Sequence analysis of porothramycin biosynthetic gene cluster

Abstract The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosyn-thetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPSmodules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methyla-tion of anthranilate unit occurs prior to the condensation reac-tion, while modifications of branched proline derivative, oxi-dation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltrans-ferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransfer-ase gene por18 homologous to orf19 from anthramycin bio-synthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as sug-gested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analy-sis of the product in the S. albus culture broth