Minimal Streptomyces sp. strain C5 daunorubicin polyketide biosynthesis genes required for aklanonic acid biosynthesis

Rajgarhia, Vineet; Strohl, William R.

doi:10.1128/jb.179.8.2690-2696.1997

Cited by 68 publications

(71 citation statements)

References 37 publications

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“…The exact functions of these genes, which are involved in the biosynthesis of doxorubicin and daunomycin, respectively, are not known. However, it has been speculated that DpsC is involved in selecting propionyl coenzyme A (propionyl-CoA) as the starter unit for daunorubicin biosynthesis (26). The deduced amino acid sequence encoded by aviM (ORF2) exhibits similarity to type I polyketide synthases.…”

Section: Resultsmentioning

confidence: 99%

Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57

et al. 1997

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A 65-kb region of DNA from Streptomyces viridochromogenes Tü57, containing genes encoding proteins involved in the biosynthesis of avilamycins, was isolated. The DNA sequence of a 6.4-kb fragment from this region revealed four open reading frames (ORF1 to ORF4), three of which are fully contained within the sequenced fragment. The deduced amino acid sequence of AviM, encoded by ORF2, shows 37% identity to a 6-methylsalicylic acid synthase from Penicillium patulum. Cultures of S. lividans TK24 and S. coelicolor CH999 containing plasmids with ORF2 on a 5.5-kb PstI fragment were able to produce orsellinic acid, an unreduced version of 6-methylsalicylic acid. The amino acid sequence encoded by ORF3 (AviD) is 62% identical to that of StrD, a dTDP-glucose synthase from S. griseus. The deduced amino acid sequence of AviE, encoded by ORF4, shows 55% identity to a dTDP-glucose dehydratase (StrE) from S. griseus. Gene insertional inactivation experiments of aviE abolished avilamycin production, indicating the involvement of aviE in the biosynthesis of avilamycins.The avilamycins (Fig. 1), which are produced by Streptomyces viridochromogenes Tü57, are oligosaccharide antibiotics and belong to the orthosomycin group of antibiotics (10). Avilamycins as well as other important members of the orthosomycins contain a dichloroisoeverninic acid moiety, as well as one or more orthoester linkages which are associated with carbohydrate residues (35). The compound SCH27899 shows excellent activity against gram-positive bacteria (22,32) and is presently being tested for possible use against human infectious diseases (31, 37). Avilamycins inhibit the growth of grampositive bacteria, and avilamycin A is a translation inhibitor binding to the 30S ribosomal subunit (34), but the exact mode of action of the avilamycins is not known. Avilamycins are used as an additive for animal breeding (MaxusG; Eli Lilly, Bad Homburg, Germany).Few genetic studies have been carried out on the biosynthesis of orthosomycins. In 1992, Bergh and Uhlen (5) described the cloning and analysis of a polyketide synthase encoding gene cluster of S. curacoi, the producer of curamycin. The isolated gene cluster may be involved in the biosynthesis of curamycin or possibly in the biosynthesis of a spore pigment. Besides polyketide synthase genes, no other genes of this cluster were described. We recently reported a PCR method to amplify gene fragments coding for deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratases (11), which are involved in the formation of 6-deoxyhexose moieties of different antibiotics (24). A PCR fragment was obtained by using chromosomal DNA from S. viridochromogenes Tü57 as the template. The deduced amino acid sequence of the fragment revealed similarity to known dNDP-glucose dehydratases (11). We have now used this PCR fragment as a probe to screen a cosmid library. On a cosmid hybridizing to the probe, three genes (aviD, aviE, and aviM) were detected. The disruption of aviE affected avilamycin production. Expression of the multifunctional...

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Section: Resultsmentioning

confidence: 99%

Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57

et al. 1997

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“…To accomplish this, each PacI site was added independently in a stepwise manner. For insertion of the first PacI restriction site, the NcoI-ClaI fragment of pOJ436 was subcloned into the corresponding sites of plasmid pANT841 (32), resulting in plasmid pANT841-NcoI/ClaI. QuikChange PCR-based mutagenesis (Agilent Technologies, Santa Clara, CA) was used to replace the HindIII site with a PacI site in plasmid pANT841-NcoI/ClaI by the use of primers PacI For (5=-GCCCGTTGCGCATGTTAATTAAGACCGATG GCCGGTTTG-3=) and PacI Rev (5=-CAAACCGGCCATCGGTCTTAAT TAACATGCGCAACGGGC-3=).…”

Section: Methodsmentioning

confidence: 99%

Metagenomic Analysis of Streptomyces lividans Reveals Host-Dependent Functional Expression

McMahon

Guan

Handelsman

et al. 2012

Appl Environ Microbiol

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ABSTRACTMost functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA inEscherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries inStreptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries inS. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native toS. lividans. All bioactive clones were functional inS. lividansbut not inE. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.

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“…It includes two ␤-ketoacyl synthase subunits; KS ␣ , which catalyzes the condensation between the acylthioester species and chain length factor (42) (we and others [37,54] prefer the KS ␤ designation for this gene, albeit without any evidence that the two KS proteins form a heterodimer); and an acyl carrier protein (ACP) that acts as an anchor for the polyketide chain during the various biochemical manipulations. Although the minimal PKS alone is sufficient to produce the basic carbon skeleton of the polyketide chain (21,43,44), additional enzymes, such as cyclases (60, 61) (or their equivalent, aromatases [43]) and ketoreductases, are required to fold and cyclize the poly-␤-ketoacyl chain into a (poly)cyclic structure.…”

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confidence: 99%

A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms

et al. 1997

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To examine as randomly as possible the role of the ␤-ketoacyl and acyl carrier protein (ACP) components of bacterial type II polyketide synthases (PKSs), homologs of the chain-length-factor (CLF) genes were cloned from the environmental community of microorganisms. With PCR primers derived from conserved regions of known ketosynthase (KS ␣ ) and ACP genes specifying the formation of 16-to 24-carbon polyketides, two CLF (KS ␤ ) genes were cloned from unclassified streptomycetes isolated from the soil, and two were cloned from soil DNA without the prior isolation of the parent microorganism. The sequence and deduced product of each gene were distinct from those of known KS ␤ genes and, by phylogenetic analysis, belonged to antibiotic-producing PKS gene clusters. Hybrid PKS gene cassettes were constructed with each novel KS ␤ gene substituted for the actI-ORF2 or tcmL KS ␤ subunit genes, along with the respective actI-ORF1 or tcmK KS ␣ , tcmM ACP, and tcmN cyclase genes, and were found to produce an octaketide or decaketide product characteristic of the ones known to be made by the heterologous KS ␣ gene partner. Since substantially less than 1% of the microorganisms present in soil are thought to be cultivatable by standard methods, this work demonstrates a potential way to gain access to a more extensive range of microbial molecular diversity and to biosynthetic pathways whose products can be tested for biological applications.The polyketide metabolites are a large group of structurally complex and diverse natural products, many of which are clinically valuable antibiotics or chemotherapeutic agents or have other useful pharmacological activities (48). Polyketides are synthesized by polyketide synthases (PKSs) which, like the related fatty acid synthases, are multifunctional enzyme assemblies that catalyze repeated decarboxylative condensations between enzyme-bound acylthioesters (52). The structural diversity of polyketides is a result of the different numbers and types of acyl units involved, coupled with various possibilities for enzyme-mediated reduction, dehydration, cyclization, and aromatization of the initial ␤-ketoacyl condensation products (52). Manipulation of the sequence or specificity of the PKS enzymes can lead to the production of novel secondary metabolites (28,29,33). The key to further exploitation of this approach to molecular diversity lies in an increased understanding of the genetics and biochemistry of the various PKS systems.Recent progress in the cloning and sequencing of microbial PKS genes has led to the identification of at least two architecturally different types of PKSs (28, 33). Modular type I PKSs, represented by erythromycin A (11, 17), avermectin (41), rapamycin (58), and soraphen (57), are discrete multifunctional enzymes sporting numerous unique active site domains. The domains are grouped into distinct modules that in turn control the sequential addition of acylthioester units to the growing polyketide chain and the subsequent modification of the respective condensation prod...

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Minimal Streptomyces sp. strain C5 daunorubicin polyketide biosynthesis genes required for aklanonic acid biosynthesis

Cited by 68 publications

References 37 publications

Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57

Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57

Metagenomic Analysis of Streptomyces lividans Reveals Host-Dependent Functional Expression

A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms

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