Combinatorial metabolic engineering and process optimization enables highly efficient production of L-lactic acid by acid-tolerant Saccharomyces cerevisiae.

L-lactic acid (L-LA) is widely used in the food, pharmaceutical, and cosmetic industries. In recent years, the production of L-LA using microbial fermentation has been favored. Herein, a Saccharomyces cerevisiae TAM strain tolerant to pH 2.4, was used as the starting strain. Exogenous L-lactate dehydrogenase expressing S. cerevisiae TAM strain with downregulated glycerol and ethanol synthesis pathways produced an L-LA titer of 29.8 g/L, and it increased to 50.5 g/L after carboxylic acid transport pathway modulation at the shake-flask level. Subsequently, increased energy supply and redox balancing increased the L-LA titer to 72.7 g/L in shake-flask fermentation without a neutralizer, with the yield of 0.66 g/g. Finally, optimization of the fermentation conditions, such as the seed quantity, oxygen level, and pH in a 15-L bioreactor, increased the L-LA titer to 192.3 g/L at pH 4.5, with a yield of 0.78 g/g. Overall, this study proposes an efficient L-LA bioproduction method.