Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSapproaches to rapidly screen large cool-season grass populations to identify endophyte diversity. Molecular analyses of endophyte genetic traits fromamong and between host populations allow us to explore resident endophyte incidence and diversity present in single host species. PCR with genomicDNA extracted from individual plants (seeds or tillers) is used to determine endophyte incidence within a line and to predict alkaloid chemotypes at theEAS (ergot alkaloids), LOL (lolines), IDT/LTM (indole-diterpenes) and PER (peramine) loci. The presence or absence of genes at each locus can be used topredict the likely pathway end product for a given endophyte-infected plant line. Phylogenetic analyses of housekeeping and mating-type genes are usedto infer hybrid versus nonhybrid origins as well as hybrid ancestral progenitors. Sequence analyses of alkaloid genes encoding key pathway steps provideallele copy number and can be used to determine progenitor origins to further support the phylogenetic relationships. Grass collections across multiplehost tribes have recently been evaluated and considerable endophyte chemotypic diversity was identified. Multiple endophyte species were able toindependently associate with some grass host species and often both hybrid and nonhybrid endophytes could be found within a population. In manycases, chemotypic diversity of the hybrids may have arisen from independent hybridization events and as such, this alkaloid diversity likely translates intodifferences in fitness and persistence of the host.27. Extracellular polysaccharide degrading capabilities of various Agaricus bisporus strains during compost cultivation. A. Patyshakuliyeva, J. Yuzon, R. P.de Vries. CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands.In the temperate forests of North America and Europe, basidiomycetes such as Agaricus bisporus are renowned for their ecological significance in thecycling of carbon from dead plant matter. A. bisporus is also the most widely produced mushroom in the world and has been cultivated for centuries.However, little is known about the interaction between A. bisporus and its most preferred substrate, composted plant matter. In this study, a wide array ofextracellular polysaccharide degrading enzymes was studied under semi-commercial conditions to understand the carbon nutritive needs of the fungi.Various time points were sampled from filling of the beds, vegetative growth and development and maturation of fruiting bodies. Clear correlations in theenzymatic activities were observed from different stages of development of A. bisporus between compost, casing layer and fruiting bodies. This couldsuggest that vegetative mycelia and the fruiting body divide their metabolic roles as vegetative mycelium of A. bisporus provides nutrients for the growthof fruiting bodies, while fruiting bodies aims on reproduction. This was also confirmed by identification of the expression of genes encoding plant andfungal polysaccharide modifying enzymes in compost, casing layer and fruiting bodies.28. Reconstruction of the rubrofusarin biosynthetic pathway in Saccharomyces cerevisiae. Rasmus J N Frandsen 1 , Peter Rugbjerg 1 , Michael Naesby 2 , UffeH Mortensen 1 . 1) Systems Biology - CMB, Technical University of Denmark, Kgs. Lyngby, Denmark; 2) Evolva SA, Duggingerstrasse 23. CH-4153 Reinach,Switzerland.The aromatic heptaketide rubrofusarin is a common core substructure of several fungal pigments, including rubrofusarin B, aurofusarin, nigerone,nigerasperone A, chaetochromin, ustilaginoidin and parasperone A. Compounds that are produced by a wide variety of different filamentous fungi such asFusarium graminearum, Aspergillus niger, Aspergillus parasiticus, Chaetomium gracile and Ustilaginoidea virens. Previous reverse genetics analysis of theaurofusarin biosynthetic pathway, by targeted gene replacement in F. graminearum (Fg), has resulted in the formulation of a six step biosynthetic pathwaythat includes rubrofusarin as an intermediate. In the current study we have used heterologous expression in Saccharomyces cerevisiae to test whether allthe enzymes required for biosynthesis of rubrofusarin have been identified. Successful reconstruction of the rubrofusarin pathway is dependent on theheterologous co-expression of four genes: the Fg polyketide synthase PKS12, the Fg dehydratase aurZ, the Fg O-methyltransferase aurJ and the Aspergillusfumigatus phosphopantetheine transferase npgA. To eliminate potential problems with intron splicing of the fungal genes in S. cerevisiae the requiredcoding sequences were de novo synthetized in codon optimized versions. The four genes were expressed individually from four different single copyplasmids, each with a unique auxotrophic marker. Co-expression of the codon optimized version of PKS12 with npgA did not result in production of anynew metabolites. However, surprisingly co-expression of a cDNA version of PKS12, assembled from gDNA by USER-fusion, resulted in production of theexpected product YWA1. Additional co-expression of the codon optimized dehydratase encoding aurZ gene lead to production of nor-rubrofusarin, andsubsequent introduction of the O-methyltransefase gene aurJ yielded rubrofusarin. These results support the previously proposed biosynthetic route forthe formation of rubrofusarin in F. graminearum. The utilized bottom-up approach shows that formation of rubrofusarin is dependent only on thecombined action of PKS12, AurZ and AurJ in F. graminearum, and likely also in other fungal species that produce compounds with a rubrofusarin core. Thelatter is further supported by sequence base homology searches in the available relevant fungal genome sequences.29. Expression and purification of hydrophobin fusion proteins targeted to intracellular protein bodies in T. reesei . Nina K. Aro, Marika Vitikainen, JussiJoensuu, Eero Mustalahti, Markku Saloheimo. Biotechnology, VTT Technical Research Centre, 02044 VTT, VTT, Finland.Recombinant protein production is a fast growing market area. The need for novel production platforms is growing together with the number of newapplications for recombinant proteins. The ascomycete T. reesei is an excellent producer of hydrolytic enzymes. However, heterologous protein productionin T. reesei is often suffering from low product yields due to protease degradation and inefficiency in heterologous protein secretion. We have previouslydemonstrated a novel recombinant protein production system for T. reesei using GFP as a model protein. This system uses hydrophobin, a small andamphipathic fungal protein, as a fusion tag for purification and ER retention signal for targeting the produced protein to intracellular protein bodies. TheGFP-HFBI fusion protein can be extracted from total protein lysate by aqueous two-phase separation system. We have now further optimised theexpression for GFP-HFBI fusion and demonstrated the applicability of this production concept for two additional proteins, glucose oxidase (GOX) and tissueplasminogen activator (tPA). Effect of C- and N-terminal hydrophobin fusion on productivity and extraction in two-phase separation system will bediscussed. The new production concept is aiming at widening the spectrum of recombinant proteins that can be produced efficiently in T. reesei.128