Linking Central Metabolism with Increased Pathway Flux: l -Valine Accumulation by Corynebacterium glutamicum

Abstract

ABSTRACT Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE , which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete l -valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since l -valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate. The same genes, ilvBNCD , overexpressed in an ilvA deletion mutant which is unable to synthesize l -isoleucine increased the concentration of this amino acid to 58 mM. A further dramatic increase was obtained when panBC was deleted, making the resulting mutant auxotrophic for d -pantothenate. When the resulting strain, C. glutamicum 13032ΔilvAΔpanBC with ilvBNCD overexpressed, was grown under limiting conditions it accumulated 91 mM l -valine. This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the l -valine biosynthesis pathway.