Long-term adaptive laboratory evolution of Saccharomyces cerevisiae for high-titer lactic acid production at low pH

BACKGROUND: Lactic acid is a highly versatile molecule whose increasing demand across the polymer, food, pharmaceutical, chemical, and cosmetics industries underscores its industrial and economic significance. Currently, lactic acid is predominantly produced via microbial fermentation using lactic acid bacteria facing limitations such as sensitivity to low pH, complex nutritional requirement and waste product generation during downstream processing. RESULTS: To address these challenges, we employed a genetically modified Saccharomyces cerevisiae strain capable of producing lactic acid and subjected it to long-term adaptive laboratory evolution. The strain was cultured in serial shake flask cultivations over a period of 35 months under elevating lactic acid concentrations and increasing stress to low pH. The evolved populations showed improved production of up to 250% in final lactic acid titers compared to the parental strain. The best-performing strains reached 67 g L⁻¹ at a final pH of 2.4 without pH control or 165 g L⁻¹ lactic acid at pH 3.0 with the addition of pH neutralizers, representing - to our knowledge - the highest LA titer reported in shake flask cultivations for S. cerevisiae. CONCLUSION: Overall, our results prove the great potential of long-term adaptive laboratory evolution in developing robust yeast cell factories for industrial organic acid production.