2.1 Plasmid construction
Plasmids were constructed according to Holkenbrink et al., 2018 [19]. Integration and guide RNA (gRNA) vectors were used to introduce gene expression constructs into characterized genome sites ofY. lipolytica [19].
Primers, synthetic DNA, BioBricks, and plasmids used in this study are listed in Supplementary Tables S1, S2, S3, and S4, respectively. BioBricks were amplified by PCR using Phusion U polymerase (Thermo Fisher Scientific) with the following thermal program: 98°C for 5 min, 30 cycles of (98°C for 20 s, 54°C for 30 s, 72°C for 30 s/kb), and 72°C for 4 min. After DNA electrophoresis on 1% agarose gel, BioBricks were purified using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel). The integration vectors were digested with FastDigest SfaAl (Thermo Fisher Scientific) and nicked with Nb.BsmI (New England BioLabs). Biobricks with compatible overhangs and nicked integration vectors were assembled and transformed into Escherichia colistrain DH5α via Uracil-Specific Excision Reaction (USER®) cloning. Clones containing the correct assembly were verified by PCR and Sanger sequencing.