Engineered and adapted Scheffersomyces stipitis strains with improved fermentative performance in hemicellulosic sugar solutions

The economically sustainable use of cellulosic and hemicellulosic feedstocks as substrates for microbial bioconversion to biofuels presents numerous challenges, including the presence of inhibitory compounds and difficult to ferment carbon sources.  The yeast Scheffersomyces stipitis has the native capacity to ferment glucose, xylose, and a variety of other components found in cellulosic sugar solutions. Engineered strains were constructed through random integration of target genes. Overexpression of a sugar transporter in conjunction with xylose utilization genes improved fermentative capacity in defined medium. Integration of a second sugar transporter gene into that genetic background yielded a strain with better performance in cellulosic sugar solutions. Next, nine matings were carried out between sets of engineered strains.  Progeny were subjected to evolutionary adaptation in corn stover hydrolysate. Seven of the nine matings yielded progeny that survived 17 serial transfers in hydrolysate. Engineered and adapted populations outperformed wild type strains in medium composed of ~94% industrial corn stover hydrolysate with ~0.5% acetic acid. Ten libraries were constructed and subjected to Illumina whole genome sequencing, including DNA from two wildtype strains, seven mated and adapted populations, and one strain generated by backcrossing a mated/adapted population to a wildtype strain. Sequence analysis is ongoing and may yield insights into the genetic determinants of fermentation performance and improved robustness. In conjunction with strain development efforts, process improvements were implemented which resulted in a 15% increase in ethanol yield and 75% reduction in the time required to reach peak ethanol in batch bioreactor culture of undiluted industrial hemicellulosic hydrolysate.