Rational Design of Cyclohexanone Monooxygenase and a Whole-Cell System for Biocatalytic Redox-Neutral Cascade Synthesis of Lactones

Whole-cell redox-neutral cascade reactions are promising for the sustainable synthesis of bulky lactones. In this study, the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAc) was rationally designed based on enhancing product release and consensus sequence design to enhance the activity and stability of the enzyme. A four-point mutant MU4 (G14A/A43G/M400L/F432I) showed improved activity and stability for the oxidation of cyclohexanone to ε-caprolactone at a high substrate loading. A newly NADPH-dependent alcohol dehydrogenase from Pseudomonas monteilii (PmADH) with high cyclohexanol oxidation activity was introduced for constructing the redox-neutral cascade for the synthesis of ε-caprolactone from cyclohexanol. To improve the intracellular environment for cascade synthesis of ε-caprolactone, CRISPR/Cas9-mediated strong promoter insertion to the endogenous catalase gene katE in the NADP self-sufficient Escherichia coli strain BK-1 (Ptrc-nadK/Ptrc-pncB) was done for mitigating the oxidative stress to obtain the strain E. coli BKE-3 harboring MU4 and PmADH. Finally, 776 mM cyclohexanol could be converted into ε-caprolactone by the BKE-3 whole-cell catalytic system with a >99% conversion rate without cyclohexanone accumulation, which was the highest record for the redox-neutral cascade synthesis of ε-caprolactone. The BKE-3 whole-cell catalytic system also showed high efficiency for the synthesis of various lactones. This work provides an efficient redox-neutral catalytic platform for the sustainable synthesis of lactones featuring high substrate loading and atomic economy.