Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions

Abstract

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.

Fig 1

Biosynthetic pathway of l-valine and by-products of C. glutamicum under oxygen deprivation. Enzymes whose genes were overexpressed are highlighted in black boxes, and enzymes whose genes were disrupted are marked with an X. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PEPC, phosphoenolpyruvate carboxylase; PC, pyruvate carboxylase; MDH, malate dehydrogenase; SDH, succinate dehydrogenase; NDH, NADH dehydrogenase; MQ, menaquinone; LDH, lactate dehydrogenase; AlaT, alanine transaminase encoded by alaT; AvtA, alanine transaminase encoded by avtA; PDH, pyruvate dehydrogenase; PQO, pyruvare:quinone oxidoreductase; CTF, CoA transferase; PTA, phosphotransacetylase; ACK, acetate kinase; AHAS, acetohydroxy acid synthase; AHAIR, acetohydroxy acid isomeroreductase (AHAIR*, NAD-preferring mutant); DHAD, dihydroxy acid dehydratase; TA, transaminase B; LeuDH, leucine dehydrogenase (L. sphaericus).

Fig 2

Relative activity of the wild-type AHAS (IlvBN) and the mutant (IlvBN^GM) in the presence of l-valine. Open circles, Val-4: IlvBN[plasmid]/IlvBN[chromosome]; gray diamonds, Val-5: IlvBN^GE[plasmid]/IlvBN[chromosome]; black squares, Val-6: IlvBN^GE[plasmid]/IlvBN^GE[chromosome] (the location of ilvBN [wild type or mutant] on each strain is shown in the brackets). The inset shows the relative activity of AHAS in the presence of lower concentrations of l-valine. The data represent averages from three independent experiments.

Fig 3

l-Valine production of Val-9 under oxygen deprivation. Black squares, l-valine; open squares, alanine; gray circles, lactate; gray triangles, acetate; gray diamonds, succinate; open circles, glucose. Each value was corrected for dilution caused by the addition of NH₃ solution and glucose throughout the reaction. The data represent averages from three independent experiments.