Zymogen frozen ez yeast transformation kit 2

Yarrowia lipolytica transformation was performed with Zymogen Frozen EZ Yeast Transformation Kit II (Zymo Research, Irvine, CA, United States). For gene deletion and integration, approximately 0.5–1 μg of linearized DNA was used for transformation, and then 150–200 μL of transformation mixture was plated on SD-ura solid media. The marker ura3 was removed as described previously (Fickers et al., 2003 (link); Wang et al., 2016 ). Selection plates were incubated at 30°C for 2–4 days. Diagnostic PCR and DNA sequencing were used to confirm gene deletion and gene integration. All primers used for identification of the engineered strains are listed in Supplementary Table 2.

Yarrowia lipolytica transformation with integrative fragments or recombinant plasmids was performed with Zymogen Frozen EZ Yeast Transformation Kit II (Zymo Research Corporation). For CRISPR plasmid transformations, the cells were transformed with plasmid and cultivated in SC-Ura liquid medium for 4 d. Then the cells were plated onto SC-Ura plates for 2 d and confirmed by sequencing. For integrative fragment transformations, approximately 2 μg of linearized DNA was used in the transformation reaction and then the cells were harvested by centrifugation at 5,000 rpm for 2 min and plated on SC agar plates without the auxotrophic compound supplemented by the fragments. Selection plates were incubated at 28°C for 2–3 days.

Knockout mutants were constructed by homologous recombination (transformation with linearized knockout cassette) and marker rescue (Cre-recombinase based uracil marker deletion) as previously described (Fickers et al., 2003 (link); Wang et al., 2016 (link)). We first constructed the strain YL-1 (PO1f-△PEX10) through the deletion of PEX10 in the Y. lipolytica PO1f. The strain YL-1was used for episomal expression of pJN44-DGA1, pJN44- DGA1-12D, pJN44-DGA1-SCD, pJN44-DGA1-15D, pJN44-DGA1-12D-SCD, pJN44-DGA1-15D-SCD, and pJN44-DGA1-12D-15D, utilizing a selective marker leucine. Then, the integrative cassette plasmid ura-△MFE1:DGA1-12D-15D was linearized (12D-15D-DGA1-MFE1-up-loxp-down), inserted into the YL-1 strain, and the transformants were screened using uracil dropout plate to create the strain YL-9. Then the plasmid pJN44-ACC1 was expressed in YL-9, resulting in YL-10. The transformation of Y. lipolytica was performed using Zymogen Frozen EZ yeast transformation kit II (Zymo Research Corporation; United States) according to the manufacturer’s instruction.