RESUMEN
New diterpenoids are accessible from non-natural FPP derivatives as substrates for an enzymatic elongation cyclization cascade using the geranylgeranyl pyrophosphate synthase (GGPPS) from Streptomyces cyaneofuscatus and the spata-13,17-diene synthase (SpS) from Streptomyces xinghaiensis. This approach led to four new biotransformation products including three new cyclododecane cores and a macrocyclic ether. For the first time, a 1,12-terpene cyclization was observed when shifting the central olefinic double bond toward the geminial methyl groups creating a nonconjugated 1,4-diene.
Asunto(s)
Transferasas Alquil y Aril , Dimetilaliltranstransferasa , Diterpenos , Streptomyces , Diterpenos/química , Diterpenos/metabolismo , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Streptomyces/enzimología , Streptomyces/química , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/química , Estructura Molecular , Ciclización , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , BiotransformaciónRESUMEN
Bacitracin is a macrocyclic peptide antibiotic that is widely used as a topical treatment for infections caused by gram-positive bacteria. Mechanistically, bacitracin targets bacteria by specifically binding to the phospholipid undecaprenyl pyrophosphate (C55PP), which plays a key role in the bacterial lipid II cycle. Recent crystallographic studies have shown that when bound to C55PP, bacitracin adopts a highly ordered amphipathic conformation. In doing so, all hydrophobic side chains align on one face of the bacitracin-C55PP complex, presumably interacting with the bacterial cell membrane. These insights led us to undertake structure-activity investigations into the individual contribution of the nonpolar amino acids found in bacitracin. To achieve this we designed, synthesized, and evaluated a series of bacitracin analogues, a number of which were found to exhibit significantly enhanced antibacterial activity against clinically relevant, drug-resistant pathogens. As for the natural product, these next-generation bacitracins were found to form stable complexes with C55PP. The structure-activity insights thus obtained serve to inform the design of C55PP-targeting antibiotics, a key and underexploited antibacterial strategy.
Asunto(s)
Antibacterianos , Bacitracina , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/química , Bacitracina/farmacología , Bacitracina/química , Relación Estructura-Actividad , Farmacorresistencia Bacteriana/efectos de los fármacos , Vancomicina/farmacología , Vancomicina/química , Vancomicina/análogos & derivados , Diseño de Fármacos , Fosfatos de Poliisoprenilo/metabolismo , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/farmacologíaRESUMEN
Eunicellane diterpenoids, containing a typical 6,10-bicycle, are bioactive compounds widely present in marine corals, but rarely found in bacteria and plants. The intrinsic macrocycle exhibits innate structural flexibility resulting in dynamic conformational changes. However, the mechanisms controlling flexibility remain unknown. The discovery of a terpene synthase, MicA, that is responsible for the biosynthesis of a nearly non-flexible eunicellane skeleton, enable us to propose a feasible theory about the flexibility in eunicellane structures. Parallel studies of all eunicellane synthases in nature discovered to date, including 2Z-geranylgeranyl diphosphate incubations and density functional theory-based Boltzmann population computations, reveale that a trans-fused bicycle with a 2Z-configuration alkene restricts conformational flexibility resulting in a nearly non-flexible eunicellane skeleton. The catalytic route and the enzymatic mechanism of MicA are also elucidated by labeling experiments, density functional theory calculations, structural analysis of the artificial intelligence-based MicA model, and mutational studies.
Asunto(s)
Transferasas Alquil y Aril , Diterpenos , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/química , Diterpenos/metabolismo , Diterpenos/química , Fosfatos de Poliisoprenilo/metabolismo , Fosfatos de Poliisoprenilo/química , Modelos MolecularesRESUMEN
Presilphiperfolan-8ß-ol synthase (BcBOT2), a substrate-promiscuous sesquiterpene cyclase (STC) of fungal origin, is capable of converting two new farnesyl pyrophosphate (FPP) derivatives modified at C7 of farnesyl pyrophosphate (FPP) bearing either a hydroxymethyl group or a methoxymethyl group. These substrates were chosen based on a computationally generated model. Biotransformations yielded five new oxygenated terpenoids. Remarkably, the formation of one of these tricyclic products can only be explained by a cationically induced migration of the methoxy group, presumably via a Meerwein-salt intermediate, unprecedented in synthetic chemistry and biosynthesis. The results show the great principle and general potential of terpene cyclases for mechanistic studies of unusual cation chemistry and for the creation of new terpene skeletons.
Asunto(s)
Sesquiterpenos , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismoRESUMEN
Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.
Asunto(s)
Transferasas Alquil y Aril , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/genética , Diterpenos/metabolismo , Diterpenos/química , Pruebas de Enzimas/métodos , Fosfatos de Poliisoprenilo/metabolismo , Fosfatos de Poliisoprenilo/química , CiclizaciónRESUMEN
Terpenoids display chemical and structural diversities as well as important biological activities. Despite their extreme variability, the range of these structures is limited by the scope of natural products that canonically derive from interconvertible five-carbon (C5) isoprene units. New approaches have recently been developed to expand their structural diversity. This review systematically explores the combinatorial biosynthesis of noncanonical building blocks via the coexpression of the canonical mevalonate (MVA) pathway and C-methyltransferases (C-MTs), or by using the lepidopteran mevalonate (LMVA) pathway. Unnatural terpenoids can be created from farnesyl diphosphate (FPP) analogs by chemobiological synthesis and terpene cyclopropanation by artificial metalloenzymes (ArMs). Advanced technologies to accelerate terpene biosynthesis are discussed. This review provides a valuable reference for increasing the diversity of valuable terpenoids and their derivatives, as well as for expanding their potential applications.
Asunto(s)
Biología Sintética , Terpenos , Terpenos/química , Terpenos/metabolismo , Biología Sintética/métodos , Ácido Mevalónico/metabolismo , Ácido Mevalónico/química , Fosfatos de Poliisoprenilo/metabolismo , Fosfatos de Poliisoprenilo/química , Sesquiterpenos/química , Sesquiterpenos/metabolismoRESUMEN
Cilagicin is a Gram-positive active antibiotic that has a dual polyprenyl phosphate binding mechanism that impedes resistance development. Here we bioinformatically screened predicted non-ribosomal polypeptide synthetase encoded structures to search for antibiotics that might similarly avoid resistance development. Synthesis and bioactivity screening of the predicted structures that we identified led to three antibiotics that are active against multidrug-resistant Gram-positive pathogens, two of which, paenilagicin and virgilagicin, did not lead to resistance even after prolonged antibiotic exposure.
Asunto(s)
Antibacterianos , Fosfatos de Poliisoprenilo , Antibacterianos/farmacología , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , FosfatosRESUMEN
Complex glycans serve essential functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane-associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates of specific isoprene unit geometry, such as undecaprenol phosphate (UndP) in bacteria. How these enzymes and substrates interact within a lipid bilayer needs further investigation. Here, we focus on a small enzyme, PglC from Campylobacter, structurally characterized for the first time in 2018 as a detergent-solubilized construct. PglC is a monotopic phosphoglycosyl transferase that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high-level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results provided a high-level view of the membrane-embedded PglC/UndP complex. The findings suggested that it is advantageous for the polyprenol phosphate to adopt a conformation in the same leaflet where the monotopic membrane protein resides as opposed to additionally disrupting the opposing leaflet of the bilayer. Further, the analysis showed that electrostatic steering acts as a major driving force contributing to the recognition and binding of both UndP and the soluble nucleotide sugar substrate. Iterative computational and experimental mutagenesis support a specific interaction of UndP with phosphoglycosyl transferase cationic residues and suggest a role for critical conformational transitions in substrate binding and specificity.
Asunto(s)
Membrana Celular , Poliprenoles , Transferasas , Ligandos , Proteínas de la Membrana , Fosfatos , Poliprenoles/metabolismo , Transferasas/química , Fosfatos de Poliisoprenilo/química , Membrana Celular/química , Bacterias/química , Bacterias/citologíaRESUMEN
Terpenoids account for more than 60% of all natural products, and their carbon skeletons originate from common isoprenoid units of different lengths such as geranyl pyrophosphate and farnesyl pyrophosphate. Here we characterize a metal-dependent, bifunctional isoprenyl diphosphate synthase from the leaf beetle Phaedon cochleariae by structural and functional analyses. Inter- and intramolecular cooperative effects in the homodimer strongly depend on the provided metal ions and regulate the biosynthetic flux of terpene precursors to either biological defence or physiological development. Strikingly, a unique chain length determination domain adapts to form geranyl or farnesyl pyrophosphate by altering enzyme symmetry and ligand affinity between both subunits. In addition, we identify an allosteric geranyl-pyrophosphate-specific binding site that shares similarity with end-product inhibition in human farnesyl pyrophosphate synthase. Our combined findings elucidate a deeply intertwined reaction mechanism in the P. cochleariae isoprenyl diphosphate synthase that integrates substrate, product and metal-ion concentrations to harness its dynamic potential.
Asunto(s)
Difosfatos , Terpenos , Humanos , Terpenos/metabolismo , Difosfatos/química , Difosfatos/metabolismo , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismoRESUMEN
We report the identification of the tnd biosynthetic cluster from the marine-derived fungus Aspergillus flavipes and the in vivo characterization of a cryptic type I diterpene synthase. The heterologous expression of the bifunctional terpene synthase led to the discovery of a diterpene backbone, talarodiene, harboring a benzo[a]cyclopenta[d]cyclooctane tricyclic fused ring system. The conversion of geranylgeranyl diphosphate to talarodiene was investigated using 13C-labeling studies, and stable isotope tracer experiments showed the biotransformation of talarodiene into talaronoid C.
Asunto(s)
Transferasas Alquil y Aril , Aspergillus , Diterpenos , Transferasas Alquil y Aril/metabolismo , Organismos Acuáticos/enzimología , Aspergillus/enzimología , Ciclooctanos , Diterpenos/metabolismo , Fosfatos de Poliisoprenilo/químicaRESUMEN
Prenyl pyrophosphate methyltransferases enhance the structural diversity of terpenoids. However, the molecular basis of their catalytic mechanisms is poorly understood. In this study, using multiple strategies, we characterized a geranyl pyrophosphate (GPP) C6-methyltransferase, BezA. Biochemical analysis revealed that BezA requires Mg2+ and solely methylates GPP. The crystal structures of BezA and its complex with S-adenosyl homocysteine were solved at 2.10 and 2.56â Å, respectively. Further analyses using site-directed mutagenesis, molecular docking, molecular dynamics simulations, and quantum mechanics/molecular mechanics calculations revealed the molecular basis of the methylation reaction. Importantly, the function of E170 as a catalytic base to complete the methylation reaction was established. We also succeeded in switching the substrate specificity by introducing a W210A substitution, resulting in an unprecedented farnesyl pyrophosphate C6-methyltransferase.
Asunto(s)
Metiltransferasas/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/metabolismo , Biocatálisis , Cristalografía por Rayos X , Teoría Funcional de la Densidad , Metiltransferasas/química , Metiltransferasas/genética , Modelos Moleculares , Estructura Molecular , Fosfatos de Poliisoprenilo/química , Sesquiterpenos/química , Streptomyces/enzimología , Especificidad por SustratoRESUMEN
A reinvestigation of the linalool synthase from Chryseobacterium polytrichastri uncovered its diterpene synthase activity, yielding polytrichastreneâ A and polytrichastrolâ A with new skeletons, besides known wanju-2,5-diene and thunbergol. The enzyme mechanism was investigated by isotopic labeling experiments and DFT calculations to explain an unusual ethyl group formation. Rationally designed exchanges of active site residues showed major functional switches, resulting for I66F in the production of five more new compounds, including polytrichastreneâ B and polytrichastrolâ B, while A87T, A192V and the double exchange A87T, A192V gave a product shift towards wanju-2,5-diene.
Asunto(s)
Chryseobacterium/enzimología , Hidroliasas/metabolismo , Fosfatos de Poliisoprenilo/biosíntesis , Teoría Funcional de la Densidad , Conformación Molecular , Fosfatos de Poliisoprenilo/químicaRESUMEN
Classical terpenoid biosynthesis involves the cyclization of the linear prenyl pyrophosphate precursors geranyl-, farnesyl-, or geranylgeranyl pyrophosphate (GPP, FPP, GGPP) and their isomers, to produce a huge number of natural compounds. Recently, it was shown for the first time that the biosynthesis of the unique homo-sesquiterpene sodorifen by Serratia plymuthica 4Rx13 involves a methylated and cyclized intermediate as the substrate of the sodorifen synthase. To further support the proposed biosynthetic pathway, we now identified the cyclic prenyl pyrophosphate intermediate pre-sodorifen pyrophosphate (PSPP). Its absolute configuration (6R,7S,9S) was determined by comparison of calculated and experimental CD-spectra of its hydrolysis product and matches with those predicted by semi-empirical quantum calculations of the reaction mechanism. In silico modeling of the reaction mechanism of the FPP C-methyltransferase (FPPMT) revealed a SN2 mechanism for the methyl transfer followed by a cyclization cascade. The cyclization of FPP to PSPP is guided by a catalytic dyad of H191 and Y39 and involves an unprecedented cyclopropyl intermediate. W46, W306, F56, and L239 form the hydrophobic binding pocket and E42 and H45 complex a magnesium cation that interacts with the diphosphate moiety of FPP. Six additional amino acids turned out to be essential for product formation and the importance of these amino acids was subsequently confirmed by site-directed mutagenesis. Our results reveal the reaction mechanism involved in methyltransferase-catalyzed cyclization and demonstrate that this coupling of C-methylation and cyclization of FPP by the FPPMT represents an alternative route of terpene biosynthesis that could increase the terpenoid diversity and structural space.
Asunto(s)
Proteínas Bacterianas/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Metiltransferasas/metabolismo , Octanos/metabolismo , Serratia/enzimología , Secuencias de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Biocatálisis , Compuestos Bicíclicos con Puentes/química , Clonación Molecular , Ciclización , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Octanos/química , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serratia/química , Serratia/genética , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Especificidad por SustratoRESUMEN
Most terpenoids are derived from the basic terpene skeletons of geranyl pyrophosphate (GPP, C10), farnesyl-PP (FPP, C15) and geranylgeranyl-PP (GGPP, C20). The trans-prenyltransferases (PTs) mediate the sequential head-to-tail condensation of an isopentenyl-PP (C5) with allylic substrates. The in silico structural comparative analyses of rice trans-PTs with 136 plant trans-PT genes allowed twelve rice PTs to be identified as GGPS_LSU (OsGGPS1), homomeric G(G)PS (OsGPS) and GGPS_SSU-II (OsGRP) in Group I; two solanesyl-PP synthase (OsSPS2 and 3) and two polyprenyl-PP synthases (OsSPS1 and 4) in Group II; and five FPSs (OsFPS1, 2, 3, 4 and 5) in Group III. Additionally, several residues in "three floors" for the chain length and several essential domains for enzymatic activities specifically varied in rice, potentiating evolutionarily rice-specific biochemical functions of twelve trans-PTs. Moreover, expression profiling and localization patterns revealed their functional compartmentation in rice. Taken together, we propose the predicted topology-based working model of rice PTs with corresponding terpene metabolites: GPP/GGPPs mainly in plastoglobuli, SPPs in stroma, PPPs in cytosol, mitochondria and chloroplast and FPPs in cytosol. Our findings could be suitably applied to metabolic engineering for producing functional terpene metabolites in rice systems.
Asunto(s)
Dimetilaliltranstransferasa/ultraestructura , Oryza/ultraestructura , Proteínas de Plantas/ultraestructura , Terpenos/metabolismo , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/genética , Regulación de la Expresión Génica de las Plantas , Oryza/química , Oryza/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , Conformación Proteica , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
Process intensification through continuous flow reactions has increased the production rates of fine chemicals and pharmaceuticals. Catalytic reactions are accelerated through an unconventional and unprecedented use of a high-performance liquid/liquid counter current chromatography system. Product generation is significantly faster than in traditional batch reactors or in segmented flow systems, which is exemplified through stereoselective phase-transfer catalyzed reactions. This methodology also enables the intensification of biocatalysis as demonstrated in high yield esterifications and in the sesquiterpene cyclase-catalyzed synthesis of sesquiterpenes from farnesyl diphosphate as high-value natural products with applications in medicine, agriculture and the fragrance industry. Product release in sesquiterpene synthases is rate limiting due to the hydrophobic nature of sesquiterpenes, but a biphasic system exposed to centrifugal forces allows for highly efficient reactions.
Asunto(s)
Liasas de Carbono-Carbono/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/metabolismo , Biocatálisis , Liasas de Carbono-Carbono/química , Estructura Molecular , Fosfatos de Poliisoprenilo/química , Sesquiterpenos/química , EstereoisomerismoRESUMEN
New sesquiterpene backbones are accessible after biotransformation of presilphiperfolan-8ß-ol synthase (BcBOT2), a fungal sesquiterpene synthase, with non-natural farnesyldiphosphates in which methyl groups are shifted by one position toward the diphosphate terminus. One of the macrocycles formed, a new germacrene A derivative, undergoes a Cope rearrangement to iso-ß-elemene. Three of the new terpenoids show olfactoric properties that range from an intense peppery note to a citrus, ozone-like, and fruity scent.
Asunto(s)
Liasas de Carbono-Carbono/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/metabolismo , Liasas de Carbono-Carbono/química , Estructura Molecular , Fosfatos de Poliisoprenilo/química , Sesquiterpenos/química , Especificidad por SustratoRESUMEN
Isoprenoid pyrophosphates are involved in protein prenylation and assume regulatory roles in cells; however, little is known about the cellular proteins that can interact with isoprenoid pyrophosphates. Here, we devised a chemical proteomic strategy, capitalizing on the use of a desthiobiotin-geranyl pyrophosphate (GPP) acyl phosphate probe for the enrichment and subsequent identification of GPP-binding proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By combining stable isotope labeling by amino acids in cell culture (SILAC) and competitive labeling with low vs high concentrations of GPP probe, with ATP vs GPP acyl phosphate probes, or with the GPP probe in the presence of different concentrations of free GPP, we uncovered a number of candidate GPP-binding proteins. We also discovered, for the first time, histone deacetylase 1 (HDAC1) as a GPP-binding protein. Furthermore, we found that the enzymatic activity of HDAC1 could be modulated by isoprenoid pyrophosphates. Together, we developed a novel chemical proteomic method for the proteome-wide discovery of GPP-binding proteins, which sets the stage for a better understanding about the biological functions of isoprenoids.
Asunto(s)
Biotina/análogos & derivados , Histona Desacetilasa 1/química , Fosfatos de Poliisoprenilo/química , Proteómica , Biotina/química , Histona Desacetilasa 1/metabolismo , Humanos , Estructura MolecularRESUMEN
The biosynthesis of bacterial cell envelope polysaccharides such as peptidoglycan relies on the use of a dedicated carrier lipid both for the assembly of precursors at the cytoplasmic face of the plasma membrane and for the translocation of lipid linked oligosaccharides across the plasma membrane into the periplasmic space. This dedicated carrier lipid, undecaprenyl phosphate, results from the dephosphorylation of undecaprenyl pyrophosphate, which is generated de novo in the cytoplasm by undecaprenyl pyrophosphate synthase and released as a by-product when newly synthesized glycans are incorporated into the existing cell envelope. The de novo synthesis of undecaprenyl pyrophosphate has been thoroughly characterized from a structural and mechanistic standpoint; however, its dephosphorylation to the active carrier lipid form, both in the course of de novo synthesis and recycling, has only been begun to be studied in depth in recent years. This review provides an overview of bacterial carrier lipid synthesis and presents the current state of knowledge regarding bacterial carrier lipid recycling.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Bacterias/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Transferasas Alquil y Aril/química , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Modelos Moleculares , Fosforilación , Fosfatos de Poliisoprenilo/química , Polisacáridos Bacterianos/biosíntesisRESUMEN
Genome sequencing and bioinformatics tools have facilitated the identification and expression of an increasing number of cryptic biosynthetic gene clusters (BGCs). However, functional analysis of all components of a metabolic pathway to precisely determine biocatalytic properties remains time-consuming and labor intensive. One way to speed this process involves microscale cell-free protein synthesis (CFPS) for direct gene to biochemical function analysis, which has rarely been applied to study multicomponent enzymatic systems in specialized metabolism. We sought to establish an in vitro transcription/translation (TT)-assay to assess assembly of cyanobacterial-derived hapalindole-type natural products (cNPs) because of their diverse bioactivity profiles and complex structural diversity. Using a CFPS system including a plasmid bearing famD2 prenyltransferase from Fischerella ambigua UTEX 1903, we showed production of the central prenylated intermediate (3GC) in the presence of exogenous geranyl-pyrophosphate (GPP) and cis-indole isonitrile. Further addition of a plasmid bearing the famC1 Stig cyclase resulted in synthesis of both FamD2 and FamC1 enzymes, which was confirmed by proteomics analysis, and catalyzed assembly of 12-epi-hapalindole U. Further combinations of Stig cyclases (FamC1-C4) produced hapalindole U and hapalindole H, while FisC identified from Fischerella sp. SAG46.79 generated 12-epi-fischerindole U. The CFPS system was further employed to screen six unnatural halogenated cis-indole isonitrile substrates using FamC1 and FisC, and the reactions were scaled-up using chemoenzymatic synthesis and identified as 5- and 6-fluoro-12-epi-hapalindole U, and 5- and 6-fluoro-12-epi-fischerindole U, respectively. This approach represents an effective, high throughput strategy to determine the functional role of biosynthetic enzymes from diverse natural product BGCs.
Asunto(s)
Biología Computacional/métodos , Cianobacterias/genética , Alcaloides Indólicos/metabolismo , Sistema Libre de Células , Cromatografía Líquida de Alta Presión , Dimetilaliltranstransferasa/genética , Alcaloides Indólicos/análisis , Indoles/análisis , Indoles/metabolismo , Familia de Multigenes , Plásmidos/genética , Plásmidos/metabolismo , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , Biosíntesis de Proteínas/genética , Espectrometría de Masas en Tándem , Transcripción Genética/genéticaRESUMEN
Non-natural terpenoids offer potential as pharmaceuticals and agrochemicals. However, their chemical syntheses are often long, complex, and not easily amenable to large-scale production. Herein, we report a modular chemoenzymatic approach to synthesize terpene analogues from diphosphorylated precursors produced in quantitative yields. Through the addition of prenyl transferases, farnesyl diphosphates, (2E,6E)-FDP and (2Z,6Z)-FDP, were isolated in greater than 80 % yields. The synthesis of 14,15-dimethyl-FDP, 12-methyl-FDP, 12-hydroxy-FDP, homo-FDP, and 15-methyl-FDP was also achieved. These modified diphosphates were used with terpene synthases to produce the unnatural sesquiterpenoid semiochemicals (S)-14,15-dimethylgermacreneâ D and (S)-12-methylgermacrene D as well as dihydroartemisinic aldehyde. This approach is applicable to the synthesis of many non-natural terpenoids, offering a scalable route free from repeated chain extensions and capricious chemical phosphorylation reactions.