Enhancing Lactic Acid Production in Komagataella phaffii 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 Komagataella phaffii (K. phaffii) by reconfiguring the synthetic pathway and a metabolite damage-repair system. First, through the introduction of lactic acid dehydrogenase (BsLDH) from Bacillus subtilis and the transporter LutP from Bacillus coagulans, 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 K. phaffii for industrial production.