Engineered Y. lipolytica for Efficient Xylose Utilization

Primary strains used in this study were Y. lipolytica A101 [61 (link)] and AJD with overexpression of YALI0E32769g—diacylglycerol acyltransferase Dga1p [40 (link)]. Both strains belong to the Department of Biotechnology and Food Microbiology at Wroclaw University of Environmental and Life Sciences, Poland. Escherichia coli DH5α was primarily used for molecular cloning. Vector pAD [62 (link)], carrying the UAS1_B16-TEF promoter, was the basis for developing new plasmids with the native XR, XDH or XK gene fragment. All plasmids and strains used in this study are listed in Additional file 1: Table S1 in the supplemental material. The all XYL genes fragments were amplified from the Y. lipolytica A101 genomic DNA. The list of primers used is shown in Additional file 1: Table S2. The PCR amplified genes were cloned into the pAD vector using SgsI and NheI/Pml1 sites, T4 DNA Ligase (Thermo Fisher Scientific) and used for transformation of E. coli. The obtained plasmids were isolated using the Plasmid Mini Kit (A&A Biotechnology, Poland), sequenced (Genomed, Poland) and digested with MssI. Linear expression cassettes were used to transform yeast according to the lithium acetate method [63 (link)]. The restriction enzymes were acquired from FastDigest Thermo Scientific.