Co-expression of an alcohol dehydrogenase and a cyclohexanone monooxygenase for cascade reactions facilitates the regeneration of the NADPH cofactor

The introduction of a three-enzyme cascade (comprising a cyclohexanone monooxygenase (CHMO), an alcohol dehydrogenase (ADH) and a lipase (CAL-A)) for the production of oligo-ε-caprolactone provided self-sufficiency with respect to NADPH-cofactor regeneration and reduced inhibiting effects on the central CHMO enzyme. For further optimization of cofactor regeneration, now a co-expression of CHMO and ADH in E. coli using a Duet™ vector was performed. This led to higher conversion values of the substrate cyclohexanol in whole-cell biocatalysis compared to an expression of both enzymes from two separate plasmids. Furthermore, a more advantageous balance of expression levels between the partial cascade enzymes was achieved via engineering of the ribosome binding site. This contributed to an even faster cofactor regeneration rate.