(317a) Automated Cellular Engineering of Saccharomyces Cerevisiae Strains with High Resistance and Production of Lactic Acid

Automated
cellular engineering of Saccharomyces
cerevisiae strains with high resistance and production of lactic acid

Yajie Wang, William H Streyer,
Tong Si, Vinh G Tran and Huimin Zhao

Enantiopure lactic acid
(LA) has broad and increased 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. It was reported in literature that heterologous
expression of stereospecific lactate dehydrogenase (LDH) genes in S. cerevisiae coupled with metabolic
engineering enabled 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). Here I will report the development and application of an automated
cellular engineering (ACE) platform for yeast multiplex genome engineering in order to obtain yeast mutants capable of producing high
titers of LA (>50 g/L) at pH 3. The ACE platform consists of a modular
framework for continuous accumulation of multiplex genome-wide mutations
(Fig.1) and a versatile integrated robotic system called Illinois Biological
Foundry for Advanced Biomanufacturing (iBioFAB).  We developed an in vivo L-LA
biosensor in S. cerevisiae using an
adapted bacterial transcription factor LIdR and its
corresponding operator O2 for screening L_-LA
overproduction at low pH. We are integrating this
screening method to the ACE platform for creation and screening of massive
genomic variants in a fully automated manner. We will characterize the low pH
resistant, LA overproduction strains using RNA-seq
and metabolic flux analysis.