Cyclocybe aegerita as a model organism for the elucidation of volatile organic compound biosynthesis pathways in fungi

Abstract

Mushrooms are part of the human diet since time immemorial, appreciated for their nutritional value and especially for their delicious flavors. Hundreds of volatile organic compounds (VOCs) have been identified in fungi contributing to the unique aroma of each species. Generally, studies on mushroom VOCs are carried out with chopped fruiting bodies of more or less one developmental stage. For determine fungal aromas for assessment of the food quality this procedure might be adequate. Nonetheless, for analysis of the biological role of fungal VOCs in context of inter alia VOC biosynthesis or fungal communication this approach can suffer from drawbacks. First of all, damaging fruiting bodies can lead to VOC artefacts due to cell disruption and the occurrence of unnatural enzymatic reactions. Furthermore, fungal VOC profiles are dynamic, changing with ongoing development. For better understanding of the biological function of fungal VOCs it is therefore helpful to know which volatile patterns are characteristic for a certain developmental stage. Against this background, an approach was developed enabling on one hand the cultivation of fungi during different developmental stages, including the growth of fruiting bodies, and on the other hand the non-invasive analysis of VOCs in the headspace (HS) of fungal cultures. These requirements were complied with modified crystallizing dishes for culture purposes and a HS-SPME-GC-MS approach to analyze the VOCs. This method was applied to analyze the volatilomes of the dikaryotic strain C. aegerita AAE-3 and four monokaryotic offspring siblings with different fruiting phenotypes throughout ten life stages. At early stages, in the HS of all tested strains alcohols and ketones, such as oct-1-en-3-ol, 2-methylbutan-1-ol and cyclopentanone, were the most prominent VOCs. Particularly counting for the dikaryon, the volatilome altered with continued fruiting body development exhibiting remarkable changes during sporulation. Here, sesquiterpenes, especially Δ6-protoilludene, α-cubebene and δ-cadinene, were the most abundant VOCs in the HS of C. aegerita AAE-3. After sporulation, the amount of sesquiterpenes decreased along with the appearance of other VOCs including octan-3-one. In contrast, less VOCs were present in the HS of the monokaryotic strains of which all were as well detectable in the HS of the dikaryon. The changes of the volatilome were the fundament for a subsequent transcriptome analysis aiming to identify enzymes involved in fungal VOC biosynthesis, especially regarding C8 VOC formation, which is, despite the fact that these substances are ubiquitous found in fungi, still barely understood. The transcriptomic study was carried out with seven developmental stages of C. aegerita AAE-3, which during the volatilome study exhibited interesting volatile patterns. Additionally, fruiting bodies (five stages) and mycelia (seven stages) samples were harvested separately to get further insights about the putative origin of the VOCs observed in the HS of C. aegerita. Combining transcriptome and volatilome data, enzymes putatively involved in the biosynthesis of C8 oxylipins in C. aegerita including lipoxygenases (LOXs), dioxygenases (DOXs), hydroperoxide lyases (HPLs), alcohol dehydrogenases (ADHs) and ene-reductases could be identified. Especially the putative DOX AAE3_13098, the putative HPLs AAE3_05330 and AAE3_09203, the putative ADHs AAE3_00054 and AAE3_06559 as well as the putative ene-reductase AAE3_15349 exhibit remarkable transcriptomic patterns making these enzymes highly interesting for future characterization studies. Furthermore, the study showed that the mycelium is probably the main source for sesquiterpenes observed during sporulation in the HS of C. aegerita AAE-3 cultures whereas changes in the C8 profile detected in late stages of development are probably due to the activity of enzymes located in the fruiting bodies.