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Directed evolution of stereo-, regio-, and chemoselective enzymes has enriched the toolbox of synthetic organic chemistry. Among the different gene mutagenesis techniques, saturation mutagenesis (S...

Ketoreductases are tools for the synthesis of chiral alcohols in industry. However, the low activity of natural enzymes often restricts their use in industrial applications. On the basis of computational analysis and previous reports, two residues (F92 and F94) probably affecting the activity of ketoreductase CgKR1 were identified. By tuning these two residues, the CgKR1-F92C/F94W variant was obtained that exhibited higher activity toward all 28 structurally diverse substrates examined than the wild-type enzyme. Among them, 13 substrates have a specific activity over 50 U mg–1 (54–775 U mg–1). Using CgKR1-F92C/F94W as a catalyst, five substrates at high loading (>100 g–1 L–1) were reduced completely in gram-scale preparative reactions. This approach provides accesses to pharmaceutically relevant chiral alcohols with high enantioselectivity (up to 99.0% ee) and high space-time yield (up to 583 g–1 L–1 day–1). Molecular dynamics simulations highlighted the crucial role of residues 92 and 94 in activity impr...

The directed evolution of enzymes promises to eliminate the long-standing limitations of biocatalysis in organic chemistry and biotechnology—the often-observed limited substrate scope, insufficient activity, and poor regioselectivity or stereoselectivity. Saturation mutagenesis at sites lining the binding pocket with formation of focused libraries has emerged as the technique of choice, but choosing the optimal size of the randomization site and reduced amino acid alphabet for minimizing the labor-determining screening effort remains a challenge. Here, we introduce structure-guided triple-code saturation mutagenesis (TCSM) by encoding three rationally chosen amino acids as building blocks in the randomization of large multiresidue sites. In contrast to conventional NNK codon degeneracy encoding all 20 canonical amino acids and requiring the screening of more than 1015 transformants for 95% library coverage, TCSM requires only small libraries not exceeding 200–800 transformants in one library. The triple c...

Biocatalytic asymmetric sulfoxidation represents a green method to prepare the useful and valuable enantiopure sulfoxides, but this method sometimes suffers from unsatisfied enantioselectivity and low productivity due to substrate and product inhibitions. Here we developed an aqueous/ionic liquid (IL) biphasic system for simultaneously enhancing the enantioselectivity and productivity of P450 monooxygenase-catalyzed asymmetric sulfoxidations of sulfides 1, 3, 5, 7, and 9, as the first example of this kind for a biooxidation. Escherichia coli (P450pyrI83H-GDH) coexpressing P450pyrI83H monooxygenase and glucose dehydrogenase was engineered for the asymmetric sulfoxidations with cofactor recycling, giving higher R-enantioselectivity than any other known P450 monooxygenases and showing high specific activities. The inhibition to the reactions and the toxicity to the cells of the substrates and products were investigated and mostly avoided by using a KP buffer/[P6,6,6,14][NTf2] biphasic reaction system, in whi...

Cascade biocatalysis via intracellular epoxidation and hydrolysis was developed as a green and efficient method for enantioselective dihydroxylation of aryl olefins to prepare chiral vicinal diols in high ee and high yield. Escherichia coli (SSP1) coexpressing styrene monooxygenase (SMO) and epoxide hydrolase SpEH was developed as a simple and efficient biocatalyst for S-enantioselective dihydroxylation of terminal aryl olefins 1a–15a to give (S)-vicinal diols 1c–15c in high ee (97.5–98.6% for 10 diols; 92.2–93.9% for 3 diols) and high yield (91–99% for 6 diols; 86–88% for 2 diols; 67% for 3 diols). Combining SMO and epoxide hydrolase StEH showing complementary regioselectivity to SpEH as a biocatalyst for the cascade biocatalysis gave rise to R-enantioselective dihydroxylation of aryl olefins, being the first example of this kind of reversing the overall enantioselectivity of cascade biocatalysis. E. coli (SST1) coexpressing SMO and StEH was also engineered as a green and efficient biocatalyst for R-dihy...