| Summary: | Lactic acid (LA) is a promising bio-based chemical that has broad applications in food, nutraceutical, and bioplastic industries. However, production of the D-form of LA (D-LA) from fermentative organisms is lacking. In this study, <i>Saccharomyces cerevisiae</i> harboring the D-lactate dehydrogenase (DLDH) gene from <i>Leuconostoc mesenteroides</i> was constructed (CEN.PK2_DLDH). To increase D-LA production, the CRISPR/Cas12a system was used for the deletion of <i>gpd1</i>, <i>gpd2</i>, and <i>adh1</i> to minimize glycerol and ethanol production. Although an improved D-LA titer was observed for both CEN.PK2_DLDHΔ<i>gpd</i> and CEN.PK2_DLDHΔ<i>gpd</i>Δ<i>adh1</i>, growth impairment was observed. To enhance the D-LA productivity, CEN.PK2_DLDHΔ<i>gpd</i> was crossed with the weak acid-tolerant <i>S. cerevisiae</i> BCC39850. The isolated hybrid2 showed a maximum D-LA concentration of 23.41 ± 1.65 g/L, equivalent to the improvement in productivity and yield by 2.2 and 1.5 folds, respectively. The simultaneous saccharification and fermentation using alkaline pretreated sugarcane bagasse by the hybrid2 led to an improved D-LA conversion yield on both the washed solid and whole slurry (0.33 and 0.24 g/g glucan). Our findings show the exploitation of natural yeast diversity and the potential strategy of gene editing combined with conventional breeding on improving the performance of <i>S. cerevisiae</i> for the production of industrially potent products.
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