Isolation and partial characterization of a cryptic polyene gene cluster in Pseudonocardia autotrophica

The polyene antibiotics, a category that includes nystatin, pimaricin, amphotericin, and candicidin, comprise a family of very promising antifungal polyketide compounds and are typically produced by soil actinomycetes. The biosynthetic gene clusters for these polyenes have been previously investigated, revealing the presence of highly similar cytochrome P450 hydroxylase (CYP) genes. Using polyene CYP-specific PCR screening with several actinomycete genomic DNAs, Pseudonocardia autotrophica was determined to contain a unique polyene-specific CYP gene. Genomic DNA library screening using the polyene-specific CYP gene probe identified a positive cosmid clone, which contained a DNA fragment of approximately 34.5 kb. The complete sequencing of this DNA fragment revealed a total of seven complete and two incomplete open reading frames, which were found to be highly similar, but still unique, when compared to previously known polyene biosynthetic genes. These results suggest that the polyene-specific screening approach may constitute an efficient method for the isolation of potentially valuable cryptic polyene biosynthetic gene clusters from various rare actinomycetes.