Rational Design of Escherichia coli for L-Isoleucine Production

Abstract

Metabolic engineering of Escherichia coli was performed to construct a 100% rationally engineered strain capable of overproducing L-isolencine, an important branched.; chain amino acid. The thrABC (encoding L-threonine biosynthetic, enzymes), ilvA :(encoding feedback-resistant threonine dehydratase), ilvIH (encoding feedback-resistant acetohydroxy acid synthase III), and ygaZH (encoding branched-chain - amino acid exporter) genes were amplified by plasmid-based overexpression. The ilvCED (encoding L-isolencine biosynthetic enzymes) and lrp (encoding global regulator Lrp) genes were also amplified by chromosomal promoter,replacement in order to further increase the flux toward i-isoleucine. The final engineered E. colt strain was able to produce 9.46 g/L of L-isolencine with a yield of 0.14 g/g of glucose by fed-hatch culture: The overall design principles described here for the production of highly regulated product should be useful in designing strains for the production of other Similar bioproducts.