Characterization of the tautomycin biosynthetic gene cluster from Streptomyces spiroverticillatus unveiling new insights into dialkylmaleic anhydride and polyketide biosynthesis

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.

FIGURE 1.

A, structures of TTM and TTN in anhydride or diacid forms, and biosynthetic origin of the dialkylmaleic anhydride by feeding experiments using ¹³C-labeled acetate and propionate. The methoxymalonate-derived unit in TTM is highlighted by the dotted oval. R, polyketide moiety of TTM or TTN. B, selected natural product inhibitors of PP-1 and PP-2A featuring a spiroketal or dialkylmaleric anhydride moiety. C, selected natural products containing a dialkylmaleic anhydride moiety.

FIGURE 2.

A, restriction map of the 130-kb DNA region from S. spiroverticillatus harboring the entire ttm gene cluster as represented by seven overlapping cosmids. B, genetic organization of the ttm gene cluster. The solid black bar indicates the DNA region sequenced. Proposed functions for individual open reading frames are coded with various patterns and summarized in Table 1. P, PstI.

FIGURE 3.

Deduced module and domain organization of TtmHIJ PKSs and a linear model for the TTM PKS templated assembly of the TTM polyketide backbone featuring four varying starter and extender units as well as key tailoring steps for TTM biosynthesis. R, H or dialkylmeric anhydride unit to emphasize that the precise timing for the TtmK-catalyzed coupling step is unknown. The AT domains are coded with various patterns to highlight their substrate specificity. KS, ketosynthase; ER, enoylreductase.

FIGURE 4.

Phylogenetic analysis of TtmHIJ AT domains and its homologs from type I PKSs that specify malonyl-CoA, methylmalonyl-CoA, methoxymalonyl-ACP, or isobutyryl-CoA. Cluster names and NCBI accession numbers for each of the AT domains are given in parentheses. The scale bar represents 0.1 amino acid substitution per position.

FIGURE 5.

Inactivation of ttmJKPRS, complementation of the ΔttmK and ΔttmS mutants, and HPLC analysis of TTM production in S. spiroverticillatus wild-type (panel I), recombinant strains SB6003 (panel II), SB6004 (panel III), SB6006 (panel V), SB6007 (panel VI), SB6008 (panel VII), and complementation strains SB6009 (panel IV), SB6011 (panel VIII). •, TTM.

FIGURE 6.

Proposed pathway for dialkylmaleic anhydride biosynthesis involving minimally TtmO, TtmP, and TtmM and its coupling with the polyketide moiety catalyzed by TtmK. R, polyketide moiety of TTM.