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

Tran, V., University of Illinois at Urbana-Champaign
Zhao, H., University of Illinois at Urbana-Champaign
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.