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Article

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

by
Shengli Wang
1,2,3,†,
Ruiqi Chen
1,2,3,†,
Lin Yuan
1,2,3,
Chenyang Zhang
4,
Dongmei Liang
3 and
Jianjun Qiao
1,2,3,*
1
Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
2
Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, China
3
Zhejiang Institute of Tianjin University (Shaoxing), Shaoxing 312300, China
4
Science Center for Future Foods, Jiangnan University, Wuxi 214122, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Fungi 2023, 9(10), 1017; https://doi.org/10.3390/jof9101017
Submission received: 14 August 2023 / Revised: 24 September 2023 / Accepted: 3 October 2023 / Published: 14 October 2023
(This article belongs to the Special Issue Molecular Genetics and Genomics of Mushroom-Forming Fungi)
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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.

1. Introduction

Sesquiterpenes are a major class of terpenoids. More than 300 types of basic skeletons have been discovered; these skeletons are widely present in plants, fungi, microorganisms, and insects [1]. Sesquiterpene structures present several acyclic, monocyclic, bicyclic, tricyclic, and tetracyclic systems [2]. Owing to their complex structures, inherent bioactivity, and aroma, sesquiterpenes have widespread applications in food [3], pharmaceutical [4], fragrance [5], fuel [6], and agricultural industries [7]. Mushroom-forming fungi are specific fungal groups with the most conspicuous fruiting bodies; for centuries, they have been used as food and traditional medicine [8]. Mushrooms tend to develop several protective strategies to protect the fruiting bodies from organism attack. For example, mushrooms can produce various structurally diverse sesquiterpenes, many of which exhibit antibacterial, antifungal, and cytotoxic activities [9,10]. These sesquiterpenes play a crucial role in inhibiting fungal growth, modifying bacterial motility, and defending against parasites [11,12]. For example, to protect against predators, Armillaria mellea can produce toxic protoilludane-type sesquiterpenes [13]. Furthermore, rufuslactone isolated from the fruiting bodies of Lactarius rufus exerts antifungal properties against some pathogenic fungi, including Alternaria alternata and Fusarium graminearum [14]. To date, various sesquiterpenoid natural products with an extensive repertoire of backbone structures have been isolated and characterized from mushrooms [15,16,17]. Elucidating their biosynthetic pathways has garnered considerable attention [18,19,20].
The biosynthesis of sesquiterpene natural products originates from the common precursor farnesyl pyrophosphate (FPP), which is derived from the C5 unit isopentenyl diphosphate and its isomer dimethylallyl diphosphate [21]. Subsequently, sesquiterpene synthases (STSs) catalyze linear FPP to generate sesquiterpene scaffolds, followed by a series of cyclization reactions and rearrangements, resulting in the synthesis of structurally diverse sesquiterpenoids [22]. To date, more than 150 STSs have been cloned, purified, and biochemically characterized from mushroom-forming fungi, including Postia placenta [18], Phanerochaete chrysosporium [23], Ganoderma lucidum [24], and Lactarius deliciosus [25]. Furthermore, the site-specific mutations and cyclization mechanisms of a few STSs have been investigated [26,27,28,29,30]. However, owing to these fungi’s complex life cycle and frequently poor growth under laboratory conditions, fungal STSs, particularly those in mushrooms (basidiomycetes), are not well studied compared with those in plants. In general, each basidiomycete contains, on average, more than 12 STS homologs [31]; this indicates that fungal STSs represent rich but largely unexploited natural resources. Over the past decade, continuous advances in sequencing technologies have led to the accumulation of a large amount of fungal genomic data, facilitating genome mining to discover new STSs.
Overall, our study aims to assemble a comprehensive dataset of experimentally characterized mushroom STSs to elucidate the relationship between STS sequences and their catalytic products, then we used the database as a reference to predict and exploit unidentified STSs from other species. Firstly, previously reported 172 STSs were collated and analyzed by phylogenetic, protein domain, and motif analyses. Then, based on the functional prediction of STSs, three new STS genes from Flammulina velutipes were experimentally characterized. Finally, we expressed β-barbatene in Saccharomyces cerevisiae and achieved the highest yield of 78.8 mg/L. On the one hand, this work offers rich functional elements to researchers for conducting synthetic biology research. On the other hand, it provides a reference for exploring new STSs with novel products, superior activity, and selectivity in nature.

2. Materials and Methods

2.1. Literature Search for Characterized STSs

The collected STSs were found by manually searching the articles published in PubMed that demonstrated the ability of STSs via in vivo or in vitro experimental characterization. The amino acid sequences and corresponding IDs were collected from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), UniProtKB (http://www.uniprot.org/), and JGI (http://www.jgi.doe.gov/) collections (date last accessed on 27 July 2023).

2.2. Phylogenetic Tree Construction

Clustal W from MEGA7 was used to perform multiple sequence alignments of the proteins of the 172 STSs. Then, MEGA7 was used to develop a phylogenetic tree using the neighbor joining method. A bootstrap of 1000 replicates was performed [32].

2.3. Analysis of Protein Motifs and Domains

The specific domains of STS proteins were identified using Pfam (http://pfam-legacy.xfam.org/) (accessed on 15 August 2023). Furthermore, the conserved motifs in the identified STS proteins were predicted using the online tool Multiple Expectation Maximization for Motif (MEME) (https://meme-suite.org/meme/doc/meme.html) (accessed on 15 August 2023) using the default parameters.

2.4. Plasmids and Strain Construction

Table S1 and Table S2 present the plasmids and strains used and constructed in this study, respectively. Table S3 lists the primers used in this study. Table S4 lists the codon-optimized gene sequences. Phanta max super-fidelity DNA polymerase (Vazyme, P505, Nanjing, China) was used for PCR amplification. The recombinant strain was constructed using our previously described method [33]. The lithium acetate method was used for yeast transformation [34].

2.5. Heterologous Expression of STSs in Yeast

The pESC-URA plasmids containing the codon-optimized FvSTS genes were transformed into an engineered S. cerevisiae strain Sc027 [35] to produce sesquiterpenes. In addition, an empty pESC-URA vector was heterologously expressed in Sc027 as a control plasmid. The recombinant yeasts were cultured in 10 mL of synthetic complete (SC) drop-out medium (20 g/L glucose, 6.7 g/L yeast nitrogen base, and 2 g/L amino acid drop-out mix) at 220 rpm and 30 °C for 18 h. Subsequently, the culture solution was inoculated into 50 mL of SC medium with an initial OD600 of 0.05 and cultured at 30 °C and 200 rpm for 30 h. Then, galactose was added at a final concentration of 10 g/L for inducible protein expression. In situ product extraction into an organic phase is a widely used method to minimize the loss due to evaporation and improve the fermentative production of sesquiterpenes [36]. Thus, 5 mL of dodecane was added to the capture product in the fermentation medium after adding galactose. After culturing for 120 h, the fermentation broth was transferred into a 50 mL tube and centrifuged at 10,000 g for 10 min. The dodecane phase was collected and dehydrated by anhydrous sodium sulfate, then filtered using a 0.22 μm filter and analyzed via gas chromatography–mass spectroscopy (GC–MS).

2.6. GC–MS Analysis

The assay products were analyzed using an 8890–7000D GC–MS system equipped with a 7693A automatic liquid sampler and flame ionization detector (Agilent Technologies, Santa Clara, CA, USA). GC analysis was performed on an HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm). One microliter of each dodecane sample was injected into the system at a split ratio of 1:10. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The temperature of the inlet and detector were set to 280 °C and 200 °C, respectively. The oven temperature was maintained at 70 °C for 2 min and then gradually increased to 300 °C at a rate of 10 °C/min. The MS scan range (m/z) was 35–350 [37]. The fermentation products were identified by comparing their MS spectra and retention times with the NIST17 library. The standard β-caryophyllene was dissolved in dodecane and used to construct the standard curves for quantification.

3. Results

3.1. Database of Characterized STSs

To obtain a comprehensive dataset of functional STSs in mushrooms, the public databases NCBI, UniProt, and JGI were searched and studies on enzymes with characteristic STS domains in mushrooms were manually reviewed. In total, 174 STSs identified from 35 basidiomycetes have been experimentally characterized in previous studies. Table 1 and Table S5 present the information on these 174 STSs, including gene name, species origin, GenBank or JGI protein ID, major and minor products, and type of cyclization reaction. Among these, the amino acid sequence information of 172 STSs was available (File S1). Apart from these functionally characterized STSs, thousands of putative STSs have been identified in sequenced mushroom genomes and transcriptomes; however, their product specificities remain unknown [17].
To understand the relationship between the STS sequences and their product structures, a protein sequence-based phylogenetic tree of the 172 STSs was constructed using the neighbor joining method with the Jones–Taylor–Thornton model and pairwise deletion with 1000 bootstrap replicates using MEGA7 software. The phylogenetic tree revealed that the STS proteins were clustered into four distinct clades: clades I, II, III, and IV (Figure 1); this finding is consistent with previous studies [16,61,62]. Clustering via sequence conservation suggested that the STSs within one specific clade catalyze the same or a related cyclization reaction. The STSs in clade I produce terpenes such as α-muurolene, germacrene A, and cadinoles via the 1,10-cyclization of (2E,6E)-FPP with the (E,E)-germacradienyl cation as an intermediate. The STSs in clade II catalyze the 1,10-ring closure of the FPP stereoisomer (3R)-nerolidyl diphosphate ((3R)-NPP) to produce the intermediate (Z,E)-germacradienyl cation. The STSs in clade III are characterized by the 1,11-cyclization of (2E,6E)-FPP via a trans-humulyl cation intermediate. Lastly, the STSs in clade IV initiate 1,6-ring closure using (3R)-NPP as a substrate to yield the intermediate (6R)-β-bisabolol cation (Figure 2) [31,38,42].

3.2. Phylogenetic Analysis

To better understand the evolutionary relationships of the STSs in each clade, phylogenetic analysis was performed separately (Figure 3). The first clade (clade I) comprised 33 representative STSs from 18 different basidiomycetes, with STSs from Craterellus cinereus (Cop1–3) and Omphalotus olearius (Omp1 and Omp3), which are known to use the 1,10-cyclization of (2E,6E)-FPP to produce sesquiterpenes derived from the (E,E)-germacradienyl cation. Cop1 and Cop2 can synthesize germacrene A as the major product, whereas Cop3, Omp1, and Omp3 can synthesize α-muurolene as the main product [31,38]. Most STSs in clade I were identified as promiscuous enzymes that produce multiple sesquiterpene scaffolds. Of these STSs, 12 STSs could synthesize α-muurolene as the main product, whereas 8 STSs could synthesize α-muurolene as a side product. Furthermore, nine STSs that can produce cadinols as the main product were identified (Figure 3a). The second clade (clade II) comprised 41 STSs from 22 species. Most of the STSs (Cop4, Omp4, Omp5a, Omp5b, etc.) in clade II could transform FPP to generate β-copaene or cadinene via its isomer (3R)-NPP. Similarly, most STSs found in this clade also generated multiple products. Furthermore, clade II was divided into three clusters, with 17 of the 18 STSs that could produce cadinene as the major product distributed in a larger cluster and 6 STSs that could synthesize viridiflorol as the main product being grouped in the other cluster. Notably, LbSTS4a could produce the acyclic sesquiterpene (E)-nerolidol and form a separate cluster within clade II (Figure 3b).
The third clade (clade III) comprised 50 STSs from 21 different species. Nearly 50% (23) of the STSs grouped in this clade could produce tricyclic sesquiterpenes, including Δ6-protoilludene, pentalenene, and aromadendrene, which are derived from the common transhumulyl cation intermediate (Figure 3c). Furthermore, most of the STSs identified in this clade could generate a single major product. For example, AvSTS01 can synthesize a sesquiterpene alcohol as a single major product, whereas PoSTS06 can synthesize a sesquiterpene hydrocarbon as a single major product [56]. Moreover, PpSTS08 can specifically produce Δ6-protoilludene [18]. The last clade (clade IV) comprised 48 representative STSs from 22 different mushroom species. The STSs found in this clade could synthesize cadinane-type sesquiterpenoids. Eight STSs appeared in the same small branch and were all specific enzymes coding a single product, γ-cadinene (Figure 3d). Interestingly, the STSs grouped in this clade could also synthesize acyclic (E)-nerolidol, α-farnesene and β-farnesene, monocyclic (Z)-α-bisabolene, bicyclic α-cuprenene, tricyclic α-santalene, α-barbatene, and β-barbatene. Therefore, the STSs that appear in this clade may be functionally diverse to synthesize different types of sesquiterpene skeletons. A simple phylogenetic analysis can offer a predictive framework for discovering more STSs from underexploited mushroom-forming fungi in nature.

3.3. Analysis of Protein Conserved Motifs

STSs have well-known conserved domains that contain aspartate-rich and NSE/DTE motifs, which play important roles in coordinating Mg2+ to facilitate the ionization of FPP/NPP in the active site [63]. First, Pfam analysis was performed to identify the specific protein domains of the 172 STSs. In total, 144 STSs were identified to contain the following conserved domains (Table 2 and Table S5): PF19086 (Terpene_syn_C_2) or PF03936 (Terpene_synth_C), which correspond to the C-terminal domain of terpene synthase. Twenty-five STSs had the domain with the Pfam ID PF06330 (TRI5), described as trichodiene synthase, which is a terpenoid cyclase domain that catalyzes the FPP cyclization to form the bicyclic sesquiterpene hydrocarbon trichodiene [64]. Two STSs from Antrodia cinnamomea, namely Tps1A and Tps2A, were characterized by the UbiA prenyltransferase domain (Pfam ID: PF01040). The other three TPSs, namely, AncA, AncC, and DS3, were characterized by the HAD_2 domain (Pfam ID: PF13419), described as a haloacid dehalogenase-like hydrolase domain. Five STSs (Tps1A, Tps2A, AncA, AncC, and DS3) appeared in the same small branch and formed a separate cluster within clade IV (Figure 3d). Furthermore, the conserved motifs of the 172 STS proteins were analyzed using MEME software. The NDxxSxxxE (NSE) motif was identified as the most conserved motif, covering 161 of the 172 STS protein sequences; in contrast, the asparagine-rich regions [D(D/E/N)xx(D/E)] had various sequences among the 172 STSs. The DExxD sequence was often observed in an appropriate position in 87 STSs. Furthermore, the DD(N)xxD sequence was identified in the aspartate-rich region of 53 STSs. Sequence conservation analysis of these STSs supports more effective site-directed mutagenesis to modulate enzyme activity and specificity and helps us to understand the cyclization mechanisms.

3.4. Characterization of FvSTSs

Previous studies have reported that F. velutipes is rich in bioactive sesquiterpenes, including sixteen cuparene-type sesquiterpenes, enokipodins with antimicrobial activity, and flammulinolides with antitumor and anticancer activities [65,66,67,68]. To better understand sesquiterpene biosynthesis in F. velutipes, candidate STS sequences in the genomic database of F. velutipes (ASM1180015v1) were searched using BLAST and the 172 STSs in our database. Twelve putative STS genes were identified, which were named FvSTS01–12 (Table S6). The twelve FvSTSs were widely distributed in the phylogenetic tree, and four STSs belonged to clade III, six to clade IV, one to clade I, and one to clade II (Figure 4). To verify the results of our bioinformatics analysis, FvSTS03, FvSTS08, and FvSTS11 were selected for experimental characterization. The codon optimization is the most critical determinant of increasing heterologous protein expression [69]. Thus, three STSs were codon-optimized for expression in S. cerevisiae and synthesized into pESC-URA vectors by GENEWIZ (Suzhou, China). Then, three plasmids were transformed into the engineered S. cerevisiae strain Sc027. GC–MS analysis (Figure 5a) revealed that FvSTS03 abundantly produced Δ-cadinol, with small amounts of minor products, including γ-muurolene, α-muurolene, β-cadinene, and α-cadinol; this is consistent with the findings of the functional evolutionary analysis. Furthermore, FvSTS08 produced β-barbatene as the main product and α-barbatene, dauca-4(11),8-diene, and α-cuprenene as side products (Figure 5b), which were also produced by the other STSs in clade IV of the phylogenetic tree. However, no products were detected in FvSTS11-expressing S. cerevisiae cultures. These results confirm the prediction capability of our method; however, the method still has some limitations.

3.5. Heterologous Production of β-Barbatene in S. cerevisiae

Studies have reported that β-barbatene can attract insects for spore dispersal and respond to herbivore infestation [70,71,72]. In this work, we obtained FvSTS08 by a bioinformatics approach, and then experimentally demonstrated that FvSTS08 can synthesize β-barbatene. To improve the heterologous production of β-barbatene in yeast, an effective strategy was developed to increase substrate FPP supply by enhancing the mevalonate (MVA) pathway and inhibiting the branch pathways (Figure S1). In a previous study, Sc027 was engineered to increase FPP supplementation by enhancing MVA. The genes dpp1 (encoding phosphatidate phosphatase) and lpp1 (encoding phosphatidate phosphatase) were reported to be responsible for converting FPP to farnesol; their knockout can enhance FPP supplementation [73]. Therefore, the genes dpp1 and lpp1 were knocked out in Sc027, generating the strain WSL01 (Figure S2). Furthermore, the plasmid pESC-FvSTS08 was transformed into strain WSL01, resulting in the strain WSL01-FvSTS08. GC−MS analysis revealed that WSL01-FvSTS08 produced higher levels of β-barbatene, with a titer of 78.8 mg/L; this was higher than that achieved using the strain Sc027-FvSTS08 (43.2 mg/L) (Figure 6a). Barbatene has two isomers: α-barbatene and β-barbatene. Interestingly, we observed a rearrangement of β-barbatene to the better-described α-isomer under different strong acid conditions (Figure 6b–d). α-Barbatene possesses considerable potential as a high-energy aviation fuel [74]. Overall, our study provides an alternative approach for producing α-barbatene.

4. Discussion

Mushroom-forming fungi are particularly well known for their ability to synthesize several structurally diverse sesquiterpenoids, many of which are used as lead compounds for new drugs owing to their diverse pharmacological activities, including anticancer, antifungal, and antibiotic effects [15]. A comprehensive understanding of fungal STSs can help elucidate the biosynthetic mechanism of sesquiterpenoids, which has gradually become the focus of attention. In the present study, we collected 174 functionally characterized fungal STSs from previous studies. Phylogenetic analysis was performed and 172 STSs were divided into four distinct clades. Similar studies have reported that basidiomycetous STSs can be divided into four distinct clades (clades I–IV) [16,41,50,61]. However, some other studies have revealed that basidiomycetous STSs can be grouped into five distinct clades (clades I–V) [18,19,31,39]. The STSs in clade V possess significant sequence similarity to those in clade IV and probably prefer to catalyze the 1,6-cyclization of NPP to generate the bisabolyl cation intermediate, which is similar to the cyclization mechanism observed in the STSs in clade IV [31,39,75].
In general, the phylogenetic tree-based classification of STS protein sequences is consistent with the classification based on the mechanisms of the cyclization reaction of their products. However, some discrepancies remain; therefore, phylogenetic analysis cannot be an accurate predictor of the product specificities of STSs. First, acyclic sesquiterpenes do not undergo cyclization; however, STSs synthesizing acyclic sesquiterpenes appeared in clades II, III, and IV. This may be because acyclic sesquiterpenes (farnesene and nerolidol) are derived from primary cations via proton loss or a reaction with water molecules in the early stage of FPP conversion, which is shared by the biosynthetic pathways of multiple sesquiterpenes. Second, sesquiterpene biosynthesis may occur via different cyclization reactions. For example, in a previous study, researchers proposed a mechanism for viridiflorol formation based on quantum chemical calculations, starting with the 1,10-cyclization of (2E,6E)-FPP [76]. Phylogenetic analysis revealed that Agr5 belongs to clade II (Figure 3b); in contrast, SiTPS was placed in Clade III (Figure 3c). This result indicates that viridiflorol biosynthesis can occur via both routes. In addition, (−)-germacrene D can be synthesized from farnesyl cations via both routes: 1,10 or 1,11-cyclization. Although each enzyme may only follow one cyclization route to form (−)-germacrene D, to date, this route remains unelucidated [77]. Third, differences are often observed between the product profiles of STSs encoded by homologous genes from the same or related species in the same clade. For example, PcSTS03 and PcSTS04 from P. chrysosporium are the closest on the evolutionary tree (Figure 3a); however, PcSTS03 predominantly produces epicubenol, whereas PcSTS04 synthesizes Δ-cadinene as the main product [23]. This may be because some STSs are promiscuous enzymes that may convert the substrate FPP into various side products via cascade reactions of hydroxylation, elimination, cyclization, and rearrangement [78,79]. Furthermore, a single substitution of an amino acid residue may significantly alter the product profiles of STSs [26,55].
Many STSs have been characterized in plants and fungi; however, information on bacterial STSs is scarce. Typical STSs comprise two conserved metal-binding motifs. The first conserved motif is the aspartate-rich region. In a previous study, the canonical form of the aspartate-rich region DDxx(D/E) was identified in 247 of 249 spermatophyte enzymes in plants [77]. However, in our database, the aspartate-rich regions of fungal STSs had various sequences (D(D/E/N)xx(D/E)). The DExxD sequence was often observed in an appropriate position in basidiomycetous STSs. The second conserved motif is called the NSE/DTE motif. The consensus sequence NDxxSxxxE was identified in 161 of the 172 fungal STSs; however, the NSE/DTE motif of plant spermatophyte STSs has various sequences ((N/D)Dxx(S/T/G)xxxE) [57]. In addition to the two conserved motifs, other characteristic conserved motifs, including DxDTT, DDxDTT, and QDxxDxxxD, are present in fungal STS sequences.
In a previous study, 30 sesquiterpenes were isolated from the solid and liquid cultures of F. velutipes, including β-cadinene and α-muurolene [46]. Furthermore, six oxygenated cuprenene derivatives were isolated from a solid culture of F. velutipes growing on cooked rice [80]. In our study, we found that FvSTS03 of F. velutipes can produce small amounts of α-muurolene and β-cadinene and that FvSTS08 of F. velutipes can produce cuprenene as a minor product. Other products such as β-barbatene and Δ-cadinol identified in this study have not been discovered from F. velutipes. This may be because the contents of these products are very low in vivo or because these products are only released under specific stress conditions.

5. Conclusions

In the present study, we collected the information of mushroom STSs with experimentally identified functions and constructed a phylogenetic tree of mushroom functional STSs based on the amino acid sequences. The catalytic products, and conserved domains and motifs of these STSs were analyzed and discussed to explore the sequence–structure–function relationships. Then, our database was applied to predict 12 putative FvSTS genes from F. velutipes and 3 FvSTS genes were experimentally verified. Finally, the product titer of 78.8 mg/L β-barbatene in S. cerevisiae was achieved through expressing FvSTS08. The approach can also be valuable for exploring other new STSs and sesquiterpenes from mushroom-forming fungi in nature.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof9101017/s1, Table S1: Overview of plasmids used in this study; Table S2: Overview of strains used in this study; Table S3: Primers used in this study; Table S4: The coding sequences of yeast codon-optimized genes; Table S5: Information and bioinformatic analysis of STSs cloned from mushroom-forming fungi; Table S6: Predicted sequence details for putative F. velutipes STSs; Figure S1: Scheme for the biosynthesis pathway of β-barbatene in S. cerevisiae; Figure S2. Preparation of DNA donor expression box of genes to be knock out; File S1: Amino acid sequence information of 172 STSs.

Author Contributions

J.Q., S.W. and R.C. conceived and designed the experiments; S.W. and R.C. performed most of the experiments; L.Y., C.Z. and D.L. provided assistance; and J.Q., S.W. and R.C. wrote the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Shaoxing “MingShiZhiXiang” Meritocrat Project (CXCQ2402376) and National Key Research and Development Project of China (2020YFA0907900).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic analysis of the functional STSs in mushroom-forming fungi. Red lines represent clade I STSs, green lines represent clade II STSs, purple lines represent clade III STSs, and blue lines represent clade IV STSs.
Figure 1. Phylogenetic analysis of the functional STSs in mushroom-forming fungi. Red lines represent clade I STSs, green lines represent clade II STSs, purple lines represent clade III STSs, and blue lines represent clade IV STSs.
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Figure 2. Reaction mechanisms underlying sesquiterpene production starting from FPP in basidiomycetes.
Figure 2. Reaction mechanisms underlying sesquiterpene production starting from FPP in basidiomycetes.
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Figure 3. The phylogeny of STSs in each clade. (a) Clade I; (b) Clade II; (c) Clade III; (d) Clade IV.
Figure 3. The phylogeny of STSs in each clade. (a) Clade I; (b) Clade II; (c) Clade III; (d) Clade IV.
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Figure 4. The phylogeny of candidate STSs in F. velutipes.
Figure 4. The phylogeny of candidate STSs in F. velutipes.
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Figure 5. GC–MS analysis of the assay products of FvSTSs in engineered yeast: (a) FvSTS03 and (b) FvSTS08. The produced sesquiterpenes were putatively annotated as 1, γ-muurolene; 2, α-muurolene; 3, β-cadinene; 4, Δ-cadinol; 5, α-cadinol; 6, α-barbatene; 7, dauca-4(11),8-diene; 8, β-barbatene; and 9, α-cuprenene.
Figure 5. GC–MS analysis of the assay products of FvSTSs in engineered yeast: (a) FvSTS03 and (b) FvSTS08. The produced sesquiterpenes were putatively annotated as 1, γ-muurolene; 2, α-muurolene; 3, β-cadinene; 4, Δ-cadinol; 5, α-cadinol; 6, α-barbatene; 7, dauca-4(11),8-diene; 8, β-barbatene; and 9, α-cuprenene.
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Figure 6. Heterologous production of β-barbatene in S. cerevisiae and gas chromatograms of the WSL01-FvSTS08 strain under different acid treatment conditions. (a) β-Barbatene production; (b) 3 M HCl treatment; (c) 3 M H2SO4 treatment; and (d) 3 M HNO3 treatment. The produced sesquiterpenes were putatively annotated as 6, α-barbatene; 7, dauca-4(11),8-diene; 8, β-barbatene; and 9, α-cuprenene. The value is the mean of three independent experiments. (** represents p < 0.01).
Figure 6. Heterologous production of β-barbatene in S. cerevisiae and gas chromatograms of the WSL01-FvSTS08 strain under different acid treatment conditions. (a) β-Barbatene production; (b) 3 M HCl treatment; (c) 3 M H2SO4 treatment; and (d) 3 M HNO3 treatment. The produced sesquiterpenes were putatively annotated as 6, α-barbatene; 7, dauca-4(11),8-diene; 8, β-barbatene; and 9, α-cuprenene. The value is the mean of three independent experiments. (** represents p < 0.01).
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Table 1. Sesquiterpene synthases cloned in mushroom-forming fungi to date.
Table 1. Sesquiterpene synthases cloned in mushroom-forming fungi to date.
SpeciesGene NameGenBank or JGI Protein IDProductsCladeReference
Lactarius
deliciosus		LdSTS1KAH9077233.11,10-di-epi-cubenolII[25]
LdSTS2KAH9033224.1NDIII
LdSTS3KAH9035467.1myrcene, trans-β-ocimene, etc.II
LdSTS6KAH9079282.1α-muurolene, γ-gurjunene, α-selinene, etc.I
LdSTS7KAH9033225.1aristoleneIII
LdSTS8KAH9071064.1NDIII
LdSTS10KAH9071064.1NDIII
LdSTS11KAH9053987.1aristoleneIII
LdSTS12KAH9033225.1NDIII
LdSTS14KAH9033227.1NDIII
Coprinus
cinereus		Cop1XP_001832573.1germacrene A; Δ-cadinene; α-muurolene;
germacrene D		I[38]
Cop2XP_001836556.1germacrene A; Δ-cadinene; α-muuroleneI
Cop3XP_001832925.1α-muurolene; germacrene A; γ-muurolene;
germacrene D; Δ-cadinene; α-copaene		I
Cop4XP_001836356.1Δ-cadinene; β-copaene; β-cubebene; sativene;
germacrene D; cubebol		II
Cop5XP_001834007.1NDIII
Cop6XP_001832549.1α-cupreneneIV
Omphalotus
olearius		Omp1JGI ID: 1311α-muuroleneI[31]
Omp2not availableNDI
Omp3JGI ID: 4636germacrene A; α-muurolene; elina-4,7-diene;
Δ-cadinene		I
Omp4JGI ID: 1447Δ-cadineneII
Omp5aJGI ID: 2392γ-cadinene; epi-zonarene; germacrene AII
Omp5bJGI ID: 2393γ-cadinene; germacrene AII
Omp6JGI ID: 4774Δ6-protoilludeneIII
Omp7JGI ID: 2271Δ6-protoilludene; pentaleneneIII
Omp8not availableNDIV
Omp9JGI ID: 3258α-barbatene; β-barbateneIV
Omp10JGI ID: 3981(E)-dauca-4(11),8-diene; dauceneIV
Stereum
hirsutum		ShSTS1JGI ID: 159379β-barbatene; α-barbateneIV[39]
ShSTS11JGI ID: 128017Δ-cadinene; α-cubebeneII
ShSTS18JGI ID: 25180Δ6-protoilludeneIII
ShSTS15JGI ID: 64702Δ6-protoilludeneIII
ShSTS16JGI ID: 73029Δ6-protoilludeneIII
ShSTS3JGI ID: 122776α-farnesene; β-farneseneIV
ShSTS4JGI ID: 52743hirsuteneIV
ShSTS5JGI ID: 161672γ-cadineneIV
ShSTS7JGI ID: 167646Δ-cadineneI
ShSTS8JGI ID: 1463901-epi-cubenol; α-cubebeneII
ShSTS10JGI ID: 111121Δ-cadinene; germacrene DII
ShSTS12JGI ID: 111127α-cubebene; β-cubebeneII
ShSTS13JGI ID: 50042β-caryophylleneIII
ShSTS17JGI ID: 69906Δ6-protoilludeneIII
HS-HMGSnot availablehirsutene; β-caryophylleneIV[40]
Clitopillus pseudo-pinsitusCpSTS1BBH51498.1sterpureneIII[41]
CpSTS2BBH51499.1Δ-cadinene; α-cubebeneII
CpSTS3BBH51500.1Δ-cadinol; α-muurolene; γ-muurolene;
unknown sesquiterpene		I
CpSTS4BBH51501.1Δ6-protoilludeneIII
CpSTS5BBH51502.1α-muurolene; γ-muuroleneI
CpSTS6BBH51503.1pentaleneneIII
CpSTS7BBH51504.1α-farneseneIII
CpSTS8BBH51505.1alloaromadendrene; unknown sesquiterpeneII
CpSTS9BBH51506.1virifloridol; ledeneII
CpSTS11BBH51508.19-alloaromadendreneII
CpSTS12BBH51509.1virifloridol; β-elemene; ledeneII
CpSTS13BBH51510.1ledene; unknown sesquiterpeneII
CpSTS14BBH51511.1β-elemene; β-farnesene; α-farneseneIV
CpSTS15BBH51512.1NDIV
CpSTS16BBH51513.1aristolene; unknown sesquiterpeneII
CpSTS17BBH51514.1β-caryophylleneIV
CpSTS18BBH51515.1γ-cadineneIV
Antrodia
cinnamomea		AcTPS4JGI ID: 40411zonarene; α-cubebene; sibirene; γ-cadineneII[42]
AcTPS5JGI ID: 40579T-cadinol; γ-cadineneI
AcTPS7JGI ID: 36944nerolidol; α-farnesol\
AcTPS9JGI ID: 477061-epi-cubenol; sibirene; cubebol; α-cubebene;
α-farnesol; γ-muurolene		II
Tps1AKAI0942648.1(+)-(S,Z)-α-bisaboleneIV[43]
Tps2AKAI0928020.1
AncAACg006372(R)-trans-γ-monocyclofarnesolIV[44]
AncCACg006375drimane-type sesquiterpene (+)-albicanol
Boreostereum vibransBvCSKU668561.1Δ-cadinol; α-muurolene; γ-muuroleneI[45]
Lignosus
rhinocerotis		GME3634KX281943α-cadinol; germacrene D-4-olI[46]
GME3638KX281944torreyol; germacrene D-4-ol; β-cubebeneIV
GME9210KX2819451,3,4,5,6,7-hexahydro-2,5,5-trimethyl-2H-2,4a-ethanonaphthaleneIII
Agrocybe
aegerita		Agr1MN146024Δ-cadinene; α-cadinol; Δ-cadinol; α-muuroleneI[17]
Agr2MN146025viridifloreneII
Agr3MN146026Δ-cadinol; Δ-cadinene; α-muurolene; γ-muuroleneI
Agr4MN146027Δ-cadinene; epicubenol; cadina-1(6),4-diene;
β- myrcene		II
Agr5MN146028viridiflorol; viridifloreneII
Agr6MN146029Δ6-protoilludeneIII
Agr7MN146030Δ6-protoilludeneIII
Agr8MN146031γ-muurolene; β-cadinene; Δ-cadinolIII
Agr9MN146032γ-muurolene; Δ-cadinene; unknown sesquiterpenolIII
Agr10MN146033NDIII
Agr11MN146034NDIII
Coniophora
puteana		Copu1XP_007772164.1NDII[47]
Copu2XP_007771895.1β-copaene; germacrene D; cubebol; germacrene D-4-olII
Copu3XP_007765978.1cubebol; germacrene D-4-ol; Δ-cadineneII
Copu5XP_007765330.1Δ-cadinol; Δ-cadinene; cubebol; α-cadinolIV[30]
Copu9XP_007765560.1I
Piloderma
croceum		Pilcr_825684JGI ID: 825684γ-cadinene; viridiflorene; β-elemeneII[17]
Galerina
marginata		Galma_104215JGI ID: 104215β-gurjuneneII
Sphaerobolus stellatusSphst_47084JGI ID: 47084viridiflorol; viridifloreneII
Dendrothele bisporaDenbi1_816208JGI ID: 816208viridiflorol; viridifloreneII
Denbi1_659367JGI ID: 659367Δ6-protoilludeneIII
Heterobasidion annosumHEtan2_454193XP_009550163.1Δ6-protoilludeneIII
Hypholoma sublateritiumHypsu1_138665A0A0D2L718.1Δ6-protoilludeneIII
Armillaria
gallica		Pro1MT277003.1Δ6-protoilludeneIII[48]
Hypholoma
fasciculare		Hfas94aMK287936.1α-humulene; β-caryophylleneIII[49]
Hfas94bMK287937.1α-humulene; β-caryophylleneIV
Hfas255not availableND\
Hfas344MK287938.1unknown sesquiterpeneIII
Cerrena
unicolor		Cun3817JGI ID: 3817γ-cadineneIV[50]
Cun5155JGI ID: 5155aromadendreneIII
Cun3157JGI ID: 3157β-cubebene; germacrene D; epicubenol; Δ-cadineneII
Cun3158JGI ID: 3158Δ-cadinene; germacrene D; β-cubebene; γ-amorpheneII
Cun0773JGI ID: 0773germacrene DI
Cun7050JGI ID: 7050Δ-cadinolI
Cun0716JGI ID: 0716Δ-cadinol; α-muuroleneI
Cun0759JGI ID: 0759α-muuroleneI
Cun3574JGI ID: 3574α-copaeneI
Cun9106JGI ID: 9106unknown sesquiterpeneIV
Postia
placenta		PpSTS01XP_024337827.1α-muurolene; Δ-cadinene; β-elemeneI[18]
PpSTS03A0A348B781.1γ-cadinene; α-cadinene; Δ-cadinene; β-elemeneI
PpSTS06A0A348B782.1α-gurjunene; bicycloelemene; bicyclogermacreneI
PpSTS08A0A348B784.1Δ6-protoilludeneIII
PpSTS09A0A348B785.1unknown sesquiterpeneIII
PpSTS10XP_024334632.1Δ-cadinene; β-copaene; sativene; sesquisabineneII
PpSTS14A0A348B788.1pentalenene; caryophylleneIII
PpSTS29A0A348B794.1unknown sesquiterpeneIV
Fomitopsis pinicolaFompi1JGI ID: 84944α-cupreneneIV[31]
Phanerodontia chrysosporiumPcSTS01BCX55496.1α-muurolene; Δ-cadinene; γ-muurolene; α-muurololI[23]
PcSTS02BCX55497.1Δ-cadinene; β-copaene; β-farnesene; cadina-1(6),4-dieneII
PcSTS03BCX55498.1epicubenolII
PcSTS04BCX55499.1Δ-cadinene; β-farnesene; β-copaene; epicubenolII
PcSTS06BCX55500.1β-barbatene; α-barbateneIV
PcSTS08BCX55502.1(E)-α-bisaboleneIV
PcSTS11BCX55504.1α-santaleneIV
Steccherinum ochraceumA8411not availablehirsuteneIV[19]
Ganoderma sinensisGsSTS43PIL26225γ-cadineneIV[24]
GsSTS45aUDP19925NDIV
GsSTS45bUDP19925γ-cadineneIV
GsSTS26MT584777.1gleenol; di-epi-1,10-cubenol; Ʈ-muurololIII[51]
GsSTS27OP094045III
GS02363PIL35634α-cadinol; Δ-cadinene; γ-cadinene; T-cadinolI[52]
GS14272PIL24516α-muuroleneI[53]
GS11330not availableα-cupreneneIV
Ganoderma
lucidum		GL26009not availableα-muurolene; γ-muuroleneI[54]
GLSTS6UDP19923γ-cadineneIV[24]
Termitomyces sp. J132STC4KNZ72568.1(+)-intermedeol; α-selinene; β-selineneIV[55]
STC9KAG5341349γ-cadineneIV
STC15KNZ74377.1(+)-germacrene D-4-ol; γ-cadinene; Δ-cadinene;
α-cadinene; β-elemene		I
Agaricus
bisporus		AbSTS05LC712879cadina-1,4-diene; cadina-1(6),4-diene; Δ-cadinene;
zonarene; epicubenol; cadin-4-en-10-ol		II[56]
AbSTS07LC712880Δ-cadinene; epizonareneIV
AbSTS09LC712881(Z)-α-bisaboleneIV
Auriscalpium vulgareAvSTS01LC712882unknown sesquiterpeneIII
AvSTS03LC712883Δ6-protoilludeneIII
AvSTS06LC712885(E)-nerolidolIII
AvSTS07LC712886(E)-nerolidolIII
AvSTS09LC712887cadina-1,4-diene; cadina-1(6),4-diene; Δ-cadinene;
β-copaene; zonarene; epicubenol; cadin-4-en-10-ol		II
Lepista nudaLnSTS01LC712891Δ6-protoilludeneIII
LnSTS02LC712892Δ6-protoilludeneIII
LnSTS04LC719126pleostene; isobazzaneneIII
LnSTS09LC712895cadina-1,4-diene; cadina-1(6),4-diene; Δ-cadinene;
β-copaene; zonarene; epicubenol; cadin-4-en-10-ol		II
LnSTS19LC712898(E)-nerolidolIV
LnSTS20LC712899β-barbateneIV
LnSTS25LC712901unknown sesquiterpeneIV
LnSTS27LC712902acora-3(7),14-dieneIV
Pleurotus
ostreatus		PoSTS01LC712903α-muurolene; isobazzanene; Δ-cadinene; α-muurololI
PoSTS02LC712904α-muurolene; Δ-cadinene; α-muurolol; zonareneI
PoSTS03LC712905α-muurolene; Δ-cadinene; isobazzanene; α-muurololI
PoSTS05LC712906cadina-1,4-diene; cadina-1(6),4-diene; Δ-cadinene;
β-farnesene; zonarene; epicubenol; cadin-4-en-10-ol		II
PoSTS06LC712907pleosteneIII
PoSTS11LC712908(E)-nerolidolIV
PoSTS16LC712909α-cupreneneIV
Trametes
versicolor		TvSTS01LC712910Δ-cadinene; cadin-4-en-10-ol; Ʈ-muurololI
TvSTS05LC712912cadina-1,4-diene; cadina-1(6),4-diene; Δ-cadinene;
β-copaene; azoarene; epicubenol; cadin-4-en-10-ol		II
TvSTS06LC712913cadina-1,4-diene; cadina-1(6),4-diene; Δ-cadinene;
β-copaene; β-farnesene; zonarene; epicubenol		III
TvSTS07LC712914Δ6-protoilludeneIII
TvSTS12LC712917γ-cadineneIV
TvSTS14LC712919β-barbatene; α-barbateneIV
TvSTS16LC712920dauca-4(11),8-diene; isobazzaneneIV
Irpex lacteusIIISJGI ID: Il4946iltremulanol AIII[57]
Serendipita
indica		SiTPSJGI ID: 77541viridiflorolIII[58]
Termitomyces sp. T153DS3not availableunknown sesquiterpeneIV[59]
Laccaria
bicolor		LbSTS4aXP_001887869.1(E)-nerolidolII[60]
LbSTS6XP_001885710.1α-cuprenene; α-cupareneIV
ND: no product detected; “\”: not analyzed in this study.
Table 2. Conserved motifs and domains of STSs.
Table 2. Conserved motifs and domains of STSs.
Sequence NameMotif I
D(D/E/N)xx(D/E)		Motif II
(NDxxSxxxE)		Domains (Pfam ID)
LdSTS1DELSDNDLYSYNMEPF19086
LdSTS2DEYTDQDLYSYNNEPF19086
LdSTS3DEVSDNDVYSYNMEPF19086
LdSTS6DNVSDNDIFSYNVEPF19086; PF03936; PF06330
LdSTS7DEYTDNDLYSYNIEPF19086
LdSTS8DEFSDNDIASYNVEPF19086
LdSTS10DEFSENDIASYNVEPF19086
LdSTS11DEYTDNDLYSYNIEPF19086
LdSTS12DEYTDNDLYSYNVEPF19086
LdSTS14DEFTDNDMYSYNIEPF19086
Cop1DNLSDNDIFSFNVEPF19086
Cop2DDWLDNDIFSFNREPF19086
Cop3DNISDNDIFSYNVEPF19086; PF03936; PF06330; PF19035
Cop4DEISDNDVYSYDMEPF19086; PF03936
Cop5DYFFDNDAYSWNVEPF19086; PF03936
Cop6DDAFQNDLLSFYKEPF06330
Omp1DNLTDNDIYSFNIEPF19086
Omp2DNLSDNDIFSYNVEPF19086
Omp3DEVSDNDIFSYNVEPF19086
Omp4DEVSDNDVYSYNKEPF19086
Omp5aDELSDNDVYSYNVEPF19086
Omp5bDEVSDNDVYSYNVEPF19086
Omp6DEYSDNDLCSYNVEPF19086
Omp7DEYSDNDTASYNYEPF19086
Omp8DDVFENDIMSFYKEPF06330
Omp9DDVFENDVLSFYKEPF06330
Omp10DDIFPNDVLSFYKEPF06330
ShSTS1DDSLENDLMSFYKEPF06330
ShSTS11DEISDNDVYSYNVEPF19086
ShSTS18DEYSDQDICSYNVEPF19086
ShSTS15DEHSDNDIVSYNIEPF19086
ShSTS16DEYSDNDIVSYNLEPF19086
ShSTS3DDWVDNEASSYVKEPF19086
ShSTS4DDYIDNDFFSYLKEPF19086
ShSTS5DDLSDNDLCSFNKEPF19086
ShSTS7DDWTDNDIFSYNVEPF19086
ShSTS8DEISDNDIYSYDMEPF19086
ShSTS10DEISDNDVYSYKVEPF19086
ShSTS12DEISDQDVYSYSMEPF19086
ShSTS13DDILDNDTFSYRREPF19086
ShSTS17DEHSDSDIVSWNLEPF19086
HS-HMGSDDYIDNDFFSYLKEPF19086; PF01154
CpSTS1DEYTDNDMCSYKKEPF19086
CpSTS2DELSDNDVYSYDMEPF19086
CpSTS3DNISDNDIFSYNVEPF19086
CpSTS4DEYTDNDLCSFRNEPF19086
CpSTS5DNLSDNDIFSYNVEPF19086
CpSTS6DEYSDNDLYSYNVEPF19086
CpSTS7DEITENDVFSFKVEPF19086
CpSTS8DEYTDNDVYSYNMEPF19086
CpSTS9DEYTDNDLFSYNMEPF19086
CpSTS11DEATDNDIHSYNMEPF19086
CpSTS12DEYTDNDLYSYNMEPF19086
CpSTS13DETTDNDIQSYNMEPF19086
CpSTS14DDYILNDIYSYKVEPF19086
CpSTS15DDLMENDLFSYRKEPF19086
CpSTS16DESSDNDIHSYNMEPF19086
CpSTS17DDIIENDLFSYRVEPF19086
CpSTS18DDLSDNDLCSFNKEPF19086; PF03936; PF06330;
AcTPS4DEVSDNDVYSYNMEPF19086
AcTPS5DDWTDNDVLSYNAEPF19086; PF03936
AcTPS9DEISDNDLYSYNMEPF19086
Tps1ADIEGDQDFPDIEFDPF01040
Tps2ADVAGDQDFPDIEFDPF01040
AncADDRIEDDFTDDPF13419
AncCDDKIEDDFTDDPF13419
BvCSDNISDNDVFSYNVEPF19086; PF03936
GME3634DDWTDNDVLSYNAEPF19086
GME3638DDWSDNDLFSYNVEna
GME9210DEYSDNDIVSYNVEPF19086
Agr1DNLSDNDIFSYSVEPF19086; PF03936
Agr2DEVTDNDLYSYNMEPF19086
Agr3DNISDNDIFSYNVEPF19086; PF03936
Agr4DEVSDNDVYSYDMEPF19086
Agr5DEYTDNDLVSYNMEPF19086
Agr6DEHTDNDLCSYNVEPF19086
Agr7DEWSDNDLCSYNVEPF19086
Agr8DEYTDNDMHSYVREPF19086; PF03936
Agr9DEYTDNDIDSYAMEPF19086
Agr10DECADnana
Agr11DEYTDnaPF19086
Copu1DELTDNDVYSYNMEPF19086
Copu2DDLTDNDVFSYNREPF19086
Copu3DELSDNDVYSYNMEPF19086
Copu5DDWSDNDVFSYNKEPF19086; PF03936
Copu9DDWLDNDIFSYNKEPF19086
Pilcr_825684DELTDNDLFSYNREPF19086
Galma_104215DEFTDNDLFSYDMEPF19086
Sphst_47084DEYTDNDLFSYNSPF19086
Denbi1_816208DEFTDNDLFSYNMEPF19086
Denbi1_659367DEHSDNDLCSYNVEPF19086
Hetan2_454193DEYSDNDIASYNLEPF19086
Hypsu1_138665DEHTDNDLCSYKVEPF19086; PF03936
Pro1DEYSDNDVVSYNLEPF19086; PF03936
Hfas94aDEYTDNDMHSYGLEPF19086
Hfas94bDEDLDNDLISYTKEPF19086
Hfas344DEYTDNDMHSYALEPF19086
Cun3817DDLSDNDLCSFNKEPF19086; PF03936
Cun5155DEHSDNDLFSYNVEPF19086
Cun3157DEISDNDIYSYNMEPF19086
Cun3158DEVSDNDVYSYNMEPF19086
Cun0773DDWSDNDILSYSKEPF19086
Cun7050DNISDNDIFSYNVEPF19086; PF03936
Cun0716DDWSDNDIFSFNVEPF19086
Cun0759DDWSDNDIFSYNKEPF19086
Cun3574DDWTDNDIFSYNKEPF19086
Cun9106NDDYEnaPF06148
PpSTS01DNISDNDIFSYNVEPF19086
PpSTS03DDWSDNDILSYNREPF19086; PF03936
PpSTS06DDITDNDIYSFNNEPF19086
PpSTS08DEYTDNDLVSYNREPF19086
PpSTS09DEYSDNDMLSWNVEPF19086
PpSTS10DEVSDNDVYSYNMEPF19086
PpSTS14DEYTDNDIASYNKEPF19086
PpSTS29DEPDINDILSFYKEPF06330
Fompi1DDPDINDILSFYKEPF06330
PcSTS01DNISDNDIFSYNVEPF19086; PF03936
PcSTS02DEVSDNDVYSYKMEPF19086
PcSTS03DEISDNDVYSYDMEPF19086
PcSTS04DEISDNDVYSYDMEPF19086
PcSTS06DDFEINDLLSFYKEPF06330
PcSTS08DDEAINDILSFYKEPF06330
PcSTS11DDCEINDIYSFHKEPF06330
A8411DDYIDNDLFSYAKEPF19086
GsSTS43DDLSDNDLCSFNKEPF19086; PF03936
GsSTS45aDDLSDNDLCSFNKEPF19086
GsSTS45bDDLSDNDLCSFNKEPF19086
GsSTS26DEYTDNDVASYNREPF19086
GsSTS27DEYTDNDVASYNREPF19086
GS02363DDWTDNDVLSYNAEPF19086; PF03936
GS14272DNISDNDIFSYNVEPF19086
GS11330DDLGENDILSFYKEPF06330
GL26009DDWTDNDVLSYNAEPF19086; PF03936
GLSTS6DDLSDNDLCSFNKEPF19086; PF03936
STC4DRLTDNDLYSYKKEPF19086
STC9DDLSDNDLCSFNKEPF19086; PF03936
STC15DNLSDNDIFSYNVEPF19086; PF03936
AbSTS05DEISDNDVYSYNVEPF19086
AbSTS07DDNFDNDITSFYKEPF06330
AbSTS09DDNYDNDIASFYKEPF06330
AvSTS01DEYTDNDLCSYNKEPF19086; PF03936
AvSTS03DEYSDNDIASYNLEPF19086; PF03936
AvSTS06DEFTDNDTYSYNIEPF19086
AvSTS07DEFTDNDTYSYNIEPF19086
AvSTS09DEVSDNDVYSYNMEPF19086
LnSTS01DEYSDNDLCSYNVEPF19086
LnSTS02DEHSDNDLCSYNVEPF19086
LnSTS04DEYSDNDVYSYNKEPF19086; PF03936
LnSTS09DELSDNDVYSYDMEPF19086
LnSTS19DDVDSNDLLSYHKEPF06330
LnSTS20DDMSSNDILSFHKEPF06330
LnSTS25DDTSPNDLMSFPKEPF19086; PF06330
LnSTS27DDKYFNDIMSFYKEPF06330
PoSTS01DNLSDNDIFSYNVEPF19086; PF03936
PoSTS02DDWLDNDLFSYNVEPF19086; PF03936
PoSTS03DNISDNDIFSYNVEPF19086; PF03936
PoSTS05DEVSDNDVYSYNMEPF19086
PoSTS06DEFSDNDVYSWNVEPF19086
PoSTS11EEITENDIYSYKKEPF19086
PoSTS16DDISSNDVLSFYKEPF06330
TvSTS01DNICDNDIFSYNVEPF19086
TvSTS05DEISDNDLYSYNMEPF19086
TvSTS06DEVSDNDVYSYNMEPF19086
TvSTS07DEQTDNDLLSYRKEPF19086; PF03936
TvSTS12DDLSDNDLCSFNKEPF19086; PF03936
TvSTS14DDLGGNDILSFYKEPF06330
TvSTS16DDLPGNDLLSFYKEPF06330
IIISDEYTDNDIASYNKEPF19086
SiTPSDDLMDNDVYSFDNEPF19086; PF03936
DS3DDKLEDLDTTPF13419
LbSTS4aDDITDNDVYSYGKEPF19086
LbSTS6DDVFQNDVLSFYKEPF06330
na: missing data; DDVFQ: amino acids replacements at the conserved motif active sites are underlined and in bold. DDFTDD: red color sequence indicates substitution of Motif II.
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Wang, S.; Chen, R.; Yuan, L.; Zhang, C.; Liang, D.; Qiao, J. Molecular and Functional Analyses of Characterized Sesquiterpene Synthases in Mushroom-Forming Fungi. J. Fungi 2023, 9, 1017. https://doi.org/10.3390/jof9101017

AMA Style

Wang S, Chen R, Yuan L, Zhang C, Liang D, Qiao J. Molecular and Functional Analyses of Characterized Sesquiterpene Synthases in Mushroom-Forming Fungi. Journal of Fungi. 2023; 9(10):1017. https://doi.org/10.3390/jof9101017

Chicago/Turabian Style

Wang, Shengli, Ruiqi Chen, Lin Yuan, Chenyang Zhang, Dongmei Liang, and Jianjun Qiao. 2023. "Molecular and Functional Analyses of Characterized Sesquiterpene Synthases in Mushroom-Forming Fungi" Journal of Fungi 9, no. 10: 1017. https://doi.org/10.3390/jof9101017

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