Regulation of ferruginol and cryptotanshinone biosynthesis in cell suspension cultures of Salvia miltiorrhiza

Miyasaka, Hitoshi; Nasu, Masao; Yamamoto, Toshihiko; Endo, Yukiko; Yoneda, Kyohei

doi:10.1016/0031-9422(86)88014-1

Cited by 20 publications

(7 citation statements)

References 5 publications

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“…To further strengthen the relevance of CYP76AH1 to ferruginol and tanshinone biosynthesis, we investigated ferruginol accumulation patterns. It has been reported that ferruginol accumulates along with tanshinones in S. miltiorrhiza suspension cells (29), as well as hairy roots (30). Tissue-specific analysis of whole plants demonstrated the expected accumulation of ferruginol in the rhizome, whereas none was detected in above-ground tissues, consistent with the CYP76AH1 transcript accumulation pattern noted above.…”

Section: Coincidence Of Cyp76ah1 Transcription and Ferruginol Accumulsupporting

confidence: 85%

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CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts

Guo

Zhou

Hillwig

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

325

275

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Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the main lipophilic bioactive components of the Chinese medicinal herb danshen (Salvia miltiorrhiza). Whereas the diterpene synthases responsible for the conversion of (E,E,E)-geranylgeranyl diphosphate into the abietane miltiradiene, a potential precursor to tanshinones, have been recently described, molecular characterization of further transformation of miltiradiene remains unavailable. Here we report stableisotope labeling results that demonstrate the intermediacy of miltiradiene in tanshinone biosynthesis. We further use a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrate that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo. We then build upon the previous establishment of miltiradiene production in Saccharomyces cerevisiae, with incorporation of CYP76AH1 and phyto-CYP reductase genes leading to heterologous production of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants including danshen, the results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids. Moreover, these results should facilitate the construction of microbial cell factories for the production of phytoterpenoids.phytoterpenoids biosynthesis | gene discovery | synthetic pathway | metabolic engineering

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Section: Coincidence Of Cyp76ah1 Transcription and Ferruginol Accumulsupporting

confidence: 85%

“…Isolation and Quantification of Ferruginol. Analysis of ferruginol levels was carried out much as previously described (29). Briefly, dried and powered hairy roots were extracted with ethyl acetate, with fivefold concentration of these extracts used for ferruginol quantification by GC analysis.…”

Section: Methodsmentioning

confidence: 99%

CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts

Guo

Zhou

Hillwig

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

325

275

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“…There are reports on the production of a diterpene, ferruginol as a predominant secondary metabolite and cryptotanshinone, but it is only in trace amounts in cell suspension cultures. 16,17) Also, Miyasaka et al, 18) examined the biogenetic capability of undifferentiated cultured cells of S. miltiorrhiza. They observed that two triterpenes, oleanolic acid and ursolic acid, were major components in the aerial parts of intact plants while ferruginol was only found in roots.…”

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confidence: 99%

Isolation and Quantitative Analysis of Cryptotanshinone, an Active Quinoid Diterpene Formed in Callus of <i>Salvia miltiorrhiza</i> B<small>UNGE</small>

Mulabagal

Nalawade

et al. 2003

Biological & Pharmaceutical Bulletin

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Effect of N(6)-benzyladenine (BA) on tanshinone formation in callus cultures of Salvia miltiorrhiza was examined in an attempt to increase the productivity of the medicinal compound, cryptotanshinone. Primary callus was induced by culturing leaf explants on Murashige and Skoog's (MS) basal medium supplemented with 1.0 mg l(-1) of 2,4-dichlorophenoxyacetic acid (2,4-D) in darkness. The callus proliferated further on MS basal medium containing 1.0 mg l(-1) 2,4-D and 0.5 mg l(-1) BA and was analyzed for cryptotanshinone by high performance liquid chromatography (HPLC). The HPLC results indicated that it contained small amounts of cryptotanshinone (0.26+/-0.05 mg/g dry wt). Omission of 2,4-D from the medium resulted in a marked increase in the content of cryptotanshinone in callus. The HPLC analysis revealed that the content of cryptotanshinone in the callus cultured on the MS basal medium supplemented with 0.1, 0.2, 0.5, 1.0, and 2.0 mg l(-1) of BA was significantly higher than the marketed crude drug (processed underground parts of S. miltiorrhiza). Maximum yield of cryptotanshinone (4.59+/-0.09 mg/g dry wt) was observed in the callus cultured on MS basal medium supplemented with 0.2 mg l(-1) BA for 60 d. Cryptotanshinone was isolated from callus through silica gel column chromatography followed by preparative TLC and characterized based on NMR and mass spectral data.

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“…miltiorrhiza has been examined. 83 Evidence for the structure and absolute configuration of pisiferdiol (1 14) (from Charnaecyparis pisifera) has been reported in full84 whilst the antibiotic and anti-tumour activities of pisiferic acid have been examined. 85 Extracts of Euphorbia acaulis are used in the treatment of inflammatory diseases in some folk medicines.…”

Section: Abietanesmentioning

confidence: 99%

Diterpenoids

Hanson¹

1988

Nat. Prod. Rep.

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Regulation of ferruginol and cryptotanshinone biosynthesis in cell suspension cultures of Salvia miltiorrhiza

Cited by 20 publications

References 5 publications

CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts

CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts

Isolation and Quantitative Analysis of Cryptotanshinone, an Active Quinoid Diterpene Formed in Callus of <i>Salvia miltiorrhiza</i> B<small>UNGE</small>

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