Cloning polyketide synthases

During their studies on OTA biosynthetic pathway genes, O Callaghan et al. (2003) did not only clone a polyketide synthase from A. ochraceus but cloned and sequenced several more differentially expressed cDNA fragments of yet unassigned origin from typical producers of OTA. Based on that work. University College Cork (Ireland) filed worldwide (WO 2004/072224 A2) as well as European (EP 1 592 705 A2)... 

Karolewiez, A., and Geisen, R. (2005). Cloning a part of the ochratoxin A biosynthetic gene cluster of Penicillium nordicum and characterization of the ochratoxin polyketide synthase gene. Syst. Appl. Microbiol. 28, 588-595. 

K. Raty, J. Kantola, A. Hautala, J. Hakala, K. Ylihonko, and P. Mantsala, Cloning and characterization of Streptomyces galilaeus aclacinomycins polyketide synthase (PKS) cluster, Gene, 293 (2002) 115-122. 

III. TOOLS FOR CLONING, EXPRESSING, AND MANIPULATING POLYKETIDE SYNTHASES... 

Chalcone synthase (CHS), the first plant natural product polyketide synthase (PKS) to be characterized at the molecular level (39), catalyzes the condensation of 4-coumaroyl-CoA with three molecules of malonyl-CoA to afford naringenin chalcone, a precursor of the major classes of plant flavonoids. The cloning of a novel type III pentaketide chromone synthase (PCS) from aloe (Aloe arborescens, Liliaceae) rich in aromatic polyketides, especially quinones such as aloe-emodin and emodin, resulted in... 

Marine natural products of the pederin class (mycalamides, onnamides, and theopederins) isolated from sponges are mixed biogenesis metabolites of polyketide synthase and nonribosomal synthase (1). In fact, biosynthesis gene clusters of this class have been cloned recently using metagenomic techniques from the marine sponge Theonella swinhoei (6). These compounds are... 

Cox RJ, Glod F, Hurley D, Lazarus CM, Nicholson TP, Rudd BAM, Simpson TJ, Wilkinson B, Zhang Y. Rapid cloning and expression of a fungal polyketide synthase gene involved in squalestatin biosynthesis. Chem. Commun. 2004 2260-2261. 

Type III polyketide synthases (PKSs) generate a diverse array of natural products by condensing multiple acetyl units from malonyl-CoA to specific starter substrates (1,2). The homodimeric enzymes orchestrate a series of acyltransferase, decarboxylation, condensation, cyclization, and aromatizatidn reactions at two functionally independent active sites. Due to their ability to vary either the starter molecule or the type of cyclization and the number of condensations, they are, along with terpene synthases, one of the major generators of carbon skeleton diversity in plant secondary metabolites (i). Among the starter substrates used, benzoyl-CoA is a rare starter molecule. It is utilized by bacterial type I PKSs to form soraphen A, enterocin and the wailupemycins (4). in plants, benzoyl-CoA is the starter unit for two type III PKSs, benzophenone synthase (BPS) and biphenyl synthase (BIS), both of which were cloned in our laboratory. 

We have identified a clone fi-om a cosmid library of So. cellulosum So ce26 that contains genes involved in soraphen A biosynthesis (10). Gene knockouts conducted with several restriction fiagments fi om this cosmid led to the abolition of soraphen A production in So ce26. One of these fragments was sequenced, and found to encode two incomplete modules from a Type I polyketide synthase (PKS). The coverage of the soraphen A locus was extended... 

Seow, K-T., G. Meurer, M. Gerlitz, E. Wendt-Pienkowski, C.R. Hutchinson and J. Davies (1997). A study of iterative type 11 polyketide synthases, using bacterial genes cloned from soil DNA a means to access and use genes from uncultured microorganisms. J. Bacteriol. 179 7360-7368. 

Schupp, T., Toupet, C., Engel, N. GofF, S. Cloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster from Amycdatopsis mediterranei. FEMS Microbiol. Lett. 159, 201-207 (1998). 

Tylosin [46-49] (Fig. 6), produced by Streptomyces fradiae, was one of the first antibiotics for which a comprehensive set of blocked mutants was isolated. They were used to help define the biosynthetic pathway. Whereas erythromycin is a 14-membered macrolide, tylosin has a 16-membered lactone structure. The gene for the final step of biosynthesis was cloned by reverse genetics from the protein sequence of the enzyme [46], and specific segments of surrounding DNA were found to complement other classes of blocked mutants. The formation of tylosin aglycon, protylonolide, is involved in five polyketide synthases. 

Oligomycin [63] (Fig. 13) is a 26-membered macrocyclic lactone produced by avermectin-producing Streptomyces avermitilis. The formation of oligomycin backbone requires 16 cycles of condensations. A region encoding polyketide synthase of oligomycin biosynthesis was cloned by transposon-induced mutagenesis. 

Several other polyketide synthase genes have been cloned and characterized. In their PKSs, some variations occur in the content and organization of different systems but the key features of the modular hypothesis remain unchanged [67-81]. 

Since the PKS (polyketide synthase) gene cluster for actinorhodin (act), an antibiotic produced by Streptomyces coelicolor[ 109], was cloned, more than 20 different gene clusters encoding polyketide biosynthetic enzymes have been isolated from various organisms, mostly actinomycetes, and characterized [98, 100]. Bacterial PKSs are classified into two broad types based on gene organization and biosynthetic mechanisms [98, 100, 102]. In modular PKSs (or type I), discrete multifunctional enzymes control the sequential addition of thioester units and their subsequent modification to produce macrocyclic compounds (or complex polyketides). Type I PKSs are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of the macrolactone portion of erythromycin A, an antibiotic produced by Saccharopolyspora erythraea. There are 7 different active-site domains in DEBS, but a given module contains only 3 to 6 active sites. Three domains, acyl carrier protein (ACP), acyltransferase (AT), and P-ketoacyl-ACP synthase (KS), constitute a minimum module. Some modules contain additional domains for reduction of p-carbons, e.g., P-ketoacyl-ACP reductase (KR), dehydratase (DH), and enoyl reductase (ER). The thioesterase-cyclase (TE) protein is present only at the end of module 6. 

Shah S, Xue Q, Tang L, Carney JR, Betlach M, McDaniel R (2000) Cloning, characterization and heterologous expression of a polyketide synthase and P-450 oxidase involved in the biosynthesis of the antibiotic oleandomycin. J Antibiotic 53 502-508... 

Kubota, T. et al.. Cloning of polyketide synthase genes from amphidinolide-producing marine dinoflagellate spp., Biol. Pharm. Bull. 29, 1314, 2006. 

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