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Content
1. What are fungal type I polyketide synthases (PKSs)?
2. Research questions
3. Methods of investigation
4. References
1. What are fungal type I polyketide synthases?
Type I polyketide synthase (PKS) genes mediate the production of secondary metabolites of the polyketide (PK) pathway, which include pigments, compounds involved in cell-to-cell signaling as well those which elicit antagonistic interactions between microorganisms. Ascomycete fungi contain large numbers of PKS genes, which are separated into three main groups: reducing PKS genes (HR-PKS, see Fig. 1), non-reducing PKS genes (NR-PKS, Fig. 1) and 6-methyl salicylic acid synthase genes (6-MSAS) (Kroken et al. 2003). Non-reducing PKS genes are involved in the synthesis of pigments and unreduced polyketides (e.g. aflatoxins), whereas reducing PKS genes produce a distinct set of ecologically important metabolites (e.g., signaling compounds, diverse mycotoxins, etc.), which have generated ecological and biotechnological interest.
Fig 1. Schematic representation of a highly reducing (HR) PKS and a non-reducing (NR) PKS.
Table 1: Information for the function of single PKS domains; according Schümann & Hertweck 2006 and Kroken et al. 2003
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Abbrev.
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Name
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Function
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KS
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(β-) ketoacyl synthase
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forms a new carbon-carbon bond by Claison condensation via 3 steps: acyl transfer, decarboxylation, condensation
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AT
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acyl transferase
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substrate recruitment and loading: AT primes the ACP with acetyl or propionyl moiety and extends the chain with malonyl or methylmalonyl moieties (in a chain-extending module)
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DH
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dehydratase
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reductive domain (β-keto processing reactions): hydroxyl to enoyl group; belongs to non-metal dehydratases
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MT (ME)
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methyltransferase (esterase)?
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methylation
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ER
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enoylreductase
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reductive domain (β-keto processing reactions): enoyl to alkyl group; belongs to medium chain dehydrogenase family
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KR
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ketoreductase
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reductive domain (β-keto processing reactions): keto to hydroxyl group; short-chain dehydrogenase family, ketone - to alcohol
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ACP (PP)
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acyl carrier protein
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handling of the acyl chain to the KS active site
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PSED
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peptide synthetase elongation domain
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non ribosomal peptide synthesis
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Domain A
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Domain B
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CLC
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Claisen-type cyclase
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cyclation
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2. Research questions
PKS gain interest in many ways, like biotechnological applications (finding of new metabolites for medical applications, etc.) but also for ecological fundamental research (pathogen research, organism interactions). In this report we focused on two topics: i) can we detect fungal PKS gene expression in a forest soil (section I), and ii) we had a look in the PKS gene family and their expression in the plant pathogen Ramularia collo-cygni.
3. Methods of investigation
RNA and DNA were extracted from the homogenized soil samples (each ~1 g) or the plant pathogen Ramularia collo-cygni. cDNAs were synthesized from extracted RNA according SMART technology (Clonetech), or using a simple first strand synthesis kit (e.g. Fermentas). Partial fungal type I polyketide synthase genes, spanning part of the ketoacyl synthase (KS) and acyl transferase (AT) domain, were amplified using the KAF1 (GAR KSI CAY GGI ACI GGI AC) and KAR2 (CCA YTG IGC ICC YTG ICC IGT RAA) primer pair (Amnuaykanjanasin et al. 2005). PCR products were gel-purified using the QIAquick Gel Extraction Kit (Qiagen) and cloned into the TOPO vectors using the TOPO TA Cloning kit (Invitrogen, Carlsbad, USA). Several clones were sequenced using standard M13 primers.
In case of R. collo-cygni, we designed specific primer pairs for each found PKS gene, and determined the expression in two growing phases. Moreover we compared the expression to the formation of a photodynamic toxin (rubellin).
See the both result pages for further informations!
4. References
Amnuaykanjanasin, A., Punya, J., Paungmoung, P., Rungrod, A., Tachaleat, A., Pongpattanakitshote, S., Cheevadhanarak, S., Tanticharoen, M., 2005. Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC 1067. FEMS Microbiology Letters 251:125-136.
Kroken, S., N. L. Glass, J. W. Taylor, O. C. Yoder, and B. G. Turgeon. 2003. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes. Proc. Natl. Acad. Sci. U.S.A. 100:15670-15675.
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