(28f) Improving Lactic Acid Production in Saccharomyces Cerevisiae Under Low pH By Directed Genome Evolution

Enantiopure lactic acid (LA) has broad and growing applications in the food, cosmetics and pharmaceutical industrials. Saccharomyces cerevisiae exhibits high tolerance towards organic acids and has emerged as a promising host for LA production. Coupled with metabolic engineering efforts, heterologous expression of stereospecific lactate dehydrogenase (LDH) genes in S. cerevisiae enables the production of pure _L- or _D-LA with titer as high as 142 g/L, albeit at an industrially non-preferred pH (pH 4.7). In this study, we applied RNAi assisted genome evolution (RAGE) to iteratively introduce genome-wide gain- and loss- of function modifications. We developed a high throughput screening process that could be easily adapted to automated cellular engineering platform that is conducted by a versatile integrated robotic system called Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB). During the first-round of directed evolution, we successfully identified candidate PK2M3-FA3 with mass accumulation 4 times more than the parent strain (PK2M3) in SC medium with 3.5% LA at pH 3. PK2M3 also can produce 1.7 times more _L-LA than PK2M3 in synthetic drop-out medium at pH 3. Additional rounds of evolution and screening are being performed to further improve the acidic tolerance and _L-LA production of PK2M3-FA3 and RNA-seq and metabolic flux analysis will be used to characterize the evolved mutants.