Metabolic engineering of cucurbitacins in Cucurbita pepo hairy roots

Abstract

In this paper we show that metabolic engineering in Cucurbita pepo hairy roots can be used to both effectively increase and modify cucurbitacins. Cucurbitacins are highly-oxygenated triterpenoids originally described in the Cucurbitaceae family, but have since been found in 15 taxonomically distant plant families. Cucurbitacin B, D, E and I are the most widespread amongst the Cucurbitaceae and they have both important biological and pharmacological activities. In this study C. pepo hairy roots were used as a platform to boost production and alter the structures of the afore mentioned cucurbitacins by metabolic engineering to potentially provide new or more desirable bioactivities. We report that the ability to induce cucurbitacin biosynthesis by basic Helix-Loop-Helix transcription factors is partially conserved within the Cucurbitaceae and therefore can potentially be used as a biotechnological tool to increase cucurbitacins in several genera of this family. Additionally, overexpression of a novel acyltransferase from cucurbitacin producing Iberis amara generates a hitherto undescribed acetylation at the C3-hydroxyl group of the cucurbitadienol backbone. While overexpression of the cytochromes P450 CsCYP88L2 and McCYP88L7 from Cucumis sativus and Momordica charantia (respectively), results in accumulation of new spectral feature as revealed by High resolution liquid chromatography mass spectroscopy analysis; the m/z of the new peak supports it might be a cucurbitacin hydroxylated at the C19 position in C. pepo hairy roots. Finally, this paper is a case study of how hairy roots can be used to metabolically engineer and introduce novel modifications in metabolic pathways that have not been fully elucidated.

Integrated Secondary Metabolomic and Antioxidant Ability Analysis Reveals the Accumulation Patterns of Metabolites in Momordica charantia L. of Different Cultivars

“…These compounds are organic compounds known as triterpene saponins and can regulate the host's immune function [51]. Cucurbitacin B and E are widely distributed in Cucurbitaceae with important biological and pharmacological activities [52]. Cucurbitacin E (CuE) has anti-inflammatory and anti-tumorigenic properties, which are related to its effect on the cellular cytoskeleton, mitotic pathways, and cellular autophagy [53].…”

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

“…Currently, cucurbitacin biosynthetic pathways are gradually being revealed in some cucurbitaceous plants ( Shang et al., 2014 ; Zhou et al., 2016b ; Takase et al., 2019b ; Almeida et al., 2022 ). In Cucurbita pepo , the formation of 2,3-oxidosqualene and 2,3:22,23-diepoxysqualene is mediated by three squalene epoxidases (SEs) ( Dong et al., 2018 ).…”

“…Cucurbitacins, valued for their diverse biological activities, face challenges in pharmaceutical applications due to low plant content and complex extraction procedures. Although synthetic biology shows potential for production of high-value natural drugs ( Hesami et al., 2022 ; Chu et al., 2023 ; Li et al., 2023 ), heterologous biosynthesis of cucurbitacins has been limited to the precursor Cuol ( Shang et al., 2014 ; Almeida et al., 2022 ). Challenges include unknown enzymes for modification and scarce intermediate compounds for testing of enzyme activity.…”

“…A previous study reported the construction of two engineered yeast strains, EY10 and EGY48-CpCPQ (CBS in C. pepo ), for production of Cuol (related output not reported) ( Shang et al., 2014 ; Zhou et al., 2016b ; Almeida et al., 2022 ). The construction strategy was to reuse the inducible pGAL promoter (GAL1 or GAL10), which has problems such as insufficient promoter strength, interference with galactose metabolism, and increased complexity of the in vivo expression system ( Da Silva and Srikrishnan, 2012 ; Tang et al., 2020 ).…”