Engineered Yeast Strains for Xylose Metabolism

The microorganisms, plasmids, and primers used in this study are provided in tables S1 and S2. In particular, the diploid yeast S. cerevisiae ATCC26603 and CRD3 were used as a host for testing the activities of mined XIs, mutated XIs, and artificial ancestral XIs. Strain CRD3 is a derivative of strain ATCC26603 with modified genotypes for enhancing xylose metabolism, including overexpressing the xylose kinase gene XKS1 and the pentose phosphate pathway genes TKL1, RPE1, RKI1, and TAL1, introducing a xylose specific transporter gene of GAL2^N376F, and deleting aldose reductase gene GRE3 and 4-nitrophenylphosphatase gene PHO13. The replicating plasmid pESC-URA provided by GenScript Corporation (www.genscript.com) was used for XI expression in S. cerevisiae. The pML104 plasmid–mediated CRISPR-Cas9 system was used to integrate XI genes into the S. cerevisiae chromosome (66 (link)).