2.4. Plasmid and pathway construction
All primers are listed in supplementary Table S1. All restriction enzymes were purchased from Fisher FastDigest enzymes. Plasmid miniprep, PCR clean-up, and gel DNA recoveries were using Zyppy and Zymoclean kits purchased from Zymo research. All the genes were PCR-amplified with the primers from genomic DNA of Y. lipolytica , S. cerevisiae ,E. coli , B. subtilis, Aspergillus nidulans , respectively (Supplymentary Table S1 and Table S2). All these genes were inserted into downstream of the Y. lipolytica TEF-intron promoter in the pYLXP’ vector backbone (Wong et al., 2017) at the SnaBI and KpnI site via Gibson assembly (Gibson et al., 2009). Upon sequence verification by Genewiz, the restriction enzyme Avr II,Nhe I, Not I, Cla I and Sal I (Fermentas, Thermo Fisher Scientific) were used to digest these vectors, and the donor DNA fragments were gel purified and assembled into the recipient vector containing previous pathway components in compliance with the YaliBricks subcloning protocol (Wong et al., 2017; Wong, Holdridge, Engel, & Xu, 2019). All assembled plasmids were verified by gel digestion and were subsequently transformed into the Y. lipolytica host strain Po1g ΔLeu using the lithium acetate/single-strand carrier DNA/PEG method (Chen et al., 1997). In chromosomal integration process, pYLXP’ vector assembled with functional genes was linearized by restriction enzyme NotI (Fermentas, Thermo Fisher Scientific). The linear fragment was transformed into theY. lipolytica host strain Po1g ΔLeu and cultivated on CSM-Leu minimal media (agar plate) for colony screening. The screened colony was later cultivated in YPD media and genomic DNA was extracted with Wizard genomic kits (Promega). Then the genomic samples were used as template for PCR verification of the integrated gene with gene-specific primers.
Results and discussions