Engineering Flocculation for Improved Tolerance and Production of d-Lactic Acid in Pichia pastoris

Abstract

d-lactic acid, a chiral organic acid, can enhance the thermal stability of polylactic acid plastics. Microorganisms such as the yeast <i>Pichia pastoris</i>, which lack the natural ability to produce or accumulate high amounts of d-lactic acid, have been metabolically engineered to produce it in high titers. However, tolerance to d-lactic acid remains a challenge. In this study, we demonstrate that cell flocculation improves tolerance to d-lactic acid and increases d-lactic acid production in <i>Pichia pastoris</i>. By incorporating a flocculation gene from <i>Saccharomyces cerevisiae</i> (<i>ScFLO1</i>) into <i>P. pastoris</i> KM71, we created a strain (KM71-ScFlo1) that demonstrated up to a 1.6-fold improvement in specific growth rate at high d-lactic acid concentrations. Furthermore, integrating a d-lactate dehydrogenase gene from <i>Leuconostoc pseudomesenteroides</i> (<i>Lp</i>DLDH) into KM71-ScFlo1 resulted in an engineered strain (KM71-ScFlo1-LpDLDH) that could produce d-lactic acid at a titer of 5.12 ± 0.35 g/L in 48 h, a 2.6-fold improvement over the control strain lacking <i>ScFLO1</i> expression. Transcriptomics analysis of this strain provided insights into the mechanism of increased tolerance to d-lactic acid, including the upregulations of genes involved in lactate transport and iron metabolism. Overall, our work represents an advancement in the efficient microbial production of d-lactic acid by manipulating yeast flocculation.