Cloning and characterization of the biosynthetic gene cluster of the bacterial RNA polymerase inhibitor tirandamycin from marine-derived Streptomyces sp. SCSIO1666

Abstract

Tirandamycins are bacterial RNA polymerase inhibitors holding great potential for antibacterial agent design. To elucidate the biosynthetic machinery and generate new derivatives, the tirandamycin biosynthetic gene cluster was cloned and sequenced from marine-derived Streptomyces sp. SCSIO1666. The biosynthetic gene cluster of tirandamycin spans a DNA region of ∼56kb and consists of 15 open reading frames (ORFs) which encode three type I polyketide synthases (TrdAI, AII, AIII), one non-ribosomal peptide synthetase (TrdD), one phosphopantetheinyl transferase (TrdM), one Type II thioesterase (TrdB), one FAD-dependent oxidoreductase (TrdL), one cytochrome P450 monooxygenase (TrdI), three proteins related to resistance and regulations (TrdHJK), and four proteins with unknown function (TrdCEFG). To investigate the roles of the genes played in the biosynthetic machinery, seven genes (trdAI and trdBDFHIK) were inactivated via in frame replacement with an apramycin gene cassette using λ-RED recombination technology. The ΔtrdAI and ΔtrdD mutants targeting the ketosynthase and adenylation domain of TrdAI and TrdD, respectively, abolished the production of tirandamycins, confirming their involvement in the tirandamycin biosynthesis. TrdH showed high homology to LuxR family transcriptional regulatory proteins, disruption of which abolished the production of tirandamycins, indicating that TrdH is a positive regulator for tirandamycin biosynthesis. On the other hand, TrdK showed high homology to TetR-family transcriptional regulatory proteins, disruption of which significantly increased the yields of tirandamycins almost one-fold, implicating that TrdK is a negative regulator for tirandamycin biosynthesis. Disruption of the gene trdI resulted in the accumulation of the intermediate tirandamycin C (3) and a trace amount of new product tirandamycin C2 (5). A model of tirandamycin biosynthesis was proposed based on bioinformatics analyses, gene inactivation experiments and intermediates isolated from the mutants. These findings set the stage for further study of the tirandamycin biosynthetic mechanism and rationally engineer new tirandamycin analogues.