Systematic engineering of Saccharomyces cerevisiae for D-lactic acid production with near theoretical yield.

D-lactic acid is a chiral three-carbon organic acid that can improve the thermostability of polylactic acid. Here, we systematically engineered Saccharomyces cerevisiae to produce D-lactic acid from glucose, a renewable carbon source, at near theoretical yield. Specifically, we screened D-lactate dehydrogenase (DLDH) variants from lactic acid bacteria in three different genera and identified the Leuconostoc pseudomesenteroides variant (LpDLDH) as having the highest activity in yeast. We then screened single-gene deletions to minimize the production of the side products ethanol and glycerol as well as prevent the conversion of D-lactic acid back to pyruvate. Based on the results of the DLDH screening and the single-gene deletions, we created a strain called ASc-d789M which overexpresses LpDLDH and contains deletions in glycerol pathway genes GPD1 and GPD2 and lactate dehydrogenase gene DLD1, as well as downregulation of ethanol pathway gene ADH1 using the L-methionine repressible promoter to minimize impact on growth. ASc-d789M produces D-lactic acid at a titer of 17.09 g/L in shake-flasks (yield of 0.89 g/g glucose consumed or 89% of the theoretical yield). Fed-batch fermentation resulted in D-lactic acid titer of 40.03 g/L (yield of 0.81 g/g glucose consumed). Altogether, our work represents progress towards efficient microbial production of D-lactic acid.

Sucrose-inducible heterologous expression of phytase in high cell density cultivation of the thermotolerant methylotrophic yeast Ogataea thermomethanolica.

In this study, production of fungal phytase in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 employing methanol-inducible OtAOX promoter and sucrose-inducible OtMal promoter was investigated in a high cell density fed-batch fermentation. Although a similar maximum cell concentration was obtained in both expression systems, the OtMal system gave ~2-fold higher phytase activity, specific yield, production yield, volumetric productivity and specific productivity rate compared with the OtAOX system. In addition to being more efficient, the OtMal system is more flexible because sucrose or sugarcane molasses can be utilized as less expensive carbon sources instead of glycerol in batch and fed-batch stages. Phytase yields from the OtMal system produced using sucrose or sugarcane molasses are comparable with those obtained with glycerol. We estimate the cost of phytase production by the OtMal system using sucrose or sugarcane molasses to be ~85% lower than the OtAOX system.