Tuning A Bi-Enzymatic Cascade Reaction In Escherichia Coli To Facilitate Nadph Regeneration For Epsilon-Caprolactone Production

epsilon-Caprolactone is a monomer of poly(epsilon-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the epsilon-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to epsilon-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an epsilon-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM epsilon-caprolactone with a high molar yield of 0.78 mol/mol.