Cloning and Characterization of a Squalene Synthase Gene from the Chaga Medicinal Mushroom, Inonotus obliquus (Agaricomycetes)

Abstract

Squalene synthase catalyzes the condensation of 2 molecules of farnesyl diphosphate to produce squalene, the first committed precursor for sterol, brassinosteroid, and triterpene biosynthesis. A squalene synthase gene, designated IoSQS, was isolated from Inonotus obliquus, a medicinal mushroom that produces a plethora of bioactive triterpenes. IoSQS complementary DNA was found to contain an open reading frame of 1476 bp, encoding a protein of 491 amino acids with a calculated molecular mass of 55.85 kDa. The IoSQS genomic DNA sequence consisted of 1813 bp and contained 4 exons and 3 introns. The restriction fragment polymorphisms revealed by Southern blot analysis suggested that IoSQS was a single-copy gene. Promoter analysis indicated that the 5' upstream region of IoSQS possessed various potential elements associated with physiological and environmental factors. The expression pattern of IoSQS in different stages and under methyl jasmonate treatment correlated with the accumulation of total triterpenoids and was consistent with the predicted results of the IoSQS promoter region. The N-terminal 466 residues of the hydrophilic sequence were expressed as a His-tagged protein in Escherichia coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. Squalene was detected in vitro in reaction mixture by high-performance liquid chromatography analysis. These results suggest that the IoSQS enzyme is involved in squalene production in I. obliquus.

RNA-Seq de Novo Assembly and Differential Transcriptome Analysis of Chaga (Inonotus obliquus) Cultured with Different Betulin Sources and the Regulation of Genes Involved in Terpenoid Biosynthesis

“…Two transcript variants of squalene synthase ( SQS1 and SQS2 ) were identified with a 95% sequence identity to each other ( SQS2 has an insertion at the 3′ end) (Table 2). Our SQS1 transcript sequence is identical to previously characterized SQS from I. obliquus [78].…”

“…For example, the lanosterol-derived triterpenoid inotodiol and the lupeol derivative betulinic acid are well known bioactive triterpenoids from I. obliquus [17,19,24,25,27,28,33,36,76,77]. Though several studies have shown the presence of triterpenoids in I. obliquus , only one gene, SQS, has been identified [78]. In order to increase knowledge on genes involved in triterpenoids production, a search for these genes was carried out in the transcriptome of I. obliquus .…”

“…According to the literature, only one terpenoid gene, SQS , has been characterized from I. obliquus, whereas nine genes related to terpenoids biosynthesis where identified in I. baumii [52,78]. The transcriptome generated in the current study allowed for the identification of all (17) orthologous genes involved in terpenoid metabolism.…”

“…For example, lanosterol is formed from oxide squalene by a cyclization reaction catalyzed by lanosterol synthase (LAS) and is further metabolized into triterpenoids such as inotodiol [52,99]. To date, only the SQS sequence has been identified, cloned and characterized from I. obliquus (Appendix A—Figure A1) [78].

Figure A1Proposed biosynthetic pathway leading to multiple terpenoids in I. obliquus .

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Transcriptome analysis of Clinopodium gracile (Benth.) Matsum and identification of genes related to Triterpenoid Saponin biosynthesis

“…Squalene synthase (SS; EC 2.5.1.21) is a key bifunctional enzyme in the terpenoid biosynthesis pathway; SS first catalyzes the formation of presqualene diphosphate (PSPP) from two molecules of FPP and then converts PSPP to squalene with NADPH and Mg 2+ ions [13,14]. The level of SS gene expression shows a positive correlation with the amount of triterpenes [15].…”