Abstract Description

Second-generation biofuels are attractive alternatives to environmentally damaging, non-renewable fossil fuels, as they are carbon neutral and produced from renewable lignocellulosic biomass (LCB). One of the main challenges facing LCB conversion to bioethanol is the incomplete use of all available sugars present in the biomass. To overcome this challenge, the hemicellulose fraction, consisting mostly of xylan, should be targeted for conversion in addition to the cellulose fraction. This study aimed to address this issue by developing strains of Saccharomyces cerevisiae capable of xylan degradation and efficient utilization of xylose. Natural S. cerevisiae strain isolates YI13, YI59 and FIN1 were selected for potential industrial applications due to their robust fermentation performance and enhanced ethanol production compared to laboratory strains. Efficient xylose utilization was conferred to the natural strains through engineering with a xylose isomerase (XI) gene cassette and a xylose transporter, combined with adaptive laboratory evolution (ALE) in minimal media with xylose as the sole carbon source. The xylose-utilizing strains were further engineered with cell-associated GH43 xylosidase activity and secreted xylanase activity. Enzymatic assays, growth trials on hemicellulosic substrates and fermentation on xylose and xylan were preformed to evaluate the engineered strains. Strains were successfully engineered with xylan conversion capabilities and efficient xylose utilization. Xylosidase activities were similar across the three stains with ~4 U/gDCW, while xylanase activities ranged from ~220 to ~770 U/gDCW. The final engineered version of YI13 showed the best xylose and xylan conversion, with ethanol titres of ~4.5 g/L from xylose (20 g/L) and ~ 3 g/L from xylan (40 g/L) in minimal media and maximum ethanol titres of ~7.1 g/L from xylose (20 g/L) and ~4.7 g/L from xylan (40 g/L) in YP media. This is the highest reported level of ethanol produced from polymeric xylan to date. Additionally, co-fermentation of glucose (20 g/L) and xylose (20 g/L) in YP yielded ~17.5 g/L ethanol, representing ~86 % of the theoretical maximum ethanol yield. In this study, the use of ALE and incorporation of a xylose transporter to improve xylose utilization was successfully demonstrated. A strain with notable xylosidase and xylanase activity, efficient xylose utilization and high ethanol production capabilities (from xylose and xylan) was developed. The development of S. cerevisiae strains capable of xylan utilization and fermentation brings the ideal of utilizing the full range of sugars in biomass feedstocks for large scale ethanol production closer.