Conferring Flocculation Phenotype to Pichia pastoris Improves D-lactic Acid Production and Tolerance

D-lactic acid is a three-carbon organic acid with a chiral structure and can improve the thermostability of polylactic acid. Microorganisms such as the methylotrophic yeast Pichia pastoris, which lack the natural ability to produce or accumulate high amounts of D-lactic acid, have been 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 leads to increased D-lactic acid production in Pichia pastoris. By incorporating a flocculation gene from Saccharomyces cerevisiae (ScFLO1) into P. pastoris 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 Leuconostoc pseudomesenteroides (LpDLDH) into KM71-ScFlo1 resulted in an engineered strain (KM71-ScFlo1-LpDLDH) that can produce D-lactic acid at a titer of 5.12  0.35 g/L in 48 hours , a 2.6-fold improvement over the control strain lacking ScFLO1 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. The engineered strains were pre-cultured in 5-mL aliquots of MGYH medium overnight and used to inoculate 10 mL of MGYH medium in 50 mL Corning tubes to achieve an initial optical density of 0.05 at 600 nm (OD600). The cultures were grown at 30 °C and 250 rpm in an orbital shaking incubator. When the strains reached the mid-exponential phase (approximately 21 hours), a 2-mL aliquot of each culture was collected and centrifuged for 5 minutes at 3000 x g.