Heterologous expression of cytotoxic sesquiterpenoids from the medicinal mushroom Lignosus rhinocerotis in yeast

Basidiomycete mushrooms are important sources of bioactive secondary metabolites including terpenoids, alkaloids, and polyketides [1]. Exploration of the medicinal and pharmacological potential of these underexplored mushroom species could be scientifically and biotechnologically rewarding. Lignosus rhinocerotis (Cooke) Ryvarden (orthographic variant is L. rhinocerus; common name: tiger milk mushroom) belongs to the order Polyporales (Polyporaceae family). It has been found in tropical regions in China, East Africa, Sri Lanka, Thailand, Philippines, Indonesia, Papua New Guinea, Australia, Vanuatu, and Malaysia [2–4]. Lau et al. [5] has compiled the ethnomycological uses of the sclerotia of the mushroom by different communities including the Malay, Chinese, and several sub-tribes of indigenous communities in Peninsular Malaysia including the Temuans and Semai aborigines to treat coughs, asthma, tuberculosis, and cancer; where several preparation methods of the fungus for medicinal purposes have been documented. Both aqueous (e.g., decoction) and non-aqueous preparations (e.g., tincture) have been reported, which may indicate the presence of hydrophilic and lipophilic active substances, respectively [5].

The genome and transcriptome studies of L. rhinocerotis have provided insights into the biology of this medicinal mushroom and opened up new opportunities for exploration via genomic approaches [6–9]. Transcriptomics analysis revealed genes encoding several small secreted cysteine-rich proteins (cerato-platanins and hydrophobins) and lectins were among the highest expressed genes in the sclerotium of L. rhinocerotis and may have important implications for various bioactivities relevant to human diseases [7]. On the other hand, the L. rhinocerotis genome harbours genes encoding a number of secondary metabolite biosynthetic enzymes, including 12 sesquiterpene synthases (STSs), one non-ribosomal peptide synthetase (NRPS), and a polyketide synthase (PKS) [6]. These enzymes are crucial for the biosynthesis of sesquiterpenes, non-ribosomal peptides, and polyketides, respectively. Among them, six secondary metabolite gene clusters harbouring genes encoding four STS genes and one each of NRPS and PKS were also found to express at notable levels in the L. rhinocerotis sclerotium [7].

Genome mining has emerged as a potential avenue to access the chemical diversity encoded in basidiomycete fungal genomes [10]. The large number of STS genes in the genome highlights the potential of L. rhinocerotis in producing diverse sesquiterpenoids. Many sesquiterpenoids have potent antibiotic and cytotoxic activities due to their high chemical reactivity [11]. Some renowned fungal sesquiterpenoids include the anticancer active illudins from Omphalotus olearius [12], the cytotoxic PR toxin (from Penicillium roqueforti) with robust antimicrobial activity [13], and the platelet aggregation inhibitor radulone A from Radulomyces confluens [11]. The basidiomycete STSs have been shown to be readily expressed in well-established laboratory hosts, such as Escherichia coli and Saccharomyces cerevisiae [12, 14]. This makes heterologous expression an attractive approach for accessing the basidiomycete terpenoid chemistry, as many basidiomycete fungi are relatively slow growing in the laboratory environment.

We have only begun to appreciate the diversity of STSs in basidiomycetes [10]. Nonetheless, sesquiterpene biosynthesis of Polyporales which consist of several medicinal mushrooms including Ganoderma lucidum [15], Antrodia cinnamomea [16], Trametes versicolor [17] and L. rhinocerotis (this study), remained largely unexplored. Given that S. cerevisiae has proven to be extremely useful for genome mining and characterisation of fungal secondary metabolite pathways [18] as well as for commercial production of terpenoids [19], we have chosen to use the yeast as an expression host for L. rhinocerotis STSs in this study. Guided by previous transcriptomic data, we cloned several sclerotium-expressed L. rhinocerotis STS genes for heterologous expression in a S. cerevisiae yeast system. The putative products from these STSs were identified by gas chromatography-mass spectrometry (GC–MS). Two major sesquiterpene alcohol products were isolated and further characterised structurally. The potential cytotoxic activities of the two compounds against a panel of human cancer cell lines were also examined.

Saccharomyces cerevisiae yeast has emerged as a powerful host cell for genome mining, biosynthetic studies, and production of fungal secondary metabolites, as it has many advantages [18, 19]. For expression of isoprenoid pathways, yeast readily provides the FPP substrate for STSs and has the added advantage of the ability to co-express functional cytochrome P450 monooxygenases that are often found encoded in terpene synthase gene clusters with compatible fungal cytochrome P450 reductase [40–43]. Successful expression of basidiomycete (Agaricales) STSs have been previously demonstrated in yeast [14]. However, this is the first report of the characterisation of STSs from the Polyporaceae family of mushrooms, which include several well-known medicinal mushrooms including G. lucidum and Antrodia camphorata.

Previous studies reported the expression of several sesquiterpene biosynthetic gene clusters in the genome and transcriptome of L. rhinocerotis [6, 7] but identification of their metabolic products remains elusive. In this study, we heterologously expressed three STSs namely GME3634, GME3638, and GME9210 in order to functionally characterise their metabolic products. GME9210 produced several sesquiterpenes at low abundances including 1,3,4,5,6,7-hexahydro-2,5,5-trimethyl-2H-2,4a-ethanonaphthalene (C₁₅H₂₄) while GME3638 and GME3634 encoded STSs that produce (+)-torreyol and α-cadinol, respectively, as major products. Both compounds are sesquiterpene alcohols and have been previously characterised but for the first time, the polypore L. rhinocerotis (Polyporaceae) has been shown to encode the pathways for biosynthesis of these sesquiterpene alcohols.

In this study, using the sequence and function data of the characterised STSs from C. cinereus (Cop1—6) and O. olearius (Omp1—10) as guide [12, 14], we extended the analysis by including the newly identified STSs from L. rhinocerotis as well as putative STS sequences from various Polyporaceae basidiomycetes to provide insights into the diversity and biochemical functions of STSs in L. rhinocerotis. It is interesting to note that GME3634 and GME3638 gene homologs exist as a pair in a gene cluster and are responsible for production of isomeric sesquiterpenes, (+)-torreyol and α-cadinol, with different stereochemistry. Similar paired GME3634/GME3638 homologs were also found in T. versicolor, T. cinnabarina, and D. squalens as clustered genes, suggesting that they are conserved in multiple Polyporaceae mushrooms. Their high homology and conserved synteny suggest that these STS homologs may be involved in the biosynthesis of (+)-torreyol and α-cadinol and/or related sesquiterpenes, which may serve common biological functions.

The occurrence of the major sesquiterpenic alcohols in the cultures of yeast harbouring GME3634 and GME3638 presents an interesting question whether these alcohols are the direct product of the enzymes. Wawrzyn et al. [12] suggested that (+)-torreyol from the Agaricales mushroom O. olearius is derived from α-muurolene synthesised by the α-muurolene synthase Omp1. A single P450 oxygenase gene collocated with omp1 in the biosynthetic cluster was proposed to be responsible for converting α-muurolene to (+)-torreyol [12]. GME3638, which synthesises (+)-torreyol, shares 43% identity to Omp1. However, we have not detected the major production of α-muurolene in the culture of yeast expressing GME3638, but just a minor one. Instead, we have detected germacrene D-4-ol as the second major product in addition to (+)-torreyol (Fig. 1). The same is true for GME3634, in which the gene clustered with GME3638 and produced the stereoisomer α-cadinol with germacrene D-4-ol as the second major product in yeast culture. Although we cannot completely ruled out that an endogenous yeast enzyme (e.g. a hydroxylase) could affect the end product of the two STSs observed in the yeast cultures, we proposed that GME3638 and GME3634 could produce (+)-torreyol and α-cadinol as the direct product resulting from hydrolysis of the muurolenyl or cadinenyl cation, respectively (Additional file 1: Figure S2). Further in vitro characterisation of the enzymes is required to elucidate the biosynthetic mechanism of GME3634 and GME3638.

Phylogenetic analysis indicates that GME3638 and GME3634 belong to the same clade (Clade I) together with the α-muurolene synthase Omp1, the germacrene A synthases Cop1 and Cop2 [14], as well as several putative uncharacterised STSs from closely related taxa (Fig. 3). Of note is that the germacranyl cation is the key intermediate of many cadinene group compounds (which includes muurolenes) [44, 45], while germacrene D and germacrene D-4-ol are constantly detected as a side product in the yeast strains expressing GME3634 and GME3638 (Table 1). This is congruent with the germacranyl cation being an essential biosynthetic intermediate of muurolenes and cadinenes (Additional file 1: Figure S2). In line with the previous study [12], it appears that although the STS phylogenetic analysis was unable to provide an accurate prediction of the structures of the sesquiterpene products at this stage due to the lack of characterised STSs from basidiomycetes, the grouping of the STSs into major clades gave a good indication of the initial cyclization step.

α-Cadinol is a common major constituent of various bioactive essential oils and plant extracts, such as those obtained from Lindera nacusua, Zingiber nimmonii, and Beilschmiedia madang [46–48]. A structure–activity relationship study of cadinane skeletal sesquiterpenes from Taiwania heartwood (Taiwania cryptomerioides) indicated that the presence of an equatorial hydroxyl (–OH) group at C-10 with a trans-fused configuration at the ring junction has a positive influence on their antifungal activities [36], such is the case of α-cadinol and therefore some antifungal activity may be expected. On the other hand, with a cis-fused rings feature, the torreyol belongs to the muurolene-type of compounds in contrast to the cadinene and their related cadinols which have a trans-fused ring structure [34]. Torreyol can be (+) or (−) depending if it is dextrorotatory or levorotatory; these isomers have been described by the names δ-cadinol, sesquigoyol, pilgerol, cedrelanol, and albicaulol [34]. The name torreyol is widely used due to its priority in usage. (+)-Torreyol was first isolated as white crystalline needles from the leaves of Torreya nucifera [49] and later in a few basidiomycetes including Clitocybe illudens (synonym O. olearius) grown on agar medium, Lentinus lepideus, and Xylobolus frustulatus [31, 50].

Investigations into elucidating the bioactive compounds in order to discover the scientific basis of the traditional uses of L. rhinocerotis recorded by ethnomycological surveys are currently emerging. In particular, its anticancer activities have been well explored by several research groups [9, 51, 52]. A partially purified fraction made up of two putative cytotoxic subtilisin-like fungal serine proteases has been recently reported to exhibit apoptosis-inducing activity in MCF7 cells [9] and more recently, Pushparajah et al. [52] synthesised a functional recombinant lectin-like fungal immunomodulatory protein using cDNA of L. rhinocerotis, which exhibit cytotoxicity against cancer cell lines. While the reported bioactive compounds are of high molecular weight protein molecules, here, we characterised three STSs from L. rhinocerotis using yeast heterologous system, which led to the discovery of (+)-torreyol that exhibited potent cytotoxicity against MCF7 and α-cadinol that is less active. To our knowledge, this is the first report on the bioactivity of (+)-torreyol and α-cadinol in pure form as these compounds are usually assayed in mixture. In view of the promising bioactivity of (+)-torreyol, future directions may include in-depth studies on its pharmacological activities in vivo and the underlying molecular mechanisms.

Two cytotoxic sesquiterpene alcohols, (+)-torreyol and α-cadinol, were isolated and purified from the yeast system expressing the L. rhinocerotis STSs GME3638 and GME3634, respectively. (+)-Torreyol is selectively active against MCF7 cells with IC₅₀ value of 3.5 ± 0.58 μg/ml. This study provided insights into the biocatalytic activities of basidiomycete STSs and provides a platform for further genome mining of bioactive secondary metabolites from medicinal mushrooms of the Polyporaceae family. Future work will focus on characterisation of other STSs in L. rhinocerotis as well as on elucidating the in vivo anticancer activity and mechanism(s) of action of (+)-torreyol.