Design and utility of oligonucleotide gene probes for fungal polyketide synthases

Nicholson, Thomas P.; Rudd, Brian A. M.; Dawson, Margaret; Lazarus, Colin M.; Simpson, Thomas J.; Cox, Russell J.

doi:10.1016/s1074-5521(00)90064-4

Cited by 167 publications

(93 citation statements)

References 34 publications

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“…Methods for PCR cloning of both NRPS and PKS genes have been reported, relying on the amino acid sequence conservation of the A domain in NRPS (50) and the KS domain in PKSs (32,37), respectively. While very effective, these methods suffer from the multiple nonribosomal peptide or polyketide pathways commonly existing in the genome of a given microorganism, especially in the Streptomyces species (2, 38).…”

Section: Discussionmentioning

confidence: 99%

Identification and Localization of the Gene Cluster Encoding Biosynthesis of the Antitumor Macrolactam Leinamycin in Streptomyces atroolivaceus S-140

2002

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Leinamycin (LNM), produced by Streptomyces atroolivaceus, is a thiazole-containing hybrid peptidepolyketide natural product structurally characterized with an unprecedented 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a 18-member macrolactam ring. LNM exhibits a broad spectrum of antimicrobial and antitumor activities, most significantly against tumors that are resistant to clinically important anticancer drugs, resulting from its DNA cleavage activity in the presence of a reducing agent. Using a PCR approach to clone a thiazole-forming nonribosomal peptide synthetase (NRPS) as a probe, we localized a 172-kb DNA region from S. atroolivaceus S-140 that harbors the lnm biosynthetic gene cluster. Sequence analysis of 11-kb DNA revealed three genes, lnmG, lnmH, and lnmI, and the deduced product of lnmI is characterized by domains characteristic to both NRPS and polyketide synthase (PKS). The involvement of the cloned gene cluster in LNM biosynthesis was confirmed by disrupting the lnmI gene to generate non-LNM-producing mutants and by characterizing LnmI as a hybrid NRPS-PKS megasynthetase, the NRPS module of which specifies for L-Cys and catalyzes thiazole formation. These results have now set the stage for full investigations of LNM biosynthesis and for generation of novel LNM analogs by combinatorial biosynthesis.The five-member heterocycles of thiazole or oxazole or their reduced structures of thiazoline and thiazolidine or oxazoline and oxazolidine are signature pharmacophores common to many clinically important natural products (44), such as bacitracin (26) and pristinamycin II B (4) (antibacterial) and bleomycin (12) and epothilone (35, 49) (anticancer) (Fig. 1A). They are also common structural motifs for many microbial pathogenesis factors (10), such as pyochelin from Pseudomonas aeruginosa (9, 39), vibriobactin from Vibrio cholerae (23, 52), yersiniabactin from Yersinia pestis (17, 40), mycobactin from Mycobacterium tuberculosis (41), and microcin B17 from Escherichia coli (24, 29) (Fig. 1B).Biosynthetically these heterocycles result from cyclization (Cy) of the cysteine (Cys), serine, or threonine side chain onto the proceeding carbonyl group of the peptide substrates, and two mechanisms are known for this process. One is exemplified by the maturation of microcin B17, where the heterocycleforming steps occur posttranslationally, catalyzed by the microcin B17 synthetase (29). The other is observed for nonribosomal peptide biosynthesis where the peptide elongation and heterocycle-forming steps proceed processively, catalyzed by nonribosomal peptide synthetases (NRPS). The latter enzymes are characterized by unique catalytic domains, such as the Cy, oxidization (Ox), and reduction domains, which act on the nascent peptidyl-S-NRPS intermediate to furnish the heterocycles into the resultant natural products (11,27,44,51).Leinamycin (LNM) is a novel thiazole-containing natural product produced by several Streptomyces atroolivaceus species (18,20,36). Its structure was established by spectroscopic (18,...

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Section: Discussionmentioning

confidence: 99%

Identification and Localization of the Gene Cluster Encoding Biosynthesis of the Antitumor Macrolactam Leinamycin in Streptomyces atroolivaceus S-140

2002

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“…A typical fungal PKS consists of a linear succession of ketosynthetase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoreductase (KR), acyl carrier protein (ACP), and thioesterase (TE) domains. In addition to the biochemically characterized enzymes, many other potential fungal PKSs have been identified by using degenerate-PCR approaches (5,30,36).…”

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confidence: 99%

Novel Polyketide Synthase from Nectria haematococca

Graziani

Vasnier

Daboussi

2004

Appl Environ Microbiol

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We identified a polyketide synthase (PKS) gene, pksN, from a strain of Nectria haematococca by complementing a mutant unable to synthesize a red perithecial pigment. pksN encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: ␤-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase. The pksN product groups with the Aspergillus nidulans WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, and aflatoxin biosynthetic pathways. Inactivation of pksN did not cause any visible change in fungal growth, asexual sporulation, or ascospore formation, suggesting that it is involved in a specific developmental function. We propose that pksN encodes a novel PKS required for the perithecial red pigment biosynthesis.

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“…We designed the screening primer pair (27F/27R, in Table 1) near the UbiA-like PT gene. 16 The positive mixture wells were then distributed to a LB solid medium containing chloramphenicol. The clones were screened by colony hybridization with the labeled probe.…”

Section: Isolation Of Fosmid Clones Covering Pya Biosynthetic Gene CLmentioning

confidence: 99%

Characterization of two cytochrome P450 monooxygenase genes of the pyripyropene biosynthetic gene cluster from Penicillium coprobium

Okawa

Yamamoto

et al. 2011

J Antibiot

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Pyripyropenes are potent inhibitors of acyl-CoA:cholesterol acyltransferase, which were initially discovered to be produced by Aspergillus fumigatus. Recently, Penicillium coprobium PF1169 has also found to produce pyripyropene A (PyA), which exhibits insecticidal properties. Pyripyropenes are natural hybrid products of both terpenoid and polyketide origin. In our research, based on data generated using the Genome Sequencer FLX for P. coprobium PF1169, we predicted the biosynthetic gene cluster of PyA by blast analysis comparing with polyketide synthase and prenyltransferase of other species. By screening the genomic fosmid library, nine open reading frames (ppb1 to ppb9) related to the biosynthesis of PyA were deduced. Among them, two cytochrome P450 monooxygenase genes (ppb3 and ppb4) were separately introduced into the model fungus A. oryzae. Bioconversion of certain predicted intermediates in the transformants has elucidated the manner of hydroxylation in the biosynthetic pathway by the expressed products of these two genes (P450-1 and P450-2). That is, P450-1 exhibits monooxygenase activity and plays the hydroxylation role at C-11 of pyripyropene E. While P450-2 plays an active role in the hydroxylation of C-7 and C-13 of pyripyropene O. Keywords: cytochrome P450 monooxygenase; Penicillium coprobium; polyketide synthase; prenyltransferase; pyripyropene INTRODUCTION Pyripyropene A (PyA) has been found to be produced by Penicillium coprobium PF1169 1 and exhibits insecticidal properties. 2 The isomers of pyripyropenes A to L, initially isolated from the fermentation broth of Aspergillus fumigatus FO-1289FO- in 1993 3), have been regarded as potent inhibitors of acyl-CoA:cholesterol acyltransferase, the enzyme responsible for intracellular esterification of cholesterol. It has been reported that PyA displays insecticidal activity against Helicoverpa zea larvae. 2 The isomers of pyripyropenes A, B and D from a marine-derived fungus Aspergillus sp. GF5 were recently found to exhibit selective anti-growth properties against human umbilical vein endothelial cells. 4 Previous biochemical experiments partially delineated the PyA biosynthetic pathway. 5 Recently, the early steps of its biosynthesis in A. fumigatus FO-1289 were precisely elucidated by applying a transgenic approach with heterologous fungus. Nevertheless, hydroxylation and acetylation mechanisms for the late steps had remained unknown.We focused on cytochrome P450s, which are candidate catalysts at this hydroxylation step. Cytochrome P450 enzymes constitute a superfamily that typically catalyze the oxidation of numerous endogenous and exogenous compounds in bacteria, fungi, plants, insects and vertebrates. [6][7][8] The cytochrome acts as a terminal oxidase accepting electrons from NADPH, through NADPH cytochrome P450 reductase, or from NADH through cytochrome b 5 . The exogenous compounds metabolized in this way include numerous pesticides and insecticides. 9 In this paper, we describe the cloning and structural analysis of a 24 kb genomic DNA regi...

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Design and utility of oligonucleotide gene probes for fungal polyketide synthases

Cited by 167 publications

References 34 publications

Identification and Localization of the Gene Cluster Encoding Biosynthesis of the Antitumor Macrolactam Leinamycin in Streptomyces atroolivaceus S-140

Identification and Localization of the Gene Cluster Encoding Biosynthesis of the Antitumor Macrolactam Leinamycin in Streptomyces atroolivaceus S-140

Novel Polyketide Synthase from Nectria haematococca

Characterization of two cytochrome P450 monooxygenase genes of the pyripyropene biosynthetic gene cluster from Penicillium coprobium

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