Genetically engineered microorganism with high yield of L-isoleucine and method for producing L-isoleucine by fermentation

A method for producing L-isoleucine by fermentation, comprising:

• a) constructing a genetically engineered strain by transforming a host strain with a plasmid combination of pZA-ilvAIH and pZE-ilvCmD-ilvE-ygaZH;
• b) culturing the genetically engineered Escherichia coli strain in a fermentation medium;
• c) feeding threonine to the fermentation medium in a fed-batch manner, wherein the total amount of threonine added is 0.1% to 5% to produce L-isoleucine;
• and
• d) recovering L-isoleucine from the fermentation medium,
• wherein said genetically engineered strain comprises:
• i) a feedback-resistant ilvA gene encoding a threonine deaminase that is substantially released from feedback inhibition by L-isoleucine;
• ii) a feedback-resistant ilvIH gene encoding an acetohydroxy acid synthase Ill that is substantially released from feedback inhibition by L-isoleucine;
• iii) a mutant acetohydroxy acid reductoisomerase gene (ilvCm), wherein said mutant acetohydroxy acid reductoisomerase gene encodes an enzyme utilizing NADH as a cofactor;
• iv) a dihydroxy-acid dehydratase gene (ilvD);
• v) a branched-chain amino acid aminotransferase gene (ilvE);
• and
• vi) a branched-chain amino acid exporter gene (ygaZH),
• wherein, in step a),
• said host strain is an E. coli strain having deletions of IdhA, pta, poxB, adhE, pflB, mgsA, frdA, dadA, avtA, and ilvE genes,
• said plasmid pZA-ilvAIH is obtained by ligating the feedback-resistant ilvA gene and the feedback-resistant ilvIH gene into plasmid pZAlac;
• and
• said plasmid pZE-ilvCmD-ilvE-ygaZH is obtained by ligating ilvCm, ilvD, ilvE, and ygaZH genes into plasmid pZElac.