SEARCH · PubMed Health

Results for “Polyketides”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12Linked to original sources

PubMedDate not supplied

Genetic control of polyketide biosynthesis in the genus Streptomyces.

The genetic control of polyketide metabolite biosynthesis in Streptomyces sp. producing actinorhodin, daunorubicin, erythromycin, spiramycin, tetracenomycin and tylosin is reviewed. Several examples of positively-acting transcriptional regulators of polyketide metabolism are known, including some two-component sensor kinase-response regulator systems. Translational and posttranslational control mechanisms are only briefly mentioned since very little is known about either of these processes. Examples of how enzyme levels and substrate supply affect polyketide metabolism also are discussed.

Amino Acid Sequence↗

PubMed1991

Expression of polyketide biosynthesis and regulatory genes in heterologous streptomycetes.

There are now several examples showing that hybrid secondary metabolites can be produced as a result of interspecies cloning of antibiotic biosynthesis genes in streptomycetes. This paper reviews examples of hybrid secondary metabolite production, and examines the underlying biochemical and regulatory principles leading to the formation of hybrid anthraquinones by recombinant anthracycline-producing streptomycetes carrying actinorhodin biosynthesis genes. An anthraquinone, aloesaponarin II, was produced by cloning the actI, actIII, actIV, and actVII genes (pANT12) of actinorhodin biosynthesis pathway from Streptomyces coelicolor in anthracycline producing streptomycetes. Streptomyces galilaeus strains 31 133 and 31 671, aclacinomycin and 2-hydroxyaklavinone producers, respectively, formed aloesaponarin II as their major polyketide product when transformed with pANT12. Subcloning experiments indicated that a 2.8-kb XhoI fragment containing only the actI and actVII loci was necessary for aloesaponarin II biosynthesis by S. galilaeus 31 133. When S. galilaeus 31 671 was transformed with the actI, actVII, and actIV genes, however, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin and 1-O-methyldesoxyerythrolaccin. When S. galilaeus 31 671 was transformed with only the intact actIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for the actIII gene, which is the reduction of the keto group at C-9 from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The effects of three regulatory loci, dauG, dnrR1, and asaA, on the production of natural and hybrid polyketides were also shown.

Anti-Bacterial Agents↗

PubMed1995

The Aspergillus parasiticus polyketide synthase gene pksA, a homolog of Aspergillus nidulans wA, is required for aflatoxin B1 biosynthesis.

Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced by Aspergillus parasiticus and Aspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway in A. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipient A. parasiticus genome at a specific locus, designated pksA. Sequence analysis suggest that pksA is a homolog of the Aspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conserved beta-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of the pksA product. No beta-ketoacyl reductase and enoyl reductase domains were found, suggesting that pksA does not encode catalytic activities for processing beta-carbon similar to those required for long chain fatty acid synthesis. The pksA gene is located in the aflatoxin pathway gene cluster and is linked to the nor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose that pksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.

Aflatoxin B1↗

PubMed2001

Polyketide synthase genes in insect- and nematode-associated fungi.

Production of polyketides is accomplished through complex enzymes known as polyketide synthases (PKS); these enzymes have highly conserved domains that might be useful in screens for PKSs in diverse groups of organisms. A degenerate PCR-based approach was used to amplify PKS fragments of the ketosynthase domain from genomic DNA of a group of insect- and nematode-associated fungi. Of 157 isolates (representing 73 genera and 144 species) screened, 92 isolates generated PCR products of predicted size (approximately 300 bp). The ability to detect PKS domains was a function of the number of different primer pairs employed in the screen. Cloning and sequencing revealed that 66 isolates had at least one unique PKS sequence; ten members of this set contained multiple PKS fragments, for a total of 76 unique PKS fragments. Since PKS genes appear to be widespread among fungi, a PCR-based screening system appears to be an efficient, directed means to identify organisms having the potential to produce polyketides.

Amino Acid Sequence↗

PubMed2001

Molecular biology of peptide and polyketide biosynthesis in cyanobacteria.

Cyanobacteria produce numerous and structurally diverse secondary metabolites, in particular nonribosomal peptide and polyketide structures. Various bioactivities could be assigned to these compounds, and some may prove useful either for development into commercial drugs or as biochemical research tools. Microcystin, a worldwide common cyanobacterial hepatotoxin, was the first metabolite whose nonribosomal biosynthesis could be confirmed by knock-out mutagenesis. The microcystin synthetase complex consists of peptide synthetases, polyketide synthases, and hybrid enzymes, and reveals a number of novel enzymatic features, signifying the potential of cyanobacterial biosynthetic systems for combinatorial biochemistry. Recent studies have shown the presence of peptide synthetase genes and polyketide synthase genes within a number of cyanobacterial genomes. This knowledge may be very valuable for future screening projects aimed at the detection of new bioactive compounds.

Cyanobacteria↗

PubMed2003

Enhanced heterologous polyketide production in Streptomyces by exploiting plasmid co-integration.

A plasmid named pSMALL was discovered in a Streptomyces coelicolor strain that significantly enhanced the levels of production of 15-methyl-6-deoxyerythronolide B, a polyketide lactone normally produced in low amounts by engineered polyketide synthase (PKS) genes. It is a co-integrate between a conventional SCP2*-derived Streptomyces expression plasmid, pJRJ2, and SCP2@, a variant of the parental SCP2* plasmid. SCP2@ has a 45-bp deletion 35 bp upstream of the start codon of the repI gene in the replication region; and this correlated with an enhanced plasmid copy number and polyketide overproduction by its derivatives. This discovery was exploited to construct pBOOST, a high-copy-number cloning vector that can be used to achieve greatly elevated (at least 25-fold), stable metabolite production by PKS genes cloned in SCP2*-derived vectors by forming co-integrates with them.

Base Sequence↗

PubMed2004

Characterization of two polyketide synthase genes in Exophiala lecanii-corni, a melanized fungus with bioremediation potential.

Exophiala lecanii-corni has significant bioremediation potential because it can degrade a wide range of volatile organic compounds. In order to identify sites for the insertion of genes that might enhance this potential, a genetic analysis of E. lecanii-corni was undertaken. Two polyketide synthase genes, ElPKS1 and ElPKS2, have now been discovered by a PCR-based strategy. ElPKS1 was isolated by a marker rescue technique. The nucleotide sequence of ElPKS1 consists of a 6576-bp open reading frame encoding a protein with 2192 amino acids, which was interrupted by a 60-bp intron near the 5' end and a 54-bp intron near the 3' end. Sequence analysis, results from disruption experiments, and physiological tests showed that ElPKS1 encoded a polyketide synthase required for melanin biosynthesis. Since ElPKS1 is non-essential, it is a desirable bioengineering target site for the insertion of native and foreign genes. The successful expression of these genes could enhance the bioremediation capability of the organism. ElPKS2 was cloned by colony hybridization screening of a partial genomic library with an ElPKS2 PCR product. ElPKS2 had a 6465-bp open reading frame that encoded 2155 amino acids and had introns of 56, 67, 54, and 71 bp. Although sequence analysis of the derived protein of ElPKS2 confirmed the polyketide synthase nature of its protein product, the function of that product remains unclear.

Amino Acid Sequence↗

PubMed2003

Crystal structure of an Acyl-ACP dehydrogenase from the FK520 polyketide biosynthetic pathway: insights into extender unit biosynthesis.

Polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as the immunosuppressants FK520 and FK506. Understanding polyketide biosynthesis at atomic resolution could present new opportunities for chemo-enzymatic synthesis of complex molecules. The crystal structure of FkbI, an enzyme involved in the biosynthesis of the methoxymalonyl extender unit of FK520, was solved to 2.1A with an R(crys) of 24.4%. FkbI has a similar fold to acyl-CoA dehydrogenases. Notwithstanding this similarity, the surface and substrate-binding site of FkbI reveal key differences from other acyl-CoA dehydrogenases, suggesting that FkbI may recognize an acyl-ACP substrate rather than an acyl-CoA substrate. This structural observation coincided the genetic experiment done by Carroll et al. J. Am. Chem. Soc., 124 (2002) 4176. Although an in vitro assay for FkbI remains elusive, the structural basis for the substrate specificity of FkbI is analyzed by a combination of sequence comparison, docking simulations and structural analysis. A biochemical mechanism for the role of FkbI in the biosynthesis of methoxymalonyl-ACP is proposed.

Acyl Coenzyme A↗

PubMed2006

Crystal structure of the polyketide cyclase AknH with bound substrate and product analogue: implications for catalytic mechanism and product stereoselectivity.

AknH is a small polyketide cyclase that catalyses the closure of the fourth carbon ring in aclacinomycin biosynthesis in Streptomyces galilaeus, converting aklanonic acid methyl ester to aklaviketone. The crystal structure analysis of this enzyme, in complex with substrate and product analogue, showed that it is closely related in fold and mechanism to the polyketide cyclase SnoaL that catalyses the corresponding reaction in the biosynthesis of nogalamycin. Similarity is also apparent at a functional level as AknH can convert nogalonic acid methyl ester, the natural substrate of SnoaL, to auraviketone in vitro and in constructs in vivo. Despite the conserved structural and mechanistic features between these enzymes, the reaction products of AknH and SnoaL are stereochemically distinct. Supplied with the same substrate, AknH yields a C9-R product, like most members of this family of polyketide cyclases, whereas the product of SnoaL has the opposite C9-S stereochemistry. A comparison of high-resolution crystal structures of the two enzymes combined with in vitro mutagenesis studies revealed two critical amino acid substitutions in the active sites, which contribute to product stereoselectivity in AknH. Replacement of residues Tyr15 and Asn51 of AknH, located in the vicinity of the main catalytic residue Asp121, by their SnoaL counter-parts phenylalanine and leucine, respectively, results in a complete loss of product stereoselectivity.

Aclarubicin↗

PubMed2000

The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units.

FK520 (ascomycin) is a macrolide produced by Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) that has immunosuppressive, neurotrophic and antifungal activities. To further elucidate the biosynthesis of this and related macrolides, we cloned and sequenced an 80kb region encompassing the FK520 gene cluster. Genes encoding the three polyketide synthase (PKS) subunits (fkbB, fkbC and fkbA), the peptide synthetase (fkbP), the 31-O-methyltransferase (fkbM), the C-9 hydroxylase (fkbD) and the 9-hydroxyl oxidase (fkbO) had the same organization as the genes reported in the FK506 gene cluster of Streptomyces sp. MA6548 (Motamedi, H., Shafiee, A., 1998. The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur. J. Biochem. 256, 528-534). Disruption of a PKS gene in the cluster using the φC31 phage vector, KC515, led to antibiotic non-producing strains, proving the identity of the cluster. Previous labeling data have indicated that FK520 biosynthesis uses novel polyketide extender units (Byrne, K.M., Shafiee, A., Nielson, J., Arison, B., Monaghan, R.L., Kaplan, L., 1993. The biosynthesis and enzymology of an immunosuppressant, immunomycin, produced by Streptomyces hygroscopicus var, ascomyceticus. Dev. Ind. Microbiol. 32, 29-45). Genes in the flanking regions of the FK520 cluster were identified that appear to be involved in synthesis of these extender units. All but two of these genes were homologous to genes with known function. In addition to a crotonyl-CoA reductase gene (fkbS), at least two other genes are proposed to be involved in biosynthesis of the atypical PKS extender unit ethylmalonyl-CoA, which accounts for the ethyl side chain on C-21 of FK520. A set of five contiguous genes (fkbGHIJK) is proposed to be involved in biosynthesis of an unusual PKS extender unit bearing an oxygen on the alpha-carbon, and leading to the 13- and 15-methoxy side chains. These putative precursor synthesis genes in the flanking regions of the FK520 cluster are not found in the flanking regions of the rapamycin cluster (Molnár, I., Aparicio, J.F., Haydock, S.F., Khaw, L.E., Schwecke, T., König, A., Staunton, J., Leadlay, P.F., 1996. Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase. Gene 169, 1-7), consistent with labeling data showing that rapamycin biosynthesis uses only malonyl and methylmalonyl extender units.

Acyl Coenzyme A↗

PubMed2002

Characterization of the biosynthetic gene cluster for the antifungal polyketide soraphen A from Sorangium cellulosum So ce26.

A genomic DNA region of over 80 kb that contains the complete biosynthetic gene cluster for the synthesis of the antifungal polyketide metabolite soraphen A was cloned from Sorangium cellulosum So ce26. The nucleotide sequence of the soraphen A gene region, including 67,523 bp was determined. Examination of this sequence led to the identification of two adjacent type I polyketide synthase (PKS) genes that encode the soraphen synthase. One of the soraphen A PKS genes includes three biosynthetic modules and the second contains five additional modules for a total of eight. The predicted substrate specificities of the acyltransferase (AT) domains, as well as the reductive loop domains identified within each module, are consistent with expectations from the structure of soraphen A. Genes were identified in the regions flanking the two soraphen synthase genes that are proposed to have roles in the biosynthesis of soraphen A. Downstream of the soraphen PKS genes is an O-methyltransferase (OMT) gene. Upstream of the soraphen PKS genes there is a gene encoding a reductase and a group of genes that are postulated to have roles in the synthesis of methoxymalonyl-acyl carrier protein (ACP). This unusual extender unit is proposed to be incorporated in two positions of the soraphen polyketide chain. One of the genes in this group contains distinct domains for an AT, an ACP, and an OMT.

Amino Acid Sequence↗

PubMed2002

Cloning and characterization of Streptomyces galilaeus aclacinomycins polyketide synthase (PKS) cluster.

We have cloned and sequenced polyketide synthase (PKS) genes from the aclacinomycin producer Streptomyces galilaeus ATCC 31,615. The sequenced 13.5-kb region contained 13 complete genes. Their organization as well as their protein sequences showed high similarity to those of other type II PKS genes. The continuous region included the genes for the minimal PKS, consisting of ketosynthase I (aknB), ketosynthase II (aknC), and acyl carrier protein (aknD). These were followed by the daunomycin dpsC and dpsD homologues (aknE2 and F, respectively), which are rare in type II PKS clusters. They are associated with the unusual starter unit, propionate, used in the biosynthesis of aklavinone, a common precursor of aclacinomycin and daunomycin. Accordingly, when aclacinomycins minimal PKS genes were substituted for those of nogalamycin in the plasmid carrying genes for auramycinone biosynthesis, aklavinone was produced in the heterologous hosts. In addition to the minimal PKS, the cloned region included the PKS genes for polyketide ketoreductase (aknA), aromatase (aknE1) and oxygenase (aknX), as well as genes putatively encoding an aklanonic acid methyl transferase (aknG) and an aklanonic acid methyl ester cyclase (aknH) for post-polyketide steps were found. Moreover, the region carried genes for an activator (aknI), a glycosyl transferase (aknK) and an epimerase (aknL) taking part in deoxysugar biosynthesis.

Aclarubicin↗

PubMed1999

Cloning and characterization of a Myxococcus xanthus cytochrome P-450 hydroxylase required for biosynthesis of the polyketide antibiotic TA.

The antibiotic TA, a complex macrocyclic polyketide of Myxococcus xanthus, is produced, like many other polyketides, through successive condensations of acetate by a type I polyketide synthase (PKS) mechanism. The chemical structure of this antibiotic and the mechanism by which it is synthesized indicate the need for several post-modification steps, such as a specific hydroxylation at C-20. Previous studies have shown that several genes, essential for TA biosynthesis, are clustered in a region of at least 36kb, which was subsequently cloned and analyzed. In this study, we report the analysis of a DNA fragment, containing a specific cytochrome P-450 hydroxylase, presumably responsible for the sole non-PKS hydroxylation at position C-20. Functional analysis of the cytochrome P-450 hydroxylase gene through specific gene disruption confirms that it is essential for the production of an active TA molecule.

Amino Acid Sequence↗

PubMed2000

The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase.

BACKGROUND: The structural and catalytic similarities between modular nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) inspired us to search for a hybrid NRPS-PKS system. The antitumor drug bleomycin (BLM) is a natural hybrid peptide-polyketide metabolite, the biosynthesis of which provides an excellent opportunity to investigate intermodular communication between NRPS and PKS modules. Here, we report the cloning, sequencing, and characterization of the BLM biosynthetic gene cluster from Streptomyces verticillus ATCC15003. RESULTS: A set of 30 genes clustered with the previously characterized blmAB resistance genes were defined by sequencing a 85-kb contiguous region of DNA from S. verticillus ATCC15003. The sequenced gene cluster consists of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module, five sugar biosynthesis genes, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The substrate specificities of individual NRPS and PKS modules were predicted based on sequence analysis, and the amino acid specificities of two NRPS modules were confirmed biochemically in vitro. The involvement of the cloned genes in BLM biosynthesis was demonstrated by bioconversion of the BLM aglycones into BLMs in Streptomyces lividans expressing a part of the gene cluster. CONCLUSION: The blm gene cluster is characterized by a hybrid NRPS-PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide-polyketide metabolites.

Amino Acid Sequence↗

PubMed2000

A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis.

BACKGROUND: Polyene macrolides are a class of large macrocyclic polyketides that interact with membrane sterols, having antibiotic activity against fungi but not bacteria. Their rings include a chromophore of 3-7 conjugated double bonds which constitute the distinct polyene structure. Pimaricin is an archetype polyene, important in the food industry as a preservative to prevent mould contamination of foods, produced by Streptomyces natalensis. We set out to clone, sequence and analyse the gene cluster responsible for the biosynthesis of this tetraene. RESULTS: A large cluster of 16 open reading frames spanning 84985 bp of the S. natalensis genome has been sequenced and found to encode 13 homologous sets of enzyme activities (modules) of a polyketide synthase (PKS) distributed within five giant multienzyme proteins (PIMS0-PIMS4). The total of 60 constituent active sites, 25 of them on a single enzyme (PIMS2), make this an exceptional multienzyme system. Eleven additional genes appear to govern modification of the polyketide-derived framework and export. Disruption of the genes encoding the PKS abolished pimaricin production. CONCLUSIONS: The overall architecture of the PKS gene cluster responsible for the biosynthesis of the 26-membered polyene macrolide pimaricin has been determined. Eleven additional tailoring genes have been cloned and analysed. The availability of the PKS cluster will facilitate the generation of designer pimaricins by combinatorial biosynthesis approaches. This work represents the extensive description of a second polyene macrolide biosynthetic gene cluster after the one for the antifungal nystatin.

Amino Acid Motifs↗

PubMed2001

Novel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sga15.

BACKGROUND: Myxobacteria have been well established as a potent source for natural products with biological activity. They produce a considerable variety of compounds which represent typical polyketide structures with incorporated amino acids (e.g. the epothilons, the myxothiazols and the myxalamids). Several of these secondary metabolites are effective inhibitors of the electron transport via the respiratory chain and have been widely used. Molecular cloning and characterization of the genes governing the biosynthesis of these structures is of considerable interest, because such information adds to the limited knowledge as to how polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) interact and how they might be manipulated in order to form novel antibiotics. RESULTS: A DNA region of approximately 50000 base pairs from Stigmatella aurantiaca Sga15 was sequenced and shown by gene disruption to be involved in myxalamid biosynthesis. Sequence analysis reveals that the myxalamids are formed by a combined PKS/NRPS system. The terminal NRPS MxaA extends the assembled polyketide chain of the myxalamids with alanine. MxaA contains an N-terminal domain with homology to NAD binding proteins, which is responsible during the biogenesis for a novel type of reductive chain release giving rise to the 2-amino-propanol moiety of the myxalamids. The last module of the PKS reveals an unprecedented genetic organization; it is encoded on two genes (mxaB1 and mxaB2), subdividing the domains of one module from each other. A sequence comparison of myxobacterial acyl-transferase domains with known systems from streptomycetes and bacilli reveals that consensus sequences proposed to be specific for methylmalonyl-CoA and malonyl-CoA are not always reliable. CONCLUSIONS: The complete biosynthetic gene cluster of the myxalamid-type electron transport inhibitor from S. aurantiaca Sga15 has been cloned and analyzed. It represents one of the few examples of combined PKS/NRPS systems, the analysis and manipulation of which has the potential to generate novel hybrid structures via combinatorial biosynthesis (e.g. via module-swapping techniques). Additionally, a new type of reductive release from PKS/NRPS systems is described.

Amino Acid Sequence↗

PubMed1999

A two-plasmid system for the glycosylation of polyketide antibiotics: bioconversion of epsilon-rhodomycinone to rhodomycin D.

BACKGROUND: The biological activity of many microbial products requires the presence of one or more deoxysugar molecules attached to agylcone. This is especially prevalent among polyketides and is an important reason that the antitumor anthracycline antibiotics are avid DNA-binding drugs. The ability to make different deoxyaminosugars and attach them to the same or different aglycones in vivo would facilitate the synthesis of new anthracyclines and the quest for antitumor drugs. This is feasible using the numerous bacterial genes for deoxysugar biosynthesis that are now available. RESULTS: Production of thymidine diphospho (TDP)-L-daunosamine (dnm), the aminodeoxysugar present in the anthracycline antitumor drugs daunorubicin (DNR) and doxorubicin (DXR), and its attachment to epsilon-rhodomycinone to generate rhodomycin D has been achieved by bioconversion with a strain of Streptomyces lividans that bears two plasmids. One contained the Streptomyces peucetius dnmJVUZTQS genes plus dnmW (previously named dpsH and considered to be a polyketide cyclase gene), dnrH, which is not required for the formation of rhodomycin D, and dnrI, a regulatory gene required for expression of the dnm and drr genes. The other plasmid had genes encoding glucose-1-phosphate thymidylyltransferase and TDP-glucose-4,6-dehydratase (dnmL and dnmM, respectively, or mtmDE, their homologs from Streptomyces agrillaceus) plus the drrAB DNR/DXR resistance genes. CONCLUSIONS: The high-yielding glycosylation of the aromatic polyketide epsilon-rhodomycinone using plasmid-borne deoxysugar biosynthesis genes proves that the minimal information for L-daunosamine biosynthesis and attachment in the heterologous host is encoded by the dnmLMJVUTS genes. This is a general approach to making both known and new glycosides of anthracyclines, several of which have medically important antitumor activity.

Anthracyclines↗

PubMed2001

Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin.

BACKGROUND: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene. RESULTS: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S. verticillus. The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (k(cat)/K(m)) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP. CONCLUSIONS: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.

Amino Acid Sequence↗