Engineering Kluyveromyces marxianus for Efficient Production of l‑Lactic Acid under Low pH Conditions

Lactic acid (LA) is widely utilized in the food, pharmaceutical, and biopolymer industries. However, microbial LA production often faces challenges due to byproduct accumulation and limited acid tolerance. (i) Herein, an ARS ori-based synthetic plasmid was developed in Kluyveromyces marxianus to facilitate key enzyme screening. Bovine-derived lactate dehydrogenase (BLDH) demonstrated superior catalytic activity under high temperatures and acidic conditions. (ii) Knocking out competitive pathway genes PDC1 (pyruvate decarboxylase) and CYB2 (l-lactate: cytochrome oxidase) via genome insertion of BLDH significantly enhanced LA production and reduced byproduct accumulation. (iii) Transcriptomic analysis identified a novel acid tolerance gene, ESBP6, whose overexpression markedly improved strain survival by 41.2% and LA production by 80.2% under high-LA stress. After optimizing fermentation conditions, the optimal strain, FL3.8, produced 144.8 g/L L-LA with a yield of 0.9 mol/mol and productivity of 2.0 g/L/h. Furthermore, this strain produced 132 g/L L-LA using a synthetic lignocellulosic hydrolysate medium. This study establishes a technical foundation for LA production using K. marxianus under low pH conditions (as low as 2.97) and highlights the biotechnological potential of nonconventional microorganisms.