Cladosporium Fulvum Cladosporium Fulvum

Scott Andrew Griffiths Andrew Scott The secondary metabolome metabolome The secondary of the fungal tomato pathogen the fungal tomato of Cladosporium fulvum The secondary metabolome of the fungal tomato pathogen Cladosporium fulvum Scott Andrew Griffiths The secondary metabolome of the fungal tomato pathogen Cladosporium fulvum Scott Andrew Griffiths Thesis committee Promotors Prof. Dr P.J.G.M. de Wit Professor of Phytopathology Wageningen University Prof. Dr P.W. Crous Professor of Evolutionary Phytopathology Wageningen University Co-promotor Dr. Jérôme Collemare IRHS-INRA, Angers, France Other members Prof. Dr R. Hall, Wageningen University Prof. Dr R.A.L. Bovenberg, University of Groningen Prof. Dr R.P. de Vries, University of Utrecht Dr. T.A. van Beek, Wageningen University This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences. The secondary metabolome of the fungal tomato pathogen Cladosporium fulvum Scott Andrew Griffiths Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 2 December 2015 at 13:30 in the Aula. Scott Andrew Griffiths The secondary metabolome of the fungal tomato pathogen Cladosporium fulvum 168 pages. PhD thesis, Wageningen University, Wageningen, NL (2015) With references, with summary in English ISBN 978-94-6257-581-3 Contents Chapter 1 General introduction and thesis outline 7 Chapter 2 Secondary metabolism and biotrophic lifestyle in the tomato 21 pathogen Cladosporium fulvum Chapter 3 Regulation of secondary metabolite production in the fungal 59 tomato pathogen Cladosporium fulvum Chapter 4 Elucidation of the cladofulvin biosynthetic pathway in 95 Cladosporium fulvum reveals a cytochrome P450 monooxygenase required for dimerization of a monomeric anthraquinone Chapter 5 Activation of a repressed secondary metabolite gene cluster in the 12 9 fungus Cladosporium fulvum prevents biotrophic parasitism on tomato Chapter 6 General discussion and concluding remarks 149 Summary 159 Acknowledgements 163 About the author 165 Chapter 1 General introduction and thesis outline Chapter 1 General introduction 1.1 Fine chemicals, natural products and secondary metabolites High value organic compounds that are produced in low quantities are termed fine chemicals (FCs), the building blocks of consumer-goods, vital medicines and agrochemicals. FCs were historically produced by using organic chemistry alone, a total synthesis approach that often requires non-renewable precursors and a large amount of energy. The potential repertoire of synthetic molecules at our disposal remains fundamentally limited by our knowledge of organic 1 chemistry. Natural products (NPs) are substances produced by a living organism. Secondary metabolites (SMs) are very bioactive NPs that are predominantly biosynthesised by plants and filamentous microbes for reasons associated with competition, survival, and long-term prosperity within their respective ecological niches. Penicillin is an anti-bacterial fungal SM that not only revolutionised the treatment of infection, but forever altered our perception of natural organic compounds. Since penicillin, many thousands of SMs with diverse biological activities have been isolated from natural sources. SMs which kill or inhibit the growth of living cells have arguably become the most important, as these have provided us with medicinal drugs and agricultural pesticides. 1.1.1 Medicinal secondary metabolites and semi-synthetic derivatives Medicinal SMs include anti-infective, anti-cancer, anti-cholesterolemic, anti-parasitic, and immunosuppressive drugs (Table 1). Promising SMs are often modified using organic chemistry to yield semi-synthetic derivatives with altered biological activities. Derivatives of natural β-lactam penicillins include the penems, carbapenems, cephems, carboxypenicillins and monobactams, each altered in their spectrum of anti-microbial activity. Daunorubicin is an SM produced by Streptomyces peucetius and is the founding member of the anthracycline class of anti-cancer antibiotics (Stutzman-Engwall et al., 1992). Although daunorubicin was used to treat different types of cancer, particularly leukemias and lymphomas, its usage was limited by its side-effects, including cumulative irreversible cardiotoxicity (Bristow et al., 1978). Semi-synthetic derivatives of daunorubicin include doxorubicin, epirubicin and idarubicin, compounds that are reduced in cardiotoxicity and altered in their spectrum of anti-cancer activity. Illustrating the power of combinatorial biosynthesis, S. peucetius was genetically engineered to produce epirubicin (Madduri et al., 1998). Cholesterol lowering statins are the most commonly prescribed and lucrative family of drugs in history, with sales in the USA alone totalling an estimated $18.7 billion in 2005 (Feher et al., 2011). The first commercially available statin was lovastatin, an SM from Aspergillus terreus, which became the starting material for the semi- synthesis of simvastatin. These examples illustrate how the costs associated with derivatization of natural SMs are offset by the clinical and commercial benefits. 8 9 General introduction Table 1. Notable commercial secondary metabolites from bacteria and fungi.1 Biological activity Secondary metabolite Producer Anti-bacterial Abyssomycin Verrucosispora sp Chloramphenicol Streptomyces venezuelae Erythromycin Saccharopolyspora erythraea Gentamycin Micromonospora sp Kanamycin Streptomyces kanamyceticus Penicillin Penicillium chrysogenum 1 Streptomycin Streptomyces griseus Tetracycline Streptomyces rimosus Rifampicin Amycolatopsis mediterranei Vancomycin Amycolatopsis orientalis Anti-cancer Actinomycin D Streptomyces antibioticus Bleomycin Streptomyces verticillus Daunorubicin Streptomyces peucetius Mitomycin D Streptomyces levendulae Salinosporamide Salinosporum sp Anti-cholesterolemic Lovastatin Aspergillus terreus Pravastatin Streptomyces carbophilus Anti-fungal Amphotericin B Streptomyces nodosus Candicidin Streptomyces griseus Filipin Streptomyces filipinensis Hamycin Streptomyces pimprina Natamycin Streptomyces natalensis Nystatin Streptomyces noursei Anti-parasite Avermectin Streptomyces avermitilis Milbemycin Streptomyces hygroscopicus Growth promoter Ardacin Kibdelosporangium aridum Avoparcin Amycolatopsis orientalis Bacitracin Bacillus subtilis Bambermycin Streptomyces bambergiensis Efrotomycin Amycolatopsis lactamdurans Monensin Streptomyces cinnamonensis Salinomycin Streptomyces albus Spiramycin Streptomyces ambofaciens Tylosin Streptomyces fradiae Virginiamycin Streptomyces virginiae Herbicide Bialophus Streptomyces fradiae Immunosuppressive Cyclosporin Tolypocladium inflatum Rapamycin Streptomyces hygroscopicus Tacrolimus Streptomyces sp 1The table was adapted from (Balagurunathan & Radhakrishnan, 2010) and expanded. 9 Chapter 1 General introduction 1.1.2 Secondary metabolites as growth promoters and pesticides Anti-microbial SMs are extensively used during the rearing of livestock for human consumption, in order to treat infectious diseases and to promote growth (Table 1). Global food production is heavily dependent on the wide-scale use of highly toxic organophosphate and carbamate pesticides. The neonicotinoids are a family of synthetic insecticides that were inspired by the alkaloid nicotine, an SM produced by species of the nightshade (Solanaceae) family of plants. 1 Compared to traditional pesticides, neonicotinoids are considerably less toxic towards birds and mammals (Tomizawa & Casida, 2003). Before the recent moratorium on the use of certain neonicotinoids by the European Union and United States regulatory authorities due to their possible role in honey-bee colony collapse disorder (Blacquière et al., 2012), this family of products accounted for 80% of seed treatments and a quarter of global insecticide sales. Similarly, the pyrethroids are a family of successful synthetic and natural insecticides that are inspired and derived from pyrethrins, SMs that are produced by the flowers of pyrethrums (Elliott et al., 1978). 1.2 Biosynthesis of secondary metabolites in fungi Fungal SMs are classified into different groups based on the enzymes and precursors involved in their biosynthesis. The main groups are polyketides, non-ribosomal peptides, polyketide-peptide hybrids, terpenes and alkaloids, the products of polyketide synthases (PKSs), non-ribosomal peptide synthases (NRPSs), hybrid PKS-NRPSs, terpene cyclases (TCs) and dimethylallyl tryptophan synthetases (DMATs), respectively (Keller et al., 2005). However, these core enzymes only synthesise the first (“raw”) compound in their respective biosynthetic pathways. Additional enzymes such as hydrolases, isomerases, oxidases, reductases and transferases are usually involved in transforming raw metabolites into more structurally elaborate SMs. Such enzymes are named decorating, tailoring, or accessory enzymes and they are encoded by discreet genes. Core and decorating genes belonging to the same biosynthetic pathway are co-regulated and often co- localised to form biosynthetic gene clusters (Keller & Hohn, 1997; Keller et al., 2005; Nierman et al., 2005). These clusters may also contain pathway-specific regulators and co-regulators that control only the gene cluster in which they reside. Gene clusters without a pathway-specific activator can also be controlled by global

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