Bioluminescent Reporter Development and Testing for Cre-recombinase and CRISPR/Cas9 Activity for Selectable Marker Recovery in Scheffersomyces stipitis

Abstract

Scheffersomyces stipitis is the most efficient xylose fermenting yeast known, though it is not as well-studied as other yeast species. The ability of S. stipitis to ferment xylose makes it a potentially useful yeast in the fermenting of lignocellulos biomass into biofuels, such as ethanol. One way to make S. stipitis more effective in biofuel production is to inhibit the glucose metabolic pathway, which requires the delition of multiple genes. S. stipitis belongs to the “CUG clade,” which is a group of yeast species that translate the CUG codon as serine instead of a leucine like other eukaryotic organisms. The difference makes most genetic tools ineffective in S. stipitis. Genetic tools need to be optimized with this change before they can be used in S. stipitis. Selectable markers are one of the tools that would need to be optimized for S. stipitis, especially since they are used during gene deletions. To make sequential gene deletions, different selectable markers are used for each deletion. Therefore, the number of deletions is based on the number of selectable markers available. Having a way to recover selectable markers so they can be reused in other gene deletions is an effective way to increase the number of deletions that can be performed. The aim of this research is to test the ease of usage and the effectiveness of two strategies for recovering selectable markers: 1) Cre-recombinase or 2) CRISPR/Cas9.