CRISPR-Cas9 mediated xylose auxotrophy in Scheffersomyces stipitis

Abstract

Scheffersomyces stipitis is a top xylose fermenter. The yeast efficiently ferments xylose into ethanol, making it industrially important. In this study, I (1) create a new toolset to knock out and replace any gene in the S. stipitis genome and (2) show the phenotypic alterations in the yeast's xylose fermentation after knocking out the HXK1 gene which encodes HXK1. This experiment compares sugar utilization in wild-type and HXK1 knockout strains. The unique toolset BLINCAR (Bioluminescent Indicator Nullified by Cas9 Actuated Recombination) demonstrated that I could knock out HXK1 in S. stipitis. I tested this utilizing molecular and yeast biotechnology. The results reveal that HXK1 knockouts utilize glucose similarly to the wild type. Since knocking out HXK1 does not cause xylose auxotrophy, other glucose phosphorylation enzymes might be responsible.