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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.

Soapwort (Saponaria officinalis) is a flowering plant from the Caryophyllaceae family with a long history of human use as a traditional source of soap. Its detergent properties are because of the production of polar compounds (saponins), of which the oleanane-based triterpenoid saponins, saponariosides A and B, are the major components. Soapwort saponins have anticancer properties and are also of interest as endosomal escape enhancers for targeted tumor therapies. Intriguingly, these saponins share common structural features with the vaccine adjuvant QS-21 and, thus, represent a potential alternative supply of saponin adjuvant precursors. Here, we sequence the S. officinalis genome and, through genome mining and combinatorial expression, identify 14 enzymes that complete the biosynthetic pathway to saponarioside B. These enzymes include a noncanonical cytosolic GH1 (glycoside hydrolase family 1) transglycosidase required for the addition of D-quinovose. Our results open avenues for accessing and engineering natural and new-to-nature pharmaceuticals, drug delivery agents and potential immunostimulants.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

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.

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.

Abstract Isoflavones are a group of phenolic compounds mostly restricted to plants of the legume family, where they mediate important interactions with plant-associated microbes, including in defense from pathogens and in nodulation. Their well-studied health promoting attributes have made them a prime target for metabolic engineering, both for bioproduction of isoflavones as high-value molecules, and in biofortification of food crops. A key gene in their biosynthesis, isoflavone synthase, was identified in legumes over two decades ago, but little is known about formation of isoflavones outside of this family. Here we identify a specialized wheat-specific isoflavone synthase, TaCYP71F53, which catalyzes a different reaction from the leguminous isoflavone synthases, thus revealing an alternative path to isoflavonoid biosynthesis and providing a non-transgenic route for engineering isoflavone production in wheat. TaCYP71F53 forms part of a biosynthetic gene cluster that produces a naringenin-derived O-methylated isoflavone, 5-hydroxy-2′,4′,7-trimethoxyisoflavone, triticein. Pathogen-induced production and in vitro antimicrobial activity of triticein suggest a defense-related role for this molecule in wheat. Genomic and metabolic analyses of wheat ancestral grasses further show that the triticein gene cluster was introduced into domesticated emmer wheat through natural hybridization ~9000 years ago, and encodes a pathogen-responsive metabolic pathway that is conserved in modern bread wheat varieties.

Genetic variation for disease resistance is present in salmonid fish; however, the molecular basis is poorly understood, and biomarkers of disease susceptibility/resistance are unavailable. Previously, we selected a line of rainbow trout for high survival following standardized challenge with Flavobacterium psychrophilum (Fp), the causative agent of bacterial cold water disease. The resistant line (ARS-Fp-R) exhibits over 60 percentage points higher survival compared to a reference susceptible line (ARS-Fp-S). To gain insight into the differential host response between genetic lines, we compared the plasma proteomes from day 6 after intramuscular challenge. Pooled plasma from unhandled, PBS-injected, and Fp-injected groups were simultaneously analyzed using a TMT 6-plex label, and the relative abundance of 513 proteins was determined. Data are available via ProteomeXchange, with identifier PXD041308, and the relative protein abundance values were compared to mRNA measured from a prior, whole-body RNA-seq dataset. Our results identified a subset of differentially abundant intracellular proteins was identified, including troponin and myosin, which were not transcriptionally regulated, suggesting that these proteins were released into plasma following pathogen-induced tissue damage. A separate subset of high-abundance, secreted proteins were transcriptionally regulated in infected fish. The highest differentially expressed protein was a C1q family member (designated complement C1q-like protein 3; C1q-LP3) that was upregulated over 20-fold in the infected susceptible line while only modestly upregulated, 1.8-fold, in the infected resistant line. Validation of biomarkers was performed using immunoassays and C1q-LP3, skeletal muscle troponin C, cathelcidin 2, haptoglobin, leptin, and growth and differentiation factor 15 exhibited elevated concentration in susceptible line plasma. Complement factor H-like 1 exhibited higher abundance in the resistant line compared to the susceptible line in both control and challenged fish and thus was a baseline differentiator between lines. C1q-LP3 and STNC were elevated in Atlantic salmon plasma following experimental challenge with Fp. In summary, these findings further the understanding of the differential host response to Fp and identifies salmonid biomarkers that may have use for genetic line evaluation and on-farm health monitoring.

Introduction: Triterpenoids and saponins have a broad range of pharmacological activities. Unlike most legumes which contain mainly oleanane-type scaffold, Astragalus membranaceus contains not only oleanane-type but also cycloartane-type saponins, for which the biosynthetic pathways are unknown. Objectives: This work aims to study the function and catalytic mechanism of oxidosqualene cyclases (OSCs), one of the most important enzymes in triterpenoid biosynthesis, in A. membranaceus. Methods: Two OSC genes, AmOSC2 and AmOSC3, were cloned from A. membranaceus. Their functions were studied by heterologous expression in tobacco and yeast, together with in vivo transient expression and virus-induced gene silencing. Site-directed mutagenesis and molecular docking were used to explain the catalytic mechanism for the conserved motif. Results: AmOSC2 is a β-amyrin synthase which showed higher expression levels in underground parts. It is associated with the production of β-amyrin and soyasaponins (oleanane-type) in vivo. AmOSC3 is a cycloartenol synthase expressed in both aerial and underground parts. It is related to the synthesis of astragalosides (cycloartane-type) in the roots, and to the synthesis of cycloartenol as a plant sterol precursor. From AmOSC2/3, conserved triad motifs VFM/VFN were discovered for β-amyrin/cycloartenol synthases, respectively. The motif is a critical determinant of yield as proved by 10 variants from different OSCs, where the variant containing the conserved motif increased the yield by up to 12.8-fold. Molecular docking and mutagenesis revealed that Val, Phe and Met residues acted together to stabilize the substrate, and the cation-π interactions from Phe played the major role. Conclusion: The study provides insights into the biogenic origin of oleanane-type and cycloartane-type triterpenoids in Astragalus membranaceus. The conserved motif offers new opportunities for OSC engineering.

Societal Impact Statement Plants are our sources of oxygen, food, medicines, clothing, building materials and fuels. They are part of our history, our trade and our imaginations. Here, we investigate the potential for integration of photographs and poetry to bring plants to life and let them tell their stories, inspired by the ancient Japanese woodprint artform, surimono. The resulting ‘photo surimono’ open up new opportunities to engage with the natural world at the juxtaposition of the written and the visual, to combat the cognitive bias of plant blindness and to introduce more connected ways of thinking about plants, people and sustainability into educational programmes.

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'. [ABSTRACT FROM AUTHOR]

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. Genomics-based predictions indicate that plants harbor the ability to make a vast array of as yet undiscovered chemistry. This Perspective argues that recent advances open up the potential to harness this capability at unprecedented scale for the discovery and development of new drugs. [ABSTRACT FROM AUTHOR]

Sesterterpenoids are a relatively rare class of plant terpenes. Sesterterpene synthase (STS)-mediated cyclization of the linear C25 isoprenoid precursor geranylfarnesyl diphosphate (GFPP) defines sesterterpene scaffolds. So far only a very limited number of STSs have been characterized. The discovery of three new plant STSs is reported that produce a suite of sesterterpenes with unprecedented 6/11/5 and 6/6/7/5 fused ring systems when transiently co-expressed with a GFPP synthase in Nicotiana benthamiana. Structural elucidation, feeding experiments, and quantum chemical calculations suggest that these STSs catalyze an unusual cyclization path involving reprotonation, intramolecular 1,6 proton transfer, and concerted but asynchronous bicyclization events. The cyclization is diverted from those catalyzed by the characterized plant STSs by forming unified 15/5 bicyclic sesterterpene intermediates. Mutagenesis further revealed a conserved amino acid residue implicated in reprotonation.

No abstract available.

Sesterterpenoids are a rare terpene class harboring untapped chemodiversity and bioactivities. Their structural diversity originates primarily from the scaffold-generating sesterterpene synthases (STSs). In fungi, all six known STSs are bifunctional, containing C-terminal trans-prenyltransferase (PT) and N-terminal terpene synthase (TPS) domains. In plants, two colocalized PT and TPS gene pairs from Arabidopsis thaliana were recently reported to synthesize sesterterpenes. However, the landscape of PT and TPS genes in plant genomes is unclear. Here, using a customized algorithm for systematically searching plant genomes, we reveal a suite of physically colocalized pairs of PT and TPS genes for the biosynthesis of a large sesterterpene repertoire in the wider Brassicaceae. Transient expression of seven TPSs from A. thaliana, Capsella rubella, and Brassica oleracea in Nicotiana benthamiana yielded fungal-type sesterterpenes with tri-, tetra-, and pentacyclic scaffolds, and notably (-)-ent-quiannulatene, an enantiomer of the fungal metabolite (+)-quiannulatene. Protein and structural modeling analysis identified an amino acid site implicated in structural diversification. Mutation of this site in one STS (AtTPS19) resulted in premature termination of carbocation intermediates and accumulation of bi-, tri-, and tetracyclic sesterterpenes, revealing the cyclization path for the pentacyclic sesterterpene (-)-retigeranin B. These structural and mechanistic insights, together with phylogenetic analysis, suggest convergent evolution of plant and fungal STSs, and also indicate that the colocalized PT-TPS gene pairs in the Brassicaceae may have originated from a common ancestral gene pair present before speciation. Our findings further provide opportunities for rapid discovery and production of sesterterpenes through metabolic and protein engineering.

The genomic organization and origin of the avenacin biosynthetic gene cluster remain unknown. Here, the authors assemble the genome of diploid oat Avena strigosa, reveal the structure and organization of the consecutive genes, characterize the last two missing pathway steps, and investigate the origin of the pathway in cereals.

Social Impact Statement Plants are rich sources of drugs and other high‐value chemicals that are used by humans. Many of the plant species that produce important molecules grow in remote locations and have extensive histories of indigenous use. Global concerns about sustainable supply have in some cases led to the development of alternative methods for production using biotechnological approaches. Consideration of responsible stewardship and use of the world's plants and associated traditional knowledge for the greater human good are at the heart of the Convention on Biological Diversity and the recently implemented Nagoya Protocol. The development of fora that enable open discussion and exploration of issues relating to these aspects will be critical in endeavors to protect and preserve both the environment and present and future generations. Summary Here, we investigate the application of cross‐disciplinary approaches to explore societal perceptions of plants and their uses, focusing on high‐value chemicals. The Global Garden project engages the public, researchers, and regulators in day‐long workshops that combine science, poetry, and visual arts practice to foster participants’ skill in imagining and re‐imagining relationships between high‐value plant products, biotechnology, and social and ethical aspects of these. The project represents an intervention into discussions of science communications and public engagement, addressing the uses and benefits of arts‐based approaches to foster imaginative engagement with plant science. The workshop reported here began with real plant case studies and a discussion of the aims of scientists using them. Participants were invited to respond to the issues of relationships among plants, chemicals, and people raised by the case studies through poetry and visual artwork. The poems and artwork that were produced show variation in the participants’ imaginings of plant science. They present distinctive visions of research and innovation and of the associated ethical and social implications. This type of forum, based on creative immersion, opens up opportunities for engaging with and exploring complex relations between plant biotechnology, society, and ethics. This article offers a reflection on the uses, challenges, and implications of arts‐based approaches to research communications and public engagement that disrupts traditional knowledge transfer structures. In doing so, we frame the project within science communication pedagogies and consider public engagement a form of pedagogy.

How plant biosynthetic gene clusters (BGCs) form and evolve remains unclear. Here, via examining available genomes within and between Arabidopsis species, the authors show that the thalianol BGC has evolved recently and is still dynamically evolving through involvement of auxiliary genes and chromosomal inversions.

Secretory leucoprotease inhibitor (SLPI) has multifaceted functions, including inhibition of protease activity, antimicrobial functions, and anti-inflammatory properties. In this study, we show that SLPI plays a role in controlling pulmonary Pseudomonas aeruginosa infection. Mice lacking SLPI were highly susceptible to P. aeruginosa infection, however there was no difference in bacterial burden. Utilising a model of P. aeruginosa LPS-induced lung inflammation, human recombinant SLPI (hrSLPI) administered intraperitoneally suppressed the recruitment of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and resulted in reduced BALF and serum levels of inflammatory cytokines and chemokines. This anti-inflammatory effect of hrSLPI was similarly demonstrated in a systemic inflammation model induced by intraperitoneal injection of LPS from various bacteria or lipoteichoic acid, highlighting the broad anti-inflammatory properties of hrSLPI. Moreover, in bone-marrow-derived macrophages, hrSLPI reduced LPS-induced phosphorylation of p-IkB-α, p-IKK-α/β, p-P38, demonstrating that the anti-inflammatory effect of hrSLPI was due to the inhibition of the NFκB and MAPK pathways. In conclusion, administration of hrSLPI attenuates excessive inflammatory responses and is therefore, a promising strategy to target inflammatory diseases such as acute respiratory distress syndrome or sepsis and could potentially be used to augment antibiotic treatment.

Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response. This comprehensive discovery approach has yielded a panel of potent neutralizing antibodies which bind distinct viral epitopes including epitopes conserved in SARS-CoV-1. Structural determination of a non-ACE2 receptor blocking antibody reveals a previously undescribed binding epitope, which is unlikely to be affected by the mutations in any of the recently reported major viral variants including B.1.1.7 (from the UK), B.1.351 (from South Africa) and B.1.1.28 (from Brazil). Finally, by combining sequences of the RBD binding and neutralizing antibodies with the B cell receptor repertoire sequencing, we also describe a highly convergent early antibody response. Similar IgM-derived sequences occur within this study group and also within patient responses described by multiple independent studies published previously.

Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.

Successful forest management for multiple uses requires balancing extractive practices with maintaining biodiversity, among other important goals. Amphibians comprise an important and abundant part of the biodiversity of many forests. Previous studies have documented declines in the abundance and diversity of amphibians in harvested forests. However, only recently have studies begun to elucidate the mechanisms that underlie such declines. Here, we studied the effects of timber harvesting on survival of geographically widespread ambystomatid salamanders in three forest regions of North America. We used terrestrial enclosures in the Northeast, Midwest, and Southeast to compare amphibian survival in unharvested controls, partially harvested stands (∼25% canopy reduction), and clearcuts with coarse woody debris either retained or removed. In all regions, patterns of amphibian survival were similar, with both juvenile and adult salamanders generally having significantly lower survival in clearcuts compared with unharvested controls. Survival of juvenile salamanders in partially harvested stands was also low, but adult salamanders survived as well or better in partially harvested stands as in controls. Larger body size in juveniles was significantly correlated with recapture, irrespective of treatment, in both the Northeast and Southeast, but not in the Midwest or for adults in any region. Relatively heavier adults were more likely to be captured again in the Southeast, but relative mass was not correlated with recapture in any other regions or for juveniles. Our results suggest that increased amphibian mortality may contribute to declines of amphibian abundance and richness after forest clearcutting for the regions evaluated here. Although our results indicate that partial harvesting is compatible with survival of adult salamanders, retention of intact forest around breeding ponds would benefit all terrestrial stages of pond-breeding salamanders and represents a best management practice for the maintenance of amphibian biodiversity.

Successful forest management for multiple uses requires balancing extractive practices with maintaining biodiversity, among other important goals. Amphibians comprise an important and abundant part of the biodiversity of many forests. Previous studies have documented declines in the abundance and diversity of amphibians in harvested forests. However, only recently have studies begun to elucidate the mechanisms that underlie such declines. Here, we studied the effects of timber harvesting on survival of geographically widespread ambystomatid salamanders in three forest regions of North America. We used terrestrial enclosures in the Northeast, Midwest, and Southeast to compare amphibian survival in unharvested controls, partially harvested stands (~25% canopy reduction), and clearcuts with coarse woody debris either retained or removed. In all regions, patterns of amphibian survival were similar, with both juvenile and adult salamanders generally having significantly lower survival in clearcuts compared with unharvested controls. Survival of juvenile salamanders in partially harvested stands was also low, but adult salamanders survived as well or better in partially harvested stands as in controls. Larger body size in juveniles was significantly correlated with recapture, irrespective of treatment, in both the Northeast and Southeast, but not in the Midwest or for adults in any region. Relatively heavier adults were more likely to be captured again in the Southeast, but relative mass was not correlated with recapture in any other regions or for juveniles. Our results suggest that increased amphibian mortality may contribute to declines of amphibian abundance and richness after forest clearcutting for the regions evaluated here. Although our results indicate that partial harvesting is compatible with survival of adult salamanders, retention of intact forest around breeding ponds would benefit all terrestrial stages of pond-breeding salamanders and represents a best management practice for the maintenance of amphibian biodiversity. © 2013 Elsevier B.V.