Evolutionary mechanisms underlying polyketide diversity in bacteria: Implications from cyanobacteria

ELKE DITTMANN
Humboldt University
Institute of Biology
Department of Molecular Ecology
Chausseestr. 117
D-10115 Berlin
Germany
Elke.Dittmann@rz.hu-berlin.de
http://www.biologie.hu-berlin.de/moloeko/

References
Traitcheva N, Jenke-Kodama H, He J, Dittmann E, Hertweck C. (2007) Non-Colinear Polyketide Biosynthesis in the Aureothin and Neoaureothin Pathways: An Evolutionary Perspective. In Press
Aureothin and neoaureothin (spectinabilin) represent rare nitroaryl-substituted polyketide metabolites from Streptomyces thioluteus and Streptomyces orinoci, respectively, which only differ in the lengths of the polyene backbones. Cloning and sequencing of the 39 kb neoaureothin (nor) biosynthesis gene cluster and its comparison with the aureothin (aur) pathway genes revealed that both polyketide synthase (PKS) assembly lines are remarkably similar. In both cases the module architecture breaks with the principle of colinearity, as individual PKS modules are used in an iterative fashion. Parsimony and neighbour-joining phylogenetic studies provided insights into the evolutionary process that led to the programming of these unusual type I PKS systems and to prediction of which modules act iteratively. The iterative function of the first module in the neoaureothin pathway, NorA, was confirmed by a successful cross-complementation.

Ishida, K., Christiansen, G., Yoshida, W., Welker, M., Hertweck, C., Bonjoch, J., Börner, T., Hemscheidt, T., Dittmann, E. (2007). Biosynthetic pathway and structure analysis of aeruginoside 126A and B, cyanobacterial peptides bearing an unusual 2-carboxy-6-hydroxyoctahydroindole moiety. Chem. Biol. 14: 565-576.
Aeruginosins represent a group of peptide metabolites isolated from various cyanobacterial genera and from marine sponges that potently inhibit different types of serine proteases. Members of this family are characterized by the presence of a 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety. We have identified and fully sequenced a NRPS gene cluster in the genome of the cyanobacterium Planktothrix agardhii CYA126/8. Insertional mutagenesis of a NRPS component led to the discovery and structural elucidation of two glycopeptides that were designated aeruginoside 126A and aeruginoside 126B. One variant of the aglycone contains a 1-amino-2-(N-amidino-Delta(3)-pyrrolinyl)ethyl moiety at the C terminus, the other bears an agmatine residue. In silico analyses of the aeruginoside biosynthetic genes aerA-aerI as well as additional mutagenesis and feeding studies allowed the prediction of enzymatic steps leading to the formation of aeruginosides and the unusual Choi moiety.

Sandmann, A., Dickschat, J., Jenke-Kodama, H., Kunze, B., Dittmann, E., and Müller,
R. (2007) A type II polyketide synthase from the Gram negative bacterium Stigmatella
aurantiaca is involved in aurachin alkaloid biosynthesis. Angew. Chem. Int. Ed., 46:
2712-2716.

Jenke-Kodama, H., Börner, T., Dittmann, E. (2006) Natural Biocombinatorics in the
Polyketide Synthase Genes of the Actinobacterium Streptomyces avermitilis. PLoS
Comp. Biol. 2(10):e132.
Modular polyketide synthases (PKSs) of bacteria provide an enormous reservoir of natural chemical diversity. Studying natural biocombinatorics may aid in the development of concepts for experimental design of genes for the biosynthesis of new bioactive compounds. Here we address the question of how the modularity of biosynthetic enzymes and the prevalence of multiple gene clusters in Streptomyces drive the evolution of metabolic diversity. The phylogeny of ketosynthase (KS) domains of Streptomyces PKSs revealed that the majority of modules involved in the biosynthesis of a single compound evolved by duplication of a single ancestor module. Using Streptomyces avermitilis as a model organism, we have reconstructed the evolutionary relationships of different domain types. This analysis suggests that 65% of the modules were altered by recombinational replacements that occurred within and between biosynthetic gene clusters. The natural reprogramming of the biosynthetic pathways was unambiguously confined to domains that account for the structural diversity of the polyketide products and never observed for the KS domains. We provide examples for natural acyltransferase (AT), ketoreductase (KR), and dehydratase (DH)-KR domain replacements. Potential sites of homologous recombination could be identified in interdomain regions and within domains. Our results indicate that homologous recombination facilitated by the modularity of PKS architecture is the most important mechanism underlying polyketide diversity in bacteria.

Gross, F., Luniak, N., Gottschalk, D., Perlova, O., Gaitatzis, N., Gerth, K., Jenke-
Kodama, H., Dittmann, E. and Müller, R. (2006) 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.
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.

Jenke-Kodama, H., Sandmann, A., Müller, R., Dittmann, E. (2005) Evolutionary
Implications of Bacterial Polyketide Synthases. Mol. Biol. Evol. 22: 2027-2039.
Polyketide synthases (PKS) perform a stepwise biosynthesis of diverse carbon skeletons from simple activated carboxylic acid units. The products of the complex pathways possess a wide range of pharmaceutical properties, including antibiotic, antitumor, antifungal, and immunosuppressive activities. We have performed a comprehensive phylogenetic analysis of multimodular and iterative PKS of bacteria and fungi and of the distinct types of fatty acid synthases (FAS) from different groups of organisms based on the highly conserved ketoacyl synthase (KS) domains. Apart from enzymes that meet the classification standards we have included enzymes involved in the biosynthesis of mycolic acids, polyunsaturated fatty acids (PUFA), and glycolipids in bacteria. This study has revealed that PKS and FAS have passed through a long joint evolution process, in which modular PKS have a central position. They appear to have derived from bacterial FAS and primary iterative PKS and, in addition, share a common ancestor with animal FAS and secondary iterative PKS. Furthermore, we have carried out a phylogenomic analysis of all modular PKS that are encoded by the complete eubacterial genomes currently available in the database. The phylogenetic distribution of acyltransferase and KS domain sequences revealed that multiple gene duplications, gene losses, as well as horizontal gene transfer (HGT) have contributed to the evolution of PKS I in bacteria. The impact of these factors seems to vary considerably between the bacterial groups. Whereas in actinobacteria and cyanobacteria the majority of PKS I genes may have evolved from a common ancestor, several lines of evidence indicate that HGT has strongly contributed to the evolution of PKS I in proteobacteria. Discovery of new evolutionary links between PKS and FAS and between the different PKS pathways in bacteria may help us in understanding the selective advantage that has led to the evolution of multiple secondary metabolite biosyntheses within individual bacteria.

Articles in Books/Comments
Jenke-Kodama, H., Müller, R. Dittmann, E. (2007) Evolutionary mechanisms underlying secondary metabolite diversity. In: Petersen, F. (ed.) Natural Products as Drugs. Birkhäuser Verlag/Novartis. In Press.
The enormous chemical diversity and the broad range of biological activities of secondary metabolites raise many questions about their role in nature and the specific traits leading to their evolution. The answers to these questions will not only be of fundamental interest but may also provide lessons that could help to improve the screening protocols of pharmaceutical companies and strategies for rational secondary metabolite engineering. In this review, we try to dissect evolutionary principles leading to the emergence, distribution, diversification and selection of genes involved in secondary metabolite biosyntheses. We give an overview about recent insights into the evolution of the different types of polyketide synthases (PKS) in microorganisms and plants and highlight unique mechanisms underlying polyketide diversity. Although phylogenetic and experimental data have significantly increased our knowledge about the role and evolution of secondary metabolites in the last decades there is still much dissent about the impact of natural selection. In order to understand the evolution towards metabolic diversity we therefore need more thorough investigations of the ecological role of secondary metabolites in the future.

Jenke-Kodama, H., Dittmann, E. (2005) Combinatorial polyketide biosynthesis at higher
stage. Molecular Systems Biology, Invited News and Views Comment., doi:10.1038/msb4100033
Modular polyketide synthases (PKS) of bacteria provide an amazing molecular assembly line for the biosynthesis of complex polyketides. This biosynthesis system has, since its discovery, attracted the attention of scientists and pharmaceutical companies owing to its combinatorial potential. It has provoked the idea of constructing 'unnatural' product libraries containing a myriad of compounds with all possible lengths and combinations of carbon units. The recent study by Menzella et al (2005) represents an important milestone on the way to construct such libraries, and highlights both the potential and the limits of biocombinatorial strategies for a complete reorganisation of natural enzyme units. The group at Kosan Biosciences Inc. has adopted and improved existing engineering techniques and describes for the first time a high-throughput methodology for the generation of synthetic triketides.

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