Biocatalytic Cascade Synthesis of Enantioenriched Epoxides and Triols from Biomass-Derived Synthons Driven by Specifically Designed Enzymes

Multi-enzymatic cascades exploiting engineered enzymes are a powerful tool for the tailor-made synthesis of complex molecules from simple inexpensive building blocks. In this work, we engineered the promiscuous enzyme 4-oxalocrotonate tautomerase (4-OT) into an effective aldolase with 160-fold increased activity compared to 4-OT wild type. Subsequently, we applied the evolved 4-OT variant to perform an aldol condensation, followed by an epoxidation reaction catalyzed by a previously engineered 4-OT mutant, in a one-pot two-step cascade for the synthesis of enantioenriched epoxides (up to 98 % ee) from biomass-derived starting materials. For three chosen substrates, the reaction was performed at milligram scale with product yields up to 68 % and remarkably high enantioselectivity. Furthermore, we developed a three-step enzymatic cascade involving an epoxide hydrolase for the production of chiral aromatic 1,2,3-prim,sec,sec-triols with high enantiopurity and good isolated yields. The reported one-pot, three-step cascade, with no intermediate isolation and being completely cofactor-less, provides an attractive route for the synthesis of chiral aromatic triols from biomass-based synthons.

Automated summary

Multi-enzymatic cascades using engineered enzymes are a useful tool for synthesizing complex molecules from simple starting materials. In this study, we engineered the enzyme 4-oxalocrotonate tautomerase (4-OT) to have increased aldolase activity. We then used the evolved 4-OT variant in a one-pot two-step cascade to synthesize enantioenriched epoxides from biomass-derived materials with high yields and enantioselectivity. We also developed a three-step enzymatic cascade for the production of chiral aromatic triols with high enantiopurity and good isolated yields.