Identification of key enzymes responsible for protolimonoid biosynthesis in plants: Opening the door to azadirachtin production

Limonoids are natural products made by plants belonging to the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, contribution to bitterness in citrus fruits, and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree (Azadirachta indica). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranortriterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of 4 carbons with associated furan ring formation, by an as yet unknown mechanism. Here we have mined genome and transcriptome sequence resources for 3 diverse limonoid-producing species (A. indica, Melia azedarach, and Citrus sinensis) to elucidate the early steps in limonoid biosynthesis. We identify an oxidosqualene cyclase able to produce the potential 30-carbon triterpene scaffold precursor tirucalla-7,24-dien-3β-ol from each of the 3 species. We further identify coexpressed cytochrome P450 enzymes from M. azedarach (MaCYP71CD2 and MaCYP71BQ5) and C. sinensis (CsCYP71CD1 and CsCYP71BQ4) that are capable of 3 oxidations of tirucalla-7,24-dien-3β-ol, resulting in spontaneous hemiacetal ring formation and the production of the protolimonoid melianol. Our work reports the characterization of protolimonoid biosynthetic enzymes from different plant species and supports the notion of pathway conservation between both plant families. It further paves the way for engineering crop plants with enhanced insect resistance and producing high-value limonoids for pharmaceutical and other applications by expression in heterologous hosts

A Concise and Versatile Double-Cyclization Strategy for the Highly Stereoselective Synthesis and Arylative Dimerization of Aspidosperma Alkaloids

Building cycles: A strategy for the concise, stereoselective synthesis of aspidosperma alkaloids and related structures via a common putative diiminium ion intermediate is reported. The approach enables the dimerization of aspidosperma-type structures at the sterically hindered C2 position. The intermediate is prepared in one step from the shown lactam through an electrophilic double-cyclization cascade (see scheme; Tf=trifluoromethanesulfonyl).National Institute of General Medical Sciences (U.S.) (GM074825

In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme

In vitro reconstitution of a biological complex or process normally involves assembly of multiple individually purified protein components. Here we present a strategy that couples expression and assembly of multiple gene products with functional detection in an in vitro reconstituted protein synthesis system. The strategy potentially allows experimental reconstruction of a multi-component biological complex or process using only DNA templates instead of purified proteins. We applied this strategy to bacterial transcription initiation by co-expressing genes encoding Escherichia coli RNA polymerase subunits and sigma factors in the reconstituted protein synthesis system and by coupling the synthesis and assembly of a functional RNA polymerase holoenzyme with the expression of a reporter gene. Using such a system, we demonstrated sigma-factor-dependent, promoter-specific transcription initiation. Since protein synthesis, complex formation and enzyme catalysis occur in the same in vitro reaction mixture, this reconstruction process resembles natural biosynthetic pathways and avoids time-consuming expression and purification of individual proteins. The strategy can significantly reduce the time normally required by conventional reconstitution methods, allow rapid generation and detection of genetic mutations, and provide an open and designable platform for in vitro study and intervention of complex biological processes

Complex scaffold remodeling in plant triterpene biosynthesis

Triterpenes with complex scaffold modifications are widespread in the plant kingdom. Limonoids are an exemplary family that are responsible for the bitter taste in citrus (e.g., limonin) and the active constituents of neem oil, a widely used bioinsecticide (e.g., azadirachtin). Despite the commercial value of limonoids, a complete biosynthetic route has not been described. We report the discovery of 22 enzymes, including a pair of neofunctionalized sterol isomerases, that catalyze 12 distinct reactions in the total biosynthesis of kihadalactone A and azadirone, products that bear the signature limonoid furan. These results enable access to valuable limonoids and provide a template for discovery and reconstitution of triterpene biosynthetic pathways in plants that require multiple skeletal rearrangements and oxidations

Gaining insight into reactivity differences between malonic acid half thioester (MAHT) and malonic acid half oxyesters (MAHO)

An efficient two-step synthesis of structure- and function-diverse thiophenol- and (cyclo)alkyl-derived malonic acid half thioesters (MAHTs) and phenol-derived malonic acid half oxyesters (MAHOs) has been achieved using cheap, readily available and easily handled starting materials. Our syntheses of MAHTs and MAHOs, the majority of which have not been previously reported, is readily scalable affording gram quantities of product. In a hydrogen deuterium exchange, an interesting stereoelectronic effect was observed when different aryl groups were incorporated. Significant changes in the rates of hydrogen deuterium exchange and levels of isotope incorporation were observed. By way of example, using [2H]methanol and 4-bromophenol-derived MAHO afforded only 14% [2H]-incorporation (9 minutes, k = 31) whereas the corresponding 4-bromothiophenol-derived MAHT afforded 97% [2H]-incorporation (9 minutes, k = 208). In a benchmarked procedure and comprehensive DFT study 54 ester and thioester configurations and conformations where characterised. This established in the MAHT series a sulfur-containing molecular orbital provides a path for increased delocalisation of electron-density into the enol which is unavailable in MAHOs, which facilitates keto-enol tautomerisation and consequently enhances the rate and percentage of hydrogen deuterium exchange

Siderophore-based detection of Fe(iii) and microbial pathogens

Siderophores are low-molecular-weight iron chelators that are produced and exported by bacteria, fungi and plants during periods of nutrient deprivation. The structures, biosynthetic logic, and coordination chemistry of these molecules have fascinated chemists for decades. Studies of such fundamental phenomena guide the use of siderophores and siderophore conjugates in a variety of medicinal applications that include iron-chelation therapies and drug delivery. Sensing applications constitute another important facet of siderophore-based technologies. The high affinities of siderophores for both ferric ions and siderophore receptors, proteins expressed on the cell surface that are required for ferric siderophore import, indicate that these small molecules may be employed for the selective capture of metal ions, proteins, and live bacteria. This minireview summaries progress in methods that utilize native bacterial and fungal siderophore scaffolds for the detection of Fe(III) or microbial pathogens.Massachusetts Institute of Technology. Dept. of Chemistr

Biosynthesis and Molecular Genetics of Polyketides in Marine Dinoflagellates

Marine dinoflagellates are the single most important group of algae that produce toxins, which have a global impact on human activities. The toxins are chemically diverse, and include macrolides, cyclic polyethers, spirolides and purine alkaloids. Whereas there is a multitude of studies describing the pharmacology of these toxins, there is limited or no knowledge regarding the biochemistry and molecular genetics involved in their biosynthesis. Recently, however, exciting advances have been made. Expressed sequence tag sequencing studies have revealed important insights into the transcriptomes of dinoflagellates, whereas other studies have implicated polyketide synthase genes in the biosynthesis of cyclic polyether toxins, and the molecular genetic basis for the biosynthesis of paralytic shellfish toxins has been elucidated in cyanobacteria. This review summarises the recent progress that has been made regarding the unusual genomes of dinoflagellates, the biosynthesis and molecular genetics of dinoflagellate toxins. In addition, the evolution of these metabolic pathways will be discussed, and an outlook for future research and possible applications is provided