Comparative analysis of heterologous lactate dehydrogenase expression in Ogataea polymorpha for enhanced L-Lactic acid production.

Lactic acid (LA) is a key platform chemical with broad industrial applications, including biodegradable plastics and pharmaceuticals. While microbial fermentation enables the sustainable production of optically pure L-lactic acid, challenges persist in substrate flexibility and process optimisation. Thermotolerant methylotrophic yeast Ogataea polymorpha has been tested as an alternative biocatalyst for sustainable production of lactic acid, a key platform compound for bioplastics and green manufacturing. However, the influence of lactate dehydrogenase (LDH) origin on metabolic flux redirection in this chassis has not been systematically investigated. This study presents the first comparative evaluation of heterologous LDHs from fungal, bacterial, protistan and mammalian sources expressed in O. polymorpha and assessed across four carbon substrates (glucose, xylose, glycerol, and methanol). The recombinant strains were evaluated for lactic acid production on glucose, xylose, and methanol under various aeration and pH-controlled conditions. Among the tested constructs, the LDH_Bt strain expressing bovine LDH exhibited the highest lactic acid titres from glucose (up to 72.6 g/L), particularly under neutralised conditions with CaCO₃, while LDH_Ro achieved the highest titre on xylose under high agitation with a neutraliser (9.59 g/L). Under methanol-based conditions, LDH_Pf produced the highest titre among the strains under pH-controlled, low-agitation conditions (1.12 g/L). However, the highest absolute methanol-derived titre was observed for LDH_Lp at 220 rpm without CaCO₃ supplementation (1.27 g/L). Despite having an intact ethanol biosynthesis pathway, the obtained transformants produced very low amounts of ethanol (typically below 0.3 g/L, with occasional higher values depending on fermentation conditions). Lactic acid formation from xylose, glycerol, and methanol was notably lower compared to glucose, indicating substrate-dependent redox and compartmentalisation constraints. A weak-to-moderate association between LDH transcript level and lactic acid titre was observed, suggesting that expression contributes to flux control but does not solely determine pathway efficiency. These findings demonstrate that enzyme origin and substrate context jointly define metabolic output and highlight O. polymorpha as a promising platform for thermotolerant lactic acid bioproduction.