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Functional characterization of triterpene synthases in Cibotium barometz.

ABSTRACT: Cibotium barometz (Linn.) J. Sm., a tree fern in the Dicksoniaceae family, is an economically important industrial exported plant in China and widely used in Traditional Chinese Medicine. C. barometz produces a range of bioactive triterpenes and their metabolites. However, the biosynthetic pathway of triterpenes in C. barometz remains unknown. To clarify the origin of diverse triterpenes in C. barometz, we conducted de novo transcriptome sequencing and analysis of C. barometz rhizomes and leaves to identify the candidate genes involved in C. barometz triterpene biosynthesis. Three C. barometz triterpene synthases (CbTSs) candidate genes were obtained. All of them were highly expressed in C. barometz rhizomes, consisting of the accumulation pattern of triterpenes in C. barometz. To characterize the function of these CbTSs, we constructed a squalene- and oxidosqualene-overproducing yeast chassis by overexpressing all the enzymes in the MVA pathway under the control of GAL-regulated promoter and disrupted the GAL80 gene in Saccharomyces cerevisiae simultaneously. Heterologous expressing CbTS1, CbTS2, and CbTS3 in engineering yeast strain produced cycloartenol, dammaradiene, and diploptene, respectively. Phylogenetic analysis revealed that CbTS1 belongs to oxidosqualene cyclase, while CbTS2 and CbTS3 belong to squalene cyclase. These results decipher enzymatic mechanisms underlying the origin of diverse triterpene in C. barometz.

SUBMITTER: Ji Z

PROVIDER: S-EPMC10320381 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Functional characterization of triterpene synthases in Cibotium barometz.

Ji Zhongju Z Fan Baolian B Chen Yidu Y Yue Jingyang J Chen Jiabo J Zhang Rongrong R Tong Yi Y Liu Zhongqiu Z Liang Jincai J Duan Lixin L

Synthetic and systems biotechnology 20230625 3

Cibotium barometz (Linn.) J. Sm., a tree fern in the Dicksoniaceae family, is an economically important industrial exported plant in China and widely used in Traditional Chinese Medicine. C. barometz produces a range of bioactive triterpenes and their metabolites. However, the biosynthetic pathway of triterpenes in C. barometz remains unknown. To clarify the origin of diverse triterpenes in C. barometz, we conducted de novo transcriptome sequencing and analysis ...[more]

PMID: 37416896

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Project description:BackgroundSesquiterpene lactones are characteristic metabolites of Asteraceae (or Compositae) which often display potent bioactivities and are sequestered in specialized organs such as laticifers, resin ducts, and trichomes. For characterization of sunflower sesquiterpene synthases we employed a simple method to isolate pure trichomes from anther appendages which facilitated the identification of these genes and investigation of their enzymatic functions and expression patterns during trichome development.ResultsGlandular trichomes of sunflower (Helianthus annuus L.) were isolated, and their RNA was extracted to investigate the initial steps of sesquiterpene lactone biosynthesis. Reverse transcription-PCR experiments led to the identification of three sesquiterpene synthases. By combination of in vitro and in vivo characterization of sesquiterpene synthase gene products in Escherichia coli and Saccharomyces cerevisiae, respectively, two enzymes were identified as germacrene A synthases, the key enzymes of sesquiterpene lactone biosynthesis. Due to the very low in vitro activity, the third enzyme was expressed in vivo in yeast as a thioredoxin-fusion protein for functional characterization. In in vivo assays, it was identified as a multiproduct enzyme with the volatile sesquiterpene hydrocarbon delta-cadinene as one of the two main products with alpha-muuorlene, beta-caryophyllene, alpha-humulene and alpha-copaene as minor products. The second main compound remained unidentified. For expression studies, glandular trichomes from the anther appendages of sunflower florets were isolated in particular developmental stages from the pre- to the post-secretory phase. All three sesquiterpene synthases were solely upregulated during the biosynthetically active stages of the trichomes. Expression in different aerial plant parts coincided with occurrence and maturity of trichomes. Young roots with root hairs showed expression of the sesquiterpene synthase genes as well.ConclusionThis study functionally identified sesquiterpene synthase genes predominantly expressed in sunflower trichomes. Evidence for the transcriptional regulation of sesquiterpene synthase genes in trichome cells suggest a potential use for these specialized cells for the identification of further genes involved in the biosynthesis, transport, and regulation of sesquiterpene lactones.

Dataset Information

Functional characterization of triterpene synthases in Cibotium barometz.

Publications

Functional characterization of triterpene synthases in <i>Cibotium barometz</i>.

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