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Identification and Characterization of a δ -Cadinol Synthase Potentially Involved in the Formation of Boreovibrins in Boreostereum vibrans of Basidiomycota

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ABSTRACT

Abstract: 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.

Graphical abstract: A new sesquiterpene synthase gene was cloned from the basidiomycete fungus Boreostereum vibrans and heterologously expressed in E. coli. The purified recombinant enzyme gave δ-cadinol as its principal product from farnesyl diphosphate and thereby identified as a δ-cadinol synthase (BvCS).

Electronic supplementary material: The online version of this article (doi:10.1007/s13659-016-0096-4) contains supplementary material, which is available to authorized users.

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GC–MS analyses of products formed by the recombinant BvCS and the heat-denatured enzyme as control, respectively, with FPP as substrate. Total ion chromatograms (left) and the corresponding mass spectra (right) illustrate the product peaks at 17.10 and 18.68 min matching germacrene D-4-ol and δ-cadinol, respectively, in mass fragmentation patterns included in the database
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Fig3: GC–MS analyses of products formed by the recombinant BvCS and the heat-denatured enzyme as control, respectively, with FPP as substrate. Total ion chromatograms (left) and the corresponding mass spectra (right) illustrate the product peaks at 17.10 and 18.68 min matching germacrene D-4-ol and δ-cadinol, respectively, in mass fragmentation patterns included in the database

Mentions: Next, functional expression in pET32a+/Escherichia coli BL21(DE3) was conducted to confirm the catalytic activity of BvCS. Soluble protein expression was achieved at 15 °C for 22 h with 0.1 mM IPTG (isopropyl-β-d-thiogalactopyranoside), as determined by SDS-PAGE analysis (Figure S1 in Electronic supplementary material). The crude enzyme was assayed for sesquiterpene synthase activity using 1 as a substrate under optional condition as described in Sect. 3. Based on GC–MS analyses, major product peak at 18.68 min (retention time) and minor products were observed for crude BvCS, compared with the empty vector as control (Electronic supplementary material). The purified recombinant protein was used for further characterization. After purification under native condition on the Ni–NTA Agarose, the analysis of the elute on SDS-PAGE led to detection of the main band corresponding exactly to the predicted size of the recombinant BvCS protein of approximately 61 kDa (Fig. 2). When incubated with FPP, the purified BvCS made δ-cadinol (6) as major product at 18.68 min, and minor products including germacrene D-4-ol (2) at 17.10 min, α-muurolene (4) at 15.32 min, and γ-muurolene (5) at 14.76 min, compared with the heat-denatured enzyme as control (Fig. 3, Electronic supplementary material). This is detected and characterized by GC–MS following comparison to the standards included in the database. The product peak at 18.68 min generated the dominant mass segments at m/z 161, 119, 204 and 105 perfectly matching δ-cadinol (also torreyol) in mass spectra of the database and the authentic δ-cadinol in publications [13, 25] (Electronic supplementary material).Fig. 2


Identification and Characterization of a δ -Cadinol Synthase Potentially Involved in the Formation of Boreovibrins in Boreostereum vibrans of Basidiomycota
GC–MS analyses of products formed by the recombinant BvCS and the heat-denatured enzyme as control, respectively, with FPP as substrate. Total ion chromatograms (left) and the corresponding mass spectra (right) illustrate the product peaks at 17.10 and 18.68 min matching germacrene D-4-ol and δ-cadinol, respectively, in mass fragmentation patterns included in the database
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Fig3: GC–MS analyses of products formed by the recombinant BvCS and the heat-denatured enzyme as control, respectively, with FPP as substrate. Total ion chromatograms (left) and the corresponding mass spectra (right) illustrate the product peaks at 17.10 and 18.68 min matching germacrene D-4-ol and δ-cadinol, respectively, in mass fragmentation patterns included in the database
Mentions: Next, functional expression in pET32a+/Escherichia coli BL21(DE3) was conducted to confirm the catalytic activity of BvCS. Soluble protein expression was achieved at 15 °C for 22 h with 0.1 mM IPTG (isopropyl-β-d-thiogalactopyranoside), as determined by SDS-PAGE analysis (Figure S1 in Electronic supplementary material). The crude enzyme was assayed for sesquiterpene synthase activity using 1 as a substrate under optional condition as described in Sect. 3. Based on GC–MS analyses, major product peak at 18.68 min (retention time) and minor products were observed for crude BvCS, compared with the empty vector as control (Electronic supplementary material). The purified recombinant protein was used for further characterization. After purification under native condition on the Ni–NTA Agarose, the analysis of the elute on SDS-PAGE led to detection of the main band corresponding exactly to the predicted size of the recombinant BvCS protein of approximately 61 kDa (Fig. 2). When incubated with FPP, the purified BvCS made δ-cadinol (6) as major product at 18.68 min, and minor products including germacrene D-4-ol (2) at 17.10 min, α-muurolene (4) at 15.32 min, and γ-muurolene (5) at 14.76 min, compared with the heat-denatured enzyme as control (Fig. 3, Electronic supplementary material). This is detected and characterized by GC–MS following comparison to the standards included in the database. The product peak at 18.68 min generated the dominant mass segments at m/z 161, 119, 204 and 105 perfectly matching δ-cadinol (also torreyol) in mass spectra of the database and the authentic δ-cadinol in publications [13, 25] (Electronic supplementary material).Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Abstract: 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.

Graphical abstract: A new sesquiterpene synthase gene was cloned from the basidiomycete fungus Boreostereum vibrans and heterologously expressed in E. coli. The purified recombinant enzyme gave δ-cadinol as its principal product from farnesyl diphosphate and thereby identified as a δ-cadinol synthase (BvCS).

Electronic supplementary material: The online version of this article (doi:10.1007/s13659-016-0096-4) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus