Rapid Combinatorial Coexpression of Biosynthetic Genes by Transient Expression in the Plant Host Nicotiana benthamiana

Chuang, Ling; Franke, Jakob

doi:10.1007/978-1-0716-2273-5_20

Cited by 9 publications

(9 citation statements)

References 31 publications

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“…N. benthamiana is a popular tool for elucidating plant biosynthetic pathways, thanks to the capacity to rapidly co-express multiple gene candidates without the need for multiple selection markers. 27,30,31 Eighteen of the 20 oxidase gene candidates were successfully cloned into Agrobacterium tumefaciens and co-infiltrated with AaOSC2, AaCYP71CD4, and AaCYP71BQ17 into N. benthamiana. Crude extracts of the co-expressing plants were then analyzed by LC-MS to look for consumption of melianol ( 8) and the production of new compounds.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Post-Cyclization Skeletal Rearrangements in Plant Triterpenoid Biosynthesis by a Pair of Branchpoint Isomerases

2023

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Triterpenoids possess potent biological activities, but their polycyclic skeletons are challenging to synthesize. The skeletal diversity of triterpenoids in plants is generated by oxidosqualene cyclases based on epoxide-triggered cationic rearrangement cascades. Normally, triterpenoid skeletons then remain unaltered during subsequent tailoring steps. In contrast, the highly modified triterpenoids found in Sapindales plants imply the existence of post-cyclization skeletal rearrangement enzymes that have not yet been found. We report here a biosynthetic pathway in Sapindales plants for the modification of already cyclized tirucallane triterpenoids, controlling the pathway bifurcation between different plant triterpenoid classes. Using a combination of bioinformatics, heterologous expression in plants and chemical analyses, we identified a cytochrome P450 monooxygenase and two isomerases which harness the epoxidation-rearrangement biosynthetic logic of triterpene cyclizations for modifying the tirucallane scaffold. The two isomerases share the same epoxide substrate made by the cytochrome P450 monooxygenase CYP88A154, but generate two different rearrangement products, one containing a cyclopropane ring. Our findings reveal a process for skeletal rearrangements of triterpenoids in nature that expands their scaffold diversity after the initial cyclization. In addition, the enzymes described here are crucial for the biotechnological production of limonoid, quassinoid, apoprotolimonoid, and glabretane triterpenoids.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Post-Cyclization Skeletal Rearrangements in Plant Triterpenoid Biosynthesis by a Pair of Branchpoint Isomerases

2023

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“…Nicotiana benthamiana LAB strain ( Bally et al, 2018 ) was grown from seeds in a greenhouse with 11–16 h of illumination per day and at a temperature between 21 and 23°C as described previously ( Chuang and Franke, 2022 ).…”

Section: Methodsmentioning

confidence: 99%

“…Our method for transient expression is described by Chuang and Franke (2022) . In short, selected gene candidates and booster genes were cloned into the plant expression vector pEAQ-HT ( Sainsbury et al, 2009 ) using in-fusion cloning or into the improved vector pHREAC ( Peyret et al, 2019 ) using Golden Gate cloning.…”

Section: Methodsmentioning

confidence: 99%

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Identification of early quassinoid biosynthesis in the invasive tree of heaven (Ailanthus altissima) confirms evolutionary origin from protolimonoids

et al. 2022

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The tree of heaven, Ailanthus altissima (MILL.) SWINGLE, is a globally invasive plant known to secrete allelopathic metabolites called quassinoids. Quassinoids are highly modified triterpenoids. So far, nothing has been known about the biochemical basis of quassinoid biosynthesis. Here, based on transcriptome and metabolome data of Ailanthus altissima, we present the first three steps of quassinoid biosynthesis, which are catalysed by an oxidosqualene cyclase and two cytochrome P450 monooxygenases, resulting in the formation of the protolimonoid melianol. Strikingly, these steps are identical to the first steps of the biosynthesis of limonoids, structurally different triterpenoids from sister plant families within the same order Sapindales. Our results are therefore not only important to fully understand the biosynthesis of complex triterpenoids in plants, but also confirm the long-standing hypothesis that quassinoids and limonoids share an evolutionary origin. In addition, our transcriptome data for Ailanthus altissima will be beneficial to other researchers investigating the physiology and ecology of this invasive tree.

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“…The discovery of biosynthetic genes in plants often involves the screening of large pools of gene candidates derived from sequencing studies [93][94][95][96]. Hence, efficient approaches are needed to select the most promising gene candidates, particularly for large gene families such as CYPs.…”

Section: Phylogenetic Distribution Of Triterpenoid-and Steroid-modify...mentioning

confidence: 99%

Cytochrome P450 monooxygenase-mediated tailoring of triterpenoids and steroids in plants

Malhotra¹,

Franke²

2022

Beilstein J. Org. Chem.

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The cytochrome P450 monooxygenase (CYP) superfamily comprises hemethiolate enzymes that perform remarkable regio- and stereospecific oxidative chemistry. As such, CYPs are key agents for the structural and functional tailoring of triterpenoids, one of the largest classes of plant natural products with widespread applications in pharmaceuticals, food, cosmetics, and agricultural industries. In this review, we provide a full overview of 149 functionally characterised CYPs involved in the biosynthesis of triterpenoids and steroids in primary as well as in specialised metabolism. We describe the phylogenetic distribution of triterpenoid- and steroid-modifying CYPs across the plant CYPome, present a structure-based summary of their reactions, and highlight recent examples of particular interest to the field. Our review therefore provides a comprehensive up-to-date picture of CYPs involved in the biosynthesis of triterpenoids and steroids in plants as a starting point for future research.

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Rapid Combinatorial Coexpression of Biosynthetic Genes by Transient Expression in the Plant Host Nicotiana benthamiana

Cited by 9 publications

References 31 publications

Post-Cyclization Skeletal Rearrangements in Plant Triterpenoid Biosynthesis by a Pair of Branchpoint Isomerases

Post-Cyclization Skeletal Rearrangements in Plant Triterpenoid Biosynthesis by a Pair of Branchpoint Isomerases

Identification of early quassinoid biosynthesis in the invasive tree of heaven (Ailanthus altissima) confirms evolutionary origin from protolimonoids

Cytochrome P450 monooxygenase-mediated tailoring of triterpenoids and steroids in plants

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