S118: Tools for metabolic engineering of Scheffersomyces stipitis for improved fermentation of xylose and cellobiose

Cellulosic and hemicellulosic hydrolysates can be fermented to ethanol by various anaerobic bacteria, engineered Saccharomyces cerevisiae, or native beetle-associated yeasts belonging to the CTG clade.  CTG yeasts substitute serine for leucine when encountering a CTG codon.  Also they only enter a transient diploid phase during mating and sporulation.  Of the xylose- and cellobiose-fermenting CTG yeasts, Scheffersomyces stipitis has been the most thoroughly studied, hence have developed an advanced genetic system for S. stipitis to better understand its physiology, biochemistry and regulation. Following completion of its genomic sequence by JGI, genome-wide transcriptome analyses on seven carbon sources under aerobic and oxygen limited conditions revealed significant differences in the expression of genes for sugar transport, respiration, glycolysis, fermentation and mating factors. The latter enabled development of sporulation media for high frequency crosses. Starting from an earlier auxotrophic selection system (ura3, leu2) we developed a transformation system that allows for selection with synthetic, codon-optimized Nat1 along with Hph, and ShBle engineered to eliminate CTG codons for leucine.  We further flanked the markers with LoxP sites for excision by a re-engineered version of Cre. Using sNat1, we have integrated multiple genes of interest into the S. stipitis CBS 6054 and NRRL Y-7124 genomes.  We subsequently mated selected and adapted strains for improved performance on hemicellulose hydrolysates. Mated strains were adapted to increasing concentrations of industrial hemicellulosic hydrolysates, and more resilient mutants were selected.  Resulting strains have demonstrated significant gains in the capacity of S. stipitis to ferment industrial hemicellulosic hydrolysates to ethanol.