Biosynthesis of Sesquiterpenes in Basidiomycetes: A Review

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.


Introduction
Fungi are widely distributed in various ecosystems of the Earth. Based on highthroughput 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

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

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] (Figures 2 and 3 then converted to sesquiterpene skeletal end-products under the action of STS. The reaction mechanism is divided into four categories [29][30][31] (Figures 2 and 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. 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. 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.

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.

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 synthe-sized 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.

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.

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.

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.

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 ∆ 6 -protoilludene (11) by C1,11 cyclization.

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.

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.

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].

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 heteroexpressed in E. coli. Viridiflorol (49) was synthesized by C1,10 cyclization from SiTPS, using FPP as the precursor.

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.).

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. 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.

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].

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 growthpromoting 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].

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].

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 ∆6-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). 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 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].

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.