Bleomycin Biosynthesis inStreptomyces verticillusATCC15003: A Model of Hybrid Peptide and Polyketide Biosynthesis

Shen, Ben; Du, Liangcheng; Sánchez, César; Chen, Meim; Edwards, Daniel

doi:10.1006/bioo.1998.1131

Cited by 39 publications

(30 citation statements)

References 45 publications

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“…We have cloned the blm gene cluster from S. verticillus and proposed that BlmIV and BlmIII are involved in the formation of the bithiazole moiety on the basis that both NRPS-1 and NRPS-0 contain a Cy domain (Fig. 2C) [7,8]. However, to convert a thiazoline into a thiazole apparently requires an additional oxidation domain^an McbC homolog, in functional analogy to the microcin B17 synthase.…”

Section: Resultsmentioning

confidence: 99%

“…2C), was ampli¢ed from the blm gene cluster [7,8] (GenBank accession number AF210249) by PCR using a forward primer of 5P-ATATGGATCCAC-GAGCGCCCGGCCCACGCCGACA-3P (the BamHI site is underlined) and a reverse primer of 5P-ATTGTCGACT-CACGTGCCGGTTCACGGGGCCTC-3P (the SalI site is underlined). The PCR-ampli¢ed 2814-bp BamHI/SalI fragment was cloned into the same sites of pET28a (Novagen, Madison, WI, USA) to yield pBS15.…”

Section: Methodsmentioning

confidence: 99%

“…We have previously proposed that the NRPS-2, NRPS-1 (encoded by blmIV) and NRPS-0 modules (encoded by blmIII) are involved in the biosynthesis of the bithiazole moiety of BLM in Streptomyces verticillus ATCC15003 [7,8]. We report here the identi¢cation of an oxidation (Ox) domain within blmIII, overexpressions of the blmIII-Ox domain and blmIII gene in Escherichia coli, and the puri¢cation and characterization of the BlmIII-Ox and BlmIII proteins.…”

Section: Introductionmentioning

confidence: 95%

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An oxidation domain in the BlmIII non-ribosomal peptide synthetase probably catalyzing thiazole formation in the biosynthesis of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003

2000

FEMS Microbiology Letters

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We have previously proposed that the BlmIV and BlmIII non-ribosomal peptide synthetases are involved in the formation of the bithiazole moiety of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003. We report here the identification and characterization of an oxidation domain in BlmIII. The oxidation domain shows local homology to a family of oxidoreductases and is present in all thiazole-forming non-ribosomal peptide synthetase modules known to date. Both the blmIII-Ox domain and blmIII gene were expressed in Escherichia coli, and the resulting BlmIII-Ox and BlmIII proteins were purified to homogeneity. The oxidation domain contains one molar equivalent of non-covalently bound FMN as a prosthetic group. These results provide experimental evidence for an oxidation domain within non-ribosomal peptide synthetases, suggesting that BlmIII-Ox probably catalyzes the thiazoline to thiazole oxidation in bleomycin biosynthesis. ß

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

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An oxidation domain in the BlmIII non-ribosomal peptide synthetase probably catalyzing thiazole formation in the biosynthesis of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003

2000

FEMS Microbiology Letters

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“…Such a resistance mechanism is in fact well established in antibiotic-producing actinomycetes, for example, BlmA, the bleomycin-binding protein from Streptomyces verticillus (42). Given the fact that antibiotic production genes have invariably been found to be clustered in one region of the microbial chromosome and to consist of structural, resistance, and regulatory genes, we adopted a strategy to clone the sgc gene cluster by mapping a putative C-1027 structural gene to the previously cloned cagA gene, considered a resistance gene that encodes the C-1027 apoprotein.…”

Section: Discussionmentioning

confidence: 99%

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

Liu

Shen

2000

Antimicrob Agents Chemother

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108

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C-1027, the most potent member of the enediyne antitumor antibiotic family, is produced by Streptomyces globisporus C-1027 and consists of an apoprotein (encoded by the cagA gene) and a nonpeptidic chromophore. The C-1027 chromophore could be viewed as being derived biosynthetically from a benzoxazolinate, a deoxyamino hexose, a ␤-amino acid, and an enediyne core. By adopting a strategy for cloning of the C-1027 biosynthesis gene cluster by mapping a putative dNDP-glucose 4,6-dehydratase (NGDH) gene to cagA, we have localized 75 kb of contiguous DNA from S. globisporus. DNA sequence analysis of two regions of the cloned gene cluster revealed two genes, sgcA and sgcB, that encode an NGDH enzyme and a transmembrane efflux protein, respectively, and confirmed that the cagA gene resides approximately 14 kb upstream of the sgcAB locus. The involvement of the cloned gene cluster in C-1027 biosynthesis was demonstrated by disrupting the sgcA gene to generate C-1027-nonproducing mutants and by complementing the sgcA mutants in vivo to restore C-1027 production. These results represent the first cloning of a gene cluster for enediyne antitumor antibiotic biosynthesis and provide a starting point for future genetic and biochemical investigations of C-1027 biosynthesis.

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“…Feeding experiments with isotopelabeled precursors and isolation of biosynthetic intermediates suggested that the BLM aglycone is derived from nine amino acids and one acetate, with methionine serving as two methyl donors (16). Cloning and analysis of the BLM, and more recently tallysomycin, and zorbamycin biosynthetic gene clus-ters suggested that this family of compounds is formed via a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS)-catalyzed enzymology from amino acid and carboxylic acid precursors (10,12,14,17,18).…”

mentioning

confidence: 99%

In Vivo Manipulation of the Bleomycin Biosynthetic Gene Cluster in Streptomyces verticillus ATCC15003 Revealing New Insights into Its Biosynthetic Pathway

Galm

Wang

Wendt-Pienkowski

et al. 2008

Journal of Biological Chemistry

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Bleomycin (BLM), an important clinically used antitumor compound, and its analogs are challenging to prepare by chemical synthesis. Genetic engineering of the biosynthetic pathway in the producer strain would provide an efficient and convenient method of generating new derivatives of this complex molecule in vivo. However, the BLM producing Streptomyces verticillus ATCC15003 has been refractory to all means of introducing plasmid DNA into its cells for nearly two decades. Several years after cloning and identification of the bleomycin biosynthetic gene cluster, this study demonstrates, for the first time, genetic accessibility of this pharmaceutically relevant producer strain by intergeneric Escherichia coli-Streptomyces conjugation. Gene replacement and in-frame deletion mutants were created by RED-mediated PCR targeting mutagenesis, and the secondary metabolite profile of the resultant mutants confirmed the identity of the BLM biosynthetic gene cluster and established its boundaries. Ultimately, the in-frame blmD deletion mutant strain S. verticillus SB5 resulted in the production of a bleomycin intermediate. The structure of this compound, decarbamoyl-BLM, was elucidated, and its DNA cleavage activity was compared with the parent compounds.The bleomycins (BLMs) 3 (1, 2) are important clinically used hybrid peptide-polyketide antitumor compounds. Combined with other agents, the BLMs are used clinically for the treatment of several types of tumors and marketed under the trade name Blenoxane, with BLM A2 and B2 as the principle constituents (3). Early development of drug resistance and cumulative pulmonary toxicity are the major limitations of BLMs in chemotherapy (3, 4). Therefore, it is an important research goal to develop strategies to produce novel BLM analogs by microbial fermentation, particularly those unavailable or extremely difficult to prepare by chemical synthesis.The BLMs are thought to exert their biological effects through a sequence-selective, metal-dependent oxidative cleavage of DNA and RNA in the presence of oxygen (5, 6). They can be dissected into four functional domains: (i) the metal-binding domain, which consists of the pyrimidoblamic acid subunit along with the adjacent ␤-hydroxyl histidine; (ii) the bithiazole and C-terminal amine, which confers the majority of BLM-DNA affinity; (iii) the (2S,3S,4R)-4-amino-3-hydroxy-2-methylpentanoic acid linker subunit, which plays an important role in the efficiency of DNA cleavage by BLMs; and (iv) the carbamoylated disaccharide moiety (4). The exact functional role of the sugars and the attached carbamoyl group remains controversial. They possibly contribute to cell recognition, cellular uptake of BLMs, metal ion coordination, and/or DNA affinity (4, 6).The investigation of biosynthetic pathways and generation of new natural product analogs by genetic engineering critically depends on the availability of tools for recombinant DNA work in the producing organism. In general, this can be achieved by the development of an expedient genetic system for ...

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Bleomycin Biosynthesis inStreptomyces verticillusATCC15003: A Model of Hybrid Peptide and Polyketide Biosynthesis

Cited by 39 publications

References 45 publications

An oxidation domain in the BlmIII non-ribosomal peptide synthetase probably catalyzing thiazole formation in the biosynthesis of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003

An oxidation domain in the BlmIII non-ribosomal peptide synthetase probably catalyzing thiazole formation in the biosynthesis of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003

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

In Vivo Manipulation of the Bleomycin Biosynthetic Gene Cluster in Streptomyces verticillus ATCC15003 Revealing New Insights into Its Biosynthetic Pathway

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