RESUMEN
All organisms produce specialized organic molecules, ranging from small volatile chemicals to large gene-encoded peptides, that have evolved to provide them with diverse cellular and ecological functions. As natural products, they are broadly applied in medicine, agriculture and nutrition. The rapid accumulation of genomic information has revealed that the metabolic capacity of virtually all organisms is vastly underappreciated. Pioneered mainly in bacteria and fungi, genome mining technologies are accelerating metabolite discovery. Recent efforts are now being expanded to all life forms, including protists, plants and animals, and new integrative omics technologies are enabling the increasingly effective mining of this molecular diversity.
Asunto(s)
Bacterias/genética , Productos Biológicos/química , Descubrimiento de Drogas/métodos , Hongos/genética , Genoma , Genómica/métodos , Plantas/genética , Animales , Productos Biológicos/metabolismo , Productos Biológicos/uso terapéutico , Biología Computacional/métodos , HumanosRESUMEN
Sea sponges are the largest marine source of small-molecule natural products described to date. Sponge-derived molecules, such as the chemotherapeutic eribulin, the calcium-channel blocker manoalide, and antimalarial compound kalihinol A, are renowned for their impressive medicinal, chemical, and biological properties. Sponges contain microbiomes that control the production of many natural products isolated from these marine invertebrates. In fact, all genomic studies to date investigating the metabolic origins of sponge-derived small molecules concluded that microbes-not the sponge animal host-are the biosynthetic producers. However, early cell-sorting studies suggested the sponge animal host may play a role particularly in the production of terpenoid molecules. To investigate the genetic underpinnings of sponge terpenoid biosynthesis, we sequenced the metagenome and transcriptome of an isonitrile sesquiterpenoid-containing sponge of the order Bubarida. Using bioinformatic searches and biochemical validation, we identified a group of type I terpene synthases (TSs) from this sponge and multiple other species, the first of this enzyme class characterized from the sponge holobiome. The Bubarida TS-associated contigs consist of intron-containing genes homologous to sponge genes and feature GC percentage and coverage consistent with other eukaryotic sequences. We identified and characterized TS homologs from five different sponge species isolated from geographically distant locations, thereby suggesting a broad distribution amongst sponges. This work sheds light on the role of sponges in secondary metabolite production and speaks to the possibility that other sponge-specific molecules originate from the animal host.
Asunto(s)
Productos Biológicos , Microbiota , Poríferos , Animales , Poríferos/genética , Organismos Acuáticos/genética , Microbiota/genética , Metagenoma , FilogeniaRESUMEN
Octocorals are major contributors of terpenoid chemical diversity in the ocean. Natural products from other sessile marine animals are primarily biosynthesized by symbiotic microbes rather than by the host. Here, we challenge this long-standing paradigm by describing a monophyletic lineage of animal-encoded terpene cyclases (TCs) ubiquitous in octocorals. We characterized 15 TC enzymes from nine genera, several of which produce precursors of iconic coral-specific terpenoids, such as pseudopterosin, lophotoxin and eleutherobin. X-ray crystallography revealed that coral TCs share conserved active site residues and structural features with bacterial TCs. The identification of coral TCs enabled the targeted identification of the enzyme that constructs the coral-exclusive capnellane scaffold. Several TC genes are colocalized with genes that encode enzymes known to modify terpenes. This work presents an example of biosynthetic capacity in the kingdom Animalia that rivals the chemical complexity generated by plants, unlocking the biotechnological potential of octocorals for biomedical applications.
Asunto(s)
Antozoos , Animales , Bacterias/genética , Terpenos/químicaRESUMEN
Multidomain enzymes orchestrate two or more catalytic activities to carry out metabolic transformations with increased control and speed. Here, we report the design and development of a genome-mining approach for targeted discovery of biochemical transformations through the analysis of co-occurring enzyme domains (CO-ED) in a single protein. CO-ED was designed to identify unannotated multifunctional enzymes for functional characterization and discovery based on the premise that linked enzyme domains have evolved to function collaboratively. Guided by CO-ED, we targeted an unannotated predicted ThiF-nitroreductase di-domain enzyme found in more than 50 proteobacteria. Through heterologous expression and biochemical reconstitution, we discovered a series of natural products containing the rare oxazolone heterocycle and characterized their biosynthesis. Notably, we identified the di-domain enzyme as an oxazolone synthetase, validating CO-ED-guided genome mining as a methodology with potential broad utility for both the discovery of unusual enzymatic transformations and the functional annotation of multidomain enzymes.
Asunto(s)
Oxazolona/metabolismo , Péptido Sintasas/metabolismo , Estructura Molecular , Oxazolona/química , Péptido Sintasas/químicaRESUMEN
Prodiginines, which are tripyrrole alkaloids displaying a wide array of bioactivities, occur as linear and cyclic congeners. Identification of an unclustered biosynthetic gene led to the discovery of the enzyme responsible for catalyzing the regiospecific C-H activation and cyclization of prodigiosin to cycloprodigiosin in Pseudoalteromonas rubra. This enzyme is related to alkylglycerol monooxygenase and unrelated to RedG, the Rieske oxygenase that produces cyclized prodiginines in Streptomyces, implying convergent evolution.
Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Prodigiosina/metabolismo , Pseudoalteromonas/enzimología , Catálisis , Ciclización , Evolución Molecular , Indoles/metabolismo , Oxidación-Reducción , Prodigiosina/análogos & derivados , Pseudoalteromonas/genética , Pirroles/metabolismo , Streptomyces/enzimología , Streptomyces/genéticaRESUMEN
The globally distributed heterotrophic dinoflagellate Noctiluca scintillans (Macartney) Kofoid & Swezy is well known for its dense blooms and prominent displays of bioluminescence. Intriguingly, along the west coast of the USA its blooms are not bioluminescent. We investigated the basis for the regional loss of bioluminescence using molecular, cellular and biochemical analyses of isolates from different geographic regions. Prominent differences of the non-bioluminescent strains were: (1) the fused luciferase and luciferin binding protein gene (lcf/lbp) was present but its transcripts were undetectable; (2) lcf/lbp contained multiple potentially deleterious mutations; (3) the substrate luciferin was absent, based on the lack of luciferin blue autofluorescence and the absence of luciferin derived metabolites; (4) although the cells possessed scintillons, the vesicles that contain the luminescent chemistry, electron microscopy revealed additional scintillon-like vesicles with an atypical internal structure; (5) cells isolated from the California coast were 43% smaller in size than bioluminescent cells from the Gulf of Mexico. Phylogenetic analyses based on the large subunit of rDNA did not show divergence of the non-bioluminescent population in relation to other bioluminescent N. scintillans from the Pacific Ocean and Arabian Sea. Our study demonstrates that gene silencing and the lack of the luciferin substrate have resulted in the loss of a significant dinoflagellate functional trait over large spatial scales in the ocean. As the bioluminescence system of dinoflagellates is well characterized, non-bioluminescent N. scintillans is an ideal model to explore the evolutionary and ecological mechanisms that lead to intraspecific functional divergence in natural dinoflagellate populations.
RESUMEN
Engineered polyketide synthases (PKSs) are promising synthetic biology platforms for the production of chemicals with diverse applications. The dehydratase (DH) domain within modular type I PKSs generates an α,ß-unsaturated bond in nascent polyketide intermediates through a dehydration reaction. Several crystal structures of DH domains have been solved, providing important structural insights into substrate selection and dehydration. Here, we present two DH domain structures from two chemically diverse PKSs. The first DH domain, isolated from the third module in the borrelidin PKS, is specific towards a trans-cyclopentane-carboxylate-containing polyketide substrate. The second DH domain, isolated from the first module in the fluvirucin B1 PKS, accepts an amide-containing polyketide intermediate. Sequence-structure analysis of these domains, in addition to previously published DH structures, display many significant similarities and key differences pertaining to substrate selection. The two major differences between BorA DH M3, FluA DH M1 and other DH domains are found in regions of unmodeled residues or residues containing high B-factors. These two regions are located between α3-ß11 and ß7-α2. From the catalytic Asp located in α3 to a conserved Pro in ß11, the residues between them form part of the bottom of the substrate-binding cavity responsible for binding to acyl-ACP intermediates.
Asunto(s)
Sintasas Poliquetidas/química , Sitios de Unión , Alcoholes Grasos/química , Alcoholes Grasos/metabolismo , Modelos Moleculares , Sintasas Poliquetidas/metabolismo , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high-throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high-throughput mass-spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click-assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochromeâ P450s in cell lysate, microsomes, and bacteria. Using this approach, a cytochrome P450BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.
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Bioensayo/métodos , Sistema Enzimático del Citocromo P-450/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Catálisis , Desarrollo de Medicamentos , Espectrometría de Masas , Especificidad por SustratoRESUMEN
Euphorbiaceae are an important source of medically important diterpenoids, such as the anticancer drug ingenol-3-angelate and the antiretroviral drug prostratin. However, extraction from the genetically intractable natural producers is often limited by the small quantities produced, while the organic synthesis of terpene-derived drugs is challenging and similarly low-yielding. While transplanting the biosynthetic pathway into a heterologous host has proven successful for some drugs, it has been largely unsuccessful for diterpenoids due to their elaborate biosynthetic pathways and lack of genetic resources and tools for gene discovery. We engineered casbene precursor production in S. cerevisiae, verified the ability of six Euphorbia lathyris and Jatropha curcas cytochrome P450s to oxidize casbene, and optimized the expression of these P450s and an alcohol dehydrogenase to generate jolkinol C, achieving ~800mg/L of jolkinol C and over 1g/L total oxidized casbanes in millititer plates, the highest titer of oxidized diterpenes in yeast reported to date. This strain enables the semisynthesis of biologically active jolkinol C derivatives and will be an important tool in the elucidation of the biosynthetic pathways for ingenanes, tiglianes, and lathyranes. These findings demonstrate the ability of S. cerevisiae to produce oxidized drug precursors in quantities that are sufficient for drug development and pathway discovery.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Diterpenos/metabolismo , Euphorbia/genética , Jatropha/genética , Microorganismos Modificados Genéticamente , Proteínas de Plantas , Saccharomyces cerevisiae , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Euphorbia/enzimología , Jatropha/enzimología , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Mass spectrometry imaging (MSI) has primarily been applied in localizing biomolecules within biological matrices. Although well-suited, the application of MSI for comparing thousands of spatially defined spotted samples has been limited. One reason for this is a lack of suitable and accessible data processing tools for the analysis of large arrayed MSI sample sets. The OpenMSI Arrayed Analysis Toolkit (OMAAT) is a software package that addresses the challenges of analyzing spatially defined samples in MSI data sets. OMAAT is written in Python and is integrated with OpenMSI ( http://openmsi.nersc.gov ), a platform for storing, sharing, and analyzing MSI data. By using a web-based python notebook (Jupyter), OMAAT is accessible to anyone without programming experience yet allows experienced users to leverage all features. OMAAT was evaluated by analyzing an MSI data set of a high-throughput glycoside hydrolase activity screen comprising 384 samples arrayed onto a NIMS surface at a 450 µm spacing, decreasing analysis time >100-fold while maintaining robust spot-finding. The utility of OMAAT was demonstrated for screening metabolic activities of different sized soil particles, including hydrolysis of sugars, revealing a pattern of size dependent activities. These results introduce OMAAT as an effective toolkit for analyzing spatially defined samples in MSI. OMAAT runs on all major operating systems, and the source code can be obtained from the following GitHub repository: https://github.com/biorack/omaat .
Asunto(s)
Análisis de Datos , Espectrometría de Masas/métodos , Programas Informáticos , Conjuntos de Datos como Asunto , Glicósido Hidrolasas , Tamaño de la Partícula , Suelo/químicaRESUMEN
Cells modulate lipid metabolism in order to maintain membrane homeostasis. Here we use a metabolic engineering approach to manipulate the stoichiometry of fatty acid unsaturation, a regulator of cell membrane fluidity, in Saccharomyces cerevisiae. Unexpectedly, reduced lipid unsaturation triggered cell-cell adhesion (flocculation), a phenomenon characteristic of industrial yeast but uncommon in laboratory strains. We find that ER lipid saturation sensors induce expression of FLO1 - encoding a cell wall polysaccharide binding protein - independently of its canonical regulator. In wild-type cells, Flo1p-dependent flocculation occurs under oxygen-limited growth, which reduces unsaturated lipid synthesis and thus serves as the environmental trigger for flocculation. Transcriptional analysis shows that FLO1 is one of the most highly induced genes in response to changes in lipid unsaturation, and that the set of membrane fluidity-sensitive genes is globally activated as part of the cell's long-term response to hypoxia during fermentation. Our results show how the lipid homeostasis machinery of budding yeast is adapted to carry out a broad response to an environmental stimulus important in biotechnology.
Asunto(s)
Membrana Celular , Ingeniería Genética , Lectinas de Unión a Manosa , Fluidez de la Membrana/genética , Lípidos de la Membrana , Oxígeno/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Membrana Celular/genética , Membrana Celular/metabolismo , Floculación , Lectinas de Unión a Manosa/biosíntesis , Lectinas de Unión a Manosa/genética , Lípidos de la Membrana/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The borrelidin polyketide synthase (PKS) begins with a carboxylated substrate and, unlike typical decarboxylative loading PKSs, retains the carboxy group in the final product. The specificity and tolerance of incorporation of carboxyacyl substrate into type I PKSs have not been explored. Here, we show that the first extension module is promiscuous in its ability to extend both carboxyacyl and non-carboxyacyl substrates. However, the loading module has a requirement for substrates containing a carboxy moiety, which are not decarboxylated in situ. Thus, the loading module is the basis for the observed specific incorporation of carboxylated starter units by the borelidin PKS.
Asunto(s)
Sintasas Poliquetidas/metabolismo , Policétidos/metabolismo , Alcoholes Grasos/química , Alcoholes Grasos/metabolismo , Estructura Molecular , Sintasas Poliquetidas/genética , Policétidos/química , Especificidad por SustratoRESUMEN
Red algae or seaweeds produce highly distinctive halogenated terpenoid compounds, including the pentabromochlorinated monoterpene halomon that was once heralded as a promising anticancer agent. The first dedicated step in the biosynthesis of these natural product molecules is expected to be catalyzed by terpene synthase (TS) enzymes. Recent work has demonstrated an emerging class of type I TSs in red algal terpene biosynthesis. However, only one such enzyme from a notoriously haloterpenoid-producing red alga (Laurencia pacifica) has been functionally characterized and the product structure is not related to halogenated terpenoids. Herein, we report 10 new type I TSs from the red algae Portieria hornemannii, Plocamium pacificum, L. pacifica, and Laurencia subopposita that produce a diversity of halogenated mono- and sesquiterpenes. We used a combination of genome sequencing, terpenoid metabolomics, in vitro biochemistry, and bioinformatics to establish red algal TSs in all four species, including those associated with the selective production of key halogenated terpene precursors myrcene, trans-ß-ocimene, and germacrene D-4-ol. These results expand on a small but growing number of characterized red algal TSs and offer insight into the biosynthesis of iconic halogenated algal compounds that are not without precedence elsewhere in biology.
Asunto(s)
Transferasas Alquil y Aril , Rhodophyta , Rhodophyta/química , Terpenos/química , Monoterpenos/químicaRESUMEN
Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme, shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/análisis , Cloranfenicol O-Acetiltransferasa/química , Cloranfenicol/análisis , Cloranfenicol/química , Espectrometría de Masas/métodos , Técnicas de Sonda Molecular , Espectrometría de Fluorescencia/métodos , Activación Enzimática , Sondas Moleculares/síntesis químicaRESUMEN
Developments in computational omics technologies have provided new means to access the hidden diversity of natural products, unearthing new potential for drug discovery. In parallel, artificial intelligence approaches such as machine learning have led to exciting developments in the computational drug design field, facilitating biological activity prediction and de novo drug design for molecular targets of interest. Here, we describe current and future synergies between these developments to effectively identify drug candidates from the plethora of molecules produced by nature. We also discuss how to address key challenges in realizing the potential of these synergies, such as the need for high-quality datasets to train deep learning algorithms and appropriate strategies for algorithm validation.
Asunto(s)
Inteligencia Artificial , Productos Biológicos , Humanos , Algoritmos , Aprendizaje Automático , Descubrimiento de Drogas , Diseño de Fármacos , Productos Biológicos/farmacologíaRESUMEN
BACKGROUND: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). RESULTS: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the extent of epoxidation and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at the Leu377 site of SaGGR. We show this TLC-based screen can distinguish between fourfold differences in enzyme activity for select mutants and validated those results by GC-MS. CONCLUSIONS: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.
RESUMEN
Lepidoptera (butterflies and moths) make the six-carbon compounds homoisopentenyl pyrophosphate (HIPP) and homodimethylallyl pyrophosphate (HDMAPP) that are incorporated into 16, 17, and 18 carbon farnesyl pyrophosphate (FPP) analogues. In this work we heterologously expressed the lepidopteran modified mevalonate pathway, a propionyl-CoA ligase, and terpene cyclases in E. coli to produce several novel terpenes containing 16 carbons. Changing the terpene cyclase generated different novel terpene product profiles. To further validate the new compounds we confirmed 13C propionate was incorporated, and that the masses and fragmentation patterns were consistent with novel 16 carbon terpenes by GC-QTOF. On the basis of the available farnesyl pyrophosphate analogues lepidoptera produce, this approach should greatly expand the reachable biochemical space with applications in areas where terpenes have traditionally found uses.
Asunto(s)
Ácido Mevalónico/metabolismo , Terpenos/metabolismo , Animales , Escherichia coli/metabolismo , Lepidópteros/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/metabolismoRESUMEN
Lipid composition determines the physical properties of biological membranes and can vary substantially between and within organisms. We describe a specific role for the viscosity of energy-transducing membranes in cellular respiration. Engineering of fatty acid biosynthesis in Escherichia coli allowed us to titrate inner membrane viscosity across a 10-fold range by controlling the abundance of unsaturated or branched lipids. These fluidizing lipids tightly controlled respiratory metabolism, an effect that can be explained with a quantitative model of the electron transport chain (ETC) that features diffusion-coupled reactions between enzymes and electron carriers (quinones). Lipid unsaturation also modulated mitochondrial respiration in engineered budding yeast strains. Thus, diffusion in the ETC may serve as an evolutionary constraint for lipid composition in respiratory membranes.
Asunto(s)
Membrana Celular/metabolismo , Escherichia coli/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Lípidos de la Membrana/metabolismo , Oxígeno/metabolismo , Membrana Celular/química , Lípidos de la Membrana/química , Mitocondrias/metabolismo , ViscosidadRESUMEN
Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of â¼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.