Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSsecretion [1]. The mlcD gene encodes a putative 'HMG-CoA reductase-like protein’, and mlcE encodes a putative efflux pump. However, the function ofthese two putative proteins has not yet been confirmed. We aim to elucidate the biological basis for compactin resistance in the compactin-producingorganism. A codon-optimized version of the mlcD gene was inserted into the Saccharomyces cerevisiae genome. The constructed yeast strain was testedfor sensitivity to lovastatin, a statin structurally similar to compactin, by growing the strain on media containing lovastatin. The strain showed an increasedresistance to lovastatin compared to the wild-type strain. Furthermore, we investigated if MlcD confers the resistance by functional complementation ofthe endogenous HMG-CoA reductases in S. cerevisiae. There are two isozymes of HMG-CoA reductase in yeast, HMG1 and HMG2, both involved in thesterol biosynthetic pathway, which leads to the synthesis of ergosterol. Following deletion of HMG1 and HMG2 genes in S. cerevisiae, we inserted the mlcDgene into the knockout mutants, and tested the resulted strains for sensitivity to lovastatin. The HMG1 and HMG2 knockout mutants were unable to growon minimal media and had an increased sensitivity to lovastatin on rich media. However, insertion of the mlcD gene restored the growth of the yeastmutants and increased their resistance to lovastatin. These results show that MlcD complements the activity of the deleted HMG-CoA reductases, enablingsynthesis of ergosterol in yeast. In addition MlcD confers statin resistance by being insensitive to the inhibiting effects of statins. Reference: [1] Abe Y.,Suzuki T., Ono C., Iwamoto K., Hosobuchi M., Yoshikawa H. Mol Genet Genomics 2002, 267, 5:636-46.54. Molecular genetic characterization of secondary metabolism pathways in Asperillus species. Clay Wang 1 , Yiming Chiang 1 , Nancy Keller 3 , KennethBruno 4 , Scott Baker 4 , Chun jun Guo 1 , James Sanchez 1 , Benjamin Knox 4 , Alexandra Soukup 3 , Jin Woo Bok 3 , Manmeet Ahuja 2 , Ruth Entwistle 2 , Liz Oakley 2 ,Shu-lin Chang 1 , Hsu-Hua Yeh 1 , Mike Praseuth 1 , Berl Oakley 2 . 1) Pharma Sci & Chemistry, Univ Southern California, Los Angeles, CA; 2) Department ofMolecular Biosciences, University of Kansas; 3) Department of Medical Microbiology and Immunology and Department of Bacteriology, University ofWisconsin Madison; 4) Pacific Northwest National Laboratory.Advances in next generation DNA sequencing have provided a large number of fungal genome sequences in public databases. Within these genomes arelarge numbers of cryptic secondary metabolism pathways. Data will be presented where we use a comparative genomics approaches to identify theproducts of these cryptic pathways. Next we use a gene knock out approach to create mutants followed by isolation and characterization of intermediatesand shunt products. Using this approach we have been able to identify the products of a meroterpenoid pathway in A. terreus.55. A branched biosynthetic pathway is involved in production of roquefortine and related compounds in Penicillium chrysogenum. Hazrat Ali 1,2 , MarcoRies 3 , Jeroen Nijland 1,2 , Peter Lankhorst 4 , Thomas Hankemeier 3,5 , Roel Bovenberg 4,6 , Rob Vreeken 3,5 , Arnold Driessen 1,2* . 1) Molecular Microbiology,Groningen Biomolecular Sciences and Biotechnology Institute, Zernike Institute for Advanced Materials, University of Groningen, The Netherlands; 2)Kluyver Centre for Genomics of Industrial Fermentations, Julianalaan 67, 2628BC Delft, The Netherlands; 3) 3Division of Analytical Biosciences,Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands; 4) DSM Biotechnology Center, Alexander Fleminglaan 1, 2613 AX Delft,The Netherlands; 5) Netherlands Metabolomics Centre, Leiden University, Leiden, The Netherlands; 6) Synthetic Biology and Cell Engineering, GroningenBiomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strainswere used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of thispathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldiketopiperazine (DHTD), roquefortine D,roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptidesynthetase RoqA accepts histidine and tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to bea degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. Thedimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding the products roquefortine D and roquefortine C, respectively. This leadsto a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into meleagrin with glandicoline A and B as intermediates, involvingtwo monooxygenases (RoqM and RoqO) and a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branchedbiosynthetic pathway rather than a linear pathway as suggested in literature.56. A biosynthetic gene cluster for the antifungal metabolite phomenoic acid in the plant pathogenic fungus, Leptosphaeria maculans. Candace Elliott 1 ,Damien Callahan 2 , Daniel Schwenk 3 , Markus Nett 4 , Dirk Hoffmeister 3 , Barbara Howlett 1 . 1) School of Botany, University of Melbourne, Melbourne,Australia; 2) Metabolomics Australia, School of Botany, The University of Melbourne, Victoria 3010, Australia; 3) Friedrich-Schiller-Universität, DepartmentPharmaceutical Biology at the Hans-Knöll-Institute, Beutenbergstrasse 11a, 07745 Jena, Germany; 4) Leibniz Institute for Natural Product Research andInfection Biology e.V., Hans-Knöll-Institute, Beutenbergstrasse 11a, 07745 Jena, Germany.Phomenoic acid, a long chain aliphatic carboxylic acid, is a major metabolite produced by Leptosphaeria maculans, a fungal pathogen of Brassica napus(canola). Early biosynthetic studies suggested that the methyl group derived from S-adenosylmethionine (SAM), whereas the incorporation pattern of[13C] acetate suggested a polyketidic origin of the linear portion of phomenoic acid (Devys et al., 1984). We have used domain modelling to predict acandidate polyketide synthase (PKS) for phomenoic acid biosynthesis. Of the 15 predicted polyketide synthases (PKS) in the L. maculans genome, sevenwere reducing with the appropriate domains (KS - keto-synthase; AT - acyltransferase; DH - dehydratase; MT- methyltransferase; ER - enoylreductase; KR -ketoreductase; ACP- acyl carrier protein) for the biosynthesis of phomenoic acid. The most highly expressed of these seven genes, PKS2, was silenced to10% of that of wild type levels and the resultant mutant produced 25 times less phomenoic acid than the wild type did, indicating that PKS2 is involved inphomenoic acid biosynthesis. This gene is part of a cluster and nearby genes are co-regulated. A two-fold reduction in the expression of the adjacenttranscriptional regulator C6TF, led to at least a 20-fold reduction in expression of PKS2, as well as of other genes in the cluster (P450, YogA, RTA1 andMFS), but not of the adjacent ChoK or a hypothetical gene (Hyp). This down-regulated mutant also showed a marked reduction in phomenoic acidproduction. Phomenoic acid is toxic towards another canola pathogen Leptosphaeria biglobosa ‘canadensis’, but L. maculans and to a lesser extent thewheat pathogen, Stagonospora nodorum are more tolerant. Phomenoic acid may play a role in allowing L. maculans to outcompete other fungi in itsenvironmental niche.57. Exploring and Manipulating Pleuromutilin Production. Patrick M Hayes 1 , Russell J Cox 2 , Andy M Bailey 1 , Gary D Foster 1 . 1) School of BiologicalSciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK; 2) School of Chemistry, University of Bristol, Bristol BS8 1TS, UK.Antibiotic resistance has arisen in a significant number of human pathogens, antibiotic discovery and development is, however, currently not keepingpace. This has lead to the reinvestigation of some naturally produced antibiotic compounds which act in a manner that avoids common resistancemechanisms. Pleuromutilin is one such compound with activity against bacteria such as Methicillin Resistant Staphylococcus aureus (MRSA). Pleuromutilinis generally synthesised at a low titre by its native producer Clitopilus passeckerianus and as such research into its biosynthesis may enable yield increases.This project has taken a multifaceted approach to manipulate the Pleuromutilin biosynthetic gene cluster in a variety of fungal organisms. 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