Engineering Escherichia coli for the S-selective production of 2-hydroxyisovalerate

2-Hydroxyisovalerate (2-HIV) is a value-added chemical that is widely applied in the synthesis of bioactive compounds and polymers. Here, we report an underexplored metabolic route for the de novo production of S-type 2-HIV (S-HIV) in Escherichia coli. In particular, we identified promiscuous activity of 4-hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis towards the conversion of 2-keto-4-methyl-pentanoate (2-KMP, an immediate precursor for L-leucine) to S-HIV. Next, we designed a variant HmaS (S201F) with abolished activity for mandelate and 4-hydroxymandelate synthesis, thereby minimizing byproduct formation. Coupled with systematic optimization of the L-leucine biosynthetic pathway, we achieved de novo production of S-HIV at 8.1 ​mM (0.95 ​g/L) in shake flasks and 33.9 ​mM (4.0 ​g/L) in 2-L fed-batch fermentation. In summary, this work represents the first time to realize the efficient synthesis of S-configuration 2-HIV in metabolically engineered E. coli.