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.