Abstract
Saponins are widely distributed plant natural products with vast structural and functional diversity. They are typically composed of a hydrophobic aglycone, which is extensively decorated ...with functional groups prior to the addition of hydrophilic sugar moieties, to result in surface-active amphipathic compounds. The saponins are broadly classified as triterpenoids, steroids or steroidal glycoalkaloids, based on the aglycone structure from which they are derived. The saponins and their biosynthetic intermediates display a variety of biological activities of interest to the pharmaceutical, cosmetic and food sectors. Although their relevance in industrial applications has long been recognized, their role in plants is underexplored. Recent research on modulating native pathway flux in saponin biosynthesis has demonstrated the roles of saponins and their biosynthetic intermediates in plant growth and development. Here, we review the literature on the effects of these molecules on plant physiology, which collectively implicate them in plant primary processes. The industrial uses and potential of saponins are discussed with respect to structure and activity, highlighting the undoubted value of these molecules as therapeutics.
Terpenoids constitute a large and diverse class of natural products that serve many functions in nature. Most of the tens of thousands of the discovered terpenoids are synthesized by plants, where ...they function as primary metabolites involved in growth and development, or as secondary metabolites that optimize the interaction between the plant and its environment. Several plant terpenoids are economically important molecules that serve many applications as pharmaceuticals, pesticides, etc. Major challenges for the commercialization of plant-derived terpenoids include their low production levels in planta and the continuous demand of industry for novel molecules withnewor superior biological activities. Here, wehighlight several synthetic biology methods to enhance and diversify the production of plant terpenoids, with a foresight towards triterpenoid engineering, the least engineered class of bioactive terpenoids. Increased or cheaper production of valuable triterpenoids may be obtained by ‘classic’ metabolic engineering of plants or by heterologous production of the compounds in other plants or microbes. Novel triterpenoid structures can be generated through combinatorial biosynthesis or directed enzyme evolution approaches. In its ultimate form, synthetic biology may lead to the production of large amounts of plant triterpenoids in in vitro systems or custom-designed artificial biological systems.
Significance The terpenes are the largest class of plant natural products. This major class of compounds represents tremendous chemical diversity of which only a relatively small fraction has so far ...been accessed and used by industry. The primary drivers of terpene diversification are terpenoid synthases and cytochromes P450, which synthesize and modify terpene scaffolds. Here, focusing on these two gene families, we investigate terpene synthesis and evolution across 17 sequenced plant genomes. Our analyses shed light on the roots of terpene biosynthesis and diversification in plants. They also reveal that different genomic mechanisms of pathway assembly predominate in eudicots and monocots.
Plants produce an array of specialized metabolites, including chemicals that are important as medicines, flavors, fragrances, pigments and insecticides. The vast majority of this metabolic diversity is untapped. Here we take a systematic approach toward dissecting genetic components of plant specialized metabolism. Focusing on the terpenes, the largest class of plant natural products, we investigate the basis of terpene diversity through analysis of multiple sequenced plant genomes. The primary drivers of terpene diversification are terpenoid synthase (TS) “signature” enzymes (which generate scaffold diversity), and cytochromes P450 (CYPs), which modify and further diversify these scaffolds, so paving the way for further downstream modifications. Our systematic search of sequenced plant genomes for all TS and CYP genes reveals that distinct TS/CYP gene pairs are found together far more commonly than would be expected by chance, and that certain TS/CYP pairings predominate, providing signals for key events that are likely to have shaped terpene diversity. We recover TS/CYP gene pairs for previously characterized terpene metabolic gene clusters and demonstrate new functional pairing of TSs and CYPs within previously uncharacterized clusters. Unexpectedly, we find evidence for different mechanisms of pathway assembly in eudicots and monocots; in the former, microsyntenic blocks of TS/CYP gene pairs duplicate and provide templates for the evolution of new pathways, whereas in the latter, new pathways arise by mixing and matching of individual TS and CYP genes through dynamic genome rearrangements. This is, to our knowledge, the first documented observation of the unique pattern of TS and CYP assembly in eudicots and monocots.
Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an ...essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids.
The saikosaponins comprise oleanane- and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified ...a gene, designated CYP716Y1 , encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16α hydroxylation of oleanane- and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated β-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.
This mini review focuses on the opportunities provided by current and emerging separation techniques for mass spectrometry metabolomics. The purpose of separation technologies in metabolomics is ...primarily to reduce complexity of the heterogeneous systems studied, and to provide concentration enrichment by increasing sensitivity towards the quantification of low abundance metabolites. For this reason, a wide variety of separation systems, from column chemistries to solvent compositions and multidimensional separations, have been applied in the field. Multidimensional separations are a common method in both proteomics applications and gas chromatography mass spectrometry, allowing orthogonal separations to further reduce analytical complexity and expand peak capacity. These applications contribute to exponential increases in run times concomitant with first dimension fractionation followed by second dimension separations. Multidimensional liquid chromatography to increase peak capacity in metabolomics, when compared to the potential of running additional samples or replicates and increasing statistical confidence, mean that uptake of these methods has been minimal. In contrast, in the last 15 years there have been significant advances in the resolution and sensitivity of ion mobility spectrometry, to the point where high-resolution separation of analytes based on their collision cross section approaches chromatographic separation, with minimal loss in sensitivity. Additionally, ion mobility separations can be performed on a chromatographic timescale with little reduction in instrument duty cycle. In this review, we compare ion mobility separation to liquid chromatographic separation, highlight the history of the use of ion mobility separations in metabolomics, outline the current state-of-the-art in the field, and discuss the future outlook of the technology. “Where there is one, you’re bound to divide it. Right in two”, James Maynard Keenan.
Synthetic biology is the repurposing of biological systems for novel objectives and applications. Through the co-ordinated and balanced expression of genes, both native and those introduced from ...other organisms, resources within an industrial chassis can be siphoned for the commercial production of high-value commodities. This developing interdisciplinary field has the potential to revolutionize natural product discovery from higher plants, by providing a diverse array of tools, technologies, and strategies for exploring the large chemically complex space of plant natural products using unicellular organisms. In this review, we emphasize the key features that influence the generation of biorefineries and highlight technologies and strategic solutions that can be used to overcome engineering pitfalls with rational design. Also presented is a succinct guide to assist the selection of unicellular chassis most suited for the engineering and subsequent production of the desired natural product, in order to meet the global demand for plant natural products in a safe and sustainable manner.
•We quantified saponin content in quinoa leaves treated with methyl jasmonate.•We found that methyl jasmonate induces accumulation of saponins in quinoa leaves and induces the expression of saponin ...biosynthetic genes in quinoa.•Reference genes for qPCR analysis were identified by bioinformatics analysis of available RNA-Seq data.•We cloned three genes involved in saponin biosynthesis.•We characterized these genes by functional analyses in yeast.
Quinoa (Chenopodium quinoa Willd.) is a highly nutritious pseudocereal with an outstanding protein, vitamin, mineral and nutraceutical content. The leaves, flowers and seed coat of quinoa contain triterpenoid saponins, which impart bitterness to the grain and make them unpalatable without postharvest removal of the saponins. In this study, we quantified saponin content in quinoa leaves from Ecuadorian sweet and bitter genotypes and assessed the expression of saponin biosynthetic genes in leaf samples elicited with methyl jasmonate. We found saponin accumulation in leaves after MeJA treatment in both ecotypes tested. As no reference genes were available to perform qPCR in quinoa, we mined publicly available RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana using geNorm, NormFinder and BestKeeper algorithms. The quinoa ortholog of At2g28390 (Monensin Sensitivity 1, MON1) was stably expressed and chosen as a suitable reference gene for qPCR analysis. Candidate saponin biosynthesis genes were screened in the quinoa RNA-Seq data and subsequent functional characterization in yeast led to the identification of CqbAS1, CqCYP716A78 and CqCYP716A79. These genes were found to be induced by MeJA, suggesting this phytohormone might also modulate saponin biosynthesis in quinoa leaves. Knowledge of the saponin biosynthesis and its regulation in quinoa may aid the further development of sweet cultivars that do not require postharvest processing.
Eight CYP93E proteins catalyzing the C-24 oxidation in triterpenoid saponin biosynthesis from different legumes were analyzed in yeast, revealing differences in their microbial triterpenoid synthesis ...potential. Display omitted
•Known CYP93E2 catalyzes C-24 oxidation of β-amyrin with low efficiency in yeast.•We screened plant genome and transcriptome data and identified CYP93E orthologs.•Six extra CYP93Es were characterized, leading to a total of nine subfamily members.•The CYP93E orthologs display large variations in catalytic efficiency in yeast.•Screening homologous enzymes is a useful tool to create superior engineered hosts.
Cytochrome P450-dependent monooxygenases (P450s) belonging to the CYP93E subfamily catalyze the C-24 oxidation of the triterpene backbone during the biosynthesis of triterpenoid saponins, which are bioactive plant natural products. In our attempts to produce plant triterpenoids in the yeast Saccharomyces cerevisiae, we observed a poor in vivo catalytic efficiency of the Medicago truncatula CYP93E2. To overcome this biosynthetic bottleneck, we screened publicly available plant genome and transcriptome data for CYP93E subfamily members. Six CYP93E orthologs, exclusively from leguminous plant species, were identified and functionally characterized in S. cerevisiae. Despite the high degree of amino acid conservation, the CYP93E orthologs showed large variations in enzymatic efficiency in yeast. The CYP93E9 from Phaseolus vulgaris showed the highest activity and converted ∼80% of the accumulating in vivo produced substrate β-amyrin to the products olean-12-ene-3β,24-diol and probable 3β-hydroxy olean-12-en-24-oic acid, with a catalytic efficiency that was 61 times higher than that of the M. truncatula CYP93E2. In conclusion, we have expanded the list of functional CYP93E orthologs to a total of nine proteins and show that there are large variations in their catalytic efficiencies when expressed in a heterologous host. Although demonstrated here for the CYP93E family involved in triterpenoid saponin biosynthesis, this phenomenon is undoubtedly extendable to other enzyme families involved in natural product synthesis. Hence, screening for homologous enzymes may become a valuable synthetic biologist’s tool for engineering superior production chassis.
Triterpenoids are high-value plant metabolites with numerous applications in medicine, agriculture, food, and home and personal care products. However, plants produce triterpenoids in low abundance, ...and their complex structures make their chemical synthesis prohibitively expensive and often impossible. As such, the yeast Saccharomyces cerevisiae has been explored as an alternative means of production. An important triterpenoid is oleanolic acid because it is the precursor to many bioactive triterpenoids of commercial interest, such as QS-21 which is being evaluated as a vaccine adjuvant in clinical trials against HIV and malaria. Oleanolic acid is derived from 2,3-oxidosqualene (natively produced by yeast) via a cyclisation and a multi-step oxidation reaction, catalysed by a β-amyrin synthase and a cytochrome P450 of the CYP716A subfamily, respectively. Although many homologues have been characterised, previous studies have used arbitrarily chosen β-amyrin synthases and CYP716As to produce oleanolic acid and its derivatives in yeast. This study presents the first comprehensive comparison of β-amyrin synthase and CYP716A enzyme activities in yeast. Strains expressing different homologues are compared for production, revealing 6.3- and 4.5-fold differences in β-amyrin and oleanolic acid productivities and varying CYP716A product profiles, which are important to consider when engineering strains for the production of bioactive oleanolic acid derivatives.
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