Cloning and Heterologous Expression of the Epothilone Gene Cluster

Tang, Li; Shah, Sanjay; Chung, Loleta; Carney, John R.; Katz, Leonard; Khosla, Chaitan; Julien, Bryan

doi:10.1126/science.287.5453.640

Cited by 415 publications

(308 citation statements)

References 23 publications

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“…The initial steps of epothilone biosynthesis involve formation of a methylthiazole ring through the action of EpoB, the single NRPS module of the PKS/NRPS pathway (13,14). The loading module, EpoA, activates malonyl-CoA and forms acetyl-S-EpoA as the acyl donor for the Cy domain of EpoB ( Figure 1B) (15).…”

mentioning

confidence: 99%

Excision of the Epothilone Synthetase B Cyclization Domain and Demonstration of in Trans Condensation/Cyclodehydration Activity

2005

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The epothilones are potent anticancer natural products produced by a polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) hybrid involving proteins EpoA-F. The single NRPS module of the epothilone assembly line, EpoB, is a distinct subunit of approximately 160 kDa and consists of four successive domains: cyclization, adenylation, oxidation, and peptidyl carrier protein (Cy-AOx-PCP). The cyclization domain is responsible for introduction of the thiazoline heterocycle into the growing polyketide/nonribosomal peptide chain from the precursors malonyl-CoA and cysteine through the multiple steps of condensation, cyclization, and dehydration. This enzyme-bound thiazoline intermediate is subsequently oxidized to a thiazole by the EpoB Ox domain. The EpoB module was dissected to provide 57 kDa EpoB(Cy) and 102 kDa EpoB(A-Ox-PCP) as subunit fragments to evaluate Cy as a freestanding domain. EpoB was reconstituted by these fragments in trans to generate the methylthiazole product. Using this system, apparent kinetic constants for the upstream acyl donor EpoA(ACP) and EpoB(Cy) were determined, providing a measure of affinity for the naturally occurring interface of the amino terminus of EpoB and the EpoA carboxy terminus. Site-directed mutants in excised EpoB(Cy) were prepared and used to examine residues involved in condensation and heterocycle formation. This work demonstrates the ability to define a functional Cy domain by excision from its native NRPS module, and examine both its protein-protein interactions and mechanism of activity.

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confidence: 99%

Excision of the Epothilone Synthetase B Cyclization Domain and Demonstration of in Trans Condensation/Cyclodehydration Activity

2005

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“…These organisms have been very well studied, especially S. coelicolor (28,29) and S. avermitilis (30,31), for each of which the whole genome sequence has already been determined and extensive genomic information involving biosynthetic pathways for useful products or other metabolic pathways has been revealed. The combined application of the genomic information and regulated expression systems such as that described here could contribute to the systematic improvement of productivity in streptomycetes, for example, by enhancing the predicted rate-limiting steps of biosynthetic pathways (32)(33)(34)(35)(36)(37)(38). In the cases that the tsr gene in pSH19 is not ideal as a selection marker [e.g., the host strains are resistant to thiostrepton (3) or the addition of thiostrepton can induce a regulon of genes via the tipA system (3, 7)], an alternative selection marker that could be substituted for tsr would be preferable.…”

Section: Discussionmentioning

confidence: 99%

Hyper-inducible expression system for streptomycetes

Herai

Hashimoto

Higashibata

et al. 2004

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

109

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Streptomycetes produce useful enzymes and a wide variety of secondary metabolites with potent biological activities (e.g., antibiotics, immunosuppressors, pesticides, etc.). Despite their importance in the pharmaceutical and agrochemical fields, there have been no reports for practical expression systems in streptomycetes. Here, we developed a ''PnitA-NitR'' system for regulatory gene expression in streptomycetes based on the expression mechanism of Rhodococcus rhodochrous J1 nitrilase, which is highly induced by an inexpensive and safe inducer, -caprolactam. Heterologous protein expression experiments demonstrated that the system allowed suppressed basal expression and hyper-inducible expression, yielding target protein levels of as high as Ϸ40% of all soluble protein. Furthermore, the system functioned in important streptomycete strains. Thus, the P nitA-NitR system should be a powerful tool for improving the productivity of various useful products in streptomycetes.

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“…It is not surprising that such assembly lines could intersect and domains͞modules be mixed and matched to create a hybrid assembly line. Such hybrid assembly lines are responsible for the biosynthesis of therapeutically important molecules such as bleomycin (12), rapamycin (13), FK506 (14), and epothilone (15,16). HMWP1 is a prototypical PKS͞NRPS hybrid system with a five-domain PKS module fused to a four-domain NRPS module, and as such it may serve as a good example to deconvolute the molecular logic of a hybrid assembly line.…”

Section: Discussionmentioning

confidence: 99%

Acyl-CoA hydrolysis by the high molecular weight protein 1 subunit of yersiniabactin synthetase: Mutational evidence for a cascade of four acyl-enzyme intermediates during hydrolytic editing

Suo

Chen²,

Walsh³

2000

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

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Yersiniabactin (Ybt) synthetase is a three-subunit, 17-domain [7 domains in high molecular weight protein (HMWP)2, 9 in HMWP1, and 1 in YbtE] enzyme producing the virulence-conferring siderophore yersiniabactin in Yersinia pestis. The 350-kDa HMWP1 subunit contains a polyketide synthase module (KS-AT-MT 2-KR-ACP) and a nonribosomal peptide synthetase module (Cy3-MT3-PCP3-TE). The fulllength HMWP1 was heterologously overexpressed in Escherichia coli and purified to near homogeneity. The purified HMWP1 showed thioesterase activity toward acyl-CoAs, such as acetyl-CoA, benzoylCoA, and malonyl-CoA, with saturation kinetics and relative catalytic efficiencies of 172:50:1. A chain-releasing thioesterase (TE) activity is ascribed to the C-terminal TE domain, and this was substantiated by the fact that acyl-N-acetylcysteamines were hydrolyzed by the didomain PCP 3-TE fragment of HMWP1. However, PCP3-TE failed to hydrolyze any of the acyl-CoAs, suggesting the TE domain does not recognize CoA moiety, thus the acyl-CoA hydrolysis by HMWP1 must involve other domains. Ser-to-Ala mutants in each of the AT, ACP, PCP 3, and TE domains reduced hydrolysis rates of the two fastest substrates, acetyl-CoA and benzoyl-CoA, by more than two orders of magnitude. Thus, the acyl-CoA hydrolysis activity requires 4 of the 9 domains of HMWP1, and it is consistent with autoacylation of the AT domain active site serine and subsequent passage of the itinerant acyl chain from AT to ACP to PCP 3 to the TE domain, a cascade of four sequential acyl-enzyme intermediates, for hydrolytic turnover. This could represent an editing pathway for this polyketide synthase͞nonribosomal peptide synthetase assembly line.

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Cloning and Heterologous Expression of the Epothilone Gene Cluster

Cited by 415 publications

References 23 publications

Excision of the Epothilone Synthetase B Cyclization Domain and Demonstration of in Trans Condensation/Cyclodehydration Activity

Excision of the Epothilone Synthetase B Cyclization Domain and Demonstration of in Trans Condensation/Cyclodehydration Activity

Hyper-inducible expression system for streptomycetes

Acyl-CoA hydrolysis by the high molecular weight protein 1 subunit of yersiniabactin synthetase: Mutational evidence for a cascade of four acyl-enzyme intermediates during hydrolytic editing

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