Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSNovel transcriptomics approaches for metabolic pathway engineering target identification in Aspergillus. Peter J. Punt, Martien Caspers, MarvinSteijaert, Eric Schoen, Machtelt Braaksma. Microbiology, TNO, Zeist, Netherlands.Among filamentous fungi Aspergillus sp. are well known production host for several organic acids. These acids, traditionally being food ingredients, morerecently have gained attention as platform or building-block chemicals. These chemicals, currently mostly produced based on petrochemistry, are thestarting point for the production of a wide variety of materials, such as resins, plastics, etc. Production of these compounds via biobased routes will be amajor contribution towards a Biobased Economy. For the production of these bulk compounds robust host organisms are required, suitable for using lowcost lignocellulose-based feedstocks, resistant against adverse conditions due to inhibitory feedstock compounds and capable of coping with high productconcentrations. A. niger was shown to fulfill most of these prerequisites (Rumbold et al., 2009).Based on the extended molecular genetic toolkit systemsbiology approaches were developed for A. niger and other fungi (e.g. Braaksma et al., 2010). These approaches were followed towards production of theseplatform chemicals in A. niger, as demonstrated by the example of itaconic acid (Li et al., 2011, 2012). The recent development of novel high throughputsequence methods has led to new much more efficient transcriptomics approaches such as RNAseq. Combination of these approaches with novelexperimental design and statistical methods for targetgene identification in metabolic pathway engineering will be illustrated. Rumbold, K., van Buijsen,H.J.J., Overkamp, K.M., van Groenestijn, J.W., Punt, P.J., Werf, M.J.V.D. (2009) Microbial production host selection for converting second-generationfeedstocks into bioproducts. Microbial Cell Factories 8, art. no. 64 Braaksma, M., van den Berg, R.A., van der Werf, M.J., Punt, P.J. (2010) A Top-DownSystems Biology Approach for the Identification of Targets for Fungal Strain and Process Development. In: Cellular and Molecular Biology of FilamentousFungi. Eds: K.A. Borkovich & D.J. Ebbole ASM Press, Washington DC. pp. 25-35 Li, A., van Luijk, N., ter Beek, M., Caspers, M., Punt, P., van der Werf, M.(2011) A clone-based transcriptomics approach for the identification of genes relevant for itaconic acid production in Aspergillus. Fungal Genetics andBiology 48 (6), pp. 602-611.A new method for gene mining and enzyme discovery. Y. Huang 1,2,3 , P. Busk 1 , M. Grell 1 , H. Zhao 2,3 , L. Lange 1 . 1) Section for Sustainable Biotechnology,Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University Copenhagen, Denmark; 2) Environmental Microbiology KeyLaboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan 610041, PR China; 3) University of theChinese Academy of Sciences, Beijing 100049, PR China.Peptide pattern recognition (PPR) is a non-alignment based sequence analysis principle and methodological approach, which can simultaneouslycompare multiple sequences and find characteristic features. This method has improved the understanding of structure/function relationship for enzymeswithin the CAZY families, which would make it easier to predict the potential function of novel enzymes, creating bigger promises for industrial purposes.Mucor circinelloides, member of the former subdivision Zygomycota, can utilize complex polysaccharides such as wheat bran, corncob, xylan, CMC andavicel as substrate to produce plant cell wall degrading enzymes. Although the genome of M. circinelloides has been sequenced, only few plant cell walldegrading enzymes are annotated in this species. In the present project, PPR was applied to analyze glycoside hydrolase families (GH family) and miningfor new GH genes in M. circinellolides genome. We found 19 different genes encoding GH3, GH5, GH6, GH7, GH9, GH16, GH38, GH43, GH47 and GH125 inthe genome. Of the three GH3 encoding genes found, one was predicted by PPR to encode a b-glucosidase. We expressed this gene in Pichia pastoris andfound that the recombinant protein has high b-glucosidase activity (4884 U/mL). In this work, PPR provided targeted short cut to discovery of enzymeswith a specific activity. Not only could PPR pinpoint genes belonging to different GH families but it did also predict the enzymatic function of the genes.Increased production of fatty acids and triglycerides in Aspergillus oryzae by modifying fatty acid metabolism. Koichi Tamano 1 , Kenneth Bruno 2 , TomokoIshii 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) National Instituteof 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.27th Fungal Genetics Conference | 79