Metabolic Analysis of <i>Corynebacterium glutamicum</i> during Lactate and Succinate Productions under Oxygen Deprivation Conditions

ABSTRACTWe previously reported thatCorynebacterium glutamicumstrain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encodinggapA, 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 inC. glutamicumglucose metabolism under oxygen deprivation. In addition togapA, overexpressing pyruvate kinase-encodingpykand phosphofructokinase-encodingpfkenabled 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-encodinggpiin 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 thatgapAoverexpression 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.

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Glucose consumption rate critically depends on redox state in Corynebacterium glutamicum under oxygen deprivation

Applied Microbiology and Biotechnology ◽

10.1007/s00253-015-6540-2 ◽

2015 ◽

Vol 99 (13) ◽

pp. 5573-5582 ◽

Cited By ~ 14

Author(s):

Yota Tsuge ◽

Kimio Uematsu ◽

Shogo Yamamoto ◽

Masako Suda ◽

Hideaki Yukawa ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Redox State ◽

Consumption Rate ◽

Glucose Consumption ◽

Oxygen Deprivation ◽

Glucose Consumption Rate

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

Applied and Environmental Microbiology ◽

10.1128/aem.07056-11 ◽

2011 ◽

Vol 78 (3) ◽

pp. 865-875 ◽

Cited By ~ 79

Author(s):

Satoshi Hasegawa ◽

Kimio Uematsu ◽

Yumi Natsuma ◽

Masako Suda ◽

Kazumi Hiraga ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Glucose Consumption ◽

Redox Balance ◽

Oxygen Deprivation ◽

Limiting Factor ◽

Acetohydroxy Acid Synthase ◽

Content Type ◽

Production And Consumption ◽

Dehydrogenase Gene ◽

Valine Biosynthesis

ABSTRACTProduction ofl-valine under oxygen deprivation conditions byCorynebacterium glutamicumlacking the lactate dehydrogenase geneldhAand overexpressing thel-valine biosynthesis genesilvBNCDEwas repressed. This was attributed to imbalanced cofactor production and consumption in the overalll-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole ofl-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement forl-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded byilvCand introduction ofLysinibacillus sphaericusleucine dehydrogenase in place of endogenous transaminase B, encoded byilvE. The intracellular NADH/NAD+ratio significantly decreased, and glucose consumption andl-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 forl-valine production under oxygen deprivation conditions. Thel-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 byilvBN. The resultant strain produced 1,470 mMl-valine after 24 h with a yield of 0.63 mol mol of glucose−1, and thel-valine productivity reached 1,940 mM after 48 h.

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