Development of xylan- and xylose assimilating Saccharomyces cerevisiae using CRISPR-Cas9 technology

Abstract

Biorefineries produce a vast array of products for industrial, pharmaceutical and food applications. To allow for an enviromentally and economically sustainable production, the input biomass should not compete with crops cultivated for other purposes such as food or feed. Instead, lignocellulosic biomass from e.g. fast-growing wood and grasses has emerged as an alternative. However, one of the most important industrial microorganisms, the yeast Saccharomyces cerevisiae, is unable to efficiently degrade and utilise the polysaccharides present in the plant cell wall, including hemicellulose, a major constituent of lignocellulosic biomass. This project was aimed at developing a yeast strain able to utilise xylan, one of the most abundant hemicelluloses. A CRISPR-Cas9 system was used to introduce the hemicellulases endo-xylanase and β-xylosidase for degradation of the xylan polymer, and the XR/XDH pathway for assimilation of the xylose monomers by S. cerevisiae. The integration of the XR/XDH pathway and the hemicellulases was successful and growth analysis of the resulting strains showed ability to grow on xylose based media as well as on xylooligosaccharides, indicating a functional XR/XDH pathway and β-xylosidase enzyme. β-xylosidase activity was also demonstrated through enzymatic assays. However, capability of degrading the xylan polymer could not be demonstrated via enzymatic assays, indicating insufficient endo-xylanase activity. While further studies are required to optimise the hemicellulase expression in the constructed strain, the xylose-degrading ability of the strain was comparable to that of other genetically modified strains expressing the XR/XDH pathway. Overall, the successful development of an S. cerevisiae strain capable of efficiently degrading xylan and utilising xylose would be of great industrial value and further research on this topic should be pursued.