Genes for Production of the Enediyne Antitumor Antibiotic C-1027 in
            <i>Streptomyces globisporus</i>
            Are Clustered with the
            <i>cagA</i>
            Gene That Encodes the C-1027 Apoprotein

Enediynes are potent antitumor antibiotics that are classified as 9- or 10-membered according to the size of the enediyne core structure. However, almost nothing is known about enediyne core biosynthesis, and the determinants of 9- versus 10-membered enediyne core biosynthetic divergence remain elusive. Previous work identified enediyne-specific polyketide synthases (PKSEs) that can be phylogenetically distinguished as being involved in 9- versus 10-membered enediyne biosynthesis, suggesting that biosynthetic divergence might originate from differing PKSE chemistries. Recent in vitro studies have identified several compounds produced by the PKSE and associated thioesterase (TE), but condition-dependent product profiles make it difficult to ascertain a true catalytic difference between 9- and 10-membered PKSE-TE systems. Here we report that PKSE chemistry does not direct 9- versus 10-membered enediyne core biosynthetic divergence as revealed by comparing the products from three 9-membered and two 10-membered PKSE-TE systems under identical conditions using robust in vivo assays. Three independent experiments support a common catalytic function for 9- and 10-membered PKSEs by the production of a heptaene metabolite from: ( i ) all five cognate PKSE-TE pairs in Escherichia coli ; ( ii ) the C-1027 and calicheamicin cognate PKSE-TEs in Streptomyces lividans K4-114; and ( iii ) selected native producers of both 9- and 10-membered enediynes. Furthermore, PKSEs and TEs from different 9- and 10-membered enediyne biosynthetic machineries are freely interchangeable, revealing that 9- versus 10-membered enediyne core biosynthetic divergence occurs beyond the PKSE-TE level. These findings establish a starting point for determining the origins of this biosynthetic divergence.

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“…Bioassays against Micrococcus luteus ATCC 9431 to detect enediyne production were performed as described previously (Fig. S3) (27).…”

Section: Methodsmentioning

confidence: 99%

Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes

Horsman

Chen²,

Thorson³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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109

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“…All of the media for Streptomyces growth were prepared according to standard protocols (31). ISP-4 and tryptic soy broth were from Difco Laboratories (Detroit, MI), and modified ISP-4 is ISP-4 supplemented with 0.05% yeast extract and 0.1% tryptone (32).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Tautomycin Biosynthetic Gene Cluster from Streptomyces spiroverticillatus Unveiling New Insights into Dialkylmaleic Anhydride and Polyketide Biosynthesis

Rajski

et al. 2008

Journal of Biological Chemistry

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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.

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“…A genomic library of S. lavendulae NRRL 11002 was constructed in Super-Cos1 according to a previously published protocol (22). E. coli VCS257 and Gigapack III XL packaging extract Table S2 in the supplemental material).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Saframycin A Gene Cluster fromStreptomyces lavendulaeNRRL 11002 Revealing a Nonribosomal Peptide Synthetase System for Assembling the Unusual Tetrapeptidyl Skeleton in an Iterative Manner

et al. 2008

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Saframycin A (SFM-A), produced by Streptomyces lavendulae NRRL 11002, belongs to the tetrahydroisoquinoline family of antibiotics, and its core is structurally similar to the core of ecteinascidin 743, which is a highly potent antitumor drug isolated from a marine tunicate. In this study, the biosynthetic gene cluster for SFM-A was cloned and localized to a 62-kb contiguous DNA region. Sequence analysis revealed 30 genes that constitute the SFM-A gene cluster, encoding an unusual nonribosomal peptide synthetase (NRPS) system and tailoring enzymes and regulatory and resistance proteins. The results of substrate prediction and in vitro characterization of the adenylation specificities of this NRPS system support the hypothesis that the last module acts in an iterative manner to form a tetrapeptidyl intermediate and that the colinearity rule does not apply. Although this mechanism is different from those proposed for the SFM-A analogs SFM-Mx1 and safracin B (SAC-B), based on the high similarity of these systems, it is likely they share a common mechanism of biosynthesis as we describe here. Construction of the biosynthetic pathway of SFM-Y3, an aminated SFM-A, was achieved in the SAC-B producer (Pseudomonas fluorescens). These findings not only shed new insight on tetrahydroisoquinoline biosynthesis but also demonstrate the feasibility of engineering microorganisms to generate structurally more complex and biologically more active analogs by combinatorial biosynthesis.

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Genes for Production of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus Are Clustered with the cagA Gene That Encodes the C-1027 Apoprotein

Cited by 84 publications

References 53 publications

Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes

Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes

Characterization of the Tautomycin Biosynthetic Gene Cluster from Streptomyces spiroverticillatus Unveiling New Insights into Dialkylmaleic Anhydride and Polyketide Biosynthesis

Characterization of the Saframycin A Gene Cluster fromStreptomyces lavendulaeNRRL 11002 Revealing a Nonribosomal Peptide Synthetase System for Assembling the Unusual Tetrapeptidyl Skeleton in an Iterative Manner

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