Multistep metabolic engineered Klebsiella oxytoca for efficient l-leucine production

l-Leucine is widely applied in food, feed and medical industries. In this work, an efficient l-leucine producing strain Klebsiella oxytoca LKO-14 was constructed based on an l-valine producer K. oxytoca VKO-9. The exogenous l-leucine biosynthesis pathway was introduced to achieve l-leucine accumulation and decrease l-valine production. Modifying l-leucine transport system and optimizing copy numbers of genes including CgleuA ^M encoding l-leucine insensitive isopropylmalate synthase, EcleuCD encoding isopropylmalate isomerase, EcleuB encoding isopropylmalate dehydrogenase, NopheDH encoding phenylalanine dehydrogenase, were conducted to increase l-leucine production. The expression of budB encoding α-acetolactate synthase and EcilvD encoding dihydroxyacid dehydratase were also enhanced to improve precursor supply of l-leucine. In addition, the dihydroxy acid dehydratase from Streptococcus mutans containing an oxygen-tolerant [2Fe-2S] cluster was introduced to further enhance l-leucine production. Finally, the plasmid free and inducer independent K. oxytoca LKO-14 produced 70.1 g/L l-leucine, with a yield of 0.347 g/g and a productivity of 1.46 g/L/h, respectively.