Study Of Whole-cell Biocatalysis For ?-caprolactone Production In Escherichia Coli

?-caprolactone is an important organic chemical intermediate that can synthesize high-value polymers such as PCL.It is used for the synthesis of materials,environmental protection,medical and other fields because of its good thermoplasticity,drug permeability and biodegradability,molding processability,biocompatibility and non-toxicity.The traditional chemical synthesis of ?-caprolactone is limited due to its cumbersome operation and high expenditure.Accordingly,the microbial synthesis of?-caprolactone with high efficiency and selectivity has become a more efficient method.In this study,a route using whole-cell biocatalysis was designed to produce ??caprolactone by cyclohexanol.In this process,a cyclohexanone monooxygenase(CHMO)was combined with an alcohol dehydrogenase(ADH)to enable the direct transformation of cyclohexanol into ?-caprolactone without the need for additional cofactor(NADPH),thus achieving the self-sufficiency of NADPH in situ.In order to improve the efficiency of this cascade,the effects of different Duet plasmids(pETDuet and pRSFDuet)on the capacity of ?-caprolactone production were investigated.The results showed that s-caprolactone yield of the strain BDR-1 containing pRSF-C10A20 achieved 60%with 16 h,which was 16%higher than the strain BDT-1 containing pET-A10C10.Furthermore,in order to balance the production and consumption of intracellular NADPH at the same level as possible,the RBS sequences in ADH and CHMO expression cassettes were rationally designed to regulate the expression of the corresponding encoded enzymes.Finally,the optimal strain BDR-13 was obtained.In the experiment of catalyzing 60 mM cyclohexanol,the production of s-caprolactone of strain BDR-13 was 52.5 mM,and the yield reached 87.5%.The production of this strain was 1.7 times than that of the control strain(BDR-2).In order to further increase the yield,the strain BDR-13 was selected for fed-batch biocatalysis.Finally,the production of ?-caprolactone reached 126 mM,and the fourth batch had a conversion rate of 76%.In this study,a cascade reaction related the production of ?-caprolactone in E.coli BL21(DE3)was successfully constructed and regulated the expression level of each enzyme by rational design,which made the cofactor NADPH self-sufficient to achieve the high production of ?-caprolactone.The construction of the strain(BDR-13)provides the possibility of industrial production of ?-caprolactone,and also provides a reference for the construction of the same type of intracellular redox reaction system.