S126: Engineering improved fermentation of ethanol production in native xylose and cellobiose fermenting yeasts

Fermentation of D-xylose, L-arabinose and cellobiose for the production of renewable fuels has proven to be challenging. While there have been numerous attempts to metabolically engineer Saccharomyces cerevisiae–often using genes from native xylose fermenting yeasts–fewer laboratories have engineered native xylose and cellobiose fermenting yeasts to improve their performances. Sheffersomyces (Pichia) stipitis can ferment xylose, galactose, mannose, glucose, cellobiose and hemicellulosic oligosaccharides and hemicellulosic hydrolysates.  It produces over 60 g/l ethanol from xylose and some strains can ferment cellobiose almost as rapidly as glucose. We used expression array technology to identify genes induced during fermentation of cellulosic and hemicellulosic sugars, and we engineered critical genes for increased expression.  We have optimized codon usage in synthetic drug resistance genes and flanked them with LoxP sites to enable highly efficient transformation and subsequent excision with an engineered Cre recombinase. We have carried out repeated rounds of transformation and marker recovery using up to 15 different autologous S. stipitis genes and have obtained numerous strains of S. stipitis that show significantly better ethanol production than the best wild-type strains known. We have also developed mating and selection systems that enable rapid crosses and identification of hybrids with improved performance properties. Another yeast that natively produces ethanol from xylose and cellobiose in relatively high yields is Spathaspora passalidarum, which under appropriate conditions will actually ferment xylose faster and in higher yield than glucose. This presentation will review gene targets, global expression array results and the performance of native and engineered unconventional yeasts.