Anne Osbourn

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.

Plant specialized metabolites encompass a diverse array of bioactive compounds with significant pharmaceutical, nutraceutical, and industrial relevance, and they play vital roles in plant defense, signaling, and ecological interactions. However, their complex biosynthetic pathways and low natural yields present a major bottleneck for commercial exploitation. Synthetic biology provides new tools for the rational design and engineering of metabolic pathways to enhance specialized metabolite biosynthesis in plants as well as in microbes such as E. coli and yeast. Here, we report a synthetic biology framework utilizing Golden Gate assembly (GGA) for the modular construction and optimization of plant metabolic pathways. Golden Gate enables scarless, directional assembly of multigene constructs with high efficiency, ideal for refactoring biosynthetic gene clusters and for the systematic design of complex metabolic networks. Golden Gate assembly can efficiently join up to 10-20 fragments simultaneously in a single reaction, a significant improvement over traditional molecular biology cloning methods. We developed and validated a standardized module of promoters, untranslated regions (UTRs), coding sequences, and regulatory elements compatible with plant transformation systems. Using this platform, we successfully reconstructed the biosynthetic pathway for plant specialized metabolites by transient expression in Nicotiana benthamiana, demonstrating significant improvements in construct stability. Our results highlight the potential of Golden Gate as a cornerstone technology for plant metabolic engineering, offering a scalable and versatile strategy to enhance the production of valuable specialized metabolites and set the stage for custom metabolic rewiring aimed at sustainable production of high-value compounds.

Societal Impact Statement: Our current understanding of plants has been shaped by the entwining of different cultures. The Chilean soapbark tree, traditionally valued as a source of natural soap, was shown by serendipitous research in France in the 1900s to produce compounds that can boost the immune response to vaccines. One of these compounds, QS‐21, was approved for use in human vaccines in 2017 and is now valued at >$100,000/g because of its growing importance as a vaccine adjuvant. This review tells the story of the humble soapbark tree and its elevation to fame as the source of one of the most sought‐after naturally made immunostimulants. Summary: The Chilean soapbark tree, Quillaja saponaria Molina, grows in the central part of Chile. As its name suggests, the tree is a natural source of soap. Indeed, the name Quillaja is derived from the indigenous Chilean word ‘küllay’, which means ‘soap’. Soapbark is not the only plant to produce natural soaps. Other examples include the perennial soapwort ( Saponaria officinalis ), which grows in Europe and has historically been used as a source of gentle soap for washing delicate fabrics, including allegedly the Turin shroud; and Sapindus mukorossi (soapberry or soapnut), which grows in temperate and tropical regions of the world. The soapy properties of these plants are due to their production of glycosylated compounds known as saponins. In fact, saponins are found across the Plant Kingdom, not just in species with ‘soap’ in their names, and are one of the largest groups of plant natural products. The major saponin produced by Q. saponaria , which is known as QS‐21, is a potent immunostimulant and has been approved for use in human vaccines. QS‐21, which is the first naturally occurring plant glycoside to be used as an adjuvant, is a highly complex molecule that is produced only by Quillaja . This review covers the history of the soapbark tree, the discovery of QS‐21 and the potential for making the next generation of saponin vaccine adjuvants using engineering biology approaches.

Saponins are a class of natural products composed of an oxidized triterpene core adorned with glycosylations, ultimately giving rise to medicinally important compounds bearing bioactivity that includes, but is not limited to, anti-inflammatory, antimicrobial, antifungal, antiarrhythmic, and immunostimulatory activities. QS-21 is a prominent immunostimulatory saponin and is a critical adjuvant component of several FDA-approved vaccines. One linchpin modification in the biosynthesis and bioactivity of several saponins, including QS-21, is O-d-fucosylation via an ester linkage. In QS-21, the C28-COOH O-d-fucose residue is part of a linear oligosaccharide that is an integral component of the “core pharmacophore” responsible for its immunomodulatory activity. In this work, we performed in-depth in vitro enzymological characterization of two glycosyltransferases involved in C28-COOH O-d-fucosylation during the maturation of two saponin natural products: QsFucT from QS-21 biosynthesis and SvFucT from vaccaroside biosynthesis. QsFucT was previously shown to be a UDP-4-keto-6-deoxy-d-glucosyltransferase; our data reveal that the taxonomically distant SvFucT also functions as a UDP-4-keto-6-deoxy-d-glucosyltransferase and that both glycosyltransferases act on a triterpene acceptor with low-micromolar affinity. Substrate scope studies demonstrate that both enzymes are highly permissive with regard to both the triterpene acceptor and, unexpectedly, the UDP-sugar donor. These data also reveal that the conserved C3-OH branched trisaccharide of QS-21 and other saponins may serve an unusual biosynthetic role in protecting the C23 aldehyde from spurious reduction during biosynthesis. In addition, we crystallized and solved the structures of QsFucT and SvFucT, providing the first structural characterization of 4-keto-6-deoxy-d-glucosyltranferases in the glycosyltransferase family 1 (GT1) class of enzymes and used these structures to explore the importance of conserved residues in the active site. These data suggest that both QsFucT and SvFucT could be leveraged to rapidly explore saponin chemical space and glycodiversify these important medicinal compounds through engineered biosynthesis or in vitro enzymatic synthesis, possibly leading to novel analogs with enhanced physicochemical or pharmacological properties.

Plants produce bioactive compounds as part of their specialized metabolism, with applications in medicine, agriculture, and nutrition. The biosynthesis of a growing number of these specialized metabolites has been found to be encoded in biosynthetic gene clusters (BGCs), creating increasing demand for genome mining tools to automate their detection. plantiSMASH enables the identification of putative plant BGCs through a rule-based approach, available via both command-line and web interfaces. Here, we present plantiSMASH 2.0 ( https://plantismash.bioinformatics.nl/ ), a major update that expands and improves the original framework with revised and additional BGC detection rules (now supporting 12 BGC types), substrate prediction for selected enzyme families, and regulatory analysis through transcription factor binding site detection. The updated plantiSMASH 2.0 database includes 30,423 putative BGCs across 430 genomes. Together, these improvements make plantiSMASH 2.0 a powerful and comprehensive platform for the detection and characterization of plant biosynthetic pathways, supporting and accelerating research in plant specialized metabolism and plant natural product discovery.

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.

Recent developments in single-cell -omic and metabolite imaging technologies and the increasing availability of high-quality genome assemblies are having a transformative impact on the way research is carried out into plant specialised metabolism. Integrating these technologies into pathway discovery projects is therefore highly advantageous. Here, we present a general introduction into methods and workflows in specialised metabolism research. We review a range of recent methodologies, highlighting what they might be used for and common pitfalls which may be encountered. Finally, we provide a practical guide on how these technologies may be incorporated into a specialised metabolic pathway discovery pipeline for researchers who are new to the field.