Abstract
Type III polyketide synthases (PKS) were regarded as typical for plant secondary metabolism before they were found in microorganisms recently. Due to microbial genome sequencing efforts, more and more type III PKS are found, most of which of unknown function. In this manuscript, we report a comprehensive analysis of the phylogeny of bacterial type III PKS and report the expression of a type III PKS from the myxobacterium Sorangium cellulosum in pseudomonads. There is no precedent of a secondary metabolite that might be biosynthetically correlated to a type III PKS from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological difference compared to the wild-type strain which is why these type III PKS are assumed to be “silent” under the laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. after the heterologous expression in Escherichia coli failed. Cultures of recombinant Pseudomonas sp. harbouring SoceCHS1 turned red upon incubation and the diffusible pigment formed was identified as 2,5,7-trihydroxy-1,4-naphthoquinone, the autooxidation product of 1,3,6,8-tetrahydroxynaphthalene. The successful heterologous production of a secondary metabolite using a gene not expressed under administered laboratory conditions provides evidence for the usefulness of our approach to activate such secondary metabolite genes for the production of novel metabolites.
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References
Austin MB, et al (2004) Crystal structure of a bacterial type III polyketide synthase and enzymatic control of reactive polyketide intermediates. J Biol Chem 279:45162–45174
Austin MB, Noel JP (2003) The chalcone synthase superfamily of type III polyketide synthases. Nat Prod Rep 20:79–110
Bagdasarian M, et al. (1981) Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16:237–247
Bangera MG, Thomashow LS (1999) Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudomonas fluorescens Q2-87. J Bacteriol 181:3155–3163
Bentley SD, et al (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417:141–147
Bischoff D, et al (2001) The biosynthesis of vancomycin-type glycopeptide antibiotics—the order of the cyclization steps. Angew Chem Int Ed Engl 40:4688–4691
Blatny JM, Brautaset T, Winther-Larsen HC, Karunakaran P, Valla S (1997) Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria. Plasmid 38:35–51
Bode HB, Dickschat JS, Kroppenstedt RM, Schulz S, Müller R (2005) Biosynthesis of iso-fatty acids in myxobacteria: iso-even fatty acids are derived by alpha-oxidation from iso-odd fatty acids. J Am Chem Soc 127:532–533
Bode HB, Müller R (2005) The impact of bacterial genomics on natural product research. Angew Chem Int Ed 44:6828–6846
Carlson CA, Pierson LS, Rosen JJ, Ingraham JL (1983) Pseudomonas stutzeri and related species undergo natural transformation. J Bacteriol 153:93–99
Cortes J, Velasco J, Foster G, Blackaby AP, Rudd BAM, Wilkinson B (2002) Identification and cloning of a type III polyketide synthase required for diffusible pigment biosynthesis in Saccharopolyspora erythraea. Mol Microbiol 44:1213–1224
Cuppels DA (1986) Generation and characterization of Tn5 insertion mutations in Pseudomonas syringae pv. tomato. Appl Environ Microbiol 51:323–327
Demain AL (1999) Pharmaceutically active secondary metabolites of microorganisms. Appl Microbiol Biotechnol 52:455–463
Dickschat JS, Bode HB, Kroppenstedt RM, Müller R, Schulz S (2005) Biosynthesis of iso-fatty acids in myxobacteria. Org Biomol Chem 3:2824–2831
Dougherty TJ, Barrett JF, Pucci MJ (2002) Microbial genomics and novel antibiotic discovery: new technology to search for new drugs. Curr Pharm Des 8:1119–1135
Felsenstein J (2002) PHYLIP (phylogeny inference package). Version 3.6. Distributed by the author
Funa N, Ohnishi Y, Fujii I, Shibuya M, Ebizuka Y, Horinouchi S (1999) A new pathway for polyketide synthesis in microorganisms. Nature 400:897–899
Gerth K, Müller R (2006) Development of simple media which allow investigations into the global regulation of chivosazol biosynthesis with Sorangium cellulosum So ce56. J Biotech (in press)
Gerth K, Pradella S, Perlova O, Beyer S, Müller R (2003) Myxobacteria: Proficient producers of novel natural products with various biological activities - past and future biotechnological aspects with the focus on the genus Sorangium. J Biotechnol 106:233–253
Gillespie DE, et al (2002) Isolation of antibiotics turbomycin A and B from a metagenomic library of soil microbial DNA. Appl Environ Microbiol 68:4301–4306
Grabley S, Thiericke R (1999) The impact of natural products on drug discovery. In: Grabley S, Thiericke R (eds) Drug discovery from nature. Springer, Berlin Heidelberg New York
Gross F, Gottschalk D, Müller R (2005) Posttranslational modification of myxobacterial carrier protein domains in Pseudomonas sp. by an intrinsic phosphopantetheinyl transferase. Appl Microbiol Biotechnol 68:66–74
Hill D, et al. (1994) Cloning of genes involved in the synthesis of pyrrolnitrin from Pseudomonas fluorescens and role of pyrrolnitrin synthesis in biological control of plant disease. Appl Environ Microbiol 60:78–85
Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17:754–755
Izumikawa M, et al (2003) Expression and characterization of the type III polyketide synthase 1,3,6,8-tetrahydroxynaphthalene synthase from Streptomyces coelicolor A3(2). J Ind Microbiol Biotechnol 30:510–515
Jenke-Kodama H, Sandmann A, Müller R, Dittmann E (2005) Evolutionary implications of bacterial polyketide synthases. Mol Biol Evol 22:2037–2039
Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci 8:275–282
Kopp M, et al (2004) Critical variations of conjugational DNA transfer into secondary metabolite multiproducing Sorangium cellulosum strains So ce12 and So ce56: development of a mariner-based transposon mutagenesis system. J Biotechnol 107:29–40
MacNeil IA, et al (2001) Expression and isolation of antimicrobial small molecules from soil DNA libraries. J Mol Microbiol Biotechnol 3:301–308
Maddison WR, Maddison WP (2000) MacClade version 4.0. Sinauer Associates, Sunderland
Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratories, Cold Spring Harbor
Moore BS, Hopke JN (2001) Discovery of a new bacterial polyketide biosynthetic pathway. ChemBioChem 2:35–38
Pelzer S, et al (1999) Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908. Antimicrob Agents Chemother 43:1565–1573
Peric-Concha N, Long PF (2003) Mining the microbial metabolome: a new frontier for natural product lead discovery. Drug Discov Today 8:1078–1084
Pfeifer V, et al (2001) A polyketide synthase in glycopeptide biosynthesis — the biosynthesis of the non-proteinogenic amino acid (S)-3,5-dihydroxyphenylglycine. J Biol Chem 276:38370–38377
Pradella S, Hans A, Sproer C, Reichenbach H, Gerth K, Beyer S (2002) Characterisation, genome size and genetic manipulation of the myxobacterium Sorangium cellulosum So ce56. Arch Microbiol 178:484–492
Russell DG, Mwandumba HC, Rhoades EE (2002) Mycobacterium and the coat of many lipids. J Cell Biol 158:421–426
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sankaranarayanan R, Saxena P, Marathe UB, Gokhale RS, Shanmugam VM, Rukmini R (2004) A novel tunnel in mycobacterial type III polyketide synthase reveals the structural basis for generating diverse metabolites. Nat Struct Mol Biol 11:894–900
Saxena P, Yadav G, Mohanty D, Gokhale RS (2003) A new family of type III polyketide synthases in Mycobacterium tuberculosis. J Biol Chem 278:44780–44790
Seshime Y, Juvvadi PR, Fujii I, Kitamoto K (2005) Discovery of a novel superfamily of type III polyketide synthases in Aspergillus oryzae. Biochem Biophys Res Commun 331:253–260
Swofford D (2002) PAUP* 4.0: Phylogenetic analysis using parsimony. Sinauer Associates, Sunderland
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882
Tsai H-F, Chang YC, Washburn RG, Wheeler MH, Kwon-Chung KJ (1998) The Developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence. J Bacteriol 180:3031–3038
Ueda K, Kim KM, Beppu T, Horinouchi S (1995) Overexpression of a gene cluster encoding a chalcone synthase-like protein confers redbrown pigment production in Streptomyces griseus. J Antibiot (Tokyo) 48:638–646
Wenzel S, Müller R (2005) Recent developments towards the heterologous expression of complex bacterial natural product biosynthetic pathways. Curr Opin Biotechnol 16:594–606
Wenzel SC, Gross F, Zhang Y, Fu J, Stewart AF, Müller R (2005) Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering. Chem Biol 12:349–356
Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493
Acknowledgments
Authentic flaviolin reference substance was a kind gift from Prof. Dr. H. Anke, Kaiserlautern. The authors highly appreciate the help of D. Krug and P. Meiser in the chemical analysis of flaviolin. This work was supported by the German Ministry for Education and Research (BMB+F, BioFuture program) and the Deutsche Forschungsgemeinschaft (DFG) within the Schwerpunktprogramm “evolution of metabolic diversity”.
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Frank Gross and Nora Luniak contributed equally to this work
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Gross, F., Luniak, N., Perlova, O. et al. Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads. Arch Microbiol 185, 28–38 (2006). https://doi.org/10.1007/s00203-005-0059-3
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DOI: https://doi.org/10.1007/s00203-005-0059-3