Development of cloning and transformation systems in the Pleuromutilis-producing Basidiomycete Clitopilus passeckerianus (Pilat) Singer NRRL 3100

Cloning and transformation systems were developed in the basidiomycete Clitopilus passeckerianus NRRL 3100. Five strains of C. passeckerianus were used, namely the wild type strain (cpwt), HP76, LP1, LP2 and LP5. Genomic DNA was isolated from the five strains and used as template for polymerase chain reaction (PCR). The primers used were constructed based on GENBANK sequences of the geranyl geranyl transferase (GGT) gene, one of the genes involved in the biosynthetic pathway of pleuromutilin. A 200-bp product was obtained from primer pairs GGT2F/GGT2R and GGT3F/GGT3R using Cpwt as template. Regenerate primers based on GGT were also constructed. From the degenerate primers, five bands were obtained from Cpwt, eight bands for LP5, while no PCR products were obtained from HP76, LP1 and LP2. The 200-bp product was cloned using the TOPOTA Cloning kit. From the transformants, the recombinant plasmid pKCP was isolated and subjected to sequence analysi s using BLASTN 2.2.6. Sequence analysis revealed that the insert DNA may have been lost between the time of cloning and sequencing. Plasmid pKCP was transformed into Cpwt protoplasts. Co-transformation procedures were also performed using plasmid p PEN510, which harbors the amdS gene coding for acetamidase since pCKP has no readily distinguishable phenotype. The transformed protoplasts were regenerated on four media supplemented with 0.6M sorbitol, namely Mycological Agar (MA), Acetamide medium (ACE), Aspergillus Minimal Medium and Acetamide Phenol Red (APR). Density of growth was taken as a measure of regeneration efficiency due to numerous colonies obtained. Similar mycelial densities were obtained for the single (pKCP or pPEN510) and double transformants (pKCP and pPEN510), indicating that the protoplasts were capable of taking up both plasmids concurrently. Growth of regenerants was dense on MA and ACE due to the higher nutrient content of these two media. Faster growth rates than those of the non-protoplasted cells were also obtained by the regenerants and transformants. Production of pleuromutilin was not observed in the transformed strains. Possible antifungal activity against members of family Moniliaceae was also detected.