Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS13. Increased production of fatty acids and triglycerides in Aspergillus oryzae by modifying fatty acid metabolism. Koichi Tamano 1 , Kenneth Bruno 2 ,Tomoko Ishii 1 , Sue Karagiosis 2 , David Culley 2 , Shuang Deng 2 , James Collet 2 , Myco Umemura 1 , Hideaki Koike 1 , Scott Baker 2 , Masayuki Machida 1 . 1) NationalInstitute of Advanced Industrial Science and Technology (AIST); 2) Pacific Northwest National Laboratory (PNNL).Biofuels are attractive substitutes for petroleum based fuels. Biofuels are considered they do not contribute to global warming in the sense they arecarbon-neutral and do not increase carbons on the globe. Hydrocarbons that are synthesized by microorganisms have potential of being used as biofuelsor the source compounds. In the hydrocarbon compounds synthesized by A. oryzae, fatty acids and triglycerides are the source compounds of biodieselthat is fatty acid methyl ester. We have increased the production by modifying fatty acid metabolism with genetic engineering in A. oryzae. Firstly,enhanced-expression strategy was used for the increase. For four enzyme genes related to the synthesis of palmitic acid [C16:0-fatty acid], the individualenhanced-expression mutants were made. And the fatty acids and triglycerides in cytosol were assayed by enzyme assay kits, respectively. As a result,both fatty acids and triglycerides were most synthesized in the enhanced-expression mutant of fatty acid synthase gene at 2.1-fold and 2.2-fold more thanthe wild-type strain, respectively. Secondly, gene disruption strategy was used for the increase. Disruptants of several enzyme genes related to long-chainfatty acid synthesis were made individually. And one of them showed drastic increase in fatty acid synthesis. In the future, further increase in the synthesisis expected by utilizing genetic engineering in A. oryzae.14. Improved Properties of Thermostable Cellobiohydrolase in a Treatment of Cellulosic Material. Taija Leinonen 1 , Susanna Mäkinen 1 , Kari Juntunen 1 ,Merja Niemi 2 , Juha Rouvinen 2 , Jari Vehmaanperä 1 , Terhi Puranen 1 . 1) Roal Oy, Rajamäki, Finland; 2) University of Eastern Finland, Department ofChemistry, Joensuu, Finland.Production of biofuels i.e. bioethanol from lignocellulosic material is a promising alternative technology for using biomass as a renewable and cleansource of energy instead of consuming limited natural resources e.g. fossil fuels, and releasing increasing amounts of CO 2. Enzymatic hydrolysis isconsidered to be the most promising technology for converting cellulosic biomass into fermentable sugars. Enzymatic total hydrolysis of (ligno)cellulosicsubstrates requires at least cellobiohydrolases, endoglucanases and beta-glucosidases. Previously cloned thermostable glycoside hydrolase family 7cellobiohydrolase (CBHI) from Acremonium thermophilum was expressed in Trichoderma reesei. The purified A. thermophilum CBHI was crystallized, andthe structure of the catalytic domain of the protein was determined at a resolution of 1.8 Å revealing the overall structure of the catalytic core of theenzyme to be similar with the previously determined structures of glycoside hydrolase family 7 cellobiohydrolases. In the biomass hydrolysis experimentsthe A. thermophilum CBHI, as part of the enzyme mixture, was shown to have enhancing effect on hydrolysis yield as compared to the Trichoderma reeseienzyme. To achieve even further improvements in thermal stability and hydrolysis performance, several single and combined amino acid mutations weredesigned based on the resolved 3D-structure. The data obtained from the mutants demonstrates that thermal stability and hydrolysis performance of theA. thermophilum CBHI protein can be increased by introducing single mutations as well as mutation combinations to the molecule.15. The phytopathogenic fungus Botrytis pseudocinerea is resistant to the fungicide fenhexamid due to detoxification by a cytochrome P450monooxygenase Cyp684. Saad Azeddine, Alexis Billard, Jocelyne Bach, Catherine Lanen, Anne-Sophie Walker, Sabine Fillinger, Danièle Debieu. INRAUR1290 BIOGER CCP, avenue Lucien Brétignières F78850 Thiverval-Grignon, France.The Botrytis species complex responsible for the grey mould disease found on grapevines is composed of two species: Botrytis cinerea, to a large extent(roughly 90%), and Botrytis pseudocinera. Despite their genetic polymorphism, these species cannot be morphologically distinguished. However, they dodiffer in their response to several fungicides, especially to the sterol biosynthesis inhibitor fenhexamid. While B. cinerea is sensitive to this hydroxyanilidefungicide, B. pseudocinerea is naturally resistant. Because a strong synergism was found on B. pseudocinerea between fenhexamid and sterol 14ademethylationinhibitors (DMIs) known to inhibit Cyp51, a cytochrome P450 monooxygenase, it was hypothetized that the detoxification of fenhexamid bya cytochrome P450 monooxygenase similar to Cyp51 is involved in the resistance B. pseudocinerea displays. To test this, we sought the geneoverexpressed in the presence of fenhexamid with the highest similarity to cyp51. Taking into account the Cyp P450 classification based on homology andphylogenetic criteria, this gene, whose function remains unknown, belongs to the Cyp684 family. It was then deleted in a B. pseudocinerea strain. Cyp684knock out mutants exhibit a loss of fenhexamid resistance and synergism between DMIs and fenhexamid, showing that the Cyp684, cytochrome P450protein is responsible for B. pseudocinerea’s natural resistance to fenhexamid and is involved in fenhexamid detoxification. Although cyp684 is alsopresent in B. cinerea, which is sensitive to fenhexamid, a polymorphism was observed between the two species: in B. pseudocinerea the cyp684 promotershows a deletion of 25 bp. We are currently establishing the cyp684 expression profiles in both species in order to analyze the impact of the promoterdeletion on its expression. Metabolization studies are also being conducted to identify metabolites that would help in understanding the enzymaticfunctions of Cyp684 and to determine to what extent Botrytis sp. is sensitive to these metabolites.16. Evolutionary rewiring of ubiquitination targets in Candida albicans promotes efficient carbon assimilation in host niches. Alistair J P Brown, DoblinSandai, Zhikang Yin, Laura Selway, David Stead, Janet Walker, Michelle D Leach, Iryna Bohovych, Iuliana V Ene, Stavroula Kastora, Susan Budge, Carol AMunro, Frank C Odds, Neil A R Gow. School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.Pathogens must assimilate carbon to grow and infect their host. Interesting questions remain about the regulation of carbon assimilation in Candidaalbicans despite the wealth of knowledge about this major fungal pathogen of humans. C. albicans is classified as a Crabtree-negative yeast because itcontinues to respire in the presence glucose [J Med Vet Mycol 26, 195]. How then can C. albicans be exquisitely sensitive to sugars, down-regulatingtranscripts involved in the utilization of alternative carbon sources following exposure to 0.01% glucose [Molec Biol Cell 20, 4845]? We have now shownthat there is a significant dislocation between the transcriptome and proteome in C. albicans: glucose triggers the decay of key transcripts but the enzymesthey encode are retained. This allows the simultaneous assimilation of alternative carbon sources such as fatty acids, carboxylic acids and sugars by C.albicans. This contrasts with the situation in Saccharomyces cerevisiae where simultaneous carbon assimilation is prevented by catabolite inactivation[Arch Micro 134, 187; Arch Micro 147, 231]. We show that C. albicans has retained the molecular apparatus that mediates ubiquitin-mediated, glucoseacceleratedprotein degradation. For example, S. cerevisiae isocitrate lyase (ScIcl1) is degraded rapidly when expressed in C. albicans. However, C. albicansisocitrate lyase (CaIcl1) lacks critical ubiquitination sites that mediate this catabolite inactivation. Furthermore, other C. albicans enzymes involved ingluconeogenesis and the glyoxylate cycle appear to lack such sites, whereas glycolytic enzymes are ubiquitinated (e.g. Fba1, Pgk1, Eno1). Therefore therehas been significant rewiring of ubiquitination targets in C. albicans compared to S. cerevisiae. This metabolic flexibility probably enhances efficientcolonisation of host niches that contain complex mixtures of nutrients.27th Fungal Genetics Conference | 125