Construction of an L-isoleucine overproducing strain of Escherichia coli K-12

The genes for a threonine deaminase that is resistant to feedback inhibitio n by L-isoleucine and for an active acetohydroxyacid synthase II were intro duced by a plasmid into a L-threonine-producing recombinant strain of Esche richia coli K-12. Analysis of culture broth of the strain using C-13 nuclea r magnetic resonance suggested that alpha, beta-dihydroxy-beta-methylvalera te (DHMV) and alpha-keto-beta-methylvalerate (KMV), the third and the fourt h 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 approximate ly 2:1:1. The concentration of accumulated L-isoleucine increased by twofol d after the additional introduction of the genes for dihyroxyacid dehydrata se (DH) and transaminase-B (TA-B), and the intermediates no longer accumula ted. The resultant strain TVD5 accumulated 10 g/l of L-isoleucine from 40 g /l of glucose.