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Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.

ABSTRACT: We previously reported that Corynebacterium glutamicum strain ?ldhA?ppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159-165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD(+) ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.

SUBMITTER: Yamamoto S

PROVIDER: S-EPMC3370556 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.

Yamamoto Shogo S Gunji Wataru W Suzuki Hiroaki H Toda Hiroshi H Suda Masako M Jojima Toru T Inui Masayuki M Yukawa Hideaki H

Applied and environmental microbiology 20120413 12

We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159-165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herei ...[more]

PMID: 22504802

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Project description:Pyruvate kinase (Pyk) catalyzes the generation of pyruvate and ATP in glycolysis and functions as a key switch in the regulation of carbon flux distribution. Both the substrates and products of Pyk are involved in the tricarboxylic acid cycle, anaplerosis and energy anabolism, which places Pyk at a primary metabolic intersection. Pyks are highly conserved in most bacteria and lower eukaryotes. Corynebacterium glutamicum is an industrial workhorse for the production of various amino acids and organic acids. Although C. glutamicum was assumed to possess only one Pyk (pyk1, NCgl2008), NCgl2809 was annotated as a pyruvate kinase with an unknown role.Here, we identified that NCgl2809 was a novel pyruvate kinase (pyk2) in C. glutamicum. Complementation of the WT?pyk1?pyk2 strain with the pyk2 gene restored its growth on D-ribose, which demonstrated that Pyk2 could substitute for Pyk1 in vivo. Pyk2 was co-dependent on Mn2+ and K+ and had a higher affinity for ADP than phosphoenolpyruvate (PEP). The catalytic activity of Pyk2 was allosterically regulated by fructose 1,6-bisphosphate (FBP) activation and ATP inhibition. Furthermore, pyk2 and ldhA, which encodes L-lactate dehydrogenase, were co-transcribed as a bicistronic mRNA under aerobic conditions and pyk2 deficiency had a slight effect on the intracellular activity of Pyk. However, the mRNA level of pyk2 in the wild-type strain under oxygen deprivation was 14.24-fold higher than that under aerobic conditions. Under oxygen deprivation, pyk1 or pyk2 deficiency decreased the generation of lactic acid, and the overexpression of either pyk1 or pyk2 increased the production of lactic acid as the activity of Pyk increased. Fed-batch fermentation of the pyk2-overexpressing WT?pyk1 strain produced 60.27?±?1.40 g/L of lactic acid, which was a 47% increase compared to the parent strain under oxygen deprivation.Pyk2 functioned as a pyruvate kinase and contributed to the increased level of Pyk activity under oxygen deprivation.

Project description:To ensure economic competitiveness, bioprocesses should achieve maximum productivities enabled by high growth rates (?) and equally high substrate consumption rates (qS) as a prerequisite of sufficient carbon-to-product conversion. Both traits were investigated and improved via bioprocess engineering approaches studying the industrial work horse Corynebacterium glutamicum. Standard minimal medium CGXII with glucose as sole carbon source was supplemented with complex brain-heart-infusion (BHI) or amino acid (AA) cocktails. Maximum ? of 0.67 h-1 was exclusively observed in 37 g BHI L-1 whereas only minor growth stimulation was found after AA supplementation (? = 0.468 h-1). Increasing glucose consumption rates (qGlc) were solely observed in certain dosages of BHI (1-10 g L-1), while 37 g BHI L-1 and AA addition revealed qGlc below the reference experiments. Moreover, BHI supplementation revealed Monod-type saturation kinetics of ? (KBHI = 2.73 g BHI L-1) referring to the preference of non-AAs as key boosting nutrients. ATP-demands under reference, 1 g BHI L-1, and AA conditions were nearly constant but halved in BHI concentrations above 5 g L-1 reflecting the energetic advantage of consuming complex nutrient components in addition to "simple" building blocks such as AAs. Furthermore, C. glutamicum revealed maximum biomass per carbon yields of about 18 gCDW C-mol-1 irrespective of the medium. In AA supplementation experiments, simultaneous uptake of 17 AAs was observed, maximum individual consumption rates determined, and L-asparagine and L-glutamine were distinguished as compounds with the highest consumption rates. Employment of the expanded stoichiometric model iMG481 successfully reproduced experimental results and revealed the importance of C. glutamicum's transaminase network to compensate needs of limiting AA supply. Model-based sensitivity studies attributed the highest impact on ? to AAs with high ATP and NADPH demands such as L-tryptophan or L-phenylalanine.

Dataset Information

Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.

Publications

Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.

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