Active-site residue, domain and module swaps in modular polyketide synthases

Vecchio, F; Petković, Hrvoje; Kendrew, Steven G.; Low, Lindsey; Wilkinson, Barrie; Lill, Rachel E.; Cortés, Jesús M.; Rudd, Brian A. M.; Staunton, Jim; Leadlay, Peter F.

doi:10.1007/s10295-003-0062-0

Cited by 114 publications

(102 citation statements)

References 25 publications

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“…The AT domains of the loading module and module 2 have the characteristic motif HAFH for recognizing acetate (malonyl-CoA) extender units (ATa) (8,38). The AT domain of module 1 is predicted to incorporate a butyrate (ethylmalonyl-CoA) unit due to the presence of a VASH motif (25,35).…”

Section: Resultsmentioning

confidence: 99%

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

Jiang

Wang

Kang

et al. 2012

Appl Environ Microbiol

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Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity.

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Section: Resultsmentioning

confidence: 99%

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

Jiang

Wang

Kang

et al. 2012

Appl Environ Microbiol

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“…The biosynthesis of the polyketide backbone of meridamycin is encoded by three large ORFs designated merA, merB and merC, which are adjacent and transcribed in the same direction and which encode, respectively, the multienzyme polypeptides MerA, MerB and MerC. Examination of the encoded AT domains of MER for distinctive amino acid sequence motifs allows the chemical nature of the extender unit recruited by each AT domain (especially, malonate versus methylmalonate or ethylmalonate) to be inferred (Haydock et al, 1995;Lau et al, 1999;Reeves et al, 2002;Del Vecchio et al, 2003). Examination of the 'reductive loop' (Staunton & Weissman, 2001) domains [KR, DH, enoylreductsase (ER)] in each extension module allows the level of reduction to be deduced for that extension.…”

Section: Organization Of the Meridamycin Biosynthetic Gene Clustermentioning

confidence: 99%

Organization of the biosynthetic gene cluster in Streptomyces sp. DSM 4137 for the novel neuroprotectant polyketide meridamycin

et al. 2006

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Meridamycin is a non-immunosuppressant, FKBP-binding macrocyclic polyketide, which has major potential as a neuroprotectant in a range of neurodegenerative disorders including dementia, Parkinson's disease and ischaemic stroke. A biosynthetic cluster predicted to encode biosynthesis of meridamycin was cloned from the prolific secondary-metabolite-producing strain Streptomyces sp. DSM 4137, not previously known to produce this compound, and specific gene deletion was used to confirm the role of this cluster in the biosynthesis of meridamycin. The meridamycin modular polyketide synthase consists of 14 extension modules distributed between three giant multienzyme proteins. The terminal module is flanked by a highly unusual cytochrome P450-like domain. The characterization of the meridamycin biosynthetic locus in this readily manipulated streptomycete species opens the way to the engineering of new, altered meridamycins of potential therapeutic importance.

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“…Examination of the encoded AT domains of CON polypeptides for distinctive amino acid sequence motifs allows the chemical nature of the extender unit recruited by each AT domain (especially malonate versus methylmalonate or ethylmalonate) to be inferred (Haydock et al, 1995;Lau et al, 1999;Reeves et al, 2001;Del Vecchio et al, 2003). A signature motif for AT domains that incorporate methoxymalonate has, because of the rarity of this extension unit, hitherto eluded definition (but see below).…”

Section: Organization Of the Concanamycin Pksmentioning

confidence: 99%

“…Identification of an AT sequence motif predicting the incorporation of extension units from methoxymalonyl-ACP It has previously been found possible to distinguish the specificity of AT domains for malonyl, methylmalonyl and ethylmalonyl extender units by analysis of short sequence motifs found within these domains (Haydock et al, 1995;Lau et al, 1999;Reeves et al, 2001;Del Vecchio et al, 2003) and this has been invaluable in helping predict the products of newly cloned PKS loci, as well as providing the basis for attempts to engineer changes in active-site specificity (Reeves et al, 2001;Del Vecchio et al, 2003). However, it has not been possible so far to identify those AT domains that specifically incorporate methoxymalonyl-ACP-derived extender units.…”

Section: Organization Of the Concanamycin Pksmentioning

confidence: 99%

“…To elucidate the unusual features of concanamycin biosynthesis and pave the way to biosynthetic engineering of novel derivatives, we have therefore undertaken the isolation and characterization of the gene cluster for the biosynthesis of concanamycin A. An unanticipated discovery from this analysis was the identification of an amino acid sequence motif which clearly distinguishes acyltransferase (AT) domains of a modular PKS which recruit methylmalonate extender units from those which recruit exclusively methoxymalonate extender units, which may assist in the direct reengineering of AT active sites in PKSs (Del Vecchio et al, 2003). We also present here techniques for convenient genetic manipulation of S. neyagawaensis and demonstrate that disruption of the cloned locus abolishes concanamycin biosynthesis.…”

Section: Introductionmentioning

confidence: 99%

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Organization of the biosynthetic gene cluster for the macrolide concanamycin A in Streptomyces neyagawaensis ATCC 27449

et al. 2005

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The macrolide antibiotic concanamycin A has been identified as an exceptionally potent inhibitor of the vacuolar (V-type) ATPase. Such compounds have been mooted as the basis of a potential drug treatment for osteoporosis, since the V-ATPase is involved in the osteoclast-mediated bone resorption that underlies this common condition. To enable combinatorial engineering of altered concanamycins, the biosynthetic gene cluster governing the biosynthesis of concanamycin A has been cloned from Streptomyces neyagawaensis and shown to span a region of over 100 kbp of contiguous DNA. An efficient transformation system has been developed for S. neyagawaensis and used to demonstrate the role of the cloned locus in the formation of concanamycin A. Sequence analysis of the 28 ORFs in the region has revealed key features of the biosynthetic pathway, in particular the biosynthetic origin of portions of the backbone, which arise from the unusual polyketide building blocks ethylmalonyl-CoA and methoxymalonyl-ACP, and the origin of the pendant deoxysugar moiety 49-O-carbamoyl-29-deoxyrhamnose, as well as the presence of a modular polyketide synthase (PKS) encoded by six giant ORFs. Examination of the methoxymalonyl-specific acyltransferase (AT) domains has led to recognition of an amino acid sequence motif which can be used to distinguish methylmalonyl-CoA-from methoxymalonyl-ACP-specific AT domains in natural PKSs. INTRODUCTIONPolyketide macrolides constitute a large family of bioactive natural products, including many compounds with important clinical applications in both human and veterinary medicine. Concanamycin A (folimycin) is an unusual macrolide, first isolated from Streptomyces diastatochromogenes S-45 (Kinashi et al., 1984), whose absolute configuration was established as shown in Fig. 1 (Westley et al., 1984). Concanamycin A and closely related compounds (concanamycins B-F) are characterized by an 18-membered tetraenic macrolide ring incorporating a methyl enol ether, and by a b-hydroxyhemiacetal side chain. They are closely related to the 16-membered macrolides called bafilomycins (Goetz et al., 1985) and also share structural features with the antifungal macrodiolide, elaiophylin A (Arcamone et al., 1959) whose biosynthetic pathway we recently elucidated (Haydock et al., 2004). These unusual macrolides have been dubbed the 'plecomacrolides' (from the Greek word plectos, describing their unusually folded side chain) (Bindseil & Zeeck, 1994).The concanamycins exhibit a wide range of important biological activities: antiviral (Omura et al., 1983), antiprotozoal (Omura et al., 1983 and antineoplastic (SekiAsano et al., 1994). These activities are thought to be due, at least in part, to their potent inhibition of the vacuolar (Vtype) H + -ATPase (Muroi et al., 1993) mediated by their binding to the membrane-spanning V 0 component of the ATPase (Drose et al., 2001). Interest in these compounds has increased recently with the recognition of the importance of the V-type ATPase in osteoclast-mediated resorption of bone (Lait...

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Active-site residue, domain and module swaps in modular polyketide synthases

Cited by 114 publications

References 25 publications

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

Organization of the biosynthetic gene cluster in Streptomyces sp. DSM 4137 for the novel neuroprotectant polyketide meridamycin

Organization of the biosynthetic gene cluster for the macrolide concanamycin A in Streptomyces neyagawaensis ATCC 27449

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