Class Of Plant Natural Products Research Articles

Evolution and diversification of carboxylesterase-like [4+2] cyclases in aspidosperma and iboga alkaloid biosynthesis

Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent [4+2] cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as "underground metabolites" via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.

Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast

Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker’s yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

Reaction intensification for biocatalytic production of polyphenolic natural product di-C-β-glucosides.

Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-β-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl β-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4-0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-β-glucoside synthesis from expedient substrates.

Cytochrome P450 monooxygenase-mediated tailoring of triterpenoids and steroids in plants.

The cytochrome P450 monooxygenase (CYP) superfamily comprises hemethiolate enzymes that perform remarkable regio- and stereospecific oxidative chemistry. As such, CYPs are key agents for the structural and functional tailoring of triterpenoids, one of the largest classes of plant natural products with widespread applications in pharmaceuticals, food, cosmetics, and agricultural industries. In this review, we provide a full overview of 149 functionally characterised CYPs involved in the biosynthesis of triterpenoids and steroids in primary as well as in specialised metabolism. We describe the phylogenetic distribution of triterpenoid- and steroid-modifying CYPs across the plant CYPome, present a structure-based summary of their reactions, and highlight recent examples of particular interest to the field. Our review therefore provides a comprehensive up-to-date picture of CYPs involved in the biosynthesis of triterpenoids and steroids in plants as a starting point for future research.

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally important compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. Here we use transcriptomic analysis to identify glycosyltransferases that are upregulated in response to biosynthetic intermediates and produce plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produces a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) is critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases does not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

Mechanism and Evolution of [4+2] Cyclases in Monoterpene Indole Alkaloid Biosynthesis

Monoterpene indole alkaloids (MIAs) comprise a large and diverse class of plant natural products, many of which exhibit potent biological activities such as anticancer, antimalarial, and antihypertensive. While the biosynthetic construction of these compounds has been a subject of intensive study for the past six decades, the identification of specific pathway genes remains an active area of investigation. MIAs belonging to the aspidosperma and iboga structural classes are produced only by select plants in the Apocynaceae (dogbane) family. The most well‐known of these is Catharanthus roseus, which produces the bisindole anticancer compounds vinblastine and vincristine through a complex pathway involving more than thirty individual enzymatic steps. Recently, we characterized three carboxylesterase‐like enzymes from C. roseus and Tabernanthe iboga that catalyze regio‐ and enantiodivergent [4+2] cycloadditions to generate the aspidosperma (tabersonine synthase, TS) and iboga (catharanthine synthase, CS; coronaridine synthase, CorS) scaffolds from a common biosynthetic intermediate. While crystal structures, computational docking, and site‐directed mutagenesis provided some key insights into the catalytic mechanism and cyclization specificity of these enzymes, several important questions remained unanswered. Here, we use a combination of computation and experiment to demonstrate that the [4+2] cyclases in MIA biosynthesis employ a stepwise rather than a concerted mechanism of cyclization. We also isolate and characterize the direct product of the CorS reaction and show that it can serve as a branch point in the biosynthesis of both iboga and pseudo‐aspidosperma alkaloids. Using a phylogenetic approach, we subsequently explore the evolution and diversification of MIA cyclase function. Reconstruction and functional analysis of ancestral enzymes allows us to trace an evolutionary trajectory from carboxylesterase to TS, from TS to CorS, and from CorS to CS. We further generate and test a panel of mutants based on both extant and ancestral cyclases to probe the molecular basis for differential cyclization activity. Our results provide insight into the mechanism and evolution of a unique class of biosynthetic enzymes and offer a means by which they could be leveraged for the facile production of new MIAs.

Engineered Production of Strictosidine and Analogues in Yeast.

Monoterpene indole alkaloids (MIAs) are an expansive class of plant natural products, many of which have been named on the World Health Organization's List of Essential Medicines. Low production from native plant hosts necessitates a more reliable source of these drugs to meet global demand. Here, we report the development of a yeast-based platform for high-titer production of the universal MIA precursor, strictosidine. Our fed-batch platform produces ∼50 mg/L strictosidine, starting from the commodity chemicals geraniol and tryptamine. The microbially produced strictosidine was purified to homogeneity and characterized by NMR. Additionally, our approach enables the production of halogenated strictosidine analogues through the feeding of modified tryptamines. The MIA platform strain enables rapid access to strictosidine for reconstitution and production of downstream MIA natural products.

De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories

Isoflavonoids comprise a class of plant natural products with great nutraceutical, pharmaceutical and agricultural significance. Their low abundance in nature and structural complexity however hampers access to these phytochemicals through traditional crop-based manufacturing or chemical synthesis. Microbial bioproduction therefore represents an attractive alternative. Here, we engineer the metabolism of Saccharomyces cerevisiae to become a platform for efficient production of daidzein, a core chemical scaffold for isoflavonoid biosynthesis, and demonstrate its application towards producing bioactive glucosides from glucose, following the screening-reconstruction-application engineering framework. First, we rebuild daidzein biosynthesis in yeast and its production is then improved by 94-fold through screening biosynthetic enzymes, identifying rate-limiting steps, implementing dynamic control, engineering substrate trafficking and fine-tuning competing metabolic processes. The optimized strain produces up to 85.4 mg L−1 of daidzein and introducing plant glycosyltransferases in this strain results in production of bioactive puerarin (72.8 mg L−1) and daidzin (73.2 mg L−1). Our work provides a promising step towards developing synthetic yeast cell factories for de novo biosynthesis of value-added isoflavonoids and the multi-phased framework may be extended to engineer pathways of complex natural products in other microbial hosts.

Fermentation Strategies for Production of Pharmaceutical Terpenoids in Engineered Yeast.

Terpenoids, also known as isoprenoids, are a broad and diverse class of plant natural products with significant industrial and pharmaceutical importance. Many of these natural products have antitumor, anti-inflammatory, antibacterial, antiviral, and antimalarial effects, support transdermal absorption, prevent and treat cardiovascular diseases, and have hypoglycemic activities. Production of these compounds are generally carried out through extraction from their natural sources or chemical synthesis. However, these processes are generally unsustainable, produce low yield, and result in wasting of substantial resources, most of them limited. Microbial production of terpenoids provides a sustainable and environment-friendly alternative. In recent years, the yeast Saccharomyces cerevisiae has become a suitable cell factory for industrial terpenoid biosynthesis due to developments in omics studies (genomics, transcriptomics, metabolomics, proteomics), and mathematical modeling. Besides that, fermentation development has a significant importance on achieving high titer, yield, and productivity (TYP) of these compounds. Up to now, there have been many studies and reviews reporting metabolic strategies for terpene biosynthesis. However, fermentation strategies have not been yet comprehensively discussed in the literature. This review summarizes recent studies of recombinant production of pharmaceutically important terpenoids by engineered yeast, S. cerevisiae, with special focus on fermentation strategies to increase TYP in order to meet industrial demands to feed the pharmaceutical market. Factors affecting recombinant terpenoids production are reviewed (strain design and fermentation parameters) and types of fermentation process (batch, fed-batch, and continuous) are discussed.

Chemical Diversity of Plant Cyanogenic Glycosides: An Overview of Reported Natural Products.

Cyanogenic glycosides are an important and widespread class of plant natural products, which are however structurally less diverse than many other classes of natural products. So far, 112 naturally occurring cyanogenic glycosides have been described in the phytochemical literature. Currently, these unique compounds have been reported from more than 2500 plant species. Natural cyanogenic glycosides show variations regarding both the aglycone and the sugar part of the molecules. The predominant sugar moiety is glucose but many substitution patterns of this glucose moiety exist in nature. Regarding the aglycone moiety, four different basic classes can be distinguished, aliphatic, cyclic, aromatic, and heterocyclic aglycones. Our overview covers all cyanogenic glycosides isolated from plants and includes 33 compounds with a non-cyclic aglycone, 20 cyclopentane derivatives, 55 natural products with an aromatic aglycone, and four dihydropyridone derivatives. In the following sections, we will provide an overview about the chemical diversity known so far and mention the first source from which the respective compounds had been isolated. This review will serve as a first reference for researchers trying to find new cyanogenic glycosides and highlights some gaps in the knowledge about the exact structures of already described compounds.

Elucidation of the complete biosynthetic pathway of the main triterpene glycosylation products of Panax notoginseng using a synthetic biology platform

UDP-glycosyltransferase (UGT)-mediated glycosylation is a widespread modification of plant natural products (PNPs), which exhibit a wide range of bioactivities, and are of great pharmaceutical, ecological and agricultural significance. However, functional annotation is available for less than 2% of the family 1 UGTs, which currently has 20,000 members that are known to glycosylate several classes of PNPs. This low percentage illustrates the difficulty of experimental study and accurate prediction of their function. Here, a synthetic biology platform for elucidating the UGT-mediated glycosylation process of PNPs was established, including glycosyltransferases dependent on UDP-glucose and UDP-xylose. This platform is based on reconstructing the specific PNPs biosynthetic pathways in dedicated microbial yeast chassis by the simple method of plug-and-play. Five UGT enzymes were identified as responsible for the biosynthesis of the main glycosylation products of triterpenes in Panax notoginseng, including a novel UDP-xylose dependent glycosyltransferase enzyme for notoginsenoside R1 biosynthesis. Additionally, we constructed a yeast cell factory that yields >1 g/L of ginsenoside compound K. This platform for functional gene identification and strain engineering can serve as the basis for creating alternative sources of important natural products and thereby protecting natural plant resources.

Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins

Radiation of the plant pyridoxal 5'-phosphate (PLP)-dependent aromatic l-amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic l-amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations.

Altering potato isoprenoid metabolism increases biomass and induces early flowering

Isoprenoids constitute the largest class of plant natural products and have diverse biological functions including in plant growth and development. In potato (Solanum tuberosum), the regulatory mechanism underlying the biosynthesis of isoprenoids through the mevalonate pathway is unclear. We assessed the role of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) homologs in potato development and in the metabolic regulation of isoprenoid biosynthesis by generating transgenic lines with down-regulated expression (RNAi-hmgr) or overexpression (OE) of one (StHMGR1 or StHMGR3) or two genes, HMGR and farnesyl diphosphate synthase (FPS; StHMGR1/StFPS1 or StHMGR3/StFPS1). Levels of sterols, steroidal glycoalkaloids (SGAs), and plastidial isoprenoids were elevated in the OE-HMGR1, OE-HMGR1/FPS1, and OE-HMGR3/FPS1 lines, and these plants exhibited early flowering, increased stem height, increased biomass, and increased total tuber weight. However, OE-HMGR3 lines showed dwarfism and had the highest sterol amounts, but without an increase in SGA levels, supporting a rate-limiting role for HMGR3 in the accumulation of sterols. Potato RNAi-hmgr lines showed inhibited growth and reduced cytosolic isoprenoid levels. We also determined the relative importance of transcriptional control at regulatory points of isoprenoid precursor biosynthesis by assessing gene-metabolite correlations. These findings provide novel insights into specific end-products of the sterol pathway and could be important for crop yield and bioenergy crops.

Structure-Guided Engineering of a Scoulerine 9-O-Methyltransferase Enables the Biosynthesis of Tetrahydropalmatrubine and Tetrahydropalmatine in Yeast

Benzylisoquinoline alkaloids (BIAs) are an important class of plant natural products with diverse pharmacological properties. Microbial platforms can produce BIAs through heterologous biosynthesis ...

Changes in isovitexin-O-glycosylation during the development of young barley plants

Phenylpropanoids are a class of plant natural products that have many biological functions, including stress defence. In barley, phenylpropanoids have been described as having protective properties against excess UV-B radiation and have been linked to resistance to pathogens. Although the phenylpropanoid composition of barley has recently been addressed in more detail, the biosynthesis and regulation of this pathway have not been fully established. Barley introgression lines, such as the S42IL-population offer a set of genetically diverse plants that enable the correlation of metabolic data to distinct genetic regions on the barley genome and, subsequently, identification of relevant genes.The phenylpropanoid profiles of the first and third leaf of barley seedlings in Scarlett and four members of the S42IL-population were obtained by LC-MS. Comparison of the leaf profiles revealed a change in the glycosylation pattern of the flavone-6-C-glucoside isovitexin in the elite cultivar Scarlett. The change was characterized by the stepwise decrease in isovitexin-7-O-glucoside (saponarin) and an increase in isovitexin-2″-O-β-D-glucoside content.The lines S42IL-101-, -177 and -178 were completely devoid of isovitexin-2″-O-β-D-glucoside. Parallel glucosyltransferase assays were consistent with the observed metabolic patterns. The genetic region responsible for this metabolic effect was located on chromosome 1H between 0.21 and 15.08 cM, encompassing 505 gene candidates in the genome of the sequenced cultivar Morex. Only one of these genes displayed sequence similarity with glucosyltransferases of plant secondary metabolism that possessed the characteristic PSPG motif.

The TriForC database: a comprehensive up-to-date resource of plant triterpene biosynthesis.

Triterpenes constitute a large and important class of plant natural products with diverse structures and functions. Their biological roles range from membrane structural components over plant hormones to specialized plant defence compounds. Furthermore, triterpenes have great potential for a variety of commercial applications such as vaccine adjuvants, anti-cancer drugs, food supplements and agronomic agents. Their biosynthesis is carried out through complicated, branched pathways by multiple enzyme types that include oxidosqualene cyclases, cytochrome P450s, and UDP-glycosyltransferases. Given that the number of characterized triterpene biosynthesis enzymes has been growing fast recently, the need for a database specifically focusing on triterpene enzymology became eminent. Here, we present the TriForC database (http://bioinformatics.psb.ugent.be/triforc/), encompassing a comprehensive catalogue of triterpene biosynthesis enzymes. This highly interlinked database serves as a user-friendly access point to versatile data sets of enzyme and compound features, enabling the scanning of a complete catalogue of experimentally validated triterpene enzymes, their substrates and products, as well as the pathways they constitute in various plant species. The database can be accessed by direct browsing or through convenient search tools including keyword, BLAST, plant species and substructure options. This database will facilitate gene mining and creating genetic toolboxes for triterpene synthetic biology.

Metabolite profiling of steroidal glycosides in the rhizome of Aspidistra sichuanensis using UPLC/Q-TOF MSE

This study aimed to perform metabolite profiling of steroidal glycosides in the rhizome of Aspidistra sichuanensis using liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). Steroidal glycosides are one of the largest classes of plant natural products with structural diversity and significant bioactivities. This study summarized the fragmentation rules of six major groups of saponins from Aspidistra. The fragment ions provided a wealth of structural information indicative of the sugar types and number, connecting sequence of sugar units, and types of aglycones. Based on the exact mass information, reliable molecular formulas of the detected compounds could be predicted, and fragmentation ions generated in MSE spectra together with the retention times of the peaks and the structures of steroidal glycosides isolated from the plants belonging to the family Convallariaceae, the structures of 60 constituents were tentatively identified from A. sichuanensis. Also, 34 potential new compounds and 15 pairs of isomers were discovered.

Antibiofilm Activity of Plant Polyphenols.

In the history of human medicine, antibiotics represent epochal examples of medical progress. However, with an approaching antibiotic crisis due to the emergence and extensive spread of antimicrobial resistance among bacterial agents, as well as to increasing number of patients with chronic and recalcitrant bacterial biofilm-associated infections, the naturally occurring molecules may become new sources of antibacterial and antibiofilm drugs for clinical usage. Polyphenols represent a class of plant natural products which are important in plant defense against microbial pathogens. The main focus of the review is on the antibiofilm activities of phenolic compounds against bacteria which play an essential role in medical device biofilm-associated infections. The other, not negligible part of the review is devoted to polyphenols’ activity against bacterial agents that cause dental caries and periodontal disease.

Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities

Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the hemanthamine- and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. In this study, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4'-O-methyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products.

Generation and Functional Evaluation of Designer Monoterpene Synthases.

Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes.