Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTScalcium influx system (LACS). In the filamentous fungus Aspergillus nidulans, we identified the homologs of HACS—the voltage gated channel CchA and thestretch activated channel MidA, which formed a complex that played important roles in conidial development, hyphal polarity establishment, and cell wallcomponents in low-calcium environmental condition. In comparison, loss of FigA, a putative member of LACS, showed very severe defects in conidiationand in self-fertility during sexual development under either low or high calcium environmental condition. Interestingly, extracellular Ca2+ was unable toimprove these figA defects substantially. Most importantly, the quantitative PCR results revealed the expression of the major asexual developmentregulator brlA and sexual development regulator nsd, steA had been remarkable regulated in figA mutant. In addition, the localization of Fig1::GFPrevealed that FigA was highly accumulated at the center of septum on the mature hypha , and the sites between vesicle-metulae, between metulaephialide.These data implied that figA likely played important roles in cellular trafficking and communication during in both asexual and sexualdifferentiation in Aspergillus nidulans. *Correspondence to be addressed: linglu@njnu.edu.cn This work was financially supported by the National NaturalScience Foundation of China (NSFC) 31200057 to Z.S, 31070031 to L.L.370. The Saccharomyces cerevisiae FUS3 homologue MAKB in Aspergillus niger is a central regulator connecting differentiation and secondarymetabolite production with nutrient availability and light. Bert-Ewald Priegnitz, Ulrike Brandt, André Fleissner. Institut für Genetik, TU Braunschweig,Braunschweig, Germany.Aspergillus niger is an important cell factory in biotechnology. Productivity of this fungus is strongly influenced by the environmental growth conditionsand the subsequent morphological adaptation. In eukaryotic organism, mitogen activated protein kinase cascades are important signaling pathwaysmediating cellular responses to environmental cues. In order to gain a deeper understanding of the molecular mechanisms of cellular adaptation in A.niger, we characterized the Saccharomyces cerevisiae FUS3 MAP kinase homologue MAKB by using a combination of molecular genetics, biochemicalanalysis and light and fluorescent microscopy. We found that the absence of makb results in specific developmental defects, which strongly depend onenvironmental conditions. For example, the mutant secrets a dark pigment in a nutrient dependent manner in constant darkness, but not while growing ina dark-light-rhythm. In addition, MAKB is also involved in asexual spore formation, indicated by its accumulation in developing conidiophores and areduced sporulation of the makb deletion mutant. Detailed comparison of sporulation in the wild-type and the mutant under different growth conditionslet us to the hypothesis that two independent trigger induce conidiation: nutrient starvation and cell age or density. Interestingly, MAKB appears to beonly involved in the latter one, indicated by suppressed sporulation of the mutant under nutrient sufficient growth conditions. Taken together, theseobservations indicate that in A. niger, MAKB is intimately involved in the adaptation of secondary metabolism and cell differentiation in response toenvironmental influences.371. Engineering and characterizing protein secretion in Aspergillus niger. Y. Zheng, G. Budkewitsch, S. Bourque, J. Burai, N. Geoffrion, J. Richard Albert,E. Munro, S. Sillaots, R. Storms. Biology, Concordia University, Montreal, QC, Canada.Aspergillus niger is widely used commercially and for basic research as a host for native and foreign protein production. This is mainly because of itsability to secrete large amounts of protein into the growth medium, and carry out the eukaryotic post-translational modifications of glycosylation,proteolytic cleavage and disulfide bond formation. Although A. niger can express some native and foreign proteins at high levels, many native proteins andmost foreign proteins are expressed at very low levels. Competition with native proteins for rate limiting steps in the secretory pathway, transcription,mRNA processing and translation are bottlenecks that can limit levels of secreted protein production. Furthermore, proteins expressed using A. niger oftenrequire extensive purification, due to the presence of high levels of many native secreted proteins that can interfere with the downstream characterizationor reduce protein stability. To address these issues we have been developing recombinant promoters that support high transcription rates, engineeringstrains that produce reduced levels of native secreted protein, and identifying secretory pathway bottlenecks that limit secreted protein yields. We havecreated a set of novel expression cassettes that are capable of supporting significantly higher transcription rates than are obtained with the widely usedglucoamylase gene (glaA) promoter. We have also, engineered new "clean" expression strains that combine dramatically reduced levels of"contaminating" native extracellular protein production with increased levels of target protein expression. RNA-seq analysis and conditional geneexpression are being used to identify additional bottlenecks that limit the production of foreign proteins.372. Design of culture conditions for secondary metabolite production of fungi based on large-scale transcriptome data. M. Umemura 1 , H. Koike 2 , M.Sano 3 , N. Yamane 2 , T. Toda 2 , K. Tamano 1 , S. Ohashi 3 , M. Machida 1 . 1) Bioproduction Research Institute, National Institute of Advanced Industrial Scienceand Technology (AIST), Sapporo, Japan; 2) Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST),Tsukuba, Japan; 3) Kanazawa Institute of Technology, Kanazawa, Japan.Fungi produce a wide variety of secondary metabolites, many of which possess bioactivity against bacteria, fungi, and human diseases (ex. penicillin,micafungin, and lovastatin). Inducing the fungal productivity of secondary metabolites is requisite for study and industrial applications of the compounds.Since types and amounts of the secondary metabolites greatly change according to strains and culture conditions, controlling the productivity is difficultand has largely depended on empirical knowledge. More data-based knowledge is required especially when treating the metabolites for which lessempirical knowledge has been accumulated. In this study, we have developed an algorithm to analyze metabolic pathway activities using large-scaletranscriptome data, and applied it to 72 sets of Aspergillus oryzae transcriptome data obtained using different nutritions. Based on the classification ofmetabolic pathways and assignment of A. Oryzae genes to them by Vongsangnak et al. [BMC Genomics, 9,245 (2008)], we evaluated the correlations of thepathways and found some positive and negative correlations between primary and secondary ones. We also found that secondary metabolic pathwayswere divided into two groups depending on the type of nutrition. Combining some culture conditions based on these results, we designed the idealnutrition for inducing each two groups of secondary metabolic pathways. We are now confirming metabolic pathway activity and secondary metaboliteproductivity under the designed culture conditions using DNA microarray and LC-MS, respectively.373. The effect of the clbR overexpression on cellulose degrading enzyme production in Aspergillus aculeatus. S. Tani, A. Kawamura, E. Kunitake, J.Sumitani, T. Kawaguchi. Life & Environmental Sci, Osaka Prefecture Univ, Osaka, Japan.Aspergillus aculeatus has two pathways that control the induction of cellulase and hemicellulase genes in response to cellulose. The expression ofcarboxymethylcellulase 1 (cmc1) and FIb-xylanase (xynIb) genes was controlled by XlnR, in contrast the expression of cellobiohydrolase I (cbhI),carboxymethylcellulase 2 (cmc2), hydrocellulase (cbhIb) and FIa-xylanase (xynIa) genes was controlled by XlnR-independent signaling pathway. We havereported that ClbR, a Zn(II) 2Cys 6 transcription factor, controlled the cellulose-responsive induction of the genes regulated by both XlnR-dependent andXlnR-independent signaling pathways. Therefore, we investigated if their enzyme productions could be improved by the clbR overexpression. The clbRgene was expressed under the promoter of the translation elongation factor 1 alpha gene. Interestingly, xylanase activity in the clbR overexpressing strain(clbRox) increased 6-fold more than that in control strain. Although endoglucanase activity did not increase, its activity was kept at high level for 10 days.Peptide mass fingerprinting revealed that FIa-xylanase production was drastically increased in the clbRox strain, in contrast that hydrocellulase production27th Fungal Genetics Conference | 211