The biocatalytic cascade conversion of ethyl4-chloroacetoacetate (COBE) to ethyl (R)-4-cyano-3-hydroxybutyrate ((R)-HN) for the preparation of atorvastatinrepresents significant economic and environmental benefits,and is catalyzed by alcohol dehydrogenase and halohydrindehalogenase (HHDH). However, as the activity of HHDHis inhibited by COBE, the cascade reaction is an inefficientone-pot reaction. In this study, substrate inhibition kineticsanalysis was performed and the inhibition by COBE wasfound to be competitive reversible inhibition. Molecularsimulation analysis was used to determine the inhibitionmechanism by COBE. The results showed that COBEbound to the active center of HHDH via the formation ofhydrogen bonds with the OH groups of S132 and Y145. Site saturation mutagenesis of residues around the activesite and at the entrance of the access tunnel was performed,and two target mutant residues were identified, F136 andW249. Small focused mutagenesis on these two residueswas performed and the F136V/W249F mutant wassuccessfully found to relieve the activity inhibition ofHHDH by COBE. The half inhibiting concentration ofmutant F136V/W249F was found to be 20-fold higher thanwild-type HHDH. The efficiency of the multi-enzymaticone-pot system for the synthesis of (R)-HN from COBEusing mutant F136V/W249F was improved significantly.