Overexpression of the phosphofructokinase encoding gene is crucial for achieving high production of D-lactate in Corynebacterium glutamicum under oxygen deprivation

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

Applied and Environmental Microbiology ◽

10.1128/aem.02806-12 ◽

2012 ◽

Vol 79 (4) ◽

pp. 1250-1257 ◽

Cited By ~ 73

Author(s):

Satoshi Hasegawa ◽

Masako Suda ◽

Kimio Uematsu ◽

Yumi Natsuma ◽

Kazumi Hiraga ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Glucose Consumption ◽

Resistant Mutant ◽

Oxygen Deprivation ◽

High Yield ◽

Acetohydroxy Acid Synthase ◽

Succinate Production ◽

Content Type ◽

High Productivity ◽

By Products

ABSTRACTWe previously demonstrated efficientl-valine production by metabolically engineeredCorynebacterium glutamicumunder oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis ofl-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase fromLysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase geneldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress thel-valine yield. Eliminating these by-products therefore was deemed key to improving thel-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase geneppc, succinate production was effectively suppressed, but both glucose consumption andl-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, includinggapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higherl-valine yield and productivity. Deleting the alanine transaminase geneavtAsuppressed alanine production. The resultant strain produced 1,280 mMl-valine at a yield of 88% mol mol of glucose−1after 24 h under oxygen deprivation, a vastly improved yield over our previous best.

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