Rational metabolic engineering of Escherichia coli for L-isoleucine overproduction

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Jae Ho Shin, Ji Young Kim, Jin Hwan Park, and Sang Yup Lee

Korea advanced institute of science and technology, South Korea

Amino acids are the building blocks of proteins and are essential in all organisms. L-isoleucine, one of the branched amino acids, cannot be synthesized in humans and must be ingested. It is thus relevant that this amino acid is highly demanded in the industries of dietary supplements, pharmaceuticals and cosmetics. Conventionally, amino acids are industrially produced by bacterial strains developed from rounds of random mutagenesis. However, the metabolism of such strains is difficult to elucidate and growth may not always be efficient. Thus, the present study was aimed at constructing a 100% genetically defined L-isoleucine producing strain. TH20, a genetically defined L-threonine overproducing strain, was used as the starting strain, as L-isoleucine is synthesized from L-threonine after five enzymatic steps in vivo. The thrABC (encoding L-threonine biosynthetic enzymes), engineered ilvA (encoding feedback-resistant threonine dehydratase), engineered ilvIH (encoding feedback-resistant acetohydroxy acid synthase ), and ygaZH (encoding branched-chain amino acid exporter) genes were amplified by plasmid-based overexpression. The ilvCED (encoding L-isoleucine biosynthetic enzymes) and lrp (encoding Lrp, a global regulator) genes were also amplified by chromosomal promoter replacement to further increase the flux toward L-isoleucine. The final engineered E. coli strain was able to produce 9.46g/L of L-isoleucine with a yield of 0.14g/g of glucose by fed-batch culture. [This work was supported by the Technology Development Program to Solve Climate Changes (systems metabolic engineering for biorefineries; NRF-2012-C1AAA001-2012M1A2A2026556) and the Intelligent Synthetic Biology Center of Global Frontier Program (2011-0031963) from the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea.]