Asymmetric synthesis of chiral β-hydroxy esters, the key building blocks for many functional materials, is currently of great interest. In this study, the biocatalytic anti-Prelog reduction of methyl acetoacetate (MAA) to methyl-()-3-hydroxybutyrate (()-HBME) was successfully carried out with high enantioselectivity using the whole cell of engineered , which harbored an AcCR (carbonyl reductase) gene from sp. CCTCC M209061 and a GDH (glucose dehydrogenase) gene from 168 for the regeneration of the coenzyme. Compared with the corresponding wild strain, the engineered cells were proved to be more effective for the bio-reduction of MAA, and afforded much higher productivity. Under the optimized conditions, the product was >99.9% and the maximum yield was 85.3% after a reaction time of 10 h, which were much higher than those reported previously. In addition, the production of ()-HBME increased significantly by using a fed-batch strategy of tuning pH, with a space-time yield of approximately 265 g L d, thus the issue in previous research of relatively low substrate concentrations appears to be solved. Besides, the established bio-catalytic system was proved to be feasible up to a 150 mL scale with a large-scale relatively high substrate concentration and selectivity. For further industrial application, these results open a way to use of whole cells of engineered for challenging higher substrate concentrations of β-ketone esters enantioselective reduction reactions.