Identification and Characterization of a δ-Cadinol Synthase Potentially Involved in the Formation of Boreovibrins in Boreostereum vibrans of Basidiomycota
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Sesquiterpenoids are very common among natural products. A large number of sesquiterpene synthase genes have been cloned and functionally characterized. However, until now there is no report about the δ-cadinol synthase predominantly forming δ-cadinol (syn. torreyol) from farnesyl diphosphate. Sesquiterpenoids boreovibrins structurally similar to δ-cadinol were previously isolated from culture broths of the basidiomycete fungus Boreostereum vibrans. This led us to expect a corresponding gene coding for a δ-cadinol synthase that may be involved in the biosynthesis of boreovibrins in B. vibrans. Here we report the cloning and heterologous expression of a new sesquiterpene synthase gene from B. vibrans. The crude and purified recombinant enzymes, when incubating with farnesyl diphosphate as substrate, gave δ-cadinol as its principal product and thereby identified as a δ-cadinol synthase.
KeywordsDelta-cadinol Sesquiterpene synthase Biosynthesis GC–MS Fungi
2 Results and Discussion
Based on the putative sesquiterpene synthase sequence from B. vibrans genome draft assembly, the full-length cDNA (designated as BvCS) was recovered by RT-PCR with specific primers and contained an open reading frame of 1182 nucleotides. The sequence has been submitted to the GenBank database under accession number KU668561. The deduced amino acid sequence of BvCS had 49 % identity to α-muurolene synthase (Cop3, accession no. A8NE23) from Coprinus cinereus  and germacradienol/germacrene D synthase (accession no. KNZ73785) from Termitomyces sp. J132. Showing 22 % identity, BvCS was less related to δ-cadinene synthase (accession no. XP_007299839) from Stereum hirsutum  (Table S1 in Electronic supplementary material).
For the biosynthesis of cadalane sesquiterpenes, three alternative mechanisms were proposed and elucidated in bacteria [16, 17] and plants [25, 26]. The intermediacy of nerolidyl diphosphate (NPP) was generally accepted. Recently, however, the pathway via the protonation of the neutral intermediate 3 was demonstrated through labeling experiments to account for production of 92 % of cadalane sesquiterpenes by MtTPS5 from the plant Medicago truncatulathe, which can give 6 from 1 as its minor product . Furthermore, the germacrene D (3) pathway was proposed for the bacterial STS from Chitinophaga pinensis DSM 2588 that can afford both δ-cadinene and germacrene D-4-ol (2), although no germacrene D was present in the enzymatic products . As shown in this study, BvCS can form 6 as major product and 2 as minor product (Fig. 3). Therefore it is likely for the enzyme to follow the germacrene D pathway as that from C. pinensis DSM 2588  (Fig. 1, Electronic supplementary material), while alternative mechanisms with the intermediacy of NPP could also be possible.
In conclusion, we cloned the full-length cDNA of a new sesquiterpene synthase gene from the basidiomycete Boreostereum vibrans and expressed it in E. coli for functional characterization. Based on GC–MS analyses, the recombinant enzyme was demonstrated to mainly produce δ-cadinol from farnesyl diphosphate and thereby identified as a δ-cadinol synthase.
3 Experimental Section
3.1 Gene Cloning
Mycelia of the fungus Boreostereum vibrans was inoculated in 0.5 L modified PDB medium (potato 200.0 g, glucose 20.0 g, KH2PO4 3.0 g, MgSO4 1.5 g, citric acid 0.1 g, and thiamin hydrochloride 10 mg in 1 L of deionized water, pH 6.5), cultured at 25 °C on a rotary shaker at 140 rpm. Mycelia were activated on PDB agar plates before inoculation in liquid PDB. Total RNA was isolated from the mycelia on day 20 using the Plant RNA Mini Kit (Qiagen). The first-strand cDNA was synthesized with Superscript™ III First-strand Synthesis System (Invitrogen). The full length cDNA was obtained with specific primer pairs 5′-CCCGACCTTCTCACCATCTGT-3′ (forward) and 5′-CGCGAGGTATAGAGCACCTGT-3′ (reverse) according to the predicted gene sequence in our B. vibrans genome draft assembly. For PCR, 30 cycles of reactions were performed at the condition (95 °C, 30 s; 58 °C, 30 s; 72 °C, 100 s) with final extension at 72 °C, 10 min. The amplicons were cloned and sequenced to verify the encoding region. The sequence, designated as BvCS, has been submitted to the GenBank database under accession number KU668561.
3.2 Expression in E. coli and Enzyme Purification
The ORF of BvCS was cloned into the expression vector pET32a+ (Novagen) which was subsequently transformed into E. coli BL21(DE3) (Novagen) for a fusion expression, using the original pET32a+ as negative control. Protein expression at 37 or 15 °C with 0.1 or 0.5 mM IPTG was determined by SDS-PAGE analysis. Cells induced with 0.1 mM IPTG at 15 °C for 22 h were collected by centrifugation (8000 rpm, 4 °C, 5 min), washed by deionized water and suspended in 50 mM Tris–HCl buffer (pH 7.5) for crude enzyme preparation or in the binding buffer (20 mM Tris–HCl pH 7.5, 0.3 M NaCl, 5 mM imidazole) for enzyme purification. The cell lysate obtained by sonication on ice was then centrifuged at 12000 rpm for 10 min at 4 °C and the supernatant, containing the soluble recombinant enzyme was used for crude enzyme assay or enzyme purification. Purification of His-tagged enzymes was performed according to the Ni–NTA Agarose protocol (Qiagen) with a wash buffer (20 mM Tris–HCl pH 7.5, 0.5 M NaCl, 150 mM imidazole) and an elution buffer (20 mM Tris–HCl pH 7.5, 0.5 M NaCl, 300 mM imidazole). The elute containing the purified enzyme was immediately desalted with 50 mM Tris–HCl buffer (pH 7.5), concentrated by centrifugation at 4 °C (Amicon Ultra-15, Merck Millipore), and immediately used for activity assays. All fractions were analyzed by SDS-PAGE on 12 % polyacrylamide gel under non-reduced condition at 140 V for 1.5 h.
3.3 Enzyme Assays and GC–MS Analyses
Crude enzyme was assayed for sesquiterpene synthase activity using FPP (Sigma) of 10 μg in assay buffer (50 mM Tris–HCl pH 7.5, 10 % glycerol, 10 mM MgCl2, 1 mM dithiothreitol, 100 mM NaCl), incubating for 4 h at 30 °C, using the empty vector as control. After extraction with 2 mL hexane, the hexane phase was collected by centrifugation, dehydrated over anhydrous sodium sulfate and concentrated under a stream of air for GC–MS analysis. The purified enzyme activity was detected as described above with FPP of 5 μg, using heat-denatured enzyme as control.
GC–MS [Agilent HP6890/5973, column: 0.25 mm × 30 mm, 0.25 µm (HP-5MS)] was conducted under electron-impact (EI) mode (70 eV). The flow rate of helium carrier gas was set at 1.0 mL/min. Samples (2 μL) were injected at 80 °C. After holding the samples for 5 min at 80 °C, the column temperature was increased at 5 °C/min to 280 °C and hold for 30 min. The MS date was collected from 35 to 500 m/z. The identification of the compounds was achieved by comparing the retention time and the mass spectra with those of the standards included in the library (wiley7n.1) and the authentic compounds in publications.
This work was supported by a grant from the National Natural Science Foundation of China (21572237). We are grateful to Professor Jian-Xin Pu (Kunming Institute of Botany) for his helpful suggestion.
Compliance with Ethical Standards
Conflict of interest
The authors declare no conflict of interest.
- 11.Y.L. Yang, H. Zhou, G. Du, K.N. Feng, T. Feng, X.L. Fu, J.K. Liu, Y. Zeng, Angew. Chem. Int. Ed. (2016). doi: 10.1002/anie.201510928
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