RESUMO
The model moss species Physcomitrium patens has long been used for studying divergence of land plants spanning from bryophytes to angiosperms. In addition to its phylogenetic relationships, the limited number of differential tissues, and comparable morphology to the earliest embryophytes provide a system to represent basic plant architecture. Based on plant-fungal interactions today, it is hypothesized these kingdoms have a long-standing relationship, predating plant terrestrialization. Mortierellaceae have origins diverging from other land fungi paralleling bryophyte divergence, are related to arbuscular mycorrhizal fungi but are free-living, observed to interact with plants, and can be found in moss microbiomes globally. Due to their parallel origins, we assess here how two Mortierellaceae species, Linnemannia elongata and Benniella erionia, interact with P. patens in coculture. We also assess how Mollicute-related or Burkholderia-related endobacterial symbionts (MRE or BRE) of these fungi impact plant response. Coculture interactions are investigated through high-throughput phenomics, microscopy, RNA-sequencing, differential expression profiling, gene ontology enrichment, and comparisons among 99 other P. patens transcriptomic studies. Here we present new high-throughput approaches for measuring P. patens growth, identify novel expression of over 800 genes that are not expressed on traditional agar media, identify subtle interactions between P. patens and Mortierellaceae, and observe changes to plant-fungal interactions dependent on whether MRE or BRE are present. Our study provides insights into how plants and fungal partners may have interacted based on their communications observed today as well as identifying L. elongata and B. erionia as modern fungal endophytes with P. patens.
Assuntos
Briófitas , Bryopsida , Micorrizas , Filogenia , Endófitos/metabolismo , Análise Multinível , Proteínas de Plantas/metabolismo , Bryopsida/genética , Bryopsida/metabolismo , Briófitas/genética , Briófitas/metabolismo , Micorrizas/metabolismoRESUMO
From the perspectives of pathway evolution, discovery and engineering of plant specialized metabolism, the nature of the biosynthetic routes represents a critical aspect. Classical models depict biosynthesis typically from an end-point angle and as linear, for example, connecting central and specialized metabolism. As the number of functionally elucidated routes increased, the enzymatic foundation of complex plant chemistries became increasingly well understood. The perception of linear pathway models has been severely challenged. With a focus on plant terpenoid specialized metabolism, we review here illustrative examples supporting that plants have evolved complex networks driving chemical diversification. The completion of several diterpene, sesquiterpene and monoterpene routes shows complex formation of scaffolds and their subsequent functionalization. These networks show that branch points, including multiple sub-routes, mean that metabolic grids are the rule rather than the exception. This concept presents significant implications for biotechnological production.
Assuntos
Alquil e Aril Transferases , Diterpenos , Sesquiterpenos , Filogenia , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Diterpenos/metabolismo , Plantas/genética , Plantas/metabolismo , Sesquiterpenos/metabolismo , Terpenos/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Building sustainable platforms to produce biofuels and specialty chemicals has become an increasingly important strategy to supplement and replace fossil fuels and petrochemical-derived products. Terpenoids are the most diverse class of natural products that have many commercial roles as specialty chemicals. Poplar is a fast growing, biomassdense bioenergy crop with many species known to produce large amounts of the hemiterpene isoprene, suggesting an inherent capacity to produce significant quantities of other terpenes. Here we aimed to engineer poplar with optimized pathways to produce squalene, a triterpene commonly used in cosmetic oils, a potential biofuel candidate, and the precursor to the further diversified classes of triterpenoids and sterols. The squalene production pathways were either re-targeted from the cytosol to plastids or co-produced with lipid droplets in the cytosol. Squalene and lipid droplet co-production appeared to be toxic, which we hypothesize to be due to disruption of adventitious root formation, suggesting a need for tissue specific production. Plastidial squalene production enabled up to 0.63 mg/g fresh weight in leaf tissue, which also resulted in reductions in isoprene emission and photosynthesis. These results were also studied through a technoeconomic analysis, providing further insight into developing poplar as a production host.
Assuntos
Populus , Esqualeno , Esqualeno/metabolismo , Populus/metabolismo , Populus/genética , Populus/crescimento & desenvolvimento , Engenharia Metabólica/métodos , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/genética , Triterpenos/metabolismo , Biocombustíveis , Plastídeos/metabolismoRESUMO
Serrulatane diterpenoids are natural products found in plants from a subset of genera within the figwort family (Scrophulariaceae). Many of these compounds have been characterized as having anti-microbial properties and share a common diterpene backbone. One example, leubethanol from Texas sage (Leucophyllum frutescens) has demonstrated activity against multi-drug-resistant tuberculosis. Leubethanol is the only serrulatane diterpenoid identified from this genus; however, a range of such compounds have been found throughout the closely related Eremophila genus. Despite their potential therapeutic relevance, the biosynthesis of serrulatane diterpenoids has not been previously reported. Here we leverage the simple product profile and high accumulation of leubethanol in the roots of L. frutescens and compare tissue-specific transcriptomes with existing data from Eremophila serrulata to decipher the biosynthesis of leubethanol. A short-chain cis-prenyl transferase (LfCPT1) first produces the rare diterpene precursor nerylneryl diphosphate, which is cyclized by an unusual plastidial terpene synthase (LfTPS1) into the characteristic serrulatane diterpene backbone. Final conversion to leubethanol is catalyzed by a cytochrome P450 (CYP71D616) of the CYP71 clan. This pathway documents the presence of a short-chain cis-prenyl diphosphate synthase, previously only found in Solanaceae, which is likely involved in the biosynthesis of other known diterpene backbones in Eremophila. LfTPS1 represents neofunctionalization of a compartment-switching terpene synthase accepting a novel substrate in the plastid. Biosynthetic access to leubethanol will enable pathway discovery to more complex serrulatane diterpenoids which share this common starting structure and provide a platform for the production and diversification of this class of promising anti-microbial therapeutics in heterologous systems.
Assuntos
Diterpenos/metabolismo , Scrophulariaceae/metabolismo , Alquil e Aril Transferases/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Eremophila (Planta)/genética , Escherichia coli/genética , Neopreno/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Fosfatos de Poli-Isoprenil/metabolismo , Scrophulariaceae/genética , Nicotiana/genética , Nicotiana/metabolismo , Transferases/genética , Transferases/metabolismoRESUMO
Members of the mint family (Lamiaceae) accumulate a wide variety of industrially and medicinally relevant diterpenes. We recently sequenced leaf transcriptomes from 48 phylogenetically diverse Lamiaceae species. Here, we summarize the available chemotaxonomic and enzyme activity data for diterpene synthases (diTPSs) in the Lamiaceae and leverage the new transcriptomes to explore the diTPS sequence and functional space. Candidate genes were selected with an intent to evenly sample the sequence homology space and to focus on species in which diTPS transcripts were found, yet from which no diterpene structures have been previously reported. We functionally characterized nine class II diTPSs and 10 class I diTPSs from 11 distinct plant species and found five class II activities, including two novel activities, as well as a spectrum of class I activities. Among the class II diTPSs, we identified a neo-cleroda-4(18),13E-dienyl diphosphate synthase from Ajuga reptans, catalyzing the likely first step in the biosynthesis of a variety of insect-antifeedant compounds. Among the class I diTPSs was a palustradiene synthase from Origanum majorana, leading to the discovery of specialized diterpenes in that species. Our results provide insights into the diversification of diterpene biosynthesis in the mint family and establish a comprehensive foundation for continued investigation of diterpene biosynthesis in the Lamiaceae.
Assuntos
Alquil e Aril Transferases/metabolismo , Bases de Dados de Produtos Farmacêuticos , Diterpenos/metabolismo , Lamiaceae/enzimologia , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Lamiaceae/genética , Lamiaceae/crescimento & desenvolvimento , Filogenia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genéticaRESUMO
Vitex agnus-castus L. (Lamiaceae) is a medicinal plant historically used throughout the Mediterranean region to treat menstrual cycle disorders, and is still used today as a clinically effective treatment for premenstrual syndrome. The pharmaceutical activity of the plant extract is linked to its ability to lower prolactin levels. This feature has been attributed to the presence of dopaminergic diterpenoids that can bind to dopamine receptors in the pituitary gland. Phytochemical analyses of V. agnus-castus show that it contains an enormous array of structurally related diterpenoids and, as such, holds potential as a rich source of new dopaminergic drugs. The present work investigated the localisation and biosynthesis of diterpenoids in V. agnus-castus. With the assistance of matrix-assisted laser desorption ionisation-mass spectrometry imaging (MALDI-MSI), diterpenoids were localised to trichomes on the surface of fruit and leaves. Analysis of a trichome-specific transcriptome database, coupled with expression studies, identified seven candidate genes involved in diterpenoid biosynthesis: three class II diterpene synthases (diTPSs); three class I diTPSs; and a cytochrome P450 (CYP). Combinatorial assays of the diTPSs resulted in the formation of a range of different diterpenes that can account for several of the backbones of bioactive diterpenoids observed in V. agnus-castus. The identified CYP, VacCYP76BK1, was found to catalyse 16-hydroxylation of the diol-diterpene, peregrinol, to labd-13Z-ene-9,15,16-triol when expressed in Saccharomyces cerevisiae. Notably, this product is a potential intermediate in the biosynthetic pathway towards bioactive furan- and lactone-containing diterpenoids that are present in this species.
Assuntos
Diterpenos/metabolismo , Proteínas de Plantas/metabolismo , Vitex/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Diterpenos/análise , Perfilação da Expressão Gênica , Oxirredução , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Medicinais/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tricomas/metabolismo , Vitex/genéticaRESUMO
The mint family (Lamiaceae) is well documented as a rich source of terpene natural products. More than 200 diterpene skeletons have been reported from mints, but biosynthetic pathways are known for just a few of these. We crossreferenced chemotaxonomic data with publicly available transcriptomes to select common selfheal (Prunella vulgaris) and its highly unusual vulgarisin diterpenoids as a case study for exploring the origins of diterpene skeletal diversity in Lamiaceae. Four terpene synthases (TPS) from the TPS-a subfamily, including two localised to the plastid, were cloned and functionally characterised. Previous examples of TPS-a enzymes from Lamiaceae were cytosolic and reported to act on the 15-carbon farnesyl diphosphate. Plastidial TPS-a enzymes using the 20-carbon geranylgeranyl diphosphate are known from other plant families, having apparently arisen independently in each family. All four new enzymes were found to be active on multiple prenyl-diphosphate substrates with different chain lengths and stereochemistries. One of the new enzymes catalysed the cyclisation of geranylgeranyl diphosphate into 11-hydroxy vulgarisane, the likely biosynthetic precursor of the vulgarisins. We uncovered the pathway to a rare diterpene skeleton. Our results support an emerging paradigm of substrate and compartment switching as important aspects of TPS evolution and diversification.
Assuntos
Alquil e Aril Transferases/metabolismo , Evolução Molecular , Prunella/enzimologia , Alquil e Aril Transferases/genética , Regulação da Expressão Gênica de Plantas , Peptídeos/metabolismo , Filogenia , Folhas de Planta/genética , Raízes de Plantas/genética , Fosfatos de Poli-Isoprenil/metabolismo , Prunella/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Terpenos/química , Terpenos/metabolismo , Transcriptoma/genéticaRESUMO
The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogenase-mediated cyclization step in the biosynthetic pathway of Euphorbia factors, illustrating the origin of the intramolecular carbon-carbon bonds present in lathyrane and ingenane diterpenoids. This unconventional cyclization describes the ring closure of the macrocyclic diterpene casbene. Through transcriptomic analysis of E. lathyris L. mature seeds and in planta functional characterization, we identified three enzymes involved in the cyclization route from casbene to jolkinol C, a lathyrane diterpene. These enzymes include two cytochromes P450 from the CYP71 clan and an alcohol dehydrogenase (ADH). CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and 5-oxidation of casbene, respectively. When coupled with these P450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes dehydrogenation of the hydroxyl groups, leading to the subsequent rearrangement and cyclization. The discovery of this nonconventional cyclization may provide the key link to complete elucidation of the biosynthetic pathways of ingenol mebutate and other bioactive macrocyclic diterpenoids.
Assuntos
Antineoplásicos Fitogênicos/biossíntese , Diterpenos/metabolismo , Euphorbia/química , Fenilpropionatos/metabolismo , Proteínas de Plantas/genética , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Antineoplásicos Fitogênicos/química , Clonagem Molecular , Ciclização , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Diterpenos/química , Euphorbia/genética , Euphorbia/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Isoenzimas/genética , Isoenzimas/metabolismo , Oxirredução , Fenilpropionatos/química , Óleos de Plantas/química , Óleos de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sementes/química , Sementes/genética , Sementes/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , TranscriptomaRESUMO
Tripterygium wilfordii (Celastraceae) is a medicinal plant with anti-inflammatory and immunosuppressive properties. Identification of a vast array of unusual sesquiterpenoids, diterpenoids and triterpenoids in T. wilfordii has spurred investigations of their pharmacological properties. The tri-epoxide lactone triptolide was the first of many diterpenoids identified, attracting interest due to the spectrum of bioactivities. To probe the genetic underpinning of diterpenoid diversity, an expansion of the class II diterpene synthase (diTPS) family was recently identified in a leaf transcriptome. Following detection of triptolide and simple diterpene scaffolds in the root, we sequenced and mined the root transcriptome. This allowed identification of the root-specific complement of TPSs and an expansion in the class I diTPS family. Functional characterization of the class II diTPSs established their activities in the formation of four C-20 diphosphate intermediates, precursors of both generalized and specialized metabolism and a novel scaffold for Celastraceae. Functional pairs of the class I and II enzymes resulted in formation of three scaffolds, accounting for some of the terpenoid diversity found in T. wilfordii. The absence of activity-forming abietane-type diterpenes encouraged further testing of TPSs outside the canonical class I diTPS family. TwTPS27, close relative of mono-TPSs, was found to couple with TwTPS9, converting normal-copalyl diphosphate to miltiradiene. The phylogenetic distance to established diTPSs indicates neo-functionalization of TwTPS27 into a diTPS, a function not previously observed in the TPS-b subfamily. This example of evolutionary convergence expands the functionality of TPSs in the TPS-b family and may contribute miltiradiene to the diterpenoids of T. wilfordii.
Assuntos
Alquil e Aril Transferases/genética , Liases Intramoleculares/genética , Proteínas de Plantas/genética , Tripterygium/genética , Abietanos/química , Abietanos/metabolismo , Alquil e Aril Transferases/classificação , Alquil e Aril Transferases/metabolismo , Sequência de Aminoácidos , Diterpenos/química , Diterpenos/metabolismo , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Perfilação da Expressão Gênica/métodos , Liases Intramoleculares/metabolismo , Estrutura Molecular , Monoterpenos/química , Monoterpenos/metabolismo , Família Multigênica , Fenantrenos/química , Fenantrenos/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Homologia de Sequência de Aminoácidos , Tripterygium/enzimologiaRESUMO
ABSTRACT: Catalyzing stereo- and regio-specific oxidation of inert hydrocarbon backbones, and a range of more exotic reactions inherently difficult in formal chemical synthesis, cytochromes P450 (P450s) offer outstanding potential for biotechnological engineering. Plants and their dazzling diversity of specialized metabolites have emerged as rich repository for functional P450s with the advances of deep transcriptomics and genome wide discovery. P450s are of outstanding interest for understanding chemical diversification throughout evolution, for gaining mechanistic insights through the study of their structure-function relationship, and for exploitation in Synthetic Biology. In this review, we highlight recent developments and examples in the discovery of plant P450s involved in the biosynthesis of industrially relevant monoterpenoids, sesquiterpenoids, diterpenoids and triterpenoids, throughout 2016 and early 2017. Examples were selected to illustrate the spectrum of value from commodity chemicals, flavor and fragrance compounds to pharmacologically active terpenoids. We focus on a recently emerging theme, where P450s control metabolic bifurcations and chemical diversity of the final product profile, either within a pathway, or through neo-functionalization in related species. The implications may inform approaches for rational assembly of recombinant pathways, biotechnological production of high value terpenoids and generation of novel chemical entities.
RESUMO
Hydrophobic resin acids (RAs) are synthesized by conifer trees as part of their defense mechanisms. One of the functions of RAs in plant defense is suggested to be the perturbation of the cellular membrane. However, there is a vast diversity of chemical structures within this class of molecules, and there are no clear correlations to the molecular mechanisms behind the RA's toxicity. In this study we unravel the molecular interactions of the three closely related RAs dehydroabietic acid, neoabietic acid, and the synthetic analogue dichlorodehydroabietic acid with dipalmitoylphosphatidylcholine (DPPC) model membranes and the polar lipid extract of soybeans. The complementarity of the biophysical techniques used (NMR, DLS, NR, DSC, Cryo-TEM) allowed correlating changes at the vesicle level with changes at the molecular level and the co-localization of RAs within DPPC monolayer. Effects on DPPC membranes are correlated with the physical chemical properties of the RA and their toxicity.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Abietanos/química , Anti-Infecciosos/química , Bicamadas Lipídicas/química , Abietanos/síntese química , Abietanos/isolamento & purificação , Anti-Infecciosos/isolamento & purificação , Microscopia Crioeletrônica , Espectroscopia de Ressonância Magnética , Extratos Vegetais/química , Glycine max/química , Traqueófitas/químicaRESUMO
The development of medical applications exploiting the broad bioactivities of the diterpene therapeutic triptolide from Tripterygium wilfordii is limited by low extraction yields from the native plant. Furthermore, the extraordinarily high structural complexity prevents an economically attractive enantioselective total synthesis. An alternative production route of triptolide through engineered Saccharomyces cerevisiae (yeast) could provide a sustainable source of triptolide. A potential intermediate in the unknown biosynthetic route to triptolide is the diterpene dehydroabietic acid. Here, we report a biosynthetic route to dehydroabietic acid by transient expression of enzymes from T. wilfordii and Sitka spruce (Picea sitchensis) in Nicotiana benthamiana. The combination of diterpene synthases TwTPS9, TwTPS27, and cytochromes P450 PsCYP720B4 yielded dehydroabietic acid and a novel analog, tentatively identified as 'miltiradienic acid'. This biosynthetic pathway was reassembled in a yeast strain engineered for increased yields of the pathway intermediates, the diterpene olefins miltiradiene and dehydroabietadiene. Introduction in that strain of PsCYP720B4 in combination with two alternative NADPH-dependent cytochrome P450 reductases resulted in scalable in vivo production of dehydroabietic acid and its analog from glucose. Approaching future elucidation of the remaining biosynthetic steps to triptolide, our findings may provide an independent platform for testing of additional recombinant candidate genes, and ultimately pave the way to biotechnological production of the high value diterpenoid therapeutic.
Assuntos
Abietanos/biossíntese , Vias Biossintéticas/genética , Diterpenos/química , Fenantrenos/química , Abietanos/genética , Sistema Enzimático do Citocromo P-450/genética , Diterpenos/uso terapêutico , Compostos de Epóxi/química , Compostos de Epóxi/uso terapêutico , Glucose/química , Glucose/metabolismo , Engenharia Metabólica , Fenantrenos/uso terapêutico , Filogenia , Picea/enzimologia , Picea/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Nicotiana/enzimologia , Nicotiana/genética , Tripterygium/enzimologia , Tripterygium/genéticaRESUMO
According to the International Diabetes Federation, type 2 diabetes (T2D) has reached epidemic proportions, affecting more than 382 million people worldwide. Inhibition of protein tyrosine phosphatase-1B (PTP1B) and α-glucosidase is a recognized therapeutic approach for management of T2D and its associated complications. The lack of clinical drugs targeting PTP1B and side effects of the existing α-glucosidase drugs, emphasize the need for new drug leads for these T2D targets. In the present work, dual high-resolution PTP1B and α-glucosidase inhibition profiles of Eremophila gibbosa, E. glabra, and E. aff. drummondii "Kalgoorlie" were used for pinpointing α-glucosidase and/or PTP1B inhibitory constituents directly from the crude extracts. A subsequent targeted high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR) analysis and preparative-scale HPLC isolation led to identification of 21 metabolites from the three species, of which 16 were serrulatane-type diterpenoids (12 new) associated with either α-glucosidase and/or PTP1B inhibition. This is the first report of serrulatane-type diterpenoids as potential α-glucosidase and/or PTP1B inhibitors.
Assuntos
Inibidores de Glicosídeo Hidrolases/farmacologia , Hipoglicemiantes/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus Tipo 2 , Inibidores de Glicosídeo Hidrolases/química , Humanos , Estrutura Molecular , Scrophulariaceae , Extração em Fase Sólida , alfa-Glucosidases/efeitos dos fármacosRESUMO
Plant-derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of classâ I and II diterpene synthases, 41 of which are "new-to-nature". Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or diastereomeric counterparts. Scalable biotechnological production for four industrially relevant targets was accomplished in engineered strains of Saccharomyces cerevisiae.
Assuntos
Diterpenos/química , Diterpenos/metabolismo , Estrutura Molecular , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , EstereoisomerismoRESUMO
Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce a hydroxyl group at carbon C-8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C-9, yielding the bicyclic peregrinol diphosphate. MvELS belongs to a subgroup of the diTPS TPS-e/f clade with unusual ßα-domain architecture. MvELS is active in vitro and in vivo with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, as identified by nuclear magnetic resonance (NMR) analysis, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids.
Assuntos
Diterpenos/metabolismo , Marrubium/enzimologia , Alquil e Aril Transferases , Sequência de Aminoácidos , Escherichia coli/genética , Marrubium/química , Marrubium/genética , Dados de Sequência Molecular , Organofosfatos , Filogenia , Proteínas de Plantas , Plantas MedicinaisRESUMO
Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.
Assuntos
Clonagem Molecular/métodos , DNA , Engenharia Genética/métodos , Plantas Geneticamente Modificadas/genética , Plantas/genética , Biologia Sintética/métodos , Botânica , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eucariotos/genética , Engenharia Genética/normas , Plasmídeos , Padrões de Referência , Transcrição GênicaRESUMO
Forskolin, a complex labdane diterpenoid found in the root of Coleus forskohlii (Lamiaceae), has received attention for its broad range of pharmacological activities, yet the biosynthesis has not been elucidated. We detected forskolin in the root cork of C. forskohlii in a specialized cell type containing characteristic structures with histochemical properties consistent with oil bodies. Organelle purification and chemical analysis confirmed the localization of forskolin and of its simplest diterpene precursor backbone, (13R) manoyl oxide, to the oil bodies. The labdane diterpene backbone is typically synthesized by two successive reactions catalyzed by two distinct classes of diterpene synthases. We have recently described the identification of a small gene family of diterpene synthase candidates (CfTPSs) in C. forskohlii. Here, we report the functional characterization of four CfTPSs using in vitro and in planta assays. CfTPS2, which synthesizes the intermediate copal-8-ol diphosphate, in combination with CfTPS3 resulted in the stereospecific formation of (13R) manoyl oxide, while the combination of CfTPS1 and CfTPS3 or CfTPS4 led to formation of miltiradiene, precursor of abietane diterpenoids in C. forskohlii. Expression profiling and phylogenetic analysis of the CfTPS family further support the functional diversification and distinct roles of the individual diterpene synthases and the involvement of CfTPS1 to CfTPS4 in specialized metabolism and of CfTPS14 and CfTPS15 in general metabolism. Our findings pave the way toward the discovery of the remaining components of the pathway to forskolin, likely localized in this specialized cell type, and support a role of oil bodies as storage organelles for lipophilic bioactive metabolites.
Assuntos
Vias Biossintéticas , Coleus/citologia , Coleus/metabolismo , Colforsina/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Abietanos/química , Abietanos/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Biomassa , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Coleus/genética , Colforsina/química , Estruturas Citoplasmáticas/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Luz , Lipídeos/química , Família Multigênica , Organelas/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espalhamento de RadiaçãoRESUMO
Forskolin is a promising medicinal compound belonging to a plethora of specialized plant metabolites that constitute a rich source of bioactive high-value compounds. A major obstacle for exploitation of plant metabolites is that they often are produced in small amounts and in plants difficult to cultivate. This may result in insufficient and unreliable supply leading to fluctuating and high sales prices. Hence, substantial efforts and resources have been invested in developing sustainable and reliable supply routes based on microbial cell factories. Here, we report microbial synthesis of (13R)-manoyl oxide, a proposed intermediate in the biosynthesis of forskolin and other medically important labdane-type terpenoids. Process optimization enabled synthesis of enantiomerically pure (13R)-manoyl oxide as the sole metabolite, providing a pure compound in just two steps with a yield of 10 mg/liter. The work presented here demonstrates the value of a standardized bioengineering pipeline and the large potential of microbial cell factories as sources for sustainable synthesis of complex biochemicals.
Assuntos
Biotecnologia/métodos , Diterpenos/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica , Plantas/enzimologia , Colforsina/metabolismo , Escherichia coli/genética , Plantas/genética , Plantas/metabolismo , EstereoisomerismoRESUMO
Plants produce over 10,000 different diterpenes of specialized (secondary) metabolism, and fewer diterpenes of general (primary) metabolism. Specialized diterpenes may have functions in ecological interactions of plants with other organisms and also benefit humanity as pharmaceuticals, fragrances, resins, and other industrial bioproducts. Examples of high-value diterpenes are taxol and forskolin pharmaceuticals or ambroxide fragrances. Yields and purity of diterpenes obtained from natural sources or by chemical synthesis are often insufficient for large-volume or high-end applications. Improvement of agricultural or biotechnological diterpene production requires knowledge of biosynthetic genes and enzymes. However, specialized diterpene pathways are extremely diverse across the plant kingdom, and most specialized diterpenes are taxonomically restricted to a few plant species, genera, or families. Consequently, there is no single reference system to guide gene discovery and rapid annotation of specialized diterpene pathways. Functional diversification of genes and plasticity of enzyme functions of these pathways further complicate correct annotation. To address this challenge, we used a set of 10 different plant species to develop a general strategy for diterpene gene discovery in nonmodel systems. The approach combines metabolite-guided transcriptome resources, custom diterpene synthase (diTPS) and cytochrome P450 reference gene databases, phylogenies, and, as shown for select diTPSs, single and coupled enzyme assays using microbial and plant expression systems. In the 10 species, we identified 46 new diTPS candidates and over 400 putatively terpenoid-related P450s in a resource of nearly 1 million predicted transcripts of diterpene-accumulating tissues. Phylogenetic patterns of lineage-specific blooms of genes guided functional characterization.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Diterpenos/metabolismo , Biologia Molecular/métodos , Plantas/genética , Plantas/metabolismo , Clonagem Molecular , Mineração de Dados , Bases de Dados Genéticas , Evolução Molecular , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , TranscriptomaRESUMO
Terpenoids represent the most diverse class of natural products, with a broad spectrum of industrial relevance including applications in green solvents, flavors and fragrances, nutraceuticals, colorants, and therapeutics. They are typically challenging to extract from their natural sources, where they occur in small amounts and mixtures of related but unwanted byproducts. Formal chemical synthesis, where established, is reliant on petrochemistry. Hence, there is great interest in developing sustainable solutions to assemble biosynthetic pathways in engineered host organisms. Metabolic engineering for chemical production has largely focused on microbial hosts, yet plants offer a sustainable production platform. In addition to containing the precursor pathways that generate the terpenoid building blocks as well as the cell structures and compartments required, or tractable localization for the enzymes involved, plants may provide a low input system to produce these chemicals using carbon dioxide and sunlight only. There have been significant recent advancements in the discovery of pathways to terpenoids of interest as well as strategies to boost yields in host plants. While part of the phytochemical field is focusing on the discovery of biosynthetic pathways, this review will focus on advancements using the pathway toolbox and toward engineering plants for the production of terpenoids. We will highlight strategies currently used to produce target products, optimization of known pathways to improve yields, compartmentalization of pathways within cells, and genetic tools developed to facilitate complex engineering of biosynthetic pathways. These advancements in Synthetic Biology are bringing engineered plant systems closer to commercially relevant hosts for the bioproduction of terpenoids.