Adaptively Evolved and Multiplexed Engineered <i>Saccharomyces cerevisiae</i> for Neutralizer-Free Production of <scp>l</scp>-Lactic Acid

l-Lactic acid is a three-carbon monocarboxylic acid that has extensive applications. However, the bioproduction of l-lactic acid requires the addition of neutralizers, which significantly increases the production costs and can cause environmental pollution. To address this, a <i>Saccharomyces cerevisiae</i> mutant, TMG2, which can tolerate a lactic acid environment (pH 2.60), was obtained through adaptive laboratory evolution. Subsequently, the "push-pull-restrain" strategy was used to improve l-lactic acid production, resulting in a production of 46.8 g/L l-lactic acid. Finally, by overexpressing the transport protein pPfFNT and improving the NADH and acetyl-CoA supply, the l-lactic acid titer of strain TMG27 was improved by 33.8% to 62.6 g/L. Without neutralizers, the l-lactic acid titer reached 76.2 g/L (the fermentation pH was 2.90) with a productivity of 2.1 g/(L h) in a 5-L bioreactor, representing the highest productivity ever reported. Collectively, these results lay the foundation for the environmentally friendly bioproduction of l-lactic acid.