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Bacterial Type III Polyketide Synthases: Phylogenetic Analysis and Potential

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Bacterial Type III Polyketide Synthases: Phylogenetic Analysis and Potential Arch Microbiol (2006) 185: 28–38 DOI 10.1007/s00203-005-0059-3 ORIGINAL PAPER Frank Gross Æ Nora Luniak Æ Olena Perlova Nikolaos Gaitatzis Æ Holger Jenke-Kodama Klaus Gerth Æ Daniela Gottschalk Æ Elke Dittmann Rolf Mu¨ller Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads Received: 8 August 2005 / Revised: 7 October 2005 / Accepted: 10 November 2005 / Published online: 5 January 2006 Ó Springer-Verlag 2006 Abstract Type III polyketide synthases (PKS) were after the heterologous expression in Escherichia coli regarded as typical for plant secondary metabolism failed. Cultures of recombinant Pseudomonas sp. har- before they were found in microorganisms recently. Due bouring SoceCHS1 turned red upon incubation and the to microbial genome sequencing efforts, more and more diffusible pigment formed was identified as 2,5,7-trihy- type III PKS are found, most of which of unknown droxy-1,4-naphthoquinone, the autooxidation product function. In this manuscript, we report a comprehensive of 1,3,6,8-tetrahydroxynaphthalene. The successful het- analysis of the phylogeny of bacterial type III PKS and erologous production of a secondary metabolite using a report the expression of a type III PKS from the gene not expressed under administered laboratory con- myxobacterium Sorangium cellulosum in pseudomonads. ditions provides evidence for the usefulness of our ap- There is no precedent of a secondary metabolite that proach to activate such secondary metabolite genes for might be biosynthetically correlated to a type III PKS the production of novel metabolites. from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological Keywords Myxobacteria Æ Type III PKS Æ Silent gene Æ difference compared to the wild-type strain which is why Pseudomonads Æ Flaviolin Æ Heterologous Expression Æ these type III PKS are assumed to be ‘‘silent’’ under the Genome sequencing Æ Phylogeny laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. Introduction Frank Gross and Nora Luniak contributed equally to this work Nearly 100,000 ‘‘small molecules’’ from natural sources have been characterized until the end of the last century. F. Gross Æ N. Luniak Æ O. Perlova Æ N. Gaitatzis Æ R. Mu¨ller (&) Institut fu¨r Pharmazeutische Biotechnologie, These secondary metabolites are defined by their Universita¨t des Saarlandes, Postfach 151150, molecular weight not exceeding 2,500 Da. They are 66041 Saarbru¨cken, Germany mainly produced by plants and microorganisms, each E-mail: [email protected] group being responsible for the production of almost Tel.: +49-681-3025474 50% of the compounds (Demain 1999). Natural prod- Fax: +49-681-3025473 ucts are of immense interest because they often exhibit D. Gottschalk Æ F. Gross Æ R. Mu¨ller biological activities, such as antibiotics, cytostatics, in- Institut fu¨r Pharmazeutische Biologie, secticidals or antifungals. On the other hand, the interest Technische Universita¨t Carolo-Wilhelmina, in natural products as a source for new lead structures in Mendelsohnstr. 1, 38106 Braunschweig, Germany drug discovery has decreased in the past decades K. Gerth Æ F. Gross Æ D. Gottschalk Æ R. Mu¨ller (Grabley and Thiericke 1999; Bode and Mu¨ller 2005) Gesellschaft fu¨r Biotechnologische Forschung mbH, and as a consequence the discovery rate of novel struc- Mascheroder Weg 1, 38124 Braunschweig, Germany tural classes declined (Peric-Concha and Long 2003). In H. Jenke-Kodama Æ E. Dittmann parallel, the severe clinical problem of multiresistant Institut fu¨r Biologie, Humboldt Universita¨t, pathogens is on the rise and numerous infectious des- Chausseestr. 117, Berlin, Germany eases are in danger of becoming uncurable. It has been concluded that to control the resistant pathogens novel N. Gaitatzis antibiotics are urgently needed in the future (Dougherty Biotica Technology Ltd., Chesterford Research Park, Little Chesterford,, CB10 1XL Nr Saffron Walden, Essex, UK et al. 2002). In the lead structure identification process, 29 the probability to isolate already known compounds can oxidation to flaviolin (Ueda et al. 1995). THN is nor- be lowered by choosing new resources for the screening. mally transformed into a 1,8-dihydroxynaphthalene One possibility to do so is to take advantage of ongoing (DHN) monomer, which polymerizes to DHN-melanin. and finished microbial genome sequencing projects of The same pigment is found in fungi, but here THN is secondary metabolite producers, which show that there synthesized by an ACP-utilizing, multi-domain iterative are numerous ‘‘silent’’ biosynthetic gene clusters, clearly type I PKS (Tsai et al. 1998). In contrast to the relative indicating an untapped genetic potential to produce small RppA protein (approx. 350 – 370 amino acids in novel secondary metabolites (Bode and Mu¨ller 2005). size) the fungal enzymes are large (approx. 4,150 amino With the number of sequenced genomes rising, these acids). silent clusters could be a valuable route to new natural It was shown that RppA is involved in the biosyn- products. In addition, new methods have been estab- thesis of melanin in S. griseus, because the substrate lished to access the genomes from difficult to culture or mycelium of an inactivation mutant contained no mel- non-culturable microorganisms via metagenomic DNA anin-like pigments (Funa et al. 1999). In the meantime libraries (Peric-Concha and Long 2003) that can be used more homologues of RppA have been identified from as sources for new biosynthetic gene clusters. Although various actinomycetes, indicating the involvement of the expression of these metagenomic libraries in Esc- this enzyme in one of the general melanin biosynthetic herichia coli led to the discovery of novel substances pathways in this genus (Funa et al. 1999). At the same (MacNeil et al. 2001; Gillespie et al. 2002), this heter- time it was reported that a type III PKS (PhlD) from ologous expression host shows some disadvantages in Pseudomonas fluorescens Q2-87 synthesizes 2,4-diac- comparison to others (Wenzel and Mu¨ller 2005). E.g., etylphloroglucinol, one of the components responsible there are several factors which favor the heterologous for the biocontrol activity of this strain (Bangera and expression in Streptomyces sp. (Peric-Concha and Long Thomashow 1999). PhlD uses acetoacetyl-CoA as star- 2003) and recently it was shown that Pseudomonas sp., ter unit, adds one malonyl-CoA moiety and performs especially Pseudomonas putida KT2440, is a suitable ring closure and aromatization to form monoacetylphl- heterologous host for secondary metabolite production. oroglucinol. Although the number of bacterial type III Using the complex polyketide/nonribosomal peptide PKS is increasing with the number of sequenced bacte- myxobacterial myxochromide S biosynthetic pathway, rial genomes (Saxena et al. 2003), the physiological production yields superior to the orginal producer could function of these enzymes is unknown in most cases. be achieved in this strain (Wenzel et al. 2005). It was also This may even hold true if a reaction product can be shown that there is no need in pseudomonads to intro- obtained in vitro, but not isolated in vivo (Sankarana- duce a broad range phosphopantetheinyl transferase to rayanan et al. 2004). secure the essential posttranslational modification re- In ongoing genome sequencing projects of the Gram- quired for active carrier protein domains involved in negative myxobacteria Myxococcus xanthus DK1622 polyketide and nonribosomal peptide biosynthesis and Sorangium cellulosum So ce56 genes encoding (Gross et al. 2005). putative type III PKS were identified (one type III PKS Recently a polyketide biosynthetic pathway for the in M. xanthus and two in S. cellulosum). There are no production of small aromatic molecules was discovered plausible products corresponding to these enzymes in bacteria. The polyketide synthase (PKS) involved was known from both microorganisms, although they were classified as type III PKS. These enzymes belong to the intensely screened for secondary metabolite production, chalcone synthase (CHS) and stilbene synthase (STS) especially S. cellulosum So ce56 [(Gerth et al. 2003) and superfamily of PKS, which were only found to this date unpublished results]. This leads to the conclusion that in plants (Bangera and Thomashow 1999; Funa et al. these genes are ‘‘silent’’ under the cultivation conditions 1999; Moore and Hopke 2001). Today, this enzyme class so far exhibited in the laboratory. Here, we present the includes at least 15 functionally divergent b-ketosynth- heterologous expression and identification of the bio- ases of plant and bacterial origin (Austin et al. 2004). synthetic product of a rppA-like type III PKS from S. Type III PKS are relatively modest in size (40–47 kDa) cellulosum in Pseudomonas sp.. This demonstrates the and function as homodimer (Moore and Hopke 2001). activation of a ‘‘silent’’ gene due to heterologous CHS is ubiquitous in higher plants and is involved in the expression. In addition, a comprehensive phylogenetic first step in flavonoid biosynthesis by catalyzing the analysis of bacterial type III PKS is provided. decarboxylative addition of three malonyl-CoA extender units to a p-coumaroyl-CoA starter moiety, which is derived from L-phenylalanine (Winkel-Shirley 2001). At Materials and methods the same catalytic sites intermolecular cyclization and aromatization
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