Biosynthetic pathway of prescription cucurbitacin IIa and high-level production of key triterpenoid intermediates in engineered yeast and tobacco.

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  • Additional Information
    • Source:
      Publisher: Published by the Plant Communications Shanghai Office in association with Cell Press, an imprint of Elsevier Inc Country of Publication: China NLM ID: 101769147 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2590-3462 (Electronic) Linking ISSN: 25903462 NLM ISO Abbreviation: Plant Commun Subsets: MEDLINE
    • Publication Information:
      Original Publication: [Shanghai] : Published by the Plant Communications Shanghai Office in association with Cell Press, an imprint of Elsevier Inc., [2020]-
    • Subject Terms:
      Nicotiana*/genetics ; Nicotiana*/metabolism ; Saccharomyces cerevisiae*/genetics ; Saccharomyces cerevisiae*/metabolism ; Triterpenes*/metabolism ; Biosynthetic Pathways*; Cucurbitacins/genetics ; Cucurbitacins/metabolism ; Plants, Genetically Modified/genetics ; Metabolic Engineering/methods ; Cytochrome P-450 Enzyme System/genetics ; Cytochrome P-450 Enzyme System/metabolism ; Plant Proteins/genetics ; Plant Proteins/metabolism
    • Abstract:
      Cucurbitacin IIa is a triterpenoid isolated exclusively from Hemsleya plants and a non-steroidal anti-inflammatory drug that functions as the main ingredient of prescription Hemslecin capsules and tablets in China. Synthetic biology provides new strategies for production of such valuable cucurbitacins at a large scale; however, the biosynthetic pathway of cucurbitacin IIa has been unknown, and the heterologous production of cucurbitacins in galactose medium has been expensive and low yielding. In this study, we characterized the functions of genes encoding two squalene epoxidases (HcSE1-2), six oxidosqualene cyclases (HcOSC1-6), two CYP450s (HcCYP87D20 and HcCYP81Q59), and an acyltransferase (HcAT1) in cucurbitacin IIa biosynthesis by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana. We achieved high-level production of the key cucurbitacin precursor 11-carbonyl-20β-hydroxy-Cuol from glucose in yeast via modular engineering of the mevalonate pathway and optimization of P450 expression levels. The resulting yields of 46.41 mg/l 11-carbonyl-20β-hydroxy-Cuol and 126.47 mg/l total cucurbitacin triterpenoids in shake flasks are the highest yields yet reported from engineered microbes. Subsequently, production of 11-carbonyl-20β-hydroxy-Cuol by transient gene expression in tobacco resulted in yields of 1.28 mg/g dry weight in leaves. This work reveals the key genes involved in biosynthesis of prescription cucurbitacin IIa and demonstrates that engineered yeast cultivated with glucose can produce high yields of key triterpenoid intermediates. We describe a low-cost and highly efficient platform for rapid screening of candidate genes and high-yield production of pharmacological triterpenoids.
      (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
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    • Contributed Indexing:
      Keywords: 11-carbonyl-20β-hydroxy-Cuol; Hemsleya chinensis; Nicotiana benthamiana; OSC; Saccharomyces cerevisiae; cucurbitacin IIa; oxidosqualene cyclase
    • Accession Number:
      0 (Triterpenes)
      60137-06-6 (Cucurbitacins)
      9035-51-2 (Cytochrome P-450 Enzyme System)
      0 (Plant Proteins)
    • Publication Date:
      Date Created: 20240301 Date Completed: 20240611 Latest Revision: 20240630
    • Publication Date:
      20240630
    • Accession Number:
      PMC11211238
    • Accession Number:
      10.1016/j.xplc.2024.100835
    • Accession Number:
      38425040