Surface tethered xylosidase activity improved xylan conversion in engineered strains of Saccharomyces cerevisiae

Abstract

AbstractBACKGROUNDSecond‐generation biofuel production strategies require utilization of the largest possible fraction of the available sugars in renewable biomass. Xylose can make up as much as 35% of plant dry cell weight (DCW), primarily in the form of the hemicellulose polymer xylan. Xylo‐oligosaccharides (XOS) are breakdown products of xylan released during pre‐treatment and hydrolysis that may inhibit the fermentation process. To enable growth on xylan and the removal of inhibitory XOS, CRISPR‐Cas9 was used to confer xylanase and GH43 xylosidase activity to xylose‐assimilating Saccharomyces cerevisiae strains. Xylosidase activity was engineered to be cell free or tethered to the yeast cell surface.RESULTSGene‐integration and expression of both xylanolytic enzyme‐encoding genes was successful. Secretion yielded higher overall xylosidase activity when strains were cultivated on glucose, but cell‐associated activity was higher when strains were cultivated in the presence of xylose. Growth trials on both xylan and XOS showed that a combination of both enzymes, along with cell‐associated xylosidase production, resulted in the best growth on both substrates. The increased growth on xylan also translated to substantial improvements in ethanol production from polymeric xylan as sole carbon source.CONCLUSIONIncreased enzyme production, growth capabilities, and hemicellulosic substrate conversion due to cell‐tethered activity over secreted activity were shown for the first time. The use of a GH43 xylosidase to help avoid problematic transglycosylation for this purpose is also novel. The development of S. cerevisiae strains capable of xylan utilization and fermentation brings the industry a step closer to the ideal of utilizing the full range of sugars in biomass feedstocks for large scale ethanol production. © 2022 Society of Chemical Industry (SCI).