*4.4. Heterologous Expression of SlPKS4 and Sl4001-Sl4007 in A. nidulans A1145*

Genes *SlPKS4* and *Sl4001–Sl4007* were amplified from genomic DNA extract from *S. lamellicola* HDN13-430. Plasmids pANU, pANR, pANP with auxotrophic markers for uracil (pyrG), riboflavin (riboB) and pyridoxine (pyroA) were digested with PacI and NotI, PacI and BamHI, PacI and HindIII, respectively, and used as vectors to insert genes. The corresponding heterologous expression plasmids were obtained by in vivo yeast homologous recombination in *S. cerevisiae* Y31. The correct colonies checked by PCR were combined, and subjected to yeast miniprep to obtain the plasmids. The plasmids obtained from yeast miniprep using Plasmid Miniprep Kit (Zymo Research, Irvine, CA, USA) were introduced into the competent cells of *E. coli* XL-1. After plasmid extraction from *E. coli* to obtain transformants with a single plasmid, plasmids were sequenced to confirm identities.

For protoplast formation of *A. nidulans* A1145, the strain *A. nidulans* A1145 was first grown on CD plates at 37 ◦C for 3 days and fresh spores were collected and stored. Then, the spores were germinated in a 250 mL Erlenmeyer flask containing CD media at 37 ◦C and 180 rpm for about 8 h. Mycelia were gathered by centrifugation at 4000 rpm for 15 min, and washed by 25 mL osmotic buffer (1.2 M MgSO4, 10 mM sodium phosphate, pH 5.8). Subsequently, the mycelia were suspended into 10 mL of osmotic buffer containing 30 mg lysing enzymes from Trichodema harzianum (Sigma) and 20 mg Yatalase (TaKaRa), transferred into an empty sterile bottle, and cultured in a shaker of 28 ◦C at 80 rpm overnight to form protoplast. After the whole night, the mixture was collected in a 50 mL centrifuge tube and covered gently with isopyknic protoplast trapping buffer (0.6 M sorbitol, 0.1 M pH 7.0 Tris-HCl). After centrifugation at 4000 rpm for 15 min at 4 ◦C, protoplasts were collected in the interface of the above two buffers. The protoplasts were then transferred to a sterile 50 mL centrifuge tube and washed by 20 mL STC buffer (1.2 M sorbitol, 10 mM CaCl2, 10 mM pH 7.5 Tris-HCl). The protoplasts were resuspended in 2 mL STC buffer for transformation.

For protoplast transformation of *A. nidulans* A1145, the necessary plasmids and the corresponding empty plasmids (the desired strains were regarded as control in the following crude analysis) were added to 100 μL *A. nidulans* A1145 protoplast suspension prepared above and the mixture was incubated on ice for 60 min. Next, 600 μL of polyethylene glycol (PEG) solution (60% PEG, 50 mM calcium chloride and 50 mM pH 7.5 Tris-HCl) was added to the protoplast mixture, and the mixture was incubated at room temperature for

an additional 25 min. The mixture was spread on the regeneration solid CDS medium with appropriate supplements, including 10 mM uridine, 5 mM uracil and/or 0.5 μg/mL pyridoxine HCl and/or 2.5 μg/mL riboflavin, depending on the plasmids being transformed and incubated at 37 ◦C for around 3 days.
