Isolation and Partial Characterization of Cryptic Polyene Gene Cluster in Pseudonorcardia autotrophica

초록

The polyene antibiotics, a category which includes nystatin, pimaricin, amphotericin and candicidin, comprise a family of most promising antifungal polyketide compounds, and are typically produced by soil actinomycetes species. The major antifungal mechanism of these polyene antibiotics is believed to be the specific binding to the ergosterol present in the fungal membrane, forming a channel to the leakage of the cellular K+ and Mg2+, and then eventually leading to the fungal cell death. Although the polyene compounds are clinically used with limitation due to high toxicity and side effect, the superior antifungal activities of polyene compounds are still appreciated for the further development of the improved antkfungal drugs. The biosynthetic gene clusters for these polyenes have been previously investigated, revealing the presence of highly homologous cytochrome P450 hydroxylase (CYP) genes. Using polyene CYP-specific PCR screening with several actinomycetes genomic DNAs, the Pseudonorcardia autotrophica strain was determined to contain a unique polyene-specific CYP gene. Genomic DNA library screening using the polyene-specific CYP gene probe revealed 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 (ORFs), which were found to be highly homologous, but also unique to the 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 species.