Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS66. Analysis of polyketide synthase gene clusters in Cladonia metacorallifera genome. J.-S. Hur 1 , J. A. Kim 1 , Y. J. Koh 1 , S. Kim 2 . 1) Korean Lichen ResearchCenter, Sunchon National University, Sunchon, South Korea; 2) Wildlife Genetic Resources Center, National Institute of Biological Resources, Korea.Lichen-forming fungi produce highly diverse and unique secondary compounds such as depsides, depsidones, dibenzofurans and depsones. Thebiosynthesis of secondary metabolites is governed by polyketide synthase (PKS). However, the molecular mechanisms underlying the biosynthesis of thesemetabolites are poorly understood. Here we present analysis of the structure of the PKS gene clusters responsible for secondary metabolite production inthe recently sequenced genome of lichen-forming fungus Cladonia metacorallifera. We found 37 type I polyketide synthase genes which were composedof 19 reducing PKSs, one partial reducing PKS and 17 non-reducing PKSs. Lichen-forming fungal PKS domains shared common structure with filamentousfungal PKSs. Phylogenetic analysis shows that some lichen-forming fungal PKSs constructed an unique clade in other filamentous fungal PKS clades.67. Inhibition of benzoate 4-monooxygenase (CYP53A15) from Cohliobolus lunatus by cinnamic acid derivatives. Branka Korosec 1 , Barbara Podobnik 2 ,Sabina Berne 3 , Neja Zupanec 1 , Metka Novak 1 , Nada Krasevec 1 , Samo Turk 4 , Matej Sova 4 , Ljerka Lah 1 , Jure Stojan 3 , Stanislav Gobec 4 , Radovan Komel 1,3 . 1)National Institute of Chemistry, Ljubljana, Slovenia; 2) Lek Pharmaceuticals d.d., Verovskova 57, SI-1000 Ljubljana, Slovenia; 3) Institute of Biochemistry,Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia; 4) Chair of Pharmaceutical Chemistry, Faculty of Pharmacy, Universityof Ljubljana, Askerceva cesta 7, SI-1000 Ljubljana, Slovenia.Fungal infections cause huge economic losses in agriculture. Some of the major phytopathogens also cause serious, and very often lethal, infections inhuman and animals. Plants may be a good source of antifungals since they have to defend themselves by producing numerous secondary metabolites,such as sterols, terpens, polycosanols and phenolic compounds. Successful development of antifungal compounds, based on natural defense molecules,could prove useful in combating infectious and toxin-producing fungi in both agriculture and medicine. In recent years several promising antifungal targetshave been under exploration. One of such is also fungal CYP53, member of the family of highly conserved CYP proteins, involved in detoxification ofbenzoate, a key intermediate in metabolism of aromatic compounds in fungi. High specificity and absence of homologue in higher eukaryotes assignCYP53A15 from the filamentous fungus Cochliobolus lunatus as interesting drug target. In our latest research we explored chemical properties ofisoeugenol for ligand-based similarity searching, and the homology model of CYP53A15 of Cochliobolus lunatus, for structure-based virtual screening of acomposite chemical library. Two cinnamic acid derivatives were amongst the highest scoring compounds. In the past few years, several other reportsabout antifungal activity of cinnamic acid derivatives have been published. In order to investigate the potential inhibitory activity on benzoate 4-monooxygenase (CYP53A15) we analyzed antifungal activity of 9 commercially available, and 10 representative cinnamic acid derivatives from our library.Furthermore, to obtain more information about structure-activity relationship 26 additional cinnamic acid esters and amides were synthesized andincluded in our assays. Among 45 cinnamic acid derivatives tested, 7 compounds have shown antifungal activity against C. lunatus, A. niger and P.ostreatus in in vivo inhibition tests. Compounds with antifungal activity were further evaluated for inhibition of CYP53A15 activity with spectral bindingtitration assay and HPLC. The best two inhibitors of CYP53A15 activity showed 70% inhibition at 600 mM concentration and were selected for furtheroptimization of new lead structures.68. Higher yields of cyclodepsipetides from Scopulariopsis brevicaulis by random mutagenesis. Linda Paun 1 , ElKbir Hihlal 1 , Annemarie Kramer 2 , AntjeLabes 2 , Johannes Imhoff 2 , Frank Kempken 1 . 1) Botanical Institute, Christian-Albrechts-University, Kiel, Germany; 2) Kieler Wirkstoff-Zentrum KiWiZ atGEOMAR, Kiel, Germany.The ascomycete Scopulariopsis brevicaulis, which was isolated from the marine sponge Tethya aurantium, produces two cyclodepsipeptides,scopularides A and B [1]. Both peptides exhibit activity against several tumor cell lines. Within the EU-project MARINE FUNGI (EU FP7, 265926) one of ouraims is to enhance the production of these secondary metabolites. We are in the process to establish two ways of random mutagenesis by both UVradiation and transposon-mediated. To this end we created UV-mutants and a miniaturised screening method was developed. UV-radiation wasperformed at 312 nm and the survival rate was set to 1 %. With this method a mutant library was established. To screen these mutants for highersecondary metabolites production, we developed a screening method which includes decreased cultivation volume, fast extraction and an optimised LC-MS analysis format. Using the UV mutagenesis, we identified several mutants with a higher scopularide production in comparison to the wild type. One ofthese mutants, which produces three times more biomass and more than double the amount of scopularide A, has been used for another round ofmutation. Next generation sequencing is being employed to identify the molecular genetic basis of the observed mutations. In parallel we employtransposable elements to introduce mutants [2]. The impact of transposons on gene expression as well as their ability to cause major mutations within thegenome makes them an interesting tool for random mutagenesis [3, 4, 5]. We employ the Vader transposon in its homologous host and found that itmostly integrates within or very close to genes thus it appears to be a useful tool for transposon-mediated mutagenesis in A. niger (6). At current we try toenhance its usability by modifying the Vader element. [1] Yu, Z.; Lang, G.; Kajahn, I.; Schmaljohann, R.; Imhoff, J. J. Nat. Prod. 2008, 71, 1052-1054 [2]Braumann I, van den Berg M, & Kempken F (2007) Fungal Genet Biol 44(12):1399-1414. [3] Daboussi MJ & Capy P (2003) Annu Rev Microbiol 57:275-299.[4] Kempken F (2003) Applied Mycology and Biotechnology, Vol. 3 Fungal Genomics, eds Arora DK & Khachatourians GG (Elsevier Science Annual ReviewSeries), pp 83-99. [5] Kempken F & Kück U (1998) BioEssays 20:652-659. [6] Hihlal E, Braumann I, van den Berg M, Kempken F (2011) Appl EnvironmentMicrobiol, 77: 2332-2336.Cell Biology and Development69. Generation of pathogenic diploids from heterogeneous conidial populations of Aspergillus flavus. Farhana Runa 1 , Ignazio Carbone 1 , DeepakBhatnagar 2 , Gary Payne 1 . 1) Plant Pathology, North Carolina State University, Raleigh , NC; 2) Southern Regional Research Center, USDA, New Orleans, LA.Aspergillus flavus, a major producer of aflatoxin, has emerged as an opportunistic pathogen for a wide range of hosts. Understanding genetic variationwithin strains of A. flavus is important for controlling disease and reducing aflatoxin contamination. Because conidia of A. flavus are multinucleated buthaploid, we wanted to know if nuclear condition or ploidy of conidia could be potential sources of genetic variation. The objective of our study is to detectnuclear heterogeneity and ploidy in conidial populations of A. flavus and determine their impact on fungal ecology. In order to examine heterokaryosis,protoplast of two different auxotrophic strains in which nuclei were labeled with yellow (EYFP) and cyan (ECFP) fluorescent markers were fused. Fusantsbetween the two strains were obtained through polyethylene mediated cell fusion and selection on minimal medium, which favored the growth of thefusants over that of either parental strain. Fusants selected for further study showed heterogeneous conidial populations with nuclei predominantlyexpressing either EYFP or ECFP, or a very few expressing both EYFP+ECFP. Conidia containing nuclei expressing only EYFP+ECFP were separated byFluorescence-Activated Cell Sorting (FACS) and found to contain both yellow and cyan fluorescent proteins in the same nuclei. Further characterization ofconidia having only one nucleus, but expressing both EYFP+ECFP, showed them to be diploids. Pathogenicity assays using Galleria mellonella showed that138