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.

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.

Identification of early quassinoid biosynthesis in the invasive tree of heaven (Ailanthus altissima) confirms evolutionary origin from protolimonoids.

The tree of heaven, Ailanthus altissima (MILL.) SWINGLE, is a globally invasive plant known to secrete allelopathic metabolites called quassinoids. Quassinoids are highly modified triterpenoids. So far, nothing has been known about the biochemical basis of quassinoid biosynthesis. Here, based on transcriptome and metabolome data of Ailanthus altissima, we present the first three steps of quassinoid biosynthesis, which are catalysed by an oxidosqualene cyclase and two cytochrome P450 monooxygenases, resulting in the formation of the protolimonoid melianol. Strikingly, these steps are identical to the first steps of the biosynthesis of limonoids, structurally different triterpenoids from sister plant families within the same order Sapindales. Our results are therefore not only important to fully understand the biosynthesis of complex triterpenoids in plants, but also confirm the long-standing hypothesis that quassinoids and limonoids share an evolutionary origin. In addition, our transcriptome data for Ailanthus altissima will be beneficial to other researchers investigating the physiology and ecology of this invasive tree.

Mesoionic Carbenes in Low- to High-Valent Vanadium Chemistry.

We report the synthesis of vanadium(V) oxo complex 1 with a pincer-type dianionic mesoionic carbene (MIC) ligand L1 and the general formula [VOCl(L1)]. A comparison of the structural (SC-XRD), electronic (UV-vis), and electrochemical (cyclic voltammetry) properties of 1 with the benzimidazolinylidene congener 2 (general formula [VOCl(L2)]) shows that the MIC is a stronger donor also for early transition metals with low d-electron population. Since electrochemical studies revealed both complexes to be reversibly reduced, the stronger donor character of MICs was not only demonstrated for the vanadium(V) but also for the vanadium(IV) oxidation state by isolating the reduced vanadium(IV) complexes [Co(Cp*)2][1] and [Co(Cp*)2][2] ([Co(Cp*)2] = decamethylcobaltocenium). The electronic structures of the compounds were investigated by computational methods. Complex 1 was found to be a moderate precursor for salt metathesis reactions, showing selective reactivity toward phenolates or secondary amides, but not toward primary amides and phosphides, thiophenols, or aryls/alkyls donors. Deoxygenation with electron-rich phosphines failed to give the desired vanadium(III) complex. However, treatment of the deprotonated ligand precursor with vanadium(III) trichloride resulted in the clean formation of the corresponding MIC vanadium(III) complex 6, which undergoes a clean two-electron oxidation with organic azides yielding the corresponding imido complexes. The reaction with TMS-N3 did not afford a nitrido complex, but instead the imido complex 10. This study reveals that, contrary to popular belief, MICs are capable of supporting early transition-metal complexes in a variety of oxidation states, thus making them promising candidates for the activation of small molecules and redox catalysis.