Molecular and Functional Analyses of Characterized Sesquiterpene Synthases in Mushroom-Forming Fungi

Abstract

Sesquiterpenes are a type of abundant natural product with widespread applications in several industries. They are biosynthesized by sesquiterpene synthases (STSs). As valuable and abundant biological resources, mushroom-forming fungi are rich in new sesquiterpenes and STSs, which remain largely unexploited. In the present study, we collected information on 172 STSs from mushroom-forming fungi with experimentally characterized products from the literature and sorted them to develop a dataset. Furthermore, we analyzed and discussed the phylogenetic tree, catalytic products, and conserved motifs of STSs. Phylogenetic analysis revealed that the STSs were clustered into four clades. Furthermore, their cyclization reaction mechanism was divided into four corresponding categories. This database was used to predict 12 putative STS genes from the edible fungi Flammulina velutipes. Finally, three FvSTSs were selected to experimentally characterize their functions. FvSTS03 predominantly produced Δ-cadinol and FvSTS08 synthesized β-barbatene as the main product; these findings were consistent with those of the functional prediction analysis. A product titer of 78.8 mg/L β-barbatene was achieved in Saccharomyces cerevisiae via metabolic engineering. Our study findings will help screen or design STSs from fungi with specific product profiles as functional elements for applications in synthetic biology.

Functional Characterization of Sesquiterpene Synthases and P450 Enzymes in Flammulina velutipes for Biosynthesis of Spiro [4.5] Decane Terpene

“…velutipes genome . Recently, three STSs were selected and their functions were characterized in Saccharomyces cerevisiae . However, the mechanisms of cyclization and the terpene synthases-skeleton relationship remain largely unknown.…”

Genome Mining of Fungal Unique Trichodiene Synthase-like Sesquiterpene Synthases

“…Recent advancements in genome mining of fungal STSs have unveiled numerous novel sesquiterpenes and enzymes [7]. However, the sequence-function relationships of STSs have been barely studied.…”

“…Although related biosynthetic enzymes have been reported from plants before, this is the first time that such enzymes have been reported from fungi. The remaining eight TDTSs produced sesquiterpene skeletons: chamipinene (3), acoradiene (4), longifolene ( 5), longiborneol (6), sativene (7), α-muurolene (8), α-cuprenene (9), cedrene (10,11), and γ-bisabolene (12) (Figures 4iii-x and S6). Among them, chamipinene (3), a rare sesquiterpene, has only been reported from the plant Illicium oligandrum [23], with the related biosynthetic enzyme unknown.…”

“…Fungi represent a rich source of diverse sesquiterpenes with pharmaceutical and agricultural relevance [5], including illudin M (cytotoxic), abscisic acid (plant growth regulation), and penifulvin (insecticidal) [6]. In the past decade, over 150 fungal STSs have been functionally characterized through genome mining, resulting in the discovery of numerous novel sesquiterpenes and enzymes [7]. However, the genome mining of fungal STSs has been limited to a small number of species, such as Coprinopsis cinerea, Omphalotus olearius, and Stereum hirsutum [7][8][9][10][11].…”

“…In the past decade, over 150 fungal STSs have been functionally characterized through genome mining, resulting in the discovery of numerous novel sesquiterpenes and enzymes [7]. However, the genome mining of fungal STSs has been limited to a small number of species, such as Coprinopsis cinerea, Omphalotus olearius, and Stereum hirsutum [7][8][9][10][11]. The sequence diversity, abundance, and distribution of fungal STSs on a global scale remain elusive.…”