Heterologous Expression of Fungal Polyketide Synthases

To construct the Pks1 heterologous expression vector, SOE (splicing by overlapping extension)-PCR and yeast-based assembly approaches were used [49 (link)]. First, the constitutive gpdA promoter was introduced into the plasmid pYH-WA-pyrG as described [24 (link)] to form pYH-wA-pyrG-gpdA. Second, two PCR fragments with overlapping regions (250 bp), corresponding to the genomic region of the coding sequences of Pks1 or Pks2, were amplified from M. robertsii genomic DNA. The two fragments and the NheI-digested pYH-WA-pyrG-gpdA were purified and transformed into S. cerevisiae BJ5464-NpgA using an S. c. EasyComp Transformation Kit (Invitrogen, USA). PCR was used to screen for yeast colonies containing the target plasmids. Target plasmids were isolated using a Zymoprep (D2001) Kit (Zymo Research, USA), and confirmed with restriction enzyme digestion and sequencing. Target plasmids were linearized with SwaI and transformed into the WT A. nidulans strain LO8030 to create transformants expressing Pks1 or Pks2. The insertion of the Pks1 or Pks2 gene into the genome of A. nidulans was confirmed with PCR using a Taq Mix kit (Tiagen Biotech, China).