l -Valine Production with Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum was engineered for the production of L-valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum ⌬aceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amounts of pyruvate, L-alanine, and L-valine from glucose as the sole carbon source. Lactate or acetate was not formed. Plasmid-bound overexpression of ilvBNCE in C. glutamicum ⌬aceE resulted in an approximately 10-fold-lower intracellular pyruvate concentration (2.3 mM) and a shift of the extracellular product pattern from pyruvate and L-alanine towards L-valine. In fed-batch fermentations at high cell densities and an excess of glucose, C. glutamicum ⌬aceE(pJC4ilvBNCE) produced up to 210 mM L-valine with a volumetric productivity of 10.0 mM h ؊1 (1.17 g l ؊1 h ؊1 ) and a maximum yield of about 0.6 mol per mol (0.4 g per g) of glucose.

Conserved Pyridoxal Protein That Regulates Ile and Val Metabolism

“…A decrease in CoA availability is assumed to prevent the efficient conversion of pyruvate and 2-ketobutyrate to acetyl-CoA and propionyl-CoA, respectively, thereby leading to pyruvate and 2-ketobutyrate accumulation. A similar situation was observed by Blombach et al and Valle et al (28,29). They recently reported that the inactivation of pyruvate dehydrogenase (⌬aceE), which catalyzes the transformation of pyruvate into acetyl-CoA, led to pyruvate accumulation and L-Val excretion in Corynebacterium glutamicum and E. coli (28,29).…”

Genetic analysis of pathway regulation for enhancing branched‐chain amino acid biosynthesis in plants

“…There have only been a few reports of attempts to engineer increased valine content in bacteria. These studies in bacteria either increased the pathway flux to valine (Radmacher et al, 2002;Blombach et al, 2007), utilized mutated valine-tolerant RSUs to circumvent end-product inhibition (Elisakova et al, 2005), or combined both strategies (Park et al, 2007).…”

Enhanced Valine Production inCorynebacterium glutamicumwith Defective H+-ATPase and C-Terminal Truncated Acetohydroxyacid Synthase

“…In addition, valine production using a pyruvate dehydrogenase knockout mutant of C. glutamicum was reported recently. 8,9) According to these reports, C. glutamicum with multiple gene-knockouts (pyruvate: quinone oxidoreductase, phosphoglucose isomerase, and pyruvate dehydrogenase gene deletion) and overexpressed ilvBNCE, produced up to 410 mM valine. A combination of the pyruvate dehydrogenase gene knockout and an ATPase-defective mutation might also be effective in improving valine production.…”

“…7) Overexpression of wild-type ilvBNCE has also been reported to be effective in the production of valine. 8,9) Protein engineering studies of AHAS from Escherichia coli has emphasized the application of C-terminal truncations of its regulatory subunit for reducing the feedback inhibition of this enzyme. [10][11][12] Based on these studies, we constructed a feedback-insensitive AHAS gene with C-terminal truncation of the regulatory subunit (ilvN) to investigate valine production in C. glutamicum.…”