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.

Carotenoids (C40H56) including lycopene and beta-carotene are relatively strong antioxidants that provide benefits to human health. Here, we screened highly efficient crt variants from red yeasts to improve lycopene and beta-carotene production in Saccharomyces cerevisiae. We identified that crt variants from Sporidiobolus pararoseus TBRC-BCC 63403 isolated from rice leaf in Thailand exhibited the highest activity in term of lycopene and beta-carotene production in the context of yeast. Specifically, the phytoene desaturase SpCrtI possessed up to 4-fold higher in vivo activity based on lycopene content than the benchmark enzyme BtCrtI from Blakeslea trispora in our engineered WWY005 strain. Also, the geranylgeranyl pyrophosphate (GGPP) synthase SpCrtE, the bifunctional phytoene synthase-lycopene cyclase SpCrtYB, and SpCrtI when combined led to 7-fold improvement in beta-carotene content over the benchmark enzymes from Xanthophyllomyces dendrorhous in the laboratory strain CEN.PK2-1C. Sucrose as an alternative to glucose was found to enhance lycopene production in cells lacking GAL80. Lastly, we demonstrated a step-wise improvement in lycopene production from shake-flasks to a 5-L fermenter using the strain with GAL80 intact. Altogether, our study represents novel findings on more effective crt genes from Sp. pararoseus over the previously reported benchmark genes and their potential applications in scale-up lycopene production.