Expression of Terpene Synthases from Edible Fungi using Yeast Chassis

Sammanfattning

Secondary metabolites produced from organisms are compounds that are not directly connected to the growth of organisms but that serve many important purposes for them in nature. Terpenes and terpenoids form a section of the secondary metabolites, produced from terpene synthase (TPS) enzymes. Fungal species are highly dependent on secondary metabolites and especially the terpenoid compounds for numerous adaptive tasks, such as defense and formation of symbiotic relationships. Despite the importance of terpenes and terpenoids in fungi and the large number of fungal species compared to plant species, the corresponding TPS genes in fungal genomes are much less studied than those from plants. In this work, as part of a larger study at UCPH, unexplored TPSs from edible fungal species were studied, to contribute to the enzyme characterizations and product exploration. 31 TPSs enzymes from fungal genomes of shiitake mushroom Lentinula edodes, oyster mushroom Pleurotus ostreatus, porcini mushroom Boletus edulis, jelly fungus Auricularia subglabra and cheese fungi Penicillium roqueforti, Penicillium biforme, and Penicillium camemberti were expressed. The genes were introduced in yeast through multicopy plasmids, using the uracil-specific excision reagent (USER) cloning technique to construct the plasmids together with an inducible GAL1 promoter. The products were analyzed in vivo with headspace solid phase microextraction (HS-SPME) as well as in vitro with hexane extraction, using gas chromatography mass spectrometry (GC-MS). From the results it could be concluded that three TPSs produced mainly monoterpenes, nine TPSs mainly sesquiterpenes, and one TPS mainly diterpenes. One putative sesquiterpene without a provided name was detected as well as sesquiterpenes sinularene and myltayl-4(12)-ene which could not be found in fungal species. The monoterpene synthase (mono-TPSs) belonged in majority to the Ascomycota phylum and the sesquiterpene synthase (sesqui-TPSs) in majority to the Basidiomycota phylum. The catalytic activity of the TPS genes was traced back to the phylogenetic tree, where especially TPSs producing monoterpenes located in one cluster and TPSs producing sesquiterpenes of cadalane structures in another. An additional experiment with expression of Erg20p(N127W), a gene described to accumulate GPP in yeast cells, led to an unexpected increase in sesquiterpenes. Furthermore, a third of the transformants were induced into buffered medium (pH 6.5) to analyze the correlation between pH and enzymatic activity. The buffered induction led to an overall greater sesquiterpene production for all except the three tested inactive transformants that still showed no terpene peaks.