Production of a novel polyketide through the construction of a hybrid polyketide synthase

Kuhstoss, Stuart; Huber, M. L. B.; Turner, J. R.; Paschal, Jonathan W.; Rao, R. Nagaraja

doi:10.1016/s0378-1119(96)00565-3

Cited by 120 publications

(106 citation statements)

References 31 publications

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“…Platenolide A is the polyketide backbone of the macrolide antibiotic spiramycin and is identical in structure to the polyketide backbone of niddamycin. A genetic map of the spiramycin PKS was recently published (17), and the organization and domain content of the modules, including the unusual KS Q , are the same as those found in this study for the niddamycin cluster. The degree of genetic relatedness of these clusters awaits the release of the spiramycin PKS nucleotide sequence.…”

Section: Discussionsupporting

confidence: 70%

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Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis

1997

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The genes encoding the polyketide synthase (PKS) portion of the niddamycin biosynthetic pathway were isolated from a library of Streptomyces caelestis NRRL-2821 chromosomal DNA. Analysis of 40 kb of DNA revealed the presence of five large open reading frames (ORFs) encoding the seven modular sets of enzymatic activities required for the synthesis of a 16-membered lactone ring. The enzymatic motifs identified within each module were consistent with those predicted from the structure of niddamycin. Disruption of the second ORF of the PKS coding region eliminated niddamycin production, demonstrating that the cloned genes are involved in the biosynthesis of this compound.Niddamycin is a macrolide antibiotic which is able to bind 50S ribosomal subunits to inhibit protein synthesis. The compound was first discovered as a secondary metabolite of Streptomyces djakartensis (16) and was later found to be produced by Streptomyces caelestis NRRL-2821 (11a). The structure of niddamycin ( Fig. 1) suggests that the polyketide backbone of the macrolide ring is formed through the ordered condensation of carboxylic acid residues derived from acetate, propionate, butyrate, and perhaps glycolate (24). The disaccharide, mycaminose-isobutyrylmycarose, is attached to the macrolide ring at C-5.Macrolides belong to a class of molecules referred to as complex polyketides, which are synthesized on large, multifunctional enzymes called polyketide synthases (PKSs). The synthesis of polyketides is mechanistically similar to that of fatty acids; however, a greater variety of starter and extender carboxylic acid residues are incorporated into the growing polyketide chain, and the ␤-keto groups formed after each condensation step undergo various degrees of reduction (15,20).PKSs, in general, contain all of the enzymatic activities necessary for the sequential condensation of acyl thioesters (␤-ketoacyl acyl carrier protein synthases [KS]), acyltransferases [AT], and acyl carrier proteins [ACP]), the subsequent reduction of the ␤-keto groups (dehydratases [DH], enoylreductases [ER], and ketoreductases [KR]), and the release of the completed chain from the PKS (thioesterases [TE]). Analysis of the erythromycin PKS genes revealed that these enzymatic domains are organized into modules, each of which is responsible for one round of condensation and reduction (5, 7, 9, 10). As a result, there is a direct correlation between the number of modules contained within the erythromycin PKS and the length of the polyketide chain. In addition, the genetic order of the erythromycin PKS modules was found to be colinear with the order of biochemical reactions, allowing directed genetic alterations which produce predicted novel erythromycin derivatives (9, 11).The polyketide portion of the 16-membered macrolide niddamycin is predicted to be synthesized by a complex (type 1) PKS (15) comprising seven modules, each catalyzing one condensation reaction. It had previously been suggested that the choice of the extender coenzyme A (CoA)-thioester is determined by the ...

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Section: Discussionsupporting

confidence: 70%

“…4). Interestingly, it has recently been reported that KS Q domains are also found at the N termini of the PKSs which synthesize the other 16-membered macrolides, carbomycin, spiramycin, and tylosin (17). The function of this motif, if any, remains to be determined.…”

Section: Resultsmentioning

confidence: 99%

Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis

1997

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“…Partial proteolysis of DEBS has established that the N-terminal domain of DEBS1, corresponding to the loading domain, is specifically acylated by propionyl-CoA , indicating that this region of the PKS is responsible for choosing the starter molecule for erythromycin biosynthesis. This idea is further supported by the finding that the 'starter unit' specificity of the spiramycin PKS can be changed from acetate to propionate when its loading domain is replaced with the corresponding domain from the tylosin PKS (Kuhstoss et al, 1996). In this work, strains that lack the erythromycin PKS loading domain, in whole or in part, were constructed with the expectation that they would be incapable of producing erythromycin.…”

Section: Discussionmentioning

confidence: 91%

The loading domain of the erythromycin polyketide synthase is not essential for erythromycin biosynthesis in Saccharopolyspora erythraea

et al. 1998

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6-Deoxyerythronol ide B synthase (DEBS) is a large multifunctional enzyme that catalyses the biosynthesis of the erythromycin polyketide aglycone. DEBS is organized into six modules, each containing the enzymic domains required for a single condensation of carboxylic acid residues which make up the growing polyketide chain. Module 1 is preceded by loading acyltransferase (AT-L) and acyl carrier protein (ACP-L) domains, hypothesized to initiate polyketide chain growth with a propionate-derived moiety. Using recombinant DNA technology several mutant strains of Saccharopolyspora erythraea were constructed that lack the initial AT-L domain or that lack both the AT4 and ACP-L domains. These strains were still able to produce erythromycin, although a t much lower levels than that produced by the wild-type strain. In addition, the AT-L domain expressed as a monofunctional enzyme was able to complement the deletion of this domain from the PKS, resulting in increased levels of erythromycin production. These findings indicate that neither the initial AT-L nor the ACP-L domains are required to initiate erythromycin biosynthesis; however, without these domains the efficiency of erythromycin biosynthesis is decreased significantly. It is proposed that in these mutants the first step in erythromycin biosynthesis is the charging of KSI with propionate directly from propionyl-CoA.

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“…These two strains were derived in research programs for other natural products and are amenable to genetic manipulation (15,30,(32)(33)(34); they have been previously engineered to eliminate production of their major secondary metabolites. Table 2 shows that S. ambofaciens and S. roseosporus exconjugants expressing the lpt gene cluster from the C31 integration site produced A54145 lipopeptide factors, while strains containing an empty vector produced no lipopeptides.…”

Section: Validation Of the Lpt Gene Cluster By Heterologous Expressionmentioning

confidence: 99%

Development of a Genetic System for Combinatorial Biosynthesis of Lipopeptides in Streptomyces fradiae and Heterologous Expression of the A54145 Biosynthesis Gene Cluster

Alexander

Rock

et al. 2010

Appl Environ Microbiol

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A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the C31 or BT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu 12 ), at the expense of factors containing 3-methyl-glutamic acid (3mGlu 12 ). This provided a practical route to produce high levels of pure Glu 12 -containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.

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Production of a novel polyketide through the construction of a hybrid polyketide synthase

Cited by 120 publications

References 31 publications

Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis

Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis

The loading domain of the erythromycin polyketide synthase is not essential for erythromycin biosynthesis in Saccharopolyspora erythraea

Development of a Genetic System for Combinatorial Biosynthesis of Lipopeptides in Streptomyces fradiae and Heterologous Expression of the A54145 Biosynthesis Gene Cluster

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