Engineering of Komagataella phaffii for enhanced d-lactic acid production via deletion of a cell wall stability-related gene and optimization of cultivation conditions.

Methanol has attracted increasing attention as a sustainable one-carbon feedstock because it can be produced from carbon dioxide and hydrogen or from waste biomass. Additionally, it can be economically stored and transported as a liquid. The methylotrophic yeast Komagataella phaffii is a promising host for producing valuable biochemicals from methanol, while d-lactic acid is an important chemical used for producing stereocomplex polylactic acid; microbial processes enable d-lactic acid synthesis with high optical purity. However, most microbial d-lactic acid production processes rely on sugar substrates, and efficient production from methanol remains limited. We deleted the cell wall stability-related gene PAS_chr4_0305 in d-lactic acid-producing K. phaffii. Evaluation of d-lactic acid production capacity in flask cultures with methanol as the sole carbon source revealed that the deletion strain showed an approximately 1.20-fold increase in d-lactic acid production compared with the parent strain. Furthermore, optimization of culture conditions resulted in d-lactic acid production reaching a maximum of 25.7 g/L. Transcriptome analysis results suggested that deletion of PAS_chr4_0305 induced expression changes in alcohol metabolism- and ribosome-related genes, and these changes potentially contributed to improved methanol assimilation and d-lactic acid productivity. This study demonstrates that combining the deletion of the cell wall stability-related gene with culture condition optimization can enhance d-lactic acid production from methanol in K. phaffii, providing a novel strategy for valuable biochemical production using methylotrophic yeast.