Fermentation of Glucose, Xylose and Cellobiose By Beetle-Associated Yeasts: Capacities, Genomics and Metabolic Engineering

Simultaneous co-fermentation of glucose with other less readily utilized carbon sources such as xylose, arabinose, galactose and cellobiose is problematic for almost all microbes – including yeasts that natively ferment cellulosic and hemicellulosic sugars. Surprisingly, the ascomyceteous, beetle-associated yeast, Spathaspora passalidarum, which ferments xylose and cellobiose natively, can also co-ferment these two sugars along with glucose under low aeration conditions. S. passalidarum will simultaneously assimilate glucose and xylose aerobically; it will simultaneously co-ferment glucose, cellobiose and xylose under oxygen limitation with an ethanol yield of 0.42 g/g, and it has a specific ethanol production rate on xylose more than 3 times faster than the corresponding rate on glucose. Metabolome analysis of S. passalidarum before onset and during the fermentations of glucose and xylose showed that the concentration of glycolytic intermediates is significantly higher on xylose than on glucose. Transcriptomic analysis of S. passalidarum cultivated on glucose or xylose under aerobic conditions showed very few significant changes in transcripts other than those directly involved in xylose assimilation. Under oxygen limitation, cells transcripts showed a minimal stress response when cultivated on xylose.  S. passalidarum has two aldose reductases and two xylitol dehydrogenases with unusual kinetic properties that enable this yeast to grow on xylose under highly oxygen limited conditions. It also possesses an endoglucanase and four beta-glucosidases along with the permeases necessary to grow on cellulosic sugars. These features make S. passalidarum very attractive for SSF applications and for studying regulatory mechanisms enabling bioconversion of lignocellulosic materials by yeasts.  Like other beetle-associated yeasts such as Scheffersomyces stipitis, S. passalidarum uses an alternative codon system in which CUG codes for serine rather than leucine. Our laboratory has developed versatile genetic tools for the transformation, mating and selection of CUG yeasts, which have enabled us to overexpress sugar transporters and other genes essential for the efficient fermentation of cellulosic and hemicellulosic sugars.