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 DeltaaceE 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 DeltaaceE 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 DeltaaceE(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.

FIG. 1.

The enzymes at the phosphoenolpyruvate-pyruvate-oxaloacetate node with the biosynthetic pathway of l-valine in C. glutamicum. Abbreviations: PK, pyruvate kinase; PCx, pyruvate carboxylase; PEP, phosphoenolpyruvate; PEPCx, phosphoenolpyruvate carboxylase; PEPCk, phosphoenolpyruvate carboxykinase; AK, acetate kinase; PTA phosphotransacetylase; DHAD, dihydroxyacid dehydratase; CoA, coenzyme A; TCA, tricarboxylic acid.

FIG. 2.

Representative batch fermentation of C. glutamicum ΔaceE on CGXII medium initially containing glucose (4%), acetate (1.5%). (A) ⋄, growth; ▪, glucose; □, acetate. (B) ▵, pyruvate; ○, l-alanine; •, l-valine; ×, l-threonine. Three independent fermentations were performed, with all three showing comparable results.

FIG. 3.

l-Valine accumulation during a representative batch cultivation of C. glutamicum ΔaceE(pJC4ilvBNCE) on CGXII medium initially containing glucose (4%), acetate (1%), and BHI (0.5%). ⋄, growth; ▪, glucose; □, acetate; ▵, pyruvate; •, l-valine. Three independent fermentations were performed, with all three showing comparable results.

FIG. 4.

l-Valine accumulation during a representative fed-batch fermentation of C. glutamicum ΔaceE(pJC4ilvBNCE) on CGXII medium initially containing glucose (4%), acetate (1%), and BHI (0.5%). ⋄, growth; ▪, glucose; □, acetate; ▵, pyruvate; •, l-valine. Three independent fed-batch fermentations were performed, with all three showing comparable results.