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

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 13032⌬ilvA⌬panBC 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.

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

“…Still, glucose feeding, aeration, pH, and the medium components appeared to play important roles in controlling the yield of valine. Further genetic engineering involving ilvA knockout, ilvD introduction, and panBC knockouts seem to be useful for more efficient valine production, as reported previously 27,28) and might be applicable in combination with our results (Fig. 1).…”

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).…”

Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence ofCorynebacterium glutamicum

“…Functionally identified ATs of this organism include the N-succinyl-2,6-diaminopimelate AT (dapC) involved in the synthesis of L-lysine, of which 650,000 tonnes per year (t/y) are produced with C. glutamicum (5), as well as ilvE, encoding the branched-chain AT necessary for L-isoleucine production (26). Mutant studies further identified the genes (and respective enzymes) bioA (adenosylmethionine-8-amino-7-oxononanoate aminotransferase) (10), argD (N-acetyl-ornithine AT) (29), and pat and pdxR (involved in aromatic amino acid and pyridoxal-5Ј-phosphate synthesis, respectively) (19).…”