Heterologous Expression of Ketoreductase KRED20 Mutant in and Bioreductive Production of ()-1, 3-Butanediol.

()-1, 3-Butanediol (1, 3-BDO) is an important intermediate in the synthesis of aromatics, pheromones, insecticides, and beta-lactam antibiotics. The KRED20 is a robust NADH-dependent ketoreductase identified from sp. . We obtained a KRED20 mutant (M12) through directed evolutionary screening of KRED20, the mutant with significantly improved activity to asymmetrically reduce 4-hydroxy-2-butanone (4H2B) to ()-1, 3-BDO. So far, both KRED20 and its mutants have been expressed in intracellular in , the process of purification after intracellular expression is complicated, which leads to high cost. Here, we expressed M12 by constructing multicopy expression strains in , and the target protein yield was 302 mg/L in shake-flask fermentation and approximately 3.5 g/L in high-density fermentation. The recombinant M12 showed optimal enzyme activity at 30 °C and had high activity within a broad pH range of 6.0-8.0, and also showed high thermal stability. The recombinant M12 was further used for the reduction of 4H2B to ()-1, 3-BDO, and 98.9% yield was achieved at 4540 mM 4H2B. The crude M12 enzyme extract was found to catalyze the bioreductive production of ()-1, 3-BDO with excellent stereoselectivity (ee > 99%) and meet the production requirements. Our research shows that the M12 mutant can be used for the synthesis of ()-1, 3-BDO, and the expression system is an ideal platform for the large-scale, low-cost preparation of KRED20 or its mutants, which may have applications in industrial settings.