Enhancing Lactic Acid Production in <i>Komagataella phaffii</i> from Multiple Carbon Sources by Rewriting Synthetic Routes and Implementation of Metabolite Damage-Repair Enzymes

The production of lactic acid, a crucial platform chemical by microbial fermentation, is currently hindered by its low production efficiency. Herein, this study aims to enhance the synthetic efficiency of lactic acid in widely used <i>Komagataella phaffii</i> (<i>K. phaffii</i>) by reconfiguring the synthetic pathway and a metabolite damage-repair system. First, through the introduction of lactic acid dehydrogenase (BsLDH) from <i>Bacillus subtilis</i> and the transporter LutP from <i>Bacillus coagulans</i>, the titer of lactic acid was increased from 1.5 g/L in the original strain to 55.4 g/L using glucose. In particular, the titer was improved to 87.2 g/L by introducing metabolite damage-repair genes for NAD(P)HX detoxification and phosphate-based inhibitor elimination. In addition, the carbon source transport and metabolism pathway were strengthened, resulting in titers of 18.7 and 7.7 g/L from glycerol and methanol. Finally, the strains were scaled up in a 5 L bioreactor, achieving lactic acid titer values of 153.0, 133.2, and 37.4 g/L from glucose, glycerol, and methanol, respectively. This study significantly improved the yield of lactic acid production from low-cost carbon sources by microbial fermentation, demonstrating the potential of engineered <i>K. phaffii</i> for industrial production.