IPTG-mediated induction of lac-based gene expression in Corynebacterium glutamicum. By applying photocaged IPTG as a synthetic inducer, however, these drawbacks could be almost completely abolished. Especially for increasing numbers of parallelized expression cultures, noninvasive and spatiotemporal light induction qualifies for a precise, homogeneous, and thus higher-order control to fully automatize or optimize future biotechnological applications. Corynebacterium glutamicum represents one of the most important biotechnological platform organisms and massively contributes to the industrial production of amino acids (1-5), but it has also been engineered, for instance, for the production of lower alcohols (6-9), organic acids (10-13), diamines (14, 15), and carotenoids (16-18). However, currently applied expression setups show only moderate performance regarding both precise control and expression homogene...

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“…b Means and standard deviations of three replicates are given control by global regulators such as ArcA (Perrenoud and Sauer 2005). Overexpression of genes encoding glycolytic enzymes as applied in E. coli (Xie et al 2014;Seol et al 2015), B. subtilis ), or S. coelicolor (Borodina et al 2008) was also used for C. glutamicum, e.g., to improve the production of high value compounds like amino acids (Yamamoto et al 2012;Reddy and Wendisch 2014), alcohols (Jojima et al 2015;Yamamoto et al 2013), or the organic acid D-lactate (Tsuge et al 2015). Under these conditions or when a repressor gene of glycolysis genes was deleted, i.e., that of the DeoR-type transcriptional regulator SugR, high titers of L-lactate were formed as side effect of faster glucose utilization (Engels and Wendisch 2007;Engels et al 2008;Teramoto et al 2011).…”

Section: Discussionmentioning

confidence: 99%

Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid

Pérez-García

Peters‐Wendisch

Wendisch

2016

Appl Microbiol Biotechnol

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

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Metabolic engineering for improved production of ethanol by Corynebacterium glutamicum

Cited by 72 publications

References 30 publications

Enhanced Glucose Consumption and Organic Acid Production by Engineered Corynebacterium glutamicum Based on Analysis of a pfkB1 Deletion Mutant

Enhanced Glucose Consumption and Organic Acid Production by Engineered Corynebacterium glutamicum Based on Analysis of a pfkB1 Deletion Mutant

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid

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