Michael J. Stephenson

Oxidosqualene cyclases (OSCs) catalyse one of nature's most intricate enzyme reactions, converting the linear precursor 2,3-oxidosqualene into an array of cyclic triterpene scaffolds through sequential carbocation cascades. Predicting OSC function based on sequence is challenging beyond broad family-level classification. Here, we develop a structure-based computational framework to identify amino acid determinants of OSC product specificity. Using 169 functionally characterised OSCs, we deploy a multifaceted approach combining differential conservation along with structural information, physico-chemical properties of amino acids and binding pocket electrostatics in order to understand the determinants of product specificity. Using Arabidopsis thaliana cycloartenol synthase AtCAS as a model, we then validate our predictions through targeted mutagenesis, achieving stepwise reprogramming towards the protosteryl-type products cucurbitadienol and lanosterol, including complete product switches. Molecular dynamics simulations support a mechanism in which subtle pocket remodelling alters active-site volume, water access and proton-elimination chemistry. These findings provide a blueprint for OSC engineering.

Summary: Lanosterol synthase (LAS) and cycloartenol synthase (CAS) use 2,3‐oxidosqualene as their substrate to produce lanosterol and cycloartenol for biosynthesis of essential sterols in animals and fungi (lanosterol) and plants (cycloartenol), respectively. Although LASs are also found in plants, their evolutionary origin and the question of whether their catalytic mechanism aligns with animal/fungal LAS remain unresolved. In this study, we use QM/MM MD simulations to reveal the atomic‐level catalytic mechanisms of LASs from all three lineages. Our simulations reveal a dominant reaction path from a C8 cation intermediate to lanosterol for plant LASs, which is different from the reaction path for animal and fungal LASs. Phylogenetic and microcollinearity analyses demonstrate that plant LASs evolved from an ancestral plant CAS and are restricted to eudicots. Combining these findings with mechanistic insights, we demonstrate that plant LASs have undergone convergent evolution with their animal and fungal counterparts, independently acquiring a role in root development. This study establishes the lanosterol biosynthesis pathway as a remarkable example of convergent evolution across eukaryotes, having arisen independently in plants, animals, and fungi. By defining the role of plant LAS in root development, this research provides key molecular targets for breeding stress‐resilient crops.

The differential cyclization and rearrangement of 2,3-oxidosqualene controlled by oxidosqualene cyclases (OSCs) represents one of the most complex single enzyme transformations in nature and gives rise to a vast array of triterpenoid diversity in the plant kingdom. Here we systematically mine 599 plant genomes representing 387 species and investigate OSC diversity across different plant lineages. From the OSC sequences identified, 20 were selected for functional evaluation. Through analysis of these enzymes, we discover product profiles within clades previously believed to be functionally conserved and OSCs producing triterpenes for which no enzymatic source was known. We also discover OSCs with product profiles that yield mechanistic insights into the control of specific reaction pathways. Our study reveals lineage-specific blooms of OSC subgroups suggestive of adaptation to different environmental niches, opens up previously inaccessible chemistry and provides a framework for systematic investigations of metabolic diversification and underlying enzymatic mechanisms in the plant kingdom.

Sesquiterpene coumarins are primarily produced by plants of the Apiaceae and Asteraceae families. Farnesylation of 7-hydroxycoumarins such as umbelliferone, scopoletin or isofraxidin yield linear 7-farnesyloxycoumarins that are converted to various cyclic sesquiterpene coumarins by sesquiterpene coumarin synthases (cyclases). The terminal double bond of the linear 7-farnesyloxycoumarins is epoxidized by a sesquiterpene coumarin epoxidase. The diverse 7-(10′,11′-oxidofarnesyloxy)-coumarins produced are protonated by various sesquiterpene coumarin synthases to generate a carbocation that initiates cyclization of the farnesyl moiety (A process analogous to the carbocation cascades observed with sesquiterpene synthases and other cyclases involved in the biosynthesis of additional terpene classes, such as the triterpenes). These reaction mechanisms typically include Wagner-Meerwein rearrangements, such as hydride, methyl, and other alkyl shifts, but can also involve more complex processes including Grob fragmentations. Around 260 sesquiterpene coumarins based on 7-farnesyloxycoumarins have been described, but essentially nothing is known about the biosynthetic enzymes involved, i.e ., farnesyltransferase, sesquiterpene coumarin epoxidase and synthase. In this review, putative reaction pathways for formation of the carbon skeletons of all known 7-farnesyloxycoumarins-derived sesquiterpene coumarins are presented.

Sesquiterpene coumarins are primarily produced by plants of the Apiaceae and Asteraceae families. Farnesylation of 7-hydroxycoumarins such as umbelliferone, scopoletin or isofraxidin yield linear 7-farnesyloxycoumarins that are converted to various cyclic sesquiterpene coumarins by sesquiterpene coumarin synthases (cyclases). The terminal double bond of the linear 7-farnesyloxycoumarins is epoxidized by a sesquiterpene coumarin epoxidase. The diverse 7-(10′,11′-oxidofarnesyloxy)-coumarins produced are protonated by various sesquiterpene coumarin synthases to generate a carbocation that initiates cyclization of the farnesyl moiety (A process analogous to the carbocation cascades observed with sesquiterpene synthases and other cyclases involved in the biosynthesis of additional terpene classes, such as the triterpenes). These reaction mechanisms typically include Wagner-Meerwein rearrangements, such as hydride, methyl, and other alkyl shifts, but can also involve more complex processes including Grob fragmentations. Around 260 sesquiterpene coumarins based on 7-farnesyloxycoumarins have been described, but essentially nothing is known about the biosynthetic enzymes involved, i.e ., farnesyltransferase, sesquiterpene coumarin epoxidase and synthase. In this review, putative reaction pathways for formation of the carbon skeletons of all known 7-farnesyloxycoumarins-derived sesquiterpene coumarins are presented.

The Chilean soapbark tree ( Quillaja saponaria ) produces soap-like molecules called QS saponins that are important vaccine adjuvants. These highly valuable compounds are sourced by extraction from the bark, and their biosynthetic pathway is unknown. Here, we sequenced the Q. saponaria genome. Through genome mining and combinatorial expression in tobacco, we identified 16 pathway enzymes that together enable the production of advanced QS pathway intermediates that represent a bridgehead for adjuvant bioengineering. We further identified the enzymes needed to make QS-7, a saponin with excellent therapeutic properties and low toxicity that is present in low abundance in Q. saponaria bark extract. Our results enable the production of Q. saponaria vaccine adjuvants in tobacco and open the way for new routes to access and engineer natural and new-to-nature immunostimulants.

Triterpenes with complex scaffold modifications are widespread in the plant kingdom. Limonoids are an exemplary family that are responsible for the bitter taste in citrus (e.g., limonin) and the active constituents of neem oil, a widely used bioinsecticide (e.g., azadirachtin). Despite the commercial value of limonoids, a complete biosynthetic route has not been described. We report the discovery of 22 enzymes, including a pair of neofunctionalized sterol isomerases, that catalyze 12 distinct reactions in the total biosynthesis of kihadalactone A and azadirone, products that bear the signature limonoid furan. These results enable access to valuable limonoids and provide a template for discovery and reconstitution of triterpene biosynthetic pathways in plants that require multiple skeletal rearrangements and oxidations.

Summary: Plants produce an array of specialized metabolites with important ecological functions. The mechanisms underpinning the evolution of new biosynthetic pathways are not well‐understood. Here, we exploit available genome sequence resources to investigate triterpene biosynthesis across the Brassicaceae. Oxidosqualene cyclases (OSCs) catalyze the first committed step in triterpene biosynthesis. Systematic analysis of 13 sequenced Brassicaceae genomes was performed to identify all OSC genes. The genome neighbourhoods (GNs) around a total of 163 OSC genes were investigated to identify Pfam domains significantly enriched in these regions. All‐vs‐all comparisons of OSC neighbourhoods and phylogenomic analysis were used to investigate the sequence similarity and evolutionary relationships of the numerous candidate triterpene biosynthetic gene clusters (BGCs) observed. Functional analysis of three representative BGCs was carried out and their triterpene pathway products were elucidated. Our results indicate that plant genomes are remarkably plastic, and that dynamic GNs generate new biosynthetic pathways in different Brassicaceae lineages by shuffling the genes encoding a core palette of triterpene‐diversifying enzymes, presumably in response to strong environmental selection pressure. These results illuminate a genomic basis for diversification of plant‐specialized metabolism through natural combinatorics of enzyme families, which can be mimicked using synthetic biology to engineer diverse bioactive molecules.

Limonoids are natural products made by plants belonging to the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, contribution to bitterness in citrus fruits, and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree ( Azadirachta indica ). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranortriterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of 4 carbons with associated furan ring formation, by an as yet unknown mechanism. Here we have mined genome and transcriptome sequence resources for 3 diverse limonoid-producing species ( A. indica , Melia azedarach , and Citrus sinensis ) to elucidate the early steps in limonoid biosynthesis. We identify an oxidosqualene cyclase able to produce the potential 30-carbon triterpene scaffold precursor tirucalla-7,24-dien-3β-ol from each of the 3 species. We further identify coexpressed cytochrome P450 enzymes from M. azedarach (MaCYP71CD2 and MaCYP71BQ5) and C. sinensis (CsCYP71CD1 and CsCYP71BQ4) that are capable of 3 oxidations of tirucalla-7,24-dien-3β-ol, resulting in spontaneous hemiacetal ring formation and the production of the protolimonoid melianol. Our work reports the characterization of protolimonoid biosynthetic enzymes from different plant species and supports the notion of pathway conservation between both plant families. It further paves the way for engineering crop plants with enhanced insect resistance and producing high-value limonoids for pharmaceutical and other applications by expression in heterologous hosts.

A well-established dichotomy in the biogenesis of polycyclic triterpenes is outlined and contrasted with a recently reported novel pentacyclic scaffold.