Abstract
Tautomycin (TTM) is a highly potent and specific protein phosphatase inhibitor isolated from Streptomyces spiroverticillatus. The biological activity of TTM makes it an important lead for drug discovery, whereas its spiroketal-containing polyketide chain and rare dialkylmaleic anhydride moiety draw attention to novel biosynthetic chemistries responsible for its production. To elucidate the biosynthetic machinery associated with these novel molecular features, the ttm biosynthetic gene cluster from S. spiroverticillatus was isolated and characterized, and its involvement in TTM biosynthesis was confirmed by gene inactivation and complementation experiments. The ttm cluster was localized to a 86-kb DNA region, consisting of 20 open reading frames that encode three modular type I polyketide synthases (TtmHIJ), one type II thioesterase (TtmT), five proteins for methoxymalonyl-S-acyl carrier protein biosynthesis (Ttm-ABCDE), eight proteins for dialkylmaleic anhydride biosynthesis and regulation (TtmKLMNOPRS), as well as two additional regulatory proteins (TtmF and TtmQ) and one tailoring enzyme (TtmG). A model for TTM biosynthesis is proposed based on functional assignments from sequence analysis, which agrees well with previous feeding experiments, and has been further supported by in vivo gene inactivation experiments. These findings set the stage to fully investigate TTM biosynthesis and to biosynthetically engineer new TTM analogs.
Highlights
Tautomycin (TTM)2 is a polyketide natural product first isolated in 1987 from Streptomyces spiroverticillatus [1]
It was later realized that TTM is a potent and specific inhibitor of protein phosphatases (PPs) PP-1 and PP-2A [9]
Because the major structural differences between TTM and TTN reside in the region distal to the dialkylmaleic anhydride moiety (Fig. 1A), it has been proposed that differences in these moieties might be responsible for the PP-1 selectivity [17,18,19]
Summary
Bacterial Strains and Plasmids—Escherichia coli DH5␣ was used as the host for general subcloning. Mutant cosmids pBS6018 (⌬ttmJ), pBS6019 (⌬ttmK), pBS6021 (⌬ttmP), pBS6022 (⌬ttmR), and pBS6023 (⌬ttmS) were constructed (supplemental Table S1) and introduced into S. spiroverticillatus by conjugation from E. coli ET12567/pUZ8002 according to the literature procedure with the following modifications [31]. The desired double cross-over mutants, selected by the apramycin-resistant and kanamycin-sensitive phenotype, were isolated as SB6003 (⌬ttmJ), SB6004 (⌬ttmK), SB6006 (⌬ttmP), SB6007 (⌬ttmR), and SB6008 (⌬ttmS), whose genotypes were verified by Southern analysis A spore suspension (5 l) of the S. spiroverticillatus wild-type or recombinant strains was first inoculated into 50 ml of seed medium in a 250-ml flask and incubated at 28 °C and 250 rpm for 2 days. Deduced functions of open reading frames in the tautomycin biosynthetic gene cluster
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