Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS9. Characterization of the 3-methyl orsellinic acid gene cluster in Aspergillus nidulans. Jakob B. Nielsen 1 , Marie L. Klejnstrup 1 , Paiman K. Jamal 1 , Dorte K.Holm 1 , Michael L. Nielsen 1 , Anna M. Kabat 2 , Charlotte H. Gotfredsen 3 , Thomas O. Larsen 1 , Uffe H. Mortensen 1 . 1) Center for Microbial Biotechnology,Department of Systems Biology, Technical University of Denmark; 2) Center for Systems Microbiology, Department of Systems Biology, TechnicalUniversity of Denmark; 3) Department of Chemistry, Technical University of Denmark.With the aim of mapping the polyketome of Aspergillus nidulans we have made a library of strains, which individually overexpress PKS genes from anectopic locus. A screen of this collection on different media demonstrated that overexpression of AN6448 (pkbA) leads to increased production of 3-methyl orsellinic acid. An inspection of the DNA sequence surrounding this gene uncovered a putative gene cluster including a gene, AN6446 (pkbR), withhomology to transcription factors. Based on this observation, we decided to overexpress pkbR. A qRT-PCR analysis of this strain was used to delineate theborders of the gene cluster as well as to stimulate formation of cichorine, cichorinic acid, nidulol and a novel cichonidulol dimer, just to name a few of theproducts that we have linked to this gene cluster. Subsequent deletion of all genes in the cluster has allowed us to propose a comprehensive model for thebiosynthetic pathway of this cluster.10. Induction of sclerotia and Aspergillus section Nigri. Jens Frisvad, Lene Petersen, Ellen Lyhne, Thomas Larsen. CMB, Dept Systems Biol, Kgs. Lyngby,Denmark.The purpose of this study was to induce sclerotium production in Aspergillus niger and other black Aspergilli. Some species in Aspergillus section Nigri areknown for their production of sclerotia, especially A. carbonarius, A. tubingensis (few isolates), A. sclerotioniger, A. sclerotiicarbonarius, A. costaricaensis,A. piperis, A. japonicus, and A. aculeatus. A. heteromorphus was reported in 1955 to produce sclerotia, but this could not be confirmed in later studies.There are also un-confirmed data on sclerotium production in Aspergillus niger, but often isolates reported to produce sclerotia were not A. niger anyway.Induction of sclerotium production in Aspergillus niger is important, since this may help in inducing the perfect state in this important industrial fungus. Byscreening several media, we were able to develop some media and use some growth conditions that induced sclerotium production in Aspergillus nigerand other species hitherto not reported to produce sclerotia. Earlier French beans were suggested as inducers of sclerotium production, but we could notrepeat this with any isolate of A. niger. However by using media such as white rice and brown rice or adding different fruits to CYA (Czapek yeastautolysate agar) and incubate at 25 C we were able to induce sclerotium production in certain strains of A. niger. Old strains used for citric acid production,or full genome sequenced strains, were not induced to produce sclerotia, but several fresh strains from different foods did produce abundant sclerotia onthe different media, at 25 C, but not 37 C. One older classical citric acid producer from NRRL produced many sclerotia, however. Sclerotium producingisolates also contained aflavinines, confirmed by HPLC-DAD-MS-MS, secondary metabolites only produced in the sclerotia, and detected in A. niger for thefirst time. Other species, such as A. ibericus, A. neoniger, A. heteromorphus, A. fijiensis, A. luchuensis (formerly A. acidus), A. aculeatinus and A.saccharolyticus could also produce sclerotia on fruit media. The sclerotia contained many sclerotium-specific secondary metabolites.11. Analyzing the impact of compartmentalization on organic acid production in Aspergillus niger. Matthias G. Steiger 1,2* , Marzena L. Blumhoff 1,2,3 ,Diethard Mattanovich 1,2 , Michael Sauer 1,2 . 1) Austrian Centre of Industrial Biotechnology (ACIB GmbH), Muthgasse 11, 1190 Vienna, Austria; 2) Universityof Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 18, 1190 Vienna, Austria; 3) University of Applied Sciences FH-CampusVienna, School of Bioengineering, Muthgasse 86, 1190 Vienna, Austria.Aspergillus niger is a well-established host organism for the production of carboxylic acids. Acids like citric, gluconic and oxalic acids can already beproduced by A. niger and high titers are obtained. The formation of carboxylic acids involves the shuttling of intermediate metabolites between differentintracellular compartments and utilizes different enzymatic capabilities of the respective compartment. The knowledge about the involved shuttlingmechanisms and the localization of the necessary enzymes is still fragmentary. Using fluorescence microscopy, it is possible to characterize theintracellular localization of GFP tagged proteins and hence mitochondrial leader sequences can be functionally tested. In order to analyze the influence ofthe compartmentalization on the organic acid production, we have chosen itaconic acid as a target substance. Itaconic acid, which was selected by the USDepartment of Energy as one of the 12 building block chemicals for the industrial biotechnology, is currently produced by A. terreus. Heterologousexpression of the A. terreus cadA gene also enables the formation of itaconic acid in A. niger although only low titers are obtained. We set out tocharacterize the influence of the compartmentalization on the productivity and re-engineered the enzymatic cascade by flipping the enzymatic activities ofthe cis-aconitic acid decarboxylase and aconitase between the mitochondrion and the cytosol. We will present new leader sequences for mitochondrialtargeting in A. niger alongside with results about the positive impact of the enzymatic re-localization on the itaconic acid production.12. Subcellular localization of aphidicolin biosynthesis enzymes from Phoma betae expressed heterologously in Aspergillus oryzae. A. Ban 1 , M. Tanaka 1 ,R. Fujii 2 , A. Minami 2 , H. Oikawa 2 , T. Shintani 1 , K. Gomi 1 . 1) Graduate Sch Agriculture Sci, Tohoku Univ, Sendai, Japan; 2) Graduate Sch Sci, Hokkaido Univ,Sapporo, Japan.In recent years, a lot of biosynthesis gene clusters involving in secondary metabolite biosynthesis from filamentous fungi have been revealed, and thusthe attempts to produce these valuable metabolites at high yield have been actively made. To this end, Aspergillus oryzae is an attractive host forheterologous secondary metabolites production because of its less productivity for own secondary metabolites, which leads to the production for themetabolite of interest at a highly pure grade. Actually, the number of reports has been increasing recently, in which biosynthetic genes involved in fungalsecondary metabolite biosynthesis were heterologously overexpressed in A. oryzae. On the other hand, it would be necessary to consider the cellularcompartments where the target secondary metabolite is synthesized in filamentous fungi to produce it efficiently in the heterologous host, A. oryzae.However, currently there is very little knowledge about the spatial distribution of the biosynthesis enzymes of the secondary metabolite in fungi.Therefore, in this study, we examined the subcellular localization of the enzyme proteins encoded by a gene cluster involved in aphidicolin biosynthesisfrom Phoma betae, which were expressed as GFP-fusion proteins in A. oryzae. The gene cluster of aphidicolin in P. betae contains 4 genes encodingbiosynthesis enzymes (geranylgeranyl diphosphate synthase [GGS], aphidicolan-16b-ol synthase [ACS], cytochrome P450 monooxygense 1 [P450-1], andP450-2), a gene for transporter (TP), and a gene for transcription factor. We constructed 4 biosynthesis enzymes and the transporter that each was fusedto GFP under the a-amylase gene promoter, and introduced into A. oryzae. Similarly, the organelle marker proteins fused to RFP were also constructed andexpressed simultaneously with GFP-fusion proteins to identify the organelle where the biosynthesis enzyme was localized. Fluorescent microscopyrevealed that GGS and ACS were distributed in the cytoplasm and P450-1 was located in endoplasmic reticulum (ER). Interestingly, all of GFP-fused P450-2was not observed in ER when only P450-2 was expressed, but mostly localized in ER when coexpressed with P450-1. In addition, TP fused to GFP waslocalized mainly on the plasma membrane and also rarely observed on other organelles such as vacuole.124