Abstract 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.

Sea cucumbers are highly valued for their bioactive constituents, particularly triterpenoid saponins, which exhibit diverse pharmacological properties. The complex amphiphilic nature of saponins poses significant challenges in their extraction and purification. Their structural diversity and amphiphilicity demand carefully optimized protocols to maximize yield and purity. Moreover, the variability of saponin profiles between different sea cucumber species and tissues necessitates reproducible methods that can be scaled from small laboratory samples to larger quantities suitable for industrial applications. This chapter describes comprehensive, reproducible protocols for both small- and large-scale extraction of saponins from sea cucumber tissues, followed by chromatographic separation and bioactivity-guided fractionation. Subsequently, analytical techniques such as thin-layer chromatography (TLC), yeast growth inhibition assays, and liquid chromatography-mass spectrometry (LC-MS) are detailed. These detailed extraction, purification, and analytical protocols facilitate downstream applications such as structural characterization, quantification, and functional studies of saponins.

ABSTRACT Cereal crops rely on a wide array of specialized metabolites to defend themselves against microbial pathogens. Here, using a combination of genome-wide analysis, heterologous pathway reconstruction, structural elucidation and in planta validation, we identify two pathogen-induced diterpenoid pathways in wheat that produce diterpenoids: the new glycosylated diterpenes aspisoside A and aspisoside B, and the diterpene alcohols scutenol A and scutenol B. The pathogen-responsive nature of these pathways, together with their antimicrobial activity are consistent with a likely defensive role in wheat. These compounds are produced by two biosynthetic gene clusters, each encoding a discrete pathway, so revealing organizational separation of wheat diterpenoid-based chemical defenses. The aspisoside-producing gene cluster is syntenic with diterpenoid phytoalexin-producing clusters in rice and barley, for momilactone and hordedane biosynthesis, respectively, yet gives rise to structurally distinct phytoalexins in wheat. The scutenol cluster is syntenic with currently uncharacterized predicted biosynthetic gene clusters in barley, oat, and Brachypodium . These findings establish diterpene glycosides as a previously unrecognized component of wheat defense chemistry and provide new insights into the chemical diversification of defense-related biosynthetic gene clusters within the Poaceae.

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.

The Plant Kingdom is collectively capable of making a huge array of structurally diverse natural products. There are currently ∼5000 publicly available plant genome sequences. This represents only ∼0.6% of the estimated 500 000 species of higher plants on the planet. Even so, we are already overwhelmed with a mass of uncharacterised sequence data. In this review, we discuss the challenges and opportunities for decoding this growing body of information with the ultimate aim of harnessing the DNA-encoded chemical engineering capability of the Plant Kingdom to make next-generation therapeutics. We contextualise these advances with exemplars that connect genome-enabled discovery to therapeutic development, discuss current and future methodologies for the elucidation of enzyme functions using AI methodologies, and highlight the challenges which need to be overcome in order to accelerate the translation from plant sequence to medicines.

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.

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.

The natural products actinonin and matlystatin feature an N-hydroxy-2-pentyl-succinamyl (HPS) chemophore that facilitates metal chelation and confers their metalloproteinase inhibitory activity. Actinonin is the most potent natural inhibitor of peptide deformylase (PDF) and exerts antimicrobial and herbicidal bioactivity by disrupting protein synthesis. Here, we used a genomics-led approach to identify candidate biosynthetic gene clusters (BGCs) hypothesized to produce HPS-containing natural products. We show that one of these BGCs is on the pathogenicity megaplasmid of the plant pathogen Rhodococcus fascians and produces lydiamycin A, a macrocyclic pentapeptide. The presence of genes predicted to make an HPS-like chemophore informed the structural recharacterization of lydiamycin via NMR and crystallography to show that it features a rare 2-pentyl-succinyl chemophore. We demonstrate that lydiamycin A inhibits bacterial PDF in vitro and show that a cluster-situated PDF gene confers resistance to lydiamycin A, representing an uncommon self-immunity mechanism associated with the production of a PDF inhibitor. In planta competition assays showed that lydiamycin enhances the fitness of R. fascians during plant colonization. This study highlights how a BGC can inform the structure, biochemical target, and ecological function of a natural product.

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.

Plants produce diverse specialized metabolites with important ecological functions. It has recently become apparent that the genes for many of these pathways are not dispersed in plant genomes, but rather are arranged like beads on a string in biosynthetic gene clusters (BGCs). Pathways encoded by BGCs are as a rule dedicated linear pathways that do not form parts of wider metabolic networks. In contrast, the genes for the biosynthesis of widely distributed more ancestral metabolites such as carotenoids and anthocyanins are not clustered. Little is known about how these more recently evolved clustered pathways interact with general plant metabolism. We recently characterized a 12-gene BGC for the biosynthesis of the antimicrobial defense compound avenacin A-1, a triterpene glycoside produced by oats. Avenacin A-1 is acylated with the fluorophore N-methyl anthranilate and confers bright blue fluorescence of oat root tips under ultraviolet light. Here, we exploit a suite of >100 avenacin-deficient mutants identified by screening for reduced root fluorescence to identify genes required for the function of this paradigm BGC. Using a combination of mutant chemotyping, biochemical and molecular analysis, and genome resequencing, we identify two nonclustered genes (Sad4 and Pal2) encoding enzymes that synthesize the donors required for avenacin glycosylation and acylation (recruited from the phenylpropanoid and tryptophan pathways). Our finding of these Cluster Auxiliary Enzymes (CAEs) provides insights into the interplay between general plant metabolism and a newly evolved lineage-specific BGC.

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.

Steroidal alkaloids are FDA-approved drugs (e.g., Zytiga) and promising drug candidates/leads (e.g., cyclopamine); yet many of the ≥ 697 known steroidal alkaloid natural products remain underutilized as drugs because it can be challenging to scale their biosynthesis in their producing organisms. Cyclopamine is a steroidal alkaloid produced by corn lily (Veratrum spp.) plants, and it is an inhibitor of the Hedgehog (Hh) signaling pathway. Therefore, cyclopamine is an important drug candidate/lead to treat human diseases that are associated with dysregulated Hh signaling, such as basal cell carcinoma and acute myeloid leukemia. Cyclopamine and its semi-synthetic derivatives have been studied in (pre)clinical trials as Hh inhibitor-based drugs. However, challenges in scaling the production of cyclopamine have slowed efforts to improve its efficacy and safety profile through (bio)synthetic derivatization, often limiting drug development to synthetic analogs of cyclopamine such as the FDA-approved drugs Odomzo, Daurismo, and Erivedge. If a platform for the scalable and sustainable production of cyclopamine were established, then its (bio)synthetic derivatization, clinical development, and, ultimately, widespread distribution could be accelerated. Ongoing efforts to achieve this goal include the biosynthesis of cyclopamine in Veratrum plant cell culture and the semi-/total chemical synthesis of cyclopamine. Herein, this work advances efforts towards a promising future approach: the biosynthesis of cyclopamine in engineered microorganisms. We completed the heterologous microbial production of verazine (biosynthetic precursor to cyclopamine) from simple sugars (i.e., glucose and galactose) in engineered Saccharomyces cerevisiae (S. cerevisiae) through the inducible upregulation of the native yeast mevalonate and lanosterol biosynthetic pathways, diversion of biosynthetic flux from ergosterol (i.e., native sterol in S. cerevisiae) to cholesterol (i.e., biosynthetic precursor to verazine), and expression of a refactored five-step verazine biosynthetic pathway. The engineered S. cerevisiae strain that produced verazine contains eight heterologous enzymes sourced from seven different species. Importantly, S. cerevisiae-produced verazine was indistinguishable via liquid chromatography-mass spectrometry from both a commercial standard (Veratrum spp. plant-produced) and Nicotiana benthamiana-produced verazine. To the best of our knowledge, this is the first report describing the heterologous production of a steroidal alkaloid in an engineered yeast. Verazine production was ultimately increased through design-build-test-learn cycles to a final titer of 83 ± 3 μg/L (4.1 ± 0.1 μg/g DCW). Together, this research lays the groundwork for future microbial biosynthesis of cyclopamine, (bio)synthetic derivatives of cyclopamine, and other steroidal alkaloid natural products.

Genomics-based predictions indicate that plants harbor the ability to make a vast array of as yet undiscovered chemistry. Recent advances open up the potential to harness this capability at unprecedented scale for the discovery and development of new therapeutics.

Plants are chemical engineers par excellence. Collectively they make a vast array of structurally diverse specialized metabolites. The raw materials for building new pathways (genes encoding biosynthetic enzymes) are commonly recruited directly or indirectly from primary metabolism. Little is known about how new metabolic pathways and networks evolve in plants, or what key nodes contribute to branches that lead to the biosynthesis of diverse chemicals. Here we review the molecular mechanisms underlying the generation of biosynthetic branchpoints. We also consider examples in which new metabolites are formed through the joining of precursor molecules arising from different biosynthetic routes, a scenario that greatly increases both the diversity and complexity of specialized metabolism. Given the emerging importance of metabolic gene clustering in helping to identify new enzymes and pathways, we further cover the significance of biosynthetic gene clusters in relation to metabolic networks and dedicated biosynthetic pathways. In conclusion, an improved understanding of the branchpoints between metabolic pathways will be key in order to be able to predict and illustrate the complex structure of metabolic networks and to better understand the plasticity of plant metabolism. This article is part of the theme issue ‘The evolution of plant metabolism’.

QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure–activity relationships and understanding the mechanisms by which these compounds promote the human immune response.

This chapter reproduces the second horizon scan for bioengineering conducted by the Centre for the Study of Existential Risk in 2020, one of the most significant pieces of horizon scanning that has been undertaken in the field to-date. Identifying the top 20 emergent issues, the authors group them according to a likely timeline for their realisation, and discuss each throughout the chapter. This allows for the most notable issues that may impact the planet and humanity to be tracked, and the most pressing issues to be identified. The early identification of such issues is relevant for researchers, policy-makers, and the general public, providing an opportunity to consider what anticipatory or future action might need to be taken.

Triterpene skeletons, catalyzing by 2,3-oxidosqualene cyclases (OSCs), are essential for synthesis of steroids and triterpenoids. In japonica rice cultivars Zhonghua11, a total of 12 OsOSCs have been found. While the catalytic functions of OsOSC1, 3, 4, 9, and 10 remain unclear, the functions of the other OsOSCs have been well studied. In this study, we conducted a comprehensive analysis of 12 OSC genes within genus Oryza with the aid of 63 genomes from cultivated and wild rice. We found that OSC genes are relatively conserved within genus Oryza with a few exceptions. Collinearity analysis further suggested that, throughout the evolutionary history of genus Oryza, the OSC genes have not undergone significant rearrangements or losses. Further functional analysis of 5 uncharacterized OSCs revealed that OsOSC10 was a friedelin synthase, which affected the development of rice grains. Additionally, the reconstructed ancestral sequences of Oryza OSC3 and Oryza OSC9 had lupeol synthase and poaceatapetol synthase activity, respectively. The discovery of friedelin synthase in rice unlocks a new catalytic path and biological function of OsOSC10. The pan-genome analysis of OSCs within genus Oryza gives insights into the evolutionary trajectory and products diversity of Oryza OSCs.

The natural products actinonin and matlystatin feature an N-hydroxy-2-pentyl-succinamyl (HPS) chemophore that facilitates metal chelation and confers their metalloproteinase inhibitory activity. Actinonin is the most potent natural inhibitor of peptide deformylase (PDF) and exerts antimicrobial and herbicidal bioactivity by disrupting protein synthesis. Here, we used a genomics-led approach to identify candidate biosynthetic gene clusters (BGCs) hypothesised to produce novel HPS-containing natural products. We show that one of these BGCs is on the pathogenicity megaplasmid of the plant pathogen Rhodococcus fascians and produces lydiamycin A, a macrocyclic pentapeptide. The presence of genes predicted to make a HPS-like chemophore informed the structural recharacterisation of lydiamycin via NMR and crystallography to show it features a rare 2-pentyl-succinyl chemophore. We demonstrate that lydiamycin A inhibits bacterial PDF in vitro and show that a cluster-situated PDF gene confers resistance to lydiamycin A, representing a novel self-immunity mechanism associated with the production of a PDF inhibitor. In planta competition assays showed that lydiamycin enhances the fitness of R. fascians during plant colonisation. This study highlights how a BGC can inform the structure, biochemical target and ecological function of a natural product. ### Competing Interest Statement The authors have declared no competing interest.