Improvement of d-Lactic Acid Production in Saccharomyces cerevisiae Under Acidic Conditions by Evolutionary and Rational Metabolic Engineering.

Microbial lactic acid (LA) production under acidic fermentation conditions is favorable to reduce the production cost, but circumventing LA toxicity is a major challenge. A d-LA-producing Saccharomyces cerevisiae strain JHY5610 is generated by expressing d-lactate dehydrogenase gene (Lm. ldhA) from Leuconostoc mesenteroides, while deleting genes involved in ethanol production (ADH1, ADH2, ADH3, ADH4, and ADH5), glycerol production (GPD1 and GPD2), and degradation of d-LA (DLD1). Adaptive laboratory evolution of JHY5610 lead to a strain JHY5710 having higher LA tolerance and d-LA-production capability. Genome sequencing of JHY5710 reveal that SUR1 mutation increases LA tolerance and d-LA-production, whereas a loss-of-function mutation of ERF2 only contributes to increasing d-LA production. Introduction of both SUR1 and erf2Δ mutations into JHY5610 largely mimic the d-LA-production capability of JHY5710, suggesting that these two mutations, which could modulate sphingolipid production and protein palmitoylation, are mainly responsible for the improved d-LA production in JHY5710. JHY5710 is further improved by deleting PDC1 encoding pyruvate decarboxylase and additional integration of Lm. ldhA gene. The resulting strain JHY5730 produce up to 82.6 g L of d-LA with a yield of 0.83 g g glucose and a productivity of 1.50 g/(L · h) in fed-batch fermentation at pH 3.5.