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.

Immobilization of D-amino acid dehydrogenase (DAADH) by the assembly of peptide linker was studied for the biosynthesis of D-phenylalanine. Hybrid material of zeolitic imidazolate framework-8 (ZIF-8) combined with reduced graphene oxide (RGO) was applied for the immobilization of DAADH from Ureibacillus thermosphaericus. The recovery rate of DAADH/ZIF-8/RGO was 165.6%. DAADH/ZIF-8/RGO remained 53.4% of its initial activity at 50 °C for 10 h while the free enzyme was inactivated. DAADH/ZIF-8/RGO maintained 70.5% activity in hyperalkaline solution with pH 12. Kinetic parameters indicated that DAADH/ZIF-8/RGO had greater affinity of phenylpyruvate as V_max/K_m of DAADH/ZIF-8/RGO was 1.27-fold than free enzyme. After seven recycles, the activity of DAADH/ZIF-8/RGO remained 64.3%. Furthermore, one-step separation and in situ immobilization of DAADH by ZIF-8/RGO/Ni was carried out with 1.5-fold activity enhancement. Combining peptide linker and metal-organic framework (MOF) immobilization, thermostability and activity of the immobilized DAADH were significantly improved.