2.2 Construction of plasmids and engineered strains
Primers and plasmids used in this study are listed in Table S1 and Table S2, respectively. CYP5150L8 expression cassette was amplified with prime pair HXT7p-CYP5150L8-F/ FBA1t-CYP5150L8-R from plasmid pRS426-HXT7p-CYP5150L8-FBA1t (Wang et al., 2018). Plasmid pRS425-iGLCPR-Hygr (Lan et al., 2019) was linearized by Pme I (NEB, Beijing, China). The amplified CYP5150L8 fragment and linearized pRS425-iGLCPR-Hygr were ligated to produce plasmid pRS425-Hyg-CYP5150L8-iGLCPR according to the procedure described in SoSoo cloning kit (Tsingke, Beijing, China).
The G418-resistance gene KanMX was amplified from plasmid pUG6 (Guldener et al., 1996) with primer pair KanMX-F/KanMX-R (Table S1). A truncated URA3 promoter was adopted to drive the transcription ofKanMX (Bao et al., 2015). The BamH I linearized plasmid pRS426-HXT7p-FBA1t and KanMX expression cassette were assembled to produce plasmid pRS426-HXT7p-FBA1t-KanMX by DNA assembler (Shao et al., 2009). The Pme I linearized plasmid pRS426-HXT7p-FBA1t-KanMX and CYP5139G1 expression cassette (amplified with primer pair HXT7p-CYP5139G1-F/FBA1t-CYP5139G1-R) was ligated to produce plasmid pRS426-KanMX-CYP5139G1 according to the procedure described in SoSoo cloning kit.
The plasmid pRS425-Hyg-CYP5150L8-iGLCPR and a series of CYP gene carrying plasmids (pRS426-CYPx), which we constructed previously (Wang et al., 2018), were transformed into S. cerevisiae YL-T3 to generate corresponding engineered yeast transformants (Table S3) using standard lithium acetate protocol (Gietz et al., 2007). Plasmid pRS425-Hyg-CYP5150L8-iGLCPR and plasmid pRS426 were also transformed into YL-T3 and served as a control. Plasmids pRS425-Hyg-CYP5150L8-iGLCPR and pRS426-KanMX-CYP5139G1 were transformed into YL-T3 to generate the strain HygL8-G418G1 (Table S3).