Heterologous Expression of Ketoreductase ChKRED20 Mutant in Pichia pastoris and Bioreductive Production of (R)-1, 3-Butanediol

(<i>R</i>)-1, 3-Butanediol (1, 3-BDO) is an important intermediate in the synthesis of aromatics, pheromones, insecticides, and beta-lactam antibiotics. The <i>Ch</i>KRED20 is a robust NADH-dependent ketoreductase identified from <i>Chryseobacterium</i> sp. <i>CA49</i>. We obtained a <i>Ch</i>KRED20 mutant (M12) through directed evolutionary screening of <i>Ch</i>KRED20, the mutant with significantly improved activity to asymmetrically reduce 4-hydroxy-2-butanone (4H2B) to (<i>R</i>)-1, 3-BDO. So far, both <i>Ch</i>KRED20 and its mutants have been expressed in intracellular in <i>E. coli</i>, the process of purification after intracellular expression is complicated, which leads to high cost. Here, we expressed M12 by constructing multicopy expression strains in <i>P. pastoris</i>, 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 (<i>R</i>)-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 (<i>R</i>)-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 (<i>R</i>)-1, 3-BDO, and the <i>P. pastoris</i> expression system is an ideal platform for the large-scale, low-cost preparation of <i>Ch</i>KRED20 or its mutants, which may have applications in industrial settings.