Biosynthesis of Sesquiterpenes in Basidiomycetes: A Review

Abstract

Sesquiterpenes are common small-molecule natural products with a wide range of promising applications and are biosynthesized by sesquiterpene synthase (STS). Basidiomycetes are valuable and important biological resources. To date, hundreds of related sesquiterpenoids have been discovered in basidiomycetes, and the biosynthetic pathways of some of these compounds have been elucidated. This review summarizes 122 STSs and 2 fusion enzymes STSs identified from 26 species of basidiomycetes over the past 20 years. The biological functions of enzymes and compound structures are described, and related research is discussed.

1. Introduction

Fungi are widely distributed in various ecosystems of the Earth. Based on high-throughput sequencing methods, approximately 5.1 million species of fungi exist in nature, but only approximately 100,000 species have been discovered [1]. Basidiomycota (commonly known as basidiomycetes) is one of the major phyla of the fungal kingdom, with more than 31,000 species identified [2]. Basidiomycetes are divided into three subphyla: rusts (Puccinomycotina), smuts (Ustilagomycotina), and mushrooms (Agaricomycotina), with several taxonomic ranks below them. Sesquiterpenes are among the most structurally diverse natural products and have many applications in various industries. They contain C15 polymers composed of three isoprene units and derivatives with diverse chemical skeletons. Fungi are rich in sesquiterpenoid natural products, many of which have good biological activities, including antibacterial, antifungal, anti-inflammatory, antitumor, vascular-relaxing, immunosuppressant, and cytotoxic activities. They can be used as lead compounds for new drugs [3,4,5,6,7,8,9,10]; especially in basidiomycetes, sesquiterpenes have various pharmacological activities [11].

Basidiomycetes often produce large fruiting bodies to disperse spores; however, these fruiting bodies are constantly threatened by other organisms that feed on them [12]. As a result, basidiomycetes have evolved a number of protective strategies against threats from other organisms, one of which is the production of toxins. Basidiomycetes produce toxic sesquiterpenes, mainly as protoilludane skeleton, to protect against predators [11]. In addition, basidiomycetes often form symbiotic relationships with roots and their hosts, providing plant hormones [13,14]. For example, basidiomycetes in the genus Lactarius produce modified lactarane and protoilludane-derived sesquiterpenes that promote plant growth [15,16,17]. Sesquiterpenes isolated from basidiomycetes also exhibit pharmacological activity. For instance, hydroxymethylacylfulvene (HMAF) is a semisynthetic antitumor agent based on the naturally occurring illudin S from the mushroom Omphalotus olearius [18]. It is currently in human clinical trials because of its anti-cancer properties [19,20]. Phellinignin A and 11,12-epoxy-12β-hydroxy-1-tremulen-5-one isolated from the genus Phellinus igniarius showed high cytotoxicity to HL-60, SMMC-7721, and SW480 cancer cells [21]. 10β,12-Dihydroxy-tremulene isolated from Phellinus igniarius showed good vasodilatory activity in the experiment [9].

So far, approximately one thousand sesquiterpenoids have been reportedly obtained from basidiomycetes (Supplementary Table S1) [22,23]. Drimanes, protoilludanes, illudanes, hirstutanes, cadinanes, and tremulanes sesquiterpene skeletons are the main skeleton types of sesquiterpenoids in basidiomycetes, comprising approximately 60% of the total population (Figure 1, Supplementary Table S2. There are 79 genera of basidiomycetes that produce sesquiterpenes, and Lactarius, Xylaria, Armillaria, Phellinus, Granulobasidium, and Conocybe are the main sources (Supplementary Table S1). According to the reported genomic data, the average number of sesquiterpene genes per strain in basidiomycetes is 12, which is much higher than the average 3.5 sesquiterpene genes in ascomycetes [24]. These results suggest that basidiomycetes produce more sesquiterpenes. However, most of the compounds produced by these potentially functional genes are unknown and require further clarification.

Figure 1.

Basidiomycota sesquiterpenes classified by a skeleton.

2. Cyclization Mode of STSs

The sesquiterpene biosynthetic pathway is divided into two steps [25]. The first step is a coupling reaction that connects isoprene precursors, dimethylallyl dipyrophosphate (DMAPP) and isoprenyl dipyrophosphate (IPP), from geranyl pyrophosphate (GPP) in a head-to-tail manner, and then condenses with another molecule of IPP to generate farnesyl pyrophosphate (FPP), which is a sesquiterpenoid biosynthetic precursor and substrate for STS [26]. As the second step, FPP generates different sesquiterpene carbon skeletons through irregular coupling reactions. Typical STS contains conserved D(D/E)XXD and NSE/DTE motifs, and these amino acid residues play important roles in coordinating the stabilization of divalent metal ions at the active site for defocusing the catalytic reaction of phosphoric acid. Cyclization is initiated by the metal-ion-induced departure of inorganic pyrophosphate (PPi) to form allyl cations, facilitating the structural shift and catalyzing cyclization closure [27,28]. For cyclic sesquiterpenes, this step can be further divided into two. FPP undergoes one or more cyclizations to form intermediates, which are then converted to sesquiterpene skeletal end-products under the action of STS. The reaction mechanism is divided into four categories [29,30,31] (Figure 2 and Figure 3)—Clade I: After (2E,6E)-FPP is deionized by pyrophosphate, it electrophilically attacks the double bond at the other end and forms a 10-membered ring carbon-positive intermediate E, E-germacradienyl cation through a 1,10 cyclization reaction; Clade II: FPP is first ionized and isomerized to form (3R)-nerolidyl diphosphate ((3R)-NPP), deionized by pyrophosphate, and electrophilically attacks the double bond to form a 10-membered ring of the carbon-positive intermediate Z, E-germacradienyl cation through 1,10 cyclization; Clade III: (2E,6E)-FPP removes pyrophosphate ionization, electrophilically attacks the double bond at the other end, and undergoes 1,11 cyclization reaction 11-membered ring carbocation intermediate trans-humulyl cation; Clade IV: After (3R)-NPP is deionized by pyrophosphate, it electrophilically attacks the double bond and forms a 6-membered ring carbocation intermediate (6R)-β-bisabolol cation through a 1,6 cyclization reaction.

Figure 2.

Cyclization patterns of sesquiterpenes in basidiomycetes.

Figure 3.

Sesquiterpene biosynthesis by STS of clades I–IV in basidiomycetes. All mentioned STSs have undergone biochemical verification.

Over the past 20 years, 122 STSs and 2 fusion enzymes STSs have been discovered and identified from 26 species of basidiomycetes (Supplementary File S1), which are responsible for the biosynthesis of hundreds of sesquiterpenes in four ways. The various STSs and their catalytic production of sesquiterpenes are summarized and discussed in this review.

3. STSs in Basidiomycota

3.1. Agaricales

Agaricales is the largest mushroom-forming flora, comprising more than 400 genera and 13,000 species [32]. To date, 10 species have been experimentally identified with 58 different STSs.

3.1.1. Macrolepiota albuminosa

Macrolepiota albuminosa (Termitomyces albuminosus) is a special mushroom in China that belongs to the Agaricaceae family. Bioinformatic analysis of the genome revealed the presence of 22 terpene synthases [33], 3 of which (STC4, STC9, and STC15) were identified as STSs and heterologously expressed by Escherichia coli [34]. Using FPP as a precursor, STC4 synthesized intermedeol (1) via C1,10 cyclization, which in turn enabled germacrene D-4-ol (2) synthesization by STC15. Through C1,6 cyclization, γ-cadinene (3) was synthesized by STC9 with NPP as a substrate.

3.1.2. Coprinopsis cinerea

Coprinopsis cinerea belongs to the Psathyrellaceae family. Six STSs (Cop1–6) have been identified in this fungus [29] and heterologously expressed in Saccharomyces cerevisiae and E. coli. With FPP as the precursor, Cop1 and Cop2 synthesized germacrene A (4) by C1,10 cyclization, and Cop3 synthesized α-muurolene (5). Using NPP as the precursor, Cop4 synthesized δ-cadinene (6) by C1,10 cyclization, and Cop6 synthesized α-cuprenene (7) by C1,6 cyclization [35]. Cop5 cannot be functionally expressed in either system.

3.1.3. Taiwanofungus camphoratus

Taiwanofungus camphoratus (Antrodia cinnamomea) belongs to the mushroom family Fomitopsidaceae and is a rare medicinal fungus found in Taiwan, China. A total of 10 terpene synthases (AcTPS1–7, 9–11) have been identified in it and they are heterologously expressed in E.coli, among which three were identified as STSs (AcTPS4, AcTPS5, and AcTPS9) [31]. T-cadinol (8) was synthesized by C1,10 cyclization of AcTPS5 with FPP as a substrate, and cubebol (9) and zonarene (10) were synthesized from AcTPS9 and AcTPS4, respectively, via C1,10 cyclization with NPP as a substrate.

3.1.4. Clitopilus pseudo-pinsitus

Clitopilus pseudo-pinsitus, belonging to the family Entolomataceae, currently has 18 related STSs (CpSTS1–18) recorded [36]. Apart from the lack of conserved CpSTS10 sequences, the remaining 17 were heterologously expressed by Aspergillus oryzae, and CpSTS15 was found to be inactive. The biosynthesis of the remaining STSs can be summarized as follows: Δ⁶-protoilludene (11) was synthesized by CpSTS4 using FPP as a precursor and C1,11 cyclization. Sterpurene (12), pentalenene (13), and α-farnesene (14) were synthesized from CpSTS1, CpSTS6, and CpSTS7, respectively. After C1,10 cyclization, δ-cadinene (6) was synthesized from CpSTS2, aristolene (15) was synthesized from CpSTS16, and alloaromadendrene (16) and 9-alloaromadendrene (17) were synthesized from CpSTS8 and CpSTS11, respectively. CpSTS9 and CpSTS12 synthesized virifloridol (18), and CpSTS13 synthesized ledene (19) with NPP as the substrate. CpSTS3 synthesized α-muurolene (5) and δ-cadinol (20). CpSTS5 synthesized α-muurolene (5) with FPP as the substrate. Through C1,6 cyclization, using NPP as a precursor, CpSTS14 synthesized β-elemene (21) and β-farnesene (22), CpSTS17 synthesized β-caryophyllene (23), and CpSTS18 synthesized γ-cadinene (3).

3.1.5. Hypholoma fasciculare

Hypholoma fasciculare is a clustered fungus belonging to the family Strophariaceae. A total of 17 STSs have been previously identified in their genome using bioinformatic methods [37], of which 4 (Hfas94a, Hfas94b, Hfas255, and Hfas344) were heterologously expressed in A. oryzae. Using FPP as a precursor, Hfas94a and Hfas94b mainly synthesized α-humulene (24) through C1,11 cyclization. Hfas255 did not produce any products, and Hfas344 synthesized an oxidized sesquiterpene with spectral data similar to that of β-caryophyllene.

3.1.6. Hypholoma lateritium

Hypholoma lateritium (Hypholoma sublateritium) belongs to the same genus as H. fasciculare and is widely distributed in China. Only one STS (Hypsu1_138665) has been identified in this mushroom [38]. Using E. coli heterologous expression with FPP as the precursor, Hypsu1_138665 was cyclized by C1,11 cyclization to synthesize Δ⁶-protoilludene (11).

3.1.7. Cyclocybe aegerita

Cyclocybe aegerita (Agrocybe aegerita), also known as pioppino mushroom, is a basidiomycete belonging to the Strophariaceae family. Eleven STSs have been identified, all of which are heterologously expressed in E. coli [38]. Two of these (Agr10 and Agr11) failed to detect the product. Using NPP as the precursor, δ-cadinene (6) was synthesized from Agr1 and Agr4, and viridiflorene (25) was synthesized from Agr2 and Agr5 by C1,10 cyclization. Using FPP as a precursor, Agr3 synthesized α-muurolene (5) by C1,10 cyclization, Agr6 and Agr7 synthesized Δ⁶-protoilludene (11), and Agr8 synthesized γ-muurolene (26) by C1,11 cyclization. Agr9 synthesized an unknown sesquiterpene alcohol.

3.1.8. Armillaria gallica

Armillaria gallica is a saprophytic or parasitic fungus belonging to the Physalacriaceae family. Using bioinformatics analysis of its genome, 20 STSs were predicted [38], but only 1 (Pro1) was heterologously expressed in E. coli [39]. Using FPP as a precursor, Pro1 synthesized Δ⁶-protoilludene (11) via C1,11 cyclization.

3.1.9. Galerina marginata

Galerina marginata is a common poisonous mushroom belonging to the Hymenogastraceae family that contains amino peptides. Only one related STS (Galma_104215) has been identified and is heterologously expressed in E. coli [38]. Using NPP as the precursor, Galma_104215 synthesized β-gurjunene (27) by C1,10 cyclization.

3.1.10. Omphalotus olearius

Omphalotus olearius belongs to the family Omphalotaceae and emits green fluorescence. Ten STSs from this fungus have been identified [30]. E. coli was used for heterologous expression. With FPP as the precursor, Omp1 and Omp3 were used to synthesize α-muurolene (5) by C1,10 cyclization, and Omp5a/b was used to synthesize γ-cadinene (3); Omp6 and Omp7 synthesized Δ⁶-protoilludene (11) by C1,11 cyclization. With NPP as the precursor, Omp4 was used to synthesize δ-cadinene (6) by C1,10 cyclization, Omp9 synthesized α-barbatene (28), and Omp10 mainly guided the synthesis of (E)-dauca-4(11), 8-diene (29) by C1,6 cyclization. Omp8 is a homologue of Omp9/10 that lacks approximately 100 amino acids at its N terminus and was not functional when expressed in E. coli.

3.2. Polyporales

Polyporales contains approximately 1800 species of fungi, representing approximately 1.5% of all known fungal species [40]. At present, 8 species of fungi in this order have been identified to contain 39 different STSs and 1 fusion enzyme.

3.2.1. Lignosus rhinocerus

Lignosus rhinocerus (Lignosus rhinocerotis), also known as tiger milk mushroom, is a macrofungal belonging to the Polyporaceae family. Twelve terpene synthase genes have been found [41], seven of which are actively expressed in the sclerotium. Three STSs (GME3634, GME3638, and GME9210) were heterologously expressed by S. cerevisiae, producing nineteen, eight, and two sesquiterpenes, respectively (29 in total). Using FPP as the precursor, through C1,10 cyclization, GME3634 mainly synthesized α-cadinol (30), and GME3638 mainly synthesized torreyol (31). Using FPP as the precursor, GME9210 mainly synthesized 1,3,4,5,6,7-hexahydro-2,5,5-trimethyl-2H-2,4a-ethanonaphthalene (32) and 1-napthalenol (33).

3.2.2. Cerrena unicolor

Cerrena unicolor belongs to the Polyporaceae family. A total of 14 STSs have been found. Heterologous expression in E. coli was performed [42]. Four of these (Cun5765, Cun6114, Cun7487, and Cun0802) were not produced, and the loss of Cun6114, Cun7487, and Cun5765 activities might be caused by the difficulty in predicting introns [43]. The failure of Cun0802 gene cloning may be related to its low transcription level [44,45], and the product of Cun9106 could not be identified. The other 9 STSs produced 10 different sesquiterpenes. Using NPP as the precursor, β-cubebene (34) was synthesized by Cun3157, and δ-cadinene (6) was synthesized by Cun3158. Using FPP as a precursor, δ-cadinol (20) was synthesized by Cun7050, α-copaene (35) was synthesized by Cun3574, α-muurolene (5) was synthesized by Cun0759, γ-cadinene (3) was synthesized by Cun3817, and germacrene D (36) was synthesized by Cun0773. Aromadendrene (37) was synthesized by C1,11 cyclization of Cun5155 with FPP as the precursor; δ-cadinol (20) was synthesized by C1,6 cyclization from Cun0716 with NPP as the precursor.

3.2.3. Rhodonia placenta

Rhodonia placenta (Postia placenta), formerly known as brown rot fungus, is a common diseased wood-rot fungus belonging to the Polyporaceae family that can grow a large area of mycelium. It is known that 6 STSs have been isolated from it; they were heterologously expressed by S. cerevisiae. A total of 25 different sesquiterpenoids were synthesized with FPP or NPP precursors [46]. Using FPP as a precursor, through C1,10 cyclization, PpSTS01 successfully synthesized α-muurolene (5), δ-cadinene (6), and β-elemene (21), PpSTS03 synthesized α-cadinene (38) and γ-cadinene (3), and PpSTS06 synthesized α-gurjunene (39); Δ⁶-protoilludene (11) and pentalenene (13) were synthesized from PpSTS08 and PpSTS14, respectively, by C1,11 cyclization. δ-Cadinene (6) was synthesized from PpSTS10 by C1,10 cyclization.

3.2.4. Fomitopsis pinicola

Fomitopsis pinicola is a brown rot basidiomycete species belonging to the family Fomitopsidaceae, commonly collected from dead conifer trees. One STS (Fompi1) was identified and heterologously expressed in E. coli [30]. α-Cuprenene (7) was synthesized through C1,6 cyclization by Fompi1 with NPP as the precursor.

3.2.5. Ganoderma lucidum

Ganoderma lucidum, which belongs to the Ganodermataceae family, is a well-known medicinal fungus. However, only two STSs, GL26009 [47] and GISTS6 [48], have been isolated and identified from this fungus and expressed heterologously in E. coli. Using FPP as the precursor, GL26009 synthesized γ-muurolene (26) and α-muurolene (5), and GISTS6 synthesized γ-cadinene (3).

3.2.6. Ganoderma sinense

Ganoderma sinense is a medicinal fungus belonging to the same genus as G. lucidum in the Ganodermataceae family. At present, six STSs have been isolated and identified from this fungus and expressed in E. coli. (GS11330, GS14272, GS02363, GsSTS43, GsSTS45a, and GsSTS45b) [48,49,50]. GsSTS45a has no function; GS02363 synthesized α-cadinol (30), δ-cadinene (6), α-muurolene (5), and γ-muurolene (26); GS11330 synthesized α-cuprenene (7); GS14272 synthesized α-muurolene (5); and GsSTS43 and GsSTS45b synthesized γ-cadinene (3).

3.2.7. Phanerodontia chrysosporium

Phanerodontia chrysosporium (Phanerochaete chrysosporium) belongs to the family Phanerochaetaceae. Eleven STSs have been recorded [51], of which seven were heterologously expressed in S. cerevisiae and cultured in SDL medium. PcSTS01 synthesized γ-muurolene (26), α-muurolene (5), and δ-cadinene (6). PcSTS02 and PcSTS04 synthesized β-copaene (40),β-farnesene (22), cadina-1(6),4-diene (41), and δ-cadinene (6). Epicubenol (42) was synthesized from PcSTS03. PcSTS06 synthesized α-barbatene (28) and β-barbatene (43). (E)-α-Bisabolene (44) was synthesized from PcSTS08, and PcSTS11 synthesized α-santalene (45).

3.2.8. Steccherinum ochraceum

Steccherinum ochraceum belongs to the family Meruliaceae. Six STSs were deduced from its genome, of which fusion enzyme A8411 was heterologously expressed in A. oryzae [52]. Hirsutene (46) was synthesized from A8411.

3.3. Russulales

Russulales comprises approximately 1767 species belonging to 80 genera and 12 families [1]. Two fungi of this order have been verified to contain fifteen different STSs and one fusion enzyme.

3.3.1. Stereum hirsutum

Stereum hirsutum belongs to the family Stereaceae. Nearly 50 related sesquiterpenoids have been found [53] as well as 18 STSs (ShSTS1-18, of which ShSTS2, 6, 9, 14 have not been studied due to their high homology to other genes) and 1 fusion protein (HS-HMGS), which were functionally verified by heterologous expression in E. coli or A. oryzae [36,38,54,55]. Their synthetic routes are summarized as follows: using NPP as a substrate, via C1,10 cyclization, ShSTS10 and ShSTS11 can synthesize δ-cadinene (6), ShSTS8 can synthesize 1-epi-cubenol (47), ShSTS12 can synthesize α-cubebene (48), and ShSTS10 can synthesize germacrene D (36). Simultaneously, ShSTS1 synthesized β-barbatene (43), ShSTS3 synthesized α-farnesene (14) and β-farnesene (22), ShSTS4 synthesized hirsutene (46), and ShSTS5 synthesized γ-cadinene (3) by C1,6 cyclization. Using FPP as a substrate, through C1,11 cyclization, ShSTS13 can synthesize β-caryophyllene (23), HS-HMGS can synthesize hirsutene (46), and ShSTS15, ShSTS16, ShSTS17, and ShSTS18 can synthesize Δ⁶-protoilludene (11); ShSTS7 synthesized δ-cadinene (6) via C1,10 cyclization.

3.3.2. Heterobasidion annosum

Heterobasidion annosum belongs to the Bondarzewiaceae family of the order Russulales. It is a forest pathogen that grows on large, perennial basidiocarps. Only one STS (Hetan2_454193) was identified in this fungus [40] and was heterologously expressed in E. coli. Using FPP as the precursor, Hetan2_454193 synthesized Δ⁶-protoilludene (11) by C1,11 cyclization.

3.4. Other Basidiomycota

The basidiomycetes in this region cannot be classified by order. There are 6 species of fungi in this part, and 10 different STSs have been verified by experiments.

3.4.1. Boreostereum vibrans

Boreostereum vibrans, originally named Stereum vibrans, is a macrofungus belonging to the Gloeophyllaceae family of the order Gloeophyllales. Many sesquiterpenes have been isolated from it [56]. BvCS is heterologously expression in E. coli [57]. δ-Cadinol (20) was synthesized by C1,10 cyclization of BvCS with FPP as a precursor.

3.4.2. Sphaerobolus stellatus

Sphaerobolus stellatus belongs to the order Geastrales and family Geastraceae. One STS (Sphst_47084) has been identified in this fungus [38] and heterologously expressed in E. coli. Using NPP as the precursor, viridiflorol (49) was synthesized via C1,10 cyclization of Sphst_47084.

3.4.3. Sanghuangporus baumii

Sanghuangporus baumii belongs to the Hymenochaetaceae family of the order Hymenochaetales and is an important medicinal fungus. Only one STS has been isolated from this species and heterologously expressed by E. coli, named SbTps1 [58].

3.4.4. Coniophora puteana

Coniophora puteana belongs to the Coniophoraceae family within the order Boletales. Four STSs have been isolated from it (Copu2, 3, 5 and 9) [59,60]. β-Copaene (40) and cubebol (9) were synthesized by C1,10 cyclization from Copu2 and Copu3 with NPP as the precursor. Using FPP as the precursor, Copu5 and Copu9 synthesized δ-cadinol (20) through C1,10 cyclization.

3.4.5. Serendipita indica

Serendipita indica is an endophytic root-colonizing species belonging to the order Sebacinales and family Serendipitaceae. One STS has been recorded [61], which is hetero-expressed in E. coli. Viridiflorol (49) was synthesized by C1,10 cyclization from SiTPS, using FPP as the precursor.

3.4.6. Dendrodontia bispora

Dendrodontia bispora (Dendrothele bispora) is a basidiomycete species belonging to the Corticiaceae family in Corticiales order. Two STSs (Denbi1_659367 and Denbi1_816208) have been isolated from this fungus [38] and hetero-expressed in E. coli. Δ⁶-protoilludene (11) was synthesized by C1,11 cyclization of Denbi1_659367. Viridiflorol (49) was synthesized by Denbi1_816208.

The taxonomic data in the above content come from GBIF (Global Biodiversity Information Facility, https://www.gbif.org/, accessed on 28 June 2022). A summary of information on sesquiterpene biosynthesis in Basidiomycota is presented in Table 1.

Table 1.

Classification of STSs from Basidiomycota.

	Type of Cyclization	Precursor	Metabolite	Producer	Biochemically Verified Enzyme
Clade I	C1,10	FPP	Intermedeol (1)	Macrolepiota albuminosa	STC4
			Germacrene D-4-ol (2)	Macrolepiota albumi-nosa	STC15
			Germacrene A (4)	Coprinopsis cinerea	Cop1, Cop2
			T-Cadinol (8)	Taiwanofungus camphoratus	AcTPS5
			α-Muurolene (5)	Omphalotus olearius	Omp1, Omp3
				Cerrena unicolor	Cun0759
				Coprinopsis cinerea	Cop3
				Rhodonia placenta	PpSTS01
				Cyclocybe aegerita	Agr3
				Ganoderma sinense	GS14272
				Ganoderma lucidum	GL26009
				Phanerodontia chrysosporium	PcSTS01
				Clitopilus pseudo-pinsitus	CpSTS3, CpSTS5
			α-Cadinol (30)	Lignosus rhinoceros Ganoderma sinense	GME3634 GS02363
			Torreyol (31)	Lignosus rhinocerus	GME3638
			δ-Cadinol (20)	Cerrena unicolor	Cun7050
				Boreostereum vibrans	BvCS
				Coniophora puteana	Copu5, Copu9
				Clitopilus pseudo-pinsitus	CpSTS3
			α-Copaene (35)	Cerrena unicolor	Cun3574
			γ-Cadinene (3)	Cerrena unicolor	Cun3817
				Rhodonia placenta	PpSTS03
			Germacrene D (36)	Cerrena unicolor	Cun0773
			δ-Cadinene (6)	Rhodonia placenta	PpSTS01
				Phanerodontia chrysosporium	PcSTS01
				Stereum hirsutum	ShSTS7
			β-Elemene (21)	Rhodonia placenta	PpSTS01
			α-Cadinene (38)	Rhodonia placenta	PpSTS03
			α-Gurjunene (39) Viridiflorol (49) γ-Muurolene (26)	Rhodonia placenta Serendipita indica Ganoderma lucidum Phanerodontia chrysosporium	PpSTS06 SiTPS GL26009 PcSTS01
Clade II	C1,10	NPP	δ-Cadinene (6)	Coprinopsis cinerea	Cop4
				Cerrena unicolor	Cun3158
				Rhodonia placenta	PpSTS10
				Clitopilus pseudo-pinsitus	CpSTS2
				Cyclocybe aegerita	Agr1, Agr4
				Omphalotus olearius	Omp4
				Stereum hirsutum Phanerodontia chrysosporium	ShSTS10, ShSTS11 PcSTS02, PcSTS04
			Cubebol (9)	Taiwanofungus camphoratus Coniophora puteana	AcTPS9 Copu2, Copu3
			1-epi-Cubenol (47)	Stereum hirsutum	ShSTS8
			Zonarene (10)	Taiwanofungus camphoratus	AcTPS4
			Ledene (19)	Clitopilus pseudo-pinsitus	CpSTS13
			Virifloridol (18)	Clitopilus pseudo-pinsitus	CpSTS9, CpSTS12
			Viridiflorol (49)	Sphaerobolus stellatus	Sphst_47084
			Alloaromadendrene (16)	Clitopilus pseudo-pinsitus	CpSTS8
			9-Alloaromadendrene (17)	Clitopilus pseudo-pinsitus	CpSTS11
			Aristolene (15)	Clitopilus pseudo-pinsitus	CpSTS16
			Viridiflorene (25)	Cyclocybe aegerita	Agr2, Agr5
			γ-Cadinene (3)	Omphalotus olearius	Omp5a, Omp5b
			α-Cubebene (48)	Stereum hirsutum	ShSTS12
			β-Cubebene (34)	Cerrena unicolor	Cun3157
			Germacrene D (36)	Stereum hirsutum	ShSTS10
			β-Copaene (40)	Coniophora puteana Phanerodontia chrysosporium	Copu2, Copu3 PcSTS02, PcSTS04
			β-Gurjunene (27) Epicubenol (42) β-Farnesene (22) Cadina-1(6),4-diene (41)	Galerina marginata Phanerodontia chrysosporium Phanerodontia chrysosporium Phanerodontia chrysosporium	Galma_104215 PcSTS03 PcSTS02, PcSTS04 PcSTS02, PcSTS04
Clade III	C1,11	FPP	α-Humulene (24)	Hypholoma fasciculare	Hfas94a, Hfas94b
			Δ6-Protoilludene (11)	Hypholoma lateritium	Hypsu1_138665
				Stereum hirsutum	ShSTS15, ShSTS16, ShSTS17, ShSTS18
				Heterobasidion annosum	Hetan2_454193
				Cyclocybe aegerita	Agr6, Agr7
				Clitopilus pseudo-pinsitus	CpSTS4
				Rhodonia placenta	PpSTS08
				Omphalotus olearius	Omp6, Omp7
				Armillaria gallica	Pro1
				Dendrodontia bispora	Denbi1_659367
			Aromadendrene (37)	Cerrena unicolor	Cun5155
			β-Caryophyllene (23)	Stereum hirsutum	ShSTS13
			Pentalenene (13)	Rhodonia placenta	PpSTS14
				Clitopilus pseudo-pinsitus	CpSTS6
			Sterpurene (12)	Clitopilus pseudo-pinsitus	CpSTS1
			α-Farnesene (14)	Clitopilus pseudo-pinsitus	CpSTS7
			γ-Muurolene (26)	Cyclocybe aegerita	Agr8
			1,3,4,5,6,7-Hexahydro-2,5,5-trimethyl-2H-2,4a-ethanonaphthalene (32)	Lignosus rhinocerus	GME9210
			1-Napthalenol (33)	Lignosus rhinocerus	GME9210
			γ-Cadinene (3)	Ganoderma sinense	GsSTS43, GsSTS45b
				Ganoderma lucidum	GISTS6
Clade IV	C1,6	NPP	α-Cuprenene (7)	Coprinopsis cinerea Ganoderma sinense Fomitopsis pinicola	Cop6 GS11330 Fompi1
			α-Barbatene (28)	Omphalotus olearius Phanerodontia chrysosporium	Omp9 PcSTS06
			β-Barbatene (43)	Phanerodontia chrysosporium	PcSTS06
				Stereum hirsutum	ShSTS1
			α-Farnesene (14)	Stereum hirsutum	ShSTS3
			β-Farnesene (22)	Stereum hirsutum Clitopilus pseudo-pinsitus	ShSTS3 CpSTS14
			Hirsutene (46)	Stereum hirsutum	ShSTS4
			γ-Cadinene (3)	Termitomyces albuminosus	STC9
				Stereum hirsutum	ShSTS5
				Clitopilus pseudo-pinsitus	CpSTS18
			β-Elemene (21)	Clitopilus pseudo-pinsitus	CpSTS14
			β-Caryophyllene (23)	Clitopilus pseudo-pinsitus	CpSTS17
			(E)-Dauca-4(11),8-diene (29)	Omphalotus olearius	Omp10
			δ-Cadinol (20)	Cerrena unicolor	Cun0716
			(E)-α-Bisabolene (44)	Phanerodontia chrysosporium	PcSTS08
			α-Santalene (45)	Phanerodontia chrysosporium	PcSTS11
others			—	Coprinopsis cinerea	Cop5
			—	Clitopilus pseudo-pinsitus	CpSTS10
			—	Hypholoma fasciculare	Hfas255
			Unknown	Hypholoma fasciculare	Hfas344
			Unknown	Cyclocybe aegerita	Agr9
			—	Cyclocybe aegerita	Agr10, Agr11
			—	Omphalotus olearius	Omp8
			—	Cerrena unicolor	Cun5765, Cun6114, Cun7487, Cun0802
			Unable to identify	Cerrena unicolor	Cun9106
			—	Ganoderma sinense	GsSTS45a
			Unknown	Sanghuangporus baumii	SbTps1
			Viridiflorol (49)	Dendrodontia bispora	Denbi1_816208
			Hirsutene (46)	Stereum hirsutum	HS-HMGS
				Steccherinum ochraceum	A8411

4. Research Process and Tools for STSs in Basidiomycetes

At present, the research process of basidiomycete STSs is mainly divided into three parts (Figure 4), and the latest research tools are developed around the core steps of these three parts (genome sequencing, basidiomycete culture methods, etc.).

Figure 4.

Technical research route of STS in basidiomycetes. The STS genome mining of basidiomycetes can be divided into three steps. The first step of genome mining is mainly based on the whole gene sequence, using bioinformatics tools to mine and predict the biosynthetic gene cluster. The second step is to obtain the target gene from the biosynthetic gene cluster of STS, and then heterologously express the target gene to obtain the product. The final step is to purify and identify the product to determine whether the gene is an STS.

4.1. Long-Read Whole-Genome Sequencing

The average genome size of basidiomycetes and ascomycetes is 46 Mb and 37 Mb, respectively [62], and the genome size of archaea and bacteria is usually within 6 Mb [63]. This means that bacterial genomes can be sequenced using short-read sequencing, but long-read sequencing and proper assembly are required for fungal genomes. Inexpensive nanopore sequencing [64] has been applied to whole-genome sequencing of basidiomycetes. For example, nanopore sequencing technology was used for whole-genome sequencing of the basidiomycete Clathrus columnatus and Inonotus obliquus, and genome assembly was completed [65,66].

Although nanopore technology enables long-read sequencing and is inexpensive, the accuracy of base calling is 85–94% depending on the sequencing method [67]. However, if nanopore long-read sequencing is used in combination with short-read sequencing, it may be possible to assemble a higher quality genome, if the software that assembles the genome has this capability. This functionality is currently available for bacterial and fungi genomes, and the Pilon software can combine Illumina and Nanopore sequence data to polish assemblies [68].

4.2. Basidiomycetes Cultures

Many basidiomycetes have high requirements for their growth environment; they can only grow under specific conditions, and most of them cannot be cultivated artificially, which greatly limits isolation and identification of sesquiterpenoids in basidiomycetes. Fungal growth can be simply divided into two stages: (i) germination of fungal spores, and (ii) subsequent filamentous growth, forming a network of hyphae called mycelium [69].

The conditions that induce or inhibit the germination of fungal spores have always puzzled researchers. So far, the main research directions are growth factor induction, activator induction, co-culture, volatile organic compound induction, and physical factors [70]. Taking Agaricomycetes ectomycorrhizal (EcM) mushrooms as an example, the M factor (growth-promoting metabolites in addition to b vitamins and amino acids are essential for the growth of tree mycorrhizal fungi), as a growth factor, promotes its growth [71]. Placing EcM mushrooms together with specific tree seedlings on lipid or gel medium can also promote spore germination, although this approach often fails [71,72]. There is evidence that EcM mushroom spore germination can also be promoted when co-cultured with bacteria [73].

The growth factors that induce the growth of fungal hyphae are mostly root exudates. Studies on EcM fungi have shown that in addition to M factor, palmitic acid, stearic acid, and cytokinins, such as kinetin, zeatin, and isopentenyl aminopurine, are also growth-promoting factors. Root exudates can induce their growth [74].

At the same time, fungal gene expression is very complex and is affected by RNAi silencing [75] and trans-acting elements of genome structure [76]. Therefore, many biosynthetic gene clusters are silent. However, by adjusting the culture conditions (changing the physical conditions of the culture, adding compounds, growth factors, etc.) [70], utilizing co-culture [77], and chromatin-based transcriptional regulation [78], silenced biosynthetic gene clusters can be activated. Studies on these operations are still in preliminary stages. However, the metabolites produced by basidiomycete fungi in different growth cycles are different [79], and many genetic regulators that control fungal development also control the production of secondary metabolites [80,81]. Studies on the basidiomycetes Coprinopsis cinerea [82] and Lentinula edodes [83] have shown that gene expression differs at the developmental stages of fruiting bodies, limiting the mining of active ingredients.

Cultivation technology for basidiomycetes has always been inadequate, but in recent years, the development of new laboratory-level cultivation techniques has brought new opportunities for artificial cultivation. For example, basidiomycetes are cultivated using microfluidic culture technology [84].

4.3. Exogenous Expression Platforms and Bioinformatics Tools for Basidiomycetes STSs

Due to the complexity of basidiomycetes genes, traditional heterologous expression platforms cannot meet the functional identification of basidiomycetes STS. Although E. coli can express STS genes, it lacks a post-translational modification system to express complex proteins and entire biosynthetic pathways, and the eukaryotic expression system in yeast cannot remove introns of fungal genes [85]. At present, A. oryzae is a relatively successful heterologous expression platform, which can more accurately splice the intron of the basidiomycetes terpenoid synthase gene [37] and correctly express the entire gene cluster [86]. Ustilago maydis has also been developed as a heterologous expression platform for the production of terpenoids [87]. It offers the advantage of metabolic compatibility and potential tolerance of substances toxic to other microorganisms.

Successful characterization of the biosynthesis of basidiomycetes products requires not only genetic engineering and heterologous expression, but also metabolic analysis [88]. Bioactivity-guided methods for isolating metabolites have been gradually replaced by more sensitive methods, such as tandem mass spectrometry (MS/MS), for untargeted metabolomics data analysis, resulting in data that can be compared with known spectral databases. Researchers can also identify unknown metabolites and intermediates through the Global Natural Products Social Molecular Networking (GNPS) [88,89] and infer biosynthetic pathways. New technologies and tools are also being developed to assist in the identification of STSs in basidiomycetes. Bioinformatics techniques can be used to establish a general prediction framework for STS and to improve the accuracy of genome-based tools for predicting biosynthetic gene clusters [58], such as antiSMASH [90] and PRISM [91]. Although it can predict the monomer sequences assembled into PKS and NRPS biosynthetic lines based on module specificity, the accuracy and specificity need to be further improved, which is also the key to identifying STS [91].

5. Discussion

Sesquiterpenes play a very important role in basidiomycetes. They can attract insects for pollination [92], defend against other organisms or parasites [93], and play an important role in basidiomycete’s physiological effect. Moreover, when basidiomycetes form a symbiotic relationship with plants, these sesquiterpenoids produced by basidiomycetes can act as phytohormones [13,14]. Therefore, basidiomycetes produce many kinds of sesquiterpenes to help them better adapt to the living environment. The influence of the ambient environment on the production of sesquiterpenes by basidiomycetes includes external physical factors (light, temperature, etc.) and chemical factors (exogenous chemical substances, etc.). The changes of sesquiterpenes during basidiomycete development and their biological roles are still unclear. At present, there are studies on the changes of sesquiterpenes during the development of the fruiting bodies of the Cyclocybe aegerita AAE-3 strain. In particular, the development of the fruiting body changes resulted in greater changes during the sporulation process. In the early stage of sporulation, mainly alcohols and ketones appeared, while in the later stage of sporulation, sesquiterpenes such as Δ6-protoilludene (11), α-cubebene (48) and δ-cadinene (6) appeared. After sporulation, sesquiterpenoids decreased and other compounds appeared, mainly octan-3-one [94].

Site-specific mutations are tools to study enzyme structure, function, and catalytic mechanism, and they include single and combinatorial mutations [95]. In the study of sesquiterpene synthases of basidiomycetes, the point mutations at residues near the conserved region are mostly used. The current point mutation experiments for sesquiterpene synthase of basidiomycetes are concentrated near the conserved regions of the RY Pair and NSE Triad (Figure 5).

Figure 5.

(a) Comparison of the conserved regions of H-α1 loop and NSE/DTE motif. (b) Comparison of conserved regions of the RY Pair to the Thirteen Positions Upstream of the RY Pair. The MUSCLE algorithm was used to compare the following protein sequences: Cop3 (XP_001832925), Cop4 (XP_001836356), Cop6 (XP_001832549) in Coprinopsis cinerea; STC4 (KAH0582448), STC9 (KAH0583476), STC15 (KAG5341349) in Macrolepiota albuminosa.

Cop3, Cop4, and Cop6 were experimentally point mutated at the sites of their H-α1 loops, respectively. K233 in Cop4 and K251 in Cop3 did not play a major role in the side chain and ligand interaction network formed during active-site closure; the mutations in Cop4 (K233, H235, T236, N238, and N239) showed that the mutations in the H-α1 loop region site significantly altered the type of product, with the mutation of N239L having the greatest effect on the product; the mutation of Cop6 (C236, E237, and N240) showed that the mutation of the H-α1 loop region site did not alter the product of Cop6. Structural modeling of the Cop enzyme pointed to a potential interaction between the H-α1 loop and the conserved residues of the two metal-binding motifs (DDXXDD and NSE/DTE). Potential interactions between the conserved Asp/Glu and the Arg, Asn and Lys sites in some sesquiterpene synthases in several fungi and plants may stabilize the closed enzyme conformation by closing the H-α1 loop [96]. STC4 was transformed into germacrene A (4) synthase after the single site W335F mutation; various variants of the W314 point mutation in STC15 were unable to obtain expressed protein, and enzyme activity was reduced after the mutation of the putative C311 active site in STC9 [34]. A triple mutant Cop2(17H2) was obtained by error-prone PCR. Cop2(17H2) contains three mutations in L59H, T65A and S310Y, and the three mutations tend to make Cop2(17H2) products be specific. Moreover, compared to the original Cop2, Cop2(17H2) is more inclined to produce Germacrene D-4-ol (2) [97].

6. Conclusions

A comparison of the reported genome sequences revealed that each basidiomycete contained, on average, more than 12 STSs. Although the reasons for the existence of many STSs are unclear, it is speculated that they are closely related to their biological activities. The development of molecular tools for basidiomycetes research will allow researchers to further explore these microbial taxa. These efforts have definitely resulted in a global push for the discovery and characterization of fungal STSs, and they provide hope for the future of fungal sesquiterpenoid discovery.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof8090913/s1, Table S1: Sesquiterpene skeleton and production source of basidiomycetes from different species; Table S2: Basidiomycetes sesquiterpenoid skeleton type and the number of compounds; File S1: Gene name and amino acid sequence of sesquiterpene synthase from basidiomycetes.

Author Contributions

Data curation, J.W., X.Y., Y.D., J.Q. and C.L.; writing original draft, J.W. and X.Y.; discussion of the contents, P.W. and J.Q.; writing—review and editing, J.-M.G. and C.L.; proofreading and approval of the manuscript, C.L. All authors have read and agreed to the published version of the manuscript.

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.

Funding Statement

This work was funded by the Innovation and Development Joint Fund of the Natural Science Foundation of Shandong Province (Project No. ZR2021LSW022), the Shaanxi Key Laboratory of Natural Products & Chemical Biology Open Foundation (Project No. SXBPCB 2021001), and the Fundamental Research Funds for the Central Universities (Project No. 2572022BD03).