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