Terpenoids
constitute a structurally diverse group of natural products
with wide applications in the pharmaceutical, nutritional, flavor
and fragrance industries. Fungi are known to produce a large variety
of terpenoids, yet fungal terpene synthases remain largely unexploited.
Here, we report the sesquiterpene network and gene clusters of the
black poplar mushroom Agrocybe aegerita. Among 11
putative sesquiterpene synthases (STSs) identified in its genome,
nine are functional, including two novel synthases producing viridiflorol
and viridiflorene. On this basis, an additional 1133 STS homologues
from higher fungi have been curated and used for a sequence similarity
network to probe isofunctional STS groups. With the focus on two STS
groups, one producing viridiflorene/viridiflorol and one Δ6-protoilludene, the isofunctionality was probed and verified.
Three new Δ6-protoilludene synthases and two new
viridflorene/viridiflorol synthases from five different fungi were
correctly predicted. The study herein serves as a fundamental predictive
framework for the discovery of fungal STSs and biosynthesis of novel
terpenoids. Furthermore, it becomes clear that fungal STS function
differs between the phyla Ascomycota and Basidiomycota with the latter
phylum being more dominant in the overall number and variability.
This study aims to encourage the scientific community to further work
on fungal STS and the products, biological functions, and potential
applications of this vast source of natural products.
Background
Cyclocybe aegerita (syn. Agrocybe aegerita) is a commercially cultivated mushroom. Its archetypal agaric morphology and its ability to undergo its whole life cycle under laboratory conditions makes this fungus a well-suited model for studying fruiting body (basidiome, basidiocarp) development. To elucidate the so far barely understood biosynthesis of fungal volatiles, alterations in the transcriptome during different developmental stages of C. aegerita were analyzed and combined with changes in the volatile profile during its different fruiting stages.
Results
A transcriptomic study at seven points in time during fruiting body development of C. aegerita with seven mycelial and five fruiting body stages was conducted. Differential gene expression was observed for genes involved in fungal fruiting body formation showing interesting transcriptional patterns and correlations of these fruiting-related genes with the developmental stages. 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. Furthermore, we were able to localize the mycelium as the main source for sesquiterpenes predominant during sporulation in the headspace of C. aegerita cultures. In contrast, changes in the C8 profile detected in late stages of development are probably due to the activity of enzymes located in the fruiting bodies.
Conclusions
In this study, the combination of volatilome and transcriptome data of C. aegerita revealed interesting candidates both for functional genetics-based analysis of fruiting-related genes and for prospective enzyme characterization studies to further elucidate the so far barely understood biosynthesis of fungal C8 oxylipins.
Volatile organic compounds (VOC) are characteristic for different fungal species. However, little is known about VOC changes during development and their biological role. Therefore, we established a laboratory cultivation system in modified crystallizing dishes for analyzing VOC during fruiting body development of the dikaryotic strain Cyclocybe aegerita AAE-3 as well as four monokaryotic offspring siblings exhibiting different fruiting phenotypes. From these, VOC were extracted directly from the headspace (HS) and analyzed by means of gas chromatography-mass spectrometry (GC-MS). For all tested strains, alcohols and ketones, including oct-1-en-3-ol, 2-methylbutan-1-ol and cyclopentanone, were the dominant substances in the HS of early developmental stages. In the dikaryon, the composition of the VOC altered with ongoing fruiting body development and, even more drastically, during sporulation. At the latter stage, sesquiterpenes, especially Δ6-protoilludene, α-cubebene and δ-cadinene, were the dominant substances. After sporulation, the amount of sesquiterpenes decreased, while additional VOC, mainly octan-3-one, appeared. In the HS of the monokaryons, less VOC were present of which all were detectable in the HS of the dikaryon C. aegerita AAE-3. The results of the present study show that the volatilome of C. aegerita changes considerably depending on the developmental stage of the fruiting body.
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