Construction of an L-isoleucine overproducing strain of Escherichia coli K-12.
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 alpha, beta-dihydroxy-beta-methylvalerate (DHMV) and alpha-keto-beta-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.
Hashiguchi
K
K
Fermentation & Biotechnology Laboratories, Ajinomoto Co. Inc., Kawasaki, Japan. bldhasiguti@te2.ajinomoto.co.jp
Takesada
H
H
Suzuki
E
E
Matsui
H
H
D000095:Acetolactate Synthase / Q000235:genetics* / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D007532:Isoleucine / Q000096:biosynthesis*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D013913:Threonine Dehydratase / Q000235:genetics* / Q000378:metabolism
D000596:Amino Acids; D003470:Culture Media; D007532:Isoleucine; D000095:Acetolactate Synthase; D013913:Threonine Dehydratase