High yield production of L-isoleucine through readjusting the ratio of two direct precursors in Escherichia coli.

L-isoleucine (L-Ile) is an essential amino acid, which biosynthesis has been studied extensively. L-valine (L-Val) is a major by-product that limits L-Ile yield. Here, multiple strategies were employed to enhance L-Ile production and reduce L-Val in Escherichia coli. The biosynthetic pathway of L-Ile was divided into L-threonine (L-Thr) and L-Ile modules. In the L-Ile module, AHAS and IlvC were screened for high affinity toward 2-oxobutanoate and α-aceto-α-hydroxybutyrate, respectively, enhancing L-Ile yield. The L-Thr module was optimized via the overexpression of key enzymes, increasing 2-oxobutanoate supply. Furthermore, the deletion ofptsGandpykFand overexpression of citramalate synthase were employed to balance the precursors ratio. The engineered strain achieved a high yield of 0.40 mol L-Ile/mol glucose, a high productivity of 0.83 g/L/h, and significantly reduced L-Val accumulation, which would facilitate the subsequent separation and purification of L-Ile. This work provides a sustainable platform for the production of L-Ile derivatives.