Post-Cyclization Skeletal Rearrangements in Plant Triterpenoid Biosynthesis by a Pair of Branchpoint Isomerases.

Triterpenoids possess potent biological activities, but their polycyclic skeletons are challenging to synthesize. The skeletal diversity of triterpenoids in plants is generated by oxidosqualene cyclases based on epoxide-triggered cationic rearrangement cascades. Normally, triterpenoid skeletons then remain unaltered during subsequent tailoring steps. In contrast, the highly modified triterpenoids found in Sapindales plants imply the existence of post-cyclization skeletal rearrangement enzymes that have not yet been found. We report here a biosynthetic pathway in Sapindales plants for the modification of already cyclized tirucallane triterpenoids, controlling the pathway bifurcation between different plant triterpenoid classes. Using a combination of bioinformatics, heterologous expression in plants and chemical analyses, we identified a cytochrome P450 monooxygenase and two isomerases which harness the epoxidation-rearrangement biosynthetic logic of triterpene cyclizations for modifying the tirucallane scaffold. The two isomerases share the same epoxide substrate made by the cytochrome P450 monooxygenase CYP88A154, but generate two different rearrangement products, one containing a cyclopropane ring. Our findings reveal a process for skeletal rearrangements of triterpenoids in nature that expands their scaffold diversity after the initial cyclization. In addition, the enzymes described here are crucial for the biotechnological production of limonoid, quassinoid, apoprotolimonoid, and glabretane triterpenoids.

Isotope-Guided Metabolomics Reveals Divergent Incorporation of Valine into Different Flavor Precursor Classes in Chives.

Plants of the genus Allium such as chives, onions or garlic produce S-alk(en)yl cysteine sulfoxides as flavor precursors. Two major representatives are S-propenyl cysteine sulfoxide (isoalliin) and S-propyl cysteine sulfoxide (propiin), which only differ by a double bond in the C3 side chain. The propenyl group of isoalliin is derived from the amino acid valine, but the source of the propyl group of propiin remains unclear. Here, we present an untargeted metabolomics approach in seedlings of chives (Allium schoenoprasum) to track mass features containing sulfur and/or 13 C from labeling experiments with valine-13 C5 guided by their isotope signatures. Our data show that propiin and related propyl-bearing metabolites incorporate carbon derived from valine-13 C5 , but to a much lesser extent than isoalliin and related propenyl compounds. Our findings provide new insights into the biosynthetic pathways of flavor precursors in Allium species and open new avenues for future untargeted labeling experiments.

Structural basis of cycloaddition in biosynthesis of iboga and aspidosperma alkaloids.

Cycloaddition reactions generate chemical complexity in a single step. Here we report the crystal structures of three homologous plant-derived cyclases involved in the biosynthesis of iboga and aspidosperma alkaloids. These enzymes act on the same substrate, named angryline, to generate three distinct scaffolds. Mutational analysis reveals how these highly similar enzymes control regio- and stereo-selectivity.

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.

Sarpagan bridge enzyme has substrate-controlled cyclization and aromatization modes.

Cyclization reactions that create complex polycyclic scaffolds are hallmarks of alkaloid biosynthetic pathways. We present the discovery of three homologous cytochrome P450s from three monoterpene indole alkaloid-producing plants (Rauwolfia serpentina, Gelsemium sempervirens and Catharanthus roseus) that provide entry into two distinct alkaloid classes, the sarpagans and the ß-carbolines. Our results highlight how a common enzymatic mechanism, guided by related but structurally distinct substrates, leads to either cyclization or aromatization.

Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor.

Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET-domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS-PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli.

Gene Discovery in Gelsemium Highlights Conserved Gene Clusters in Monoterpene Indole Alkaloid Biosynthesis.

Genome mining is a routine technique in microbes for discovering biosynthetic pathways. In plants, however, genomic information is not commonly used to identify novel biosynthesis genes. Here, we present the genome of the medicinal plant and oxindole monoterpene indole alkaloid (MIA) producer Gelsemium sempervirens (Gelsemiaceae). A gene cluster from Catharanthus roseus, which is utilized at least six enzymatic steps downstream from the last common intermediate shared between the two plant alkaloid types, is found in G. sempervirens, although the corresponding enzymes act on entirely different substrates. This study provides insights into the common genomic context of MIA pathways and is an important milestone in the further elucidation of the Gelsemium oxindole alkaloid pathway.

A Pair of Bacterial Siderophores Releases and Traps an Intercellular Signal Molecule: An Unusual Case of Natural Nitrone Bioconjugation.

In microbial interactions bacteria employ diverse molecules with specific functions, such as sensing the environment, communication with other microbes or hosts, and conferring virulence. Insights into the molecular basis of bacterial communication are thus of high relevance for ecology and medicine. Targeted gene activation and in vitro studies revealed that the cell-to-cell signaling molecule and disease mediator IQS (aeruginaldehyde) of the human pathogen Pseudomonas aeruginosa and related bacteria derives from the siderophore pyochelin. Addition of IQS to bacterial cultures (Burkholderia thailandensis) showed that the signaling molecule is captured by a congener of another siderophore family, malleobactin, to form a nitrone conjugate (malleonitrone) that is active against the IQS-producer. This study uncovers complex communication processes with derailed siderophore functions, a novel nitrone bioconjugation, and a new type of antibiotic against Gram-negative bacteria.

Cyclopropanol Warhead in Malleicyprol Confers Virulence of Human- and Animal-Pathogenic Burkholderia Species.

Burkholderia species such as B. mallei and B. pseudomallei are bacterial pathogens causing fatal infections in humans and animals (glanders and melioidosis), yet knowledge on their virulence factors is limited. While pathogenic effects have been linked to a highly conserved gene locus (bur/mal) in the B. mallei group, the metabolite associated to the encoded polyketide synthase, burkholderic acid (syn. malleilactone), could not explain the observed phenotypes. By metabolic profiling and molecular network analyses of the model organism B. thailandensis, the primary products of the cryptic pathway were identified as unusual cyclopropanol-substituted polyketides. First, sulfomalleicyprols were identified as inactive precursors of burkholderic acid. Furthermore, a highly reactive upstream metabolite, malleicyprol, was discovered and obtained in two stabilized forms. Cell-based assays and a nematode infection model showed that the rare natural product confers cytotoxicity and virulence.

Isolation and characterisation of irinans, androstane-type withanolides from Physalis peruviana L.

Withanolides are steroidal lactones widespread in Nightshade plants with often potent antiproliferative activities. Additionally, the structural diversity of this compound class holds much potential for the discovery of novel biological activity. Here, we report two newly characterised withanolides, named irinans, from Physalis peruviana with highly unusual truncated backbones that resemble mammalian androstane sex hormones. Based on biomimetic chemical reactions, we propose a model that links these compounds to withanolide biosynthesis. Irinans have potent antiproliferative activities, that are however lower than those of 4ß-hydroxywithanolide E. Our work establishes androwithanolides as a new subclass of withanolides.

Dual Catalytic Activity of a Cytochrome P450 Controls Bifurcation at a Metabolic Branch Point of Alkaloid Biosynthesis in Rauwolfia serpentina.

Plants create tremendous chemical diversity from a single biosynthetic intermediate. In plant-derived ajmalan alkaloid pathways, the biosynthetic intermediate vomilenine can be transformed into the anti-arrhythmic compound ajmaline, or alternatively, can isomerize to form perakine, an alkaloid with a structurally distinct scaffold. Here we report the discovery and characterization of vinorine hydroxylase, a cytochrome P450 enzyme that hydroxylates vinorine to form vomilenine, which was found to exist as a mixture of rapidly interconverting epimers. Surprisingly, this cytochrome P450 also catalyzes the non-oxidative isomerization of the ajmaline precursor vomilenine to perakine. This unusual dual catalytic activity of vinorine hydroxylase thereby provides a control mechanism for the bifurcation of these alkaloid pathway branches. This discovery highlights the unusual catalytic functionality that has evolved in plant pathways.

Plasticity of the malleobactin pathway and its impact on siderophore action in human pathogenic bacteria.

The human pathogenic bacteria Burkholderia mallei, Burkholderia pseudomallei, and Burkholderia thailandensis harbor a highly conserved gene cluster coding for the biosynthesis of the long sought-after malleobactins. Four new, unexpected congeners of the malleobactin family that were isolated and fully characterized in this study feature unusual deviations from the parent, ornibactin-like architecture. Thus, the malleobactin non-ribosomal peptide synthetase (NRPS) has a rare flexibility that yields diverse peptide backbones, of which one candidate confers pronounced siderophore activity (EC50: 8.4 µM, CAS assay). These findings not only unveil a highly diverse assembly line but also are an important addition to the knowledgebase of the pathogens' metabolomes.

Divergolide congeners illuminate alternative reaction channels for ansamycin diversification.

Isolation and structure elucidation of six new divergolides reveal unusual ansamycin diversification reactions including formation of the unusual isobutenyl side chain from a branched polyketide synthase extender unit, azepinone ring closure, macrolide ring contraction and formation of a seco variant by a neighboring group-assisted decarboxylation.

Terpenoid biosynthesis off the beaten track: unconventional cyclases and their impact on biomimetic synthesis.

Terpene and terpenoid cyclizations are counted among the most complex chemical reactions occurring in nature and contribute crucially to the tremendous structural diversity of this largest family of natural products. Many studies were conducted at the chemical, genetic, and biochemical levels to gain mechanistic insights into these intriguing reactions that are catalyzed by terpene and terpenoid cyclases. A myriad of these enzymes have been characterized. Classical textbook knowledge divides terpene/terpenoid cyclases into two major classes according to their structure and reaction mechanism. However, recent discoveries of novel types of terpenoid cyclases illustrate that nature's enzymatic repertoire is far more diverse than initially thought. This Review outlines novel terpenoid cyclases that are out of the ordinary.

Evolution of siderophore pathways in human pathogenic bacteria.

Ornibactin and malleobactin are hydroxamate siderophores employed by human pathogenic bacteria belonging to the genus Burkholderia. Similarities in their structures and corresponding biosynthesis gene clusters strongly suggest an evolutionary relationship. Through gene coexpression and targeted gene manipulations, the malleobactin pathway was successfully morphed into an ornibactin assembly line. Such an evolutionary-guided approach has been unprecedented for nonribosomal peptide synthetases. Furthermore, the timing of amino acid acylation before peptide assembly, the absolute configuration of the ornibactin side chain, and the function of the acyl transferase were elucidated. Beyond providing a proof of principle for the rational design of siderophore pathways, a compelling model for the evolution of virulence traits is presented.

Biosynthetic code for divergolide assembly in a bacterial mangrove endophyte.

Divergolides are structurally diverse ansamycins produced by a bacterial endophyte (Streptomyces sp.) of the mangrove tree Bruguiera gymnorrhiza. By genomic analyses a gene locus coding for the divergolide pathway was detected. The div gene cluster encodes genes for the biosynthesis of 3-amino-5-hydroxybenzoate and the rare extender units ethylmalonyl-CoA and isobutylmalonyl-CoA, polyketide assembly by a modular type I polyketide synthase (PKS), and enzymes involved in tailoring reactions, such as a Baeyer-Villiger oxygenase. A detailed PKS domain analysis confirmed the stereochemical integrity of the divergolides and provided valuable new insights into the formation of the diverse aromatic chromophores. The bioinformatic analyses and the isolation and full structural elucidation of four new divergolide congeners led to a revised biosynthetic model that illustrates the formation of four different types of ansamycin chromophores from a single polyketide precursor.

Chemical tools for unpicking plant specialised metabolic pathways.

Elucidating the biochemical pathways of specialised metabolites in plants is key to enable or improve their sustainable biotechnological production. Chemical tools can greatly facilitate the discovery of biosynthetic genes and enzymes. Here, we summarise transdisciplinary approaches where methods from chemistry and chemical biology helped to overcome key challenges of pathway elucidation. Based on recent examples, we describe how state-of-the-art isotope labelling experiments can guide the selection of biosynthetic gene candidates, how affinity-based probes enable the identification of novel enzymes, how semisynthesis can improve the availability of elusive pathway intermediates, and how biomimetic reactions provide a better understanding of inherent chemical reactivity. We anticipate that a wider application of such chemical methods will accelerate the pace of pathway elucidation in plants.

Biosynthesis of the allelopathic alkaloid gramine in barley by a cryptic oxidative rearrangement.

The defensive alkaloid gramine not only protects barley and other grasses from insects but also negatively affects their palatability to ruminants. The key gene for gramine formation has remained elusive, hampering breeding initiatives. In this work, we report that a gene encoding cytochrome P450 monooxygenase CYP76M57, which we name AMI synthase (AMIS), enables the production of gramine in Nicotiana benthamiana, Arabidopsis thaliana, and Saccharomyces cerevisiae. We reconstituted gramine production in the gramine-free barley (Hordeum vulgare) variety Golden Promise and eliminated it from cultivar Tafeno by Cas-mediated gene editing. In vitro experiments unraveled that an unexpected cryptic oxidative rearrangement underlies this noncanonical conversion of an amino acid to a chain-shortened biogenic amine. The discovery of the genetic basis of gramine formation now permits tailor-made optimization of gramine-linked traits in barley by plant breeding.

Leveraging synthetic biology and metabolic engineering to overcome obstacles in plant pathway elucidation.

Major hurdles in plant biosynthetic pathway elucidation and engineering include the need for rapid testing of enzyme candidates and the lack of complex substrates that are often not accumulated in the plant, amenable to synthesis, or commercially available. Linking metabolic engineering with gene discovery in both yeast and plant holds great promise to expedite the elucidation process and, at the same time, provide a platform for the sustainable production of plant metabolites. In this review, we highlight how synthetic biology and metabolic engineering alleviated longstanding obstacles in plant pathway elucidation. Recent advances in developing these chassis that showcase established and emerging strategies in accelerating biosynthetic gene discovery will also be discussed.

A New Fluorescence Detection Method for Tryptophan- and Tyrosine-Derived Allelopathic Compounds in Barley and Lupin.

Barley (Hordeum vulgare) is one of the most widely cultivated crops for feedstock and beer production, whereas lupins (Lupinus spp.) are grown as fodder and their seeds are a source of protein. Both species produce the allelopathic alkaloids gramine and hordenine. These plant-specialized metabolites may be of economic interest for crop protection, depending on their tissue distribution. However, in high concentrations they pose a health risk to humans and animals that feed on them. This study was carried out to develop and validate a new method for monitoring these alkaloids and their related metabolites using fluorescence detection. Separation was performed on an HSS T3 column using slightly acidified water-acetonitrile eluents. Calibration plots expressed linearity over the range 0.09-100 pmol/µL for gramine. The accuracy and precision ranged from 97.8 to 123.4%, <7% RSD. The method was successfully applied in a study of the natural range of abundance of gramine, hordenine and their related metabolites, AMI, tryptophan and tyramine, in 22 barley accessions and 10 lupin species. This method provides accurate and highly sensitive chromatographic separation and detection of tryptophan- and tyrosine-derived allelochemicals and is an accessible alternative to LC-MS techniques for routine screening.