Construction of an l-isoleucine overproducing strain of escherichia coli K-12

Abstract

The genes for a threonine deaminase that is resistant to feedback inhibition by L-Isoleucine and for an active acetohydroxyacid synthase II were introduced by a plasmid into a L-threonine-producing recombinant strain of Escherichia coli K-12. Analysis of culture broth of the strain using 13C nuclear magnetic resonance suggested that α, β-dihydroxy-β-methylvalerate (DHMV) and α-keto-β-methylvalerate (KMV), the third and the fourth intermediates in the L-Isoleucine biosynthetic pathway from L-threonine, respectively, accumulated in the medium in amounts comparable to that of L-Isoleucine. The ratio of accumulated L-Isoleucine:DHMV:KMV were approximately 2:1:1. The concentration of accumulated L-Isoleucine increased by twofold after the additional introduction of the genes for dihyroxyacid dehydratase (DH) and transaminase-B (TA-B), and the intermediates no longer accumulated. The resultant strain TVD5 accumulated 10 g/l of L-Isoleucine from 40 g/l of glucose. © 1999, Taylor & Francis Group, LLC. All rights reserved.