Pesc ura

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)).

The production of human TK1 was carried out in an E. coli expression system by Genscript’s recombinant protein service. In-house production of human TK1 was done using the pESC-URA (Genscript, Piscataway, NJ) yeast expression system and a Saccharomyces cerevisiae yeast strain with a REG-1 mutation. Briefly, the coding sequence of the human TK1 (Genbank NM_003258.5) was synthesized and placed into the pESC-URA vector flanked by the Sal I restriction sites. A tag of 6 histidines was included at the C-terminus of the TK1 sequence to facilitate His-tag purification. The TK1-pESC-URA vector was introduced in electrocompetent yeast using the lithium acetate procedure and an Eporator system (Eppendorf, Hamburg, Germany) [35 ]. After electroporating, the cells were plated in synthetic complete (SC) drop-out Ura-plates (Takara Bio USA Inc, Mountain View, CA) and grown for 36 h at 30 °C. Yeast culture was scaled up to 500 ml and induced for protein expression with galactose. After 36 h, yeast was harvested and brake-open in lysis buffer with the Halt™ protease inhibitor cocktail (ThermoFisher Scientific, Waltham, MA) in a French press. Recombinant TK1 was purified from cleared lysate using NI-NTA-agarose beads columns (Qiagen, Hilden, Germany) and validated with commercially outsourced TK1 produced in E. coli by Genscript and the commercial anti TK1 antibody ab91651 in Western blot.