Metabolic Engineering of Pichia pastoris for De Novo Biosynthesis of D-Lactic Acid from Methanol

Abstract D-Lactic acid (D-LA) is an essential chiral monomer for polylactic acid (PLA), widely used in biodegradable food packaging and agricultural materials. Conventional sugar-based fermentation is constrained by competition with food resources and price volatility, highlighting the need for sustainable alternatives. Methanol, a renewable and cost-effective C1 substrate, provides thermodynamic advantages for D-LA biosynthesis but remains underutilized. In this study, Pichia pastoris was modified by systematic metabolic engineering to create an efficient methanol-D-LA conversion pathway. By reducing D-LA degradation, enhancing methanol utilization, improving acid tolerance, and eliminating competing pathways, the optimized fed-batch process achieved a D-LA titer of 12.05 g/L. Sodium citrate was employed as an auxiliary metabolic regulator to support central metabolism and process robustness. This work demonstrates methanol bioconversion as a sustainable route for D-LA production and establishes P. pastoris as a promising platform for manufacturing biobased monomers and high-value chemicals from renewable C1 feedstocks.