RESUMEN
The production of compact vectors for gene stacking is hindered by a lack of effective linkers. Here, we report that a 26-nt nucleic acid linker, NAL1, from the fungus Glarea lozoyensis and its truncated derivatives could connect two genes as a bicistron, enabling independent translation in a maize protoplast transient expression system and human 293 T cells. The optimized 9-nt NAL10 linker was then used to connect four genes driven by a bidirectional promoter; this combination was successfully used to reconstruct the astaxanthin biosynthesis pathway in transgenic maize. The short and efficient nucleic acid linker NAL10 can be widely used in multi-gene expression and synthetic biology in animals and plants.
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Plantas Modificadas Genéticamente , Biología Sintética , Zea mays , Biología Sintética/métodos , Zea mays/genética , Zea mays/metabolismo , Humanos , Plantas Modificadas Genéticamente/genética , Regiones Promotoras Genéticas/genética , Células HEK293 , Xantófilas/metabolismo , Hypocreales/genética , Hypocreales/metabolismo , Animales , Ácidos Nucleicos/genética , Expresión Génica , Vectores Genéticos/genética , Protoplastos/metabolismoRESUMEN
Xylomyrocins, a unique group of nonribosomal peptide secondary metabolites, were discovered in Paramyrothecium and Colletotrichum spp. fungi by employing a combination of high-resolution tandem mass spectrometry (HRMS/MS)-based chemometrics, comparative genome mining, gene disruption, stable isotope feeding, and chemical complementation techniques. These polyol cyclodepsipeptides all feature an unprecedented d-xylonic acid moiety as part of their macrocyclic scaffold. This biosynthon is derived from d-xylose supplied by xylooligosaccharide catabolic enzymes encoded in the xylomyrocin biosynthetic gene cluster, revealing a novel link between carbohydrate catabolism and nonribosomal peptide biosynthesis. Xylomyrocins from different fungal isolates differ in the number and nature of their amino acid building blocks that are nevertheless incorporated by orthologous nonribosomal peptide synthetase (NRPS) enzymes. Another source of structural diversity is the variable choice of the nucleophile for intramolecular macrocyclic ester formation during xylomyrocin chain termination. This nucleophile is selected from the multiple available alcohol functionalities of the polyol moiety, revealing a surprising polyspecificity for the NRPS terminal condensation domain. Some xylomyrocin congeners also feature N-methylated amino acid residues in positions where the corresponding NRPS modules lack N-methyltransferase (M) domains, providing a rare example of promiscuous methylation in the context of an NRPS with an otherwise canonical, collinear biosynthetic program.
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Depsipéptidos , Proteínas Fúngicas , Hongos , Aminoácidos/química , Metabolismo de los Hidratos de Carbono , Quimiometría , Depsipéptidos/química , Depsipéptidos/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Hongos/genética , Hongos/metabolismo , Familia de Multigenes , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Péptido Sintasas/química , AzúcaresRESUMEN
Polyketides with the isochroman-3-one pharmacophore are rare among fungal natural products as their biosynthesis requires an unorthodox S-type aromatic ring cyclization. Genome mining uncovered a conserved gene cluster in select leotiomycetous fungi that encodes the biosynthesis of cytosporones, including isochroman-3-one congeners. Combinatorial biosynthesis in total biosynthetic and biocatalytic formats in Saccharomyces cerevisiae and in vitro reconstitution of key reactions with purified enzymes revealed how cytosporone structural and bioactivity diversity is generated. The S-type acyl dihydroxyphenylacetic acid (ADA) core of cytosporones is assembled by a collaborating polyketide synthase pair. Thioesterase domain-catalyzed transesterification releases ADA esters, some of which are known Nur77 modulators. Alternatively, hydrolytic release allows C6 hydroxylation by a flavin-dependent monooxygenase, yielding a trihydroxybenzene moiety. Reduction of the C9 carbonyl by a short chain dehydrogenase/reductase initiates isochroman-3-one formation, affording cytosporones with cytotoxic and antimicrobial activity. Enoyl di- or trihydroxyphenylacetic acids are generated as shunt products, while isocroman-3,4-diones are formed by autoxidation. The cytosporone pathway offers novel polyketide biosynthetic enzymes for combinatorial synthetic biology to advance the production of "unnatural" natural products for drug discovery.
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Productos Biológicos , Policétidos , Hongos/genética , Saccharomyces cerevisiae/metabolismo , Sintasas Poliquetidas/metabolismo , Policétidos/química , Productos Biológicos/metabolismoRESUMEN
Unnatural product (uNP) nonribosomal peptides promise to be a valuable source of pharmacophores for drug discovery. However, the extremely large size and complexity of the nonribosomal peptide synthetase (NRPS) enzymes pose formidable challenges to the production of such uNPs by combinatorial biosynthesis and synthetic biology. Here we report a new NRPS dissection strategy that facilitates the engineering and heterologous production of these NRPSs. This strategy divides NRPSs into "splitting units", each forming an enzyme subunit that contains catalytically independent modules. Functional collaboration between the subunits is then facilitated by artificially duplicating, at the N-terminus of the downstream subunit, the linker - thiolation domain - linker fragment that is resident at the C-terminus of the upstream subunit. Using the suggested split site that follows a conserved motif in the linker connecting the adenylation and the thiolation domains allows cognate or chimeric splitting unit pairs to achieve productivities that match, and in many cases surpass those of hybrid chimeric enzymes, and even those of intact NRPSs, upon production in a heterologous chassis. Our strategy provides facile options for the rational engineering of fungal NRPSs and for the combinatorial reprogramming of nonribosomal peptide production.
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Péptido Sintasas , Ingeniería de Proteínas , Péptido Sintasas/metabolismo , Péptido Sintasas/química , Péptido Sintasas/genética , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Covering: 2011 up to the end of 2021.Fungal nonribosomal peptides (NRPs) and the related polyketide-nonribosomal peptide hybrid products (PK-NRPs) are a prolific source of bioactive compounds, some of which have been developed into essential drugs. The synthesis of these complex natural products (NPs) utilizes nonribosomal peptide synthetases (NRPSs), multidomain megaenzymes that assemble specific peptide products by sequential condensation of amino acids and amino acid-like substances, independent of the ribosome. NRPSs, collaborating polyketide synthase modules, and their associated tailoring enzymes involved in product maturation represent promising targets for NP structure diversification and the generation of small molecule unnatural products (uNPs) with improved or novel bioactivities. Indeed, reprogramming of NRPSs and recruiting of novel tailoring enzymes is the strategy by which nature evolves NRP products. The recent years have witnessed a rapid development in the discovery and identification of novel NRPs and PK-NRPs, and significant advances have also been made towards the engineering of fungal NRP assembly lines to generate uNP peptides. However, the intrinsic complexities of fungal NRP and PK-NRP biosynthesis, and the large size of the NRPSs still present formidable conceptual and technical challenges for the rational and efficient reprogramming of these pathways. This review examines key examples for the successful (and for some less-successful) re-engineering of fungal NRPS assembly lines to inform future efforts towards generating novel, biologically active peptides and PK-NRPs.
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Proteínas Fúngicas , Policétidos , Proteínas Fúngicas/metabolismo , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Péptidos/química , Péptido Sintasas/metabolismo , Biosíntesis de Péptidos Independientes de Ácidos NucleicosRESUMEN
Icariin (ICA) has wide applications in nutraceuticals and medicine with strong anticancer activities. However, the structural complexity and low abundance in plants of ICA lead to the unsustainable and high-cost supply from chemical synthesis and plant extraction. Here, the whole biosynthesis pathway of ICA was elucidated, then was constructed in Saccharomyces cerevisiae, including a 13-step heterologous ICA pathway from eleven kinds of plants as well as deletions or overexpression of ten yeast endogenous genes. Spatial regulation of 8-C-prenyltransferase to mitochondria and three-stage sequential control of 4'-O-methyltransferase, 3-OH rhamnosyltransferase, and 7-OH glycosyltransferase expression successfully achieved the de novo synthesis of ICA with a titer of 130 µg/L under shake-flask culture. The ICA synthesis from glucose represents the longest reconstructed pathway of flavonoid in microbe so far. This study provides a potential choice for the sustainable microbial production of number of complex flavonoids.
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Ingeniería Metabólica , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Flavonoides/genética , Glucosa/metabolismoRESUMEN
Six new squalene derived polyether glycosides, onygenaleosides A-F (1-6), that possess a 6/5 bicyclic fused ring skeleton were isolated from the cultures of Onygenales sp. YX1425, along with two known analogues (7 and 8). The planar structures of the new compounds were elucidated based on analysis of NMR and MS spectroscopy data, and the absolute configuration of 1 was determined by the advanced Mosher method and quantum chemical calculations. Compound 2 was active against Spodoptera frugiperda with an LC50 value of 193.4 ± 1.1 µg/mL.
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Insecticidas , Triterpenos , Glicósidos/farmacología , Glicósidos/química , Triterpenos/farmacología , Triterpenos/química , Insecticidas/farmacología , Espectroscopía de Resonancia Magnética/métodos , Esqueleto , Estructura MolecularRESUMEN
Mass spectrometry-based dereplication and prioritization led to the discovery of four multi-N-methylated cyclodecapeptides, auyuittuqamides E-H (1-4), from a soil-derived Sesquicillium sp. The planar structures of these compounds were elucidated based on analysis of HRESIMS and NMR data. Absolute configurations of the chiral amino acid residues were assigned by a combination of the advanced Marfey's method, chiral-phase LC-MS analysis, and J-based configuration analysis, revealing that 1-4 contain both d- and l-isomers of N-methylleucine (MeLeu). Differentiation of d- and l-MeLeu in the sequence was achieved by advanced Marfey's analysis of the diagnostic peptide fragments generated from partial hydrolysis of 1. Bioinformatic analysis identified a putative biosynthetic gene cluster (auy) for auyuittuqamides E-H, and a plausible biosynthetic pathway was proposed. These newly identified fungal cyclodecapeptides (1-4) displayed in vitro growth inhibitory activity against vancomycin-resistant Enterococcus faecium with MIC values of 8 µg/mL.
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Aminoácidos , Fragmentos de Péptidos , Aminoácidos/química , Cromatografía Liquida , Espectrometría de Masas , Estructura Molecular , Péptidos Cíclicos/análisis , Péptidos Cíclicos/químicaRESUMEN
Isocitrate lyase (ICL), as the key enzyme in the glyoxylate metabolic pathway, plays an important role in metabolic adaptation to environmental changes. In this study, metagenomic DNA from the soil and water microorganism collected from the Dongzhai Harbor Mangroves (DHM) reserve, in Haikou City, China, was high-throughput sequenced using an Illumina HiSeq 4000 platform. The icl121 gene, encoding an ICL with the highly conserved catalytic pattern IENQVSDEKQCGHQD was identified. Then, this gene was subcloned into the pET-30a vector and overexpressed in Escherichia coli BL21 (DE3) cells. The maximum enzymatic activity of the recombinant ICL121 protein is 9.47 × 102 U/mg occurring at pH 7.5 and 37°C. Furthermore, as a metalo-enzyme, ICL121 can utilize the appropriate concentrations of Mg2+, Mn2+, and Na+ ion as cofactors to exhibit high enzymatic activity. In particular, the novel metagenomic-derived icl121 gene displayed distinct salt tolerance (NaCl) and might be useful for generating salt-tolerant crops in the future.
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Isocitratoliasa , Humedales , Isocitratoliasa/química , Isocitratoliasa/genética , Isocitratoliasa/metabolismo , Escherichia coli/genética , Secuencia de Bases , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND: Advances in DNA sequencing technologies have transformed our capacity to perform life science research, decipher the dynamics of complex soil microbial communities and exploit them for plant disease management. However, soil is a complex conglomerate, which makes functional metagenomics studies very challenging. RESULTS: Metagenomes were assembled by long-read (PacBio, PB), short-read (Illumina, IL), and mixture of PB and IL (PI) sequencing of soil DNA samples were compared. Ortholog analyses and functional annotation revealed that the PI approach significantly increased the contig length of the metagenomic sequences compared to IL and enlarged the gene pool compared to PB. The PI approach also offered comparable or higher species abundance than either PB or IL alone, and showed significant advantages for studying natural product biosynthetic genes in the soil microbiomes. CONCLUSION: Our results provide an effective strategy for combining long and short-read DNA sequencing data to explore and distill the maximum information out of soil metagenomics.
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Metagenoma , Suelo , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenómica , Análisis de Secuencia de ADNRESUMEN
Understanding how antibiotic-producing bacteria deal with highly reactive chemicals will ultimately guide therapeutic strategies to combat the increasing clinical resistance crisis. Here, we uncovered a distinctive self-defense strategy featured by a secreted oxidoreductase NapU to perform extracellularly oxidative activation and conditionally overoxidative inactivation of a matured prodrug in naphthyridinomycin (NDM) biosynthesis from Streptomyces lusitanus NRRL 8034. It was suggested that formation of NDM first involves a nonribosomal peptide synthetase assembly line to generate a prodrug. After exclusion and prodrug maturation, we identified a pharmacophore-inactivated intermediate, which required reactivation by NapU via oxidative C-H bond functionalization extracellularly to afford NDM. Beyond that, NapU could further oxidatively inactivate the NDM pharmacophore to avoid self-cytotoxicity if they coexist longer than necessary. This discovery represents an amalgamation of sophisticatedly temporal and spatial shielding mode conferring self-resistance in antibiotic biosynthesis from Gram-positive bacteria.
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Antibacterianos/metabolismo , Profármacos/metabolismo , Streptomyces/metabolismo , Naftiridinas/metabolismo , Oxidación-Reducción , Oxidorreductasas/metabolismo , Péptido Sintasas/metabolismoRESUMEN
Glycosylation is a prominent strategy to optimize the pharmacokinetic and pharmacodynamic properties of drug-like small-molecule scaffolds by modulating their solubility, stability, bioavailability, and bioactivity. Glycosyltransferases applicable for "sugarcoating" various small-molecule acceptors have been isolated and characterized from plants and bacteria, but remained cryptic from filamentous fungi until recently, despite the frequent use of some fungi for whole-cell biocatalytic glycosylations. Here, we use bioinformatic and genomic tools combined with heterologous expression to identify a glycosyltransferase-methyltransferase (GT-MT) gene pair that encodes a methylglucosylation functional module in the ascomycetous fungus Beauveria bassiana The GT is the founding member of a family nonorthologous to characterized fungal enzymes. Using combinatorial biosynthetic and biocatalytic platforms, we reveal that this GT is a promiscuous enzyme that efficiently modifies a broad range of drug-like substrates, including polyketides, anthraquinones, flavonoids, and naphthalenes. It yields both O- and N-glucosides with remarkable regio- and stereospecificity, a spectrum not demonstrated for other characterized fungal enzymes. These glucosides are faithfully processed by the dedicated MT to afford 4-O-methylglucosides. The resulting "unnatural products" show increased solubility, while representative polyketide methylglucosides also display increased stability against glycoside hydrolysis. Upon methylglucosidation, specific polyketides were found to attain cancer cell line-specific antiproliferative or matrix attachment inhibitory activities. These findings will guide genome mining for fungal GTs with novel substrate and product specificities, and empower the efficient combinatorial biosynthesis of a broad range of natural and unnatural glycosides in total biosynthetic or biocatalytic formats.
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Antineoplásicos , Descubrimiento de Drogas , Hongos , Glicosiltransferasas , Metiltransferasas , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hongos/enzimología , Hongos/genética , Hongos/metabolismo , Glicosilación , Glicosiltransferasas/química , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Humanos , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Policétidos/metabolismo , Células VeroRESUMEN
Combinatorial biosynthesis with fungal polyketide synthases (PKSs) promises to produce unprecedented bioactive "unnatural" natural products (uNPs) for drug discovery. Genome mining of the dothideomycete Rhytidhysteron rufulum uncovered a collaborating highly reducing PKS (hrPKS)-nonreducing PKS (nrPKS) pair. These enzymes produce trace amounts of rare S-type benzenediol macrolactone congeners with a phenylacetate core in a heterologous host. However, subunit shuffling and domain swaps with voucher enzymes demonstrated that all PKS domains are highly productive. This contradiction led us to reveal novel programming layers exerted by the starter unit acyltransferase (SAT) and the thioesterase (TE) domains on the PKS system. First, macrocyclic vs linear product formation is dictated by the intrinsic biosynthetic program of the TE domain. Next, the chain length of the hrPKS product is strongly influenced in trans by the off-loading preferences of the nrPKS SAT domain. Last, TE domains are size-selective filters that facilitate or obstruct product formation from certain priming units. Thus, the intrinsic programs of the SAT and TE domains are both part of the extrinsic program of the hrPKS subunit and modulate the observable metaprogram of the whole PKS system. Reconstruction of SAT and TE phylogenies suggests that these domains travel different evolutionary trajectories, with the resulting divergence creating potential conflicts in the PKS metaprogram. Such conflicts often emerge in chimeric PKSs created by combinatorial biosynthesis, reducing biosynthetic efficiency or even incapacitating the system. Understanding the points of failure for such engineered biocatalysts is pivotal to advance the biosynthetic production of uNPs.
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Ascomicetos/enzimología , Proteínas Fúngicas/química , Sintasas Poliquetidas/biosíntesis , Sintasas Poliquetidas/química , Aciltransferasas/química , Secuencia de Aminoácidos , Vías Biosintéticas , Técnicas Químicas Combinatorias , Modelos Moleculares , Familia de Multigenes/genética , Fenilacetatos/química , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Tioléster Hidrolasas/químicaRESUMEN
Covering: 2014 up to the third quarter of 2019 Entomopathogens constitute a unique, specialized trophic subgroup of fungi, most of whose members belong to the order Hypocreales (class Sordariomycetes, phylum Ascomycota). These Hypocrealean Entomopathogenic Fungi (HEF) produce a large variety of secondary metabolites (SMs) and their genomes rank highly for the number of predicted, unique SM biosynthetic gene clusters. SMs from HEF have diverse roles in insect pathogenicity as virulence factors by modulating various interactions between the producer fungus and its insect host. In addition, these SMs also defend the carcass of the prey against opportunistic microbial invaders, mediate intra- and interspecies communication, and mitigate abiotic and biotic stresses. Thus, these SMs contribute to the role of HEF as commercial biopesticides in the context of integrated pest management systems, and provide lead compounds for the development of chemical pesticides for crop protection. These bioactive SMs also underpin the widespread use of certain HEF as nutraceuticals and traditional remedies, and allowed the modern pharmaceutical industry to repurpose some of these molecules as life-saving human medications. Herein, we survey the structures and biological activities of SMs described from HEF, and summarize new information on the roles of these metabolites in fungal virulence.
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Productos Biológicos/metabolismo , Hypocreales/metabolismo , Insectos/microbiología , Animales , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Hypocreales/química , Redes y Vías Metabólicas , Metaboloma , Policétidos/metabolismo , Metabolismo Secundario , Terpenos/metabolismoRESUMEN
Covering: 2014 up to the third quarter of 2019 Hypocrealean entomopathogenic fungi (HEF) produce a large variety of secondary metabolites (SMs) that are prominent virulence factors or mediate various interactions in the native niches of these organisms. Many of these SMs show insecticidal, immune system modulatory, antimicrobial, cytotoxic and other bioactivities of clinical or agricultural significance. Recent advances in whole genome sequencing technologies and bioinformatics have revealed many biosynthetic gene clusters (BGCs) potentially involved in SM production in HEF. Some of these BGCs are now well characterized, with the structures of the cognate product congeners elucidated, and the proposed biosynthetic functions of key enzymes validated. However, the vast majority of HEF BGCs are still not linked to SM products ("orphan" BGCs), including many clusters that are not expressed (silent) under routine laboratory conditions. Thus, investigations into the encoded parvome (the secondary metabolome predicted from the genome) of HEF allows the discovery of BGCs for known SMs; uncovers novel metabolites based on the BGCs; and catalogues the predicted SM biosynthetic potential of these fungi. Herein, we summarize new developments of the field, and survey the polyketide, nonribosomal peptide, terpenoid and hybrid SM BGCs encoded in the currently available 40 HEF genome sequences. Studying the encoded parvome of HEF will increase our understanding of the multifaceted roles that SMs play in biotic and abiotic interactions and will also reveal biologically active SMs that can be exploited for the discovery of human and veterinary drugs or crop protection agents.
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Genómica , Hypocreales/metabolismo , Insectos/microbiología , Metaboloma/genética , Animales , Genoma Fúngico/genética , Genómica/métodos , Hypocreales/genética , Metabolismo Secundario/genéticaRESUMEN
Seven new 4-acyl-2-aminoimidazoles, designated strepimidazoles A-G (1-7), were discovered from the endophytic Streptomyces sp. PKU-EA00015 isolated from Salvia miltiorrhiza Bunge, whose dry root "Danshen" is one of the most widely used traditional Chinese medicines. The resonance signals of the 2-aminoimidazole moiety in 1-7 were absent in the NMR spectra due to tautomerization, and the structures of 1-7 were identified after preparation of their acetylation products 1a-7a, respectively. Compounds 1-7 represent a new family of 2-aminoimidazole-containing natural products, enriching the structural diversity of natural products from endophytic origin. Compounds 1-7 showed different degrees of inhibitory activities against the plant pathogenic fungus Verticillium dahliae V991, revealing structure-activity relationships on the acyl moieties. The plant pathogenic fungus V. dahliae has been confirmed to cause serious chlorosis of cultivated S. miltiorrhiza Bunge in China. This study opens the door for further investigation of mutualistic relationships between S. miltiorrhiza Bunge and their endophytic actinomycetes and for possible antifungal agent development for biological control of V. dahliae in the future.
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Ascomicetos/efectos de los fármacos , Imidazoles/farmacología , Plantas/microbiología , Streptomyces/química , Ascomicetos/patogenicidad , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Imidazoles/química , Imidazoles/aislamiento & purificación , Medicina Tradicional China , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Análisis Espectral/métodosRESUMEN
O-Methylation modulates the pharmacokinetic and pharmacodynamic (PK/PD) properties of small-molecule natural products, affecting their bioavailability, stability, and binding to targets. Diversity-oriented combinatorial biosynthesis of new chemical entities for drug discovery and optimization of known bioactive scaffolds during drug development both demand efficient O-methyltransferase (OMT) biocatalysts with considerable substrate promiscuity and tunable regioselectivity that can be deployed in a scalable and sustainable manner. Here we demonstrate efficient total biosynthetic and biocatalytic platforms that use a pair of fungal OMTs with orthogonal regiospecificity to produce unnatural O-methylated benzenediol lactone polyketides. We show that rational, structure-guided active-site cavity engineering can reprogram the regioselectivity of these enzymes. We also characterize the interplay of engineered regioselectivity with substrate plasticity. These findings will guide combinatorial biosynthetic tailoring of unnatural products toward the generation of diverse chemical matter for drug discovery and the PK/PD optimization of bioactive scaffolds for drug development.
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Lactonas/síntesis química , Metiltransferasas/química , Policétidos/síntesis química , Sustitución de Aminoácidos , Ascomicetos/enzimología , Dominio Catalítico , Humanos , Cinética , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Ingeniería de Proteínas , Especificidad por Sustrato , Zearalenona/análogos & derivados , Zearalenona/metabolismo , Zeranol/análogos & derivados , Zeranol/metabolismoRESUMEN
Unlike most Pseudomonas, the root-associated bacterium Pseudomonas stutzeri A1501 fixes nitrogen after the horizontal acquisition of a nitrogen-fixing (nif) island. A genome-wide search for small noncoding RNAs (ncRNAs) in P. stutzeri A1501 identified the novel P. stutzeri-specific ncRNA NfiS in the core genome, whose synthesis was significantly induced under nitrogen fixation or sorbitol stress conditions. The expression of NfiS was RNA chaperone Hfq-dependent and activated by the sigma factor RpoN/global nitrogen activator NtrC/nif-specific activator NifA regulatory cascade. The nfiS-deficient mutant displayed reduced nitrogenase activity, as well as increased sensitivity to multiple stresses, such as osmotic and oxidative stresses. Secondary structure prediction and complementation studies confirmed that a stem-loop structure was essential for NfiS to regulate the nitrogenase gene nifK mRNA synthesis and thus nitrogenase activity. Microscale thermophoresis and physiological analysis showed that NfiS directly pairs with nifK mRNA and ultimately enhances nitrogenase activity by increasing the translation efficiency and the half-life of nifK mRNA. Our data also suggest structural and functional divergence of NfiS evolution in diazotrophic and nondiazotrophic backgrounds. It is proposed that NfiS was recruited by nifK mRNA as a novel regulator to integrate the horizontally acquired nif island into host global networks.
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Proteínas Bacterianas/genética , Emparejamiento Base/genética , Fijación del Nitrógeno/genética , Nitrogenasa/genética , Pseudomonas stutzeri/genética , ARN no Traducido/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Mutación , Nitrogenasa/metabolismo , Conformación de Ácido Nucleico , Pseudomonas stutzeri/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/química , Homología de Secuencia de Ácido NucleicoRESUMEN
The discovery of antibiotics from microorganisms using classic bioactivity screens suffers from heavy labor and high re-discovery rate. Recently, largely uncovered biosynthetic potentials were unveiled by new approaches, such as genetic manipulation of "silent" biosynthetic gene clusters, innovative data acquisition, and processing methods. In this work, a fast and efficient antibiotic identification pipeline based on the MALDI-TOF imaging mass spectrometry was applied to study the antifungal metabolites during the confrontation of two fungal species, Penicillium polonicum and wilt-inducing fungus Fusarium oxysporum. By visualizing the spatial distribution of metabolites directly on the microbial colony and surrounding media, we predicted the antifungal candidates before isolating pure compounds and individually testing their bioactivity, which subsequently guided the identification of target molecules using classic chromatographic methods. Via this procedure, we successfully identified two antifungal metabolites, fructigenine A and B, which belong to indole alkaloid class and were not reported for antifungal activity. Our work assigned new bioactivity to previously reported compounds and more importantly showed the efficiency of this approach towards quick discovery of bioactive compounds, which can help study the vast unexploited synthetic potential of microbial secondary metabolites.
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Antifúngicos/metabolismo , Penicillium/metabolismo , Antibacterianos/metabolismo , Fusarium/genética , Fusarium/metabolismo , Espectrometría de Masas/métodos , Familia de Multigenes/genética , Penicillium/genética , Metabolismo Secundario/genéticaRESUMEN
Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into "build-couple-pair" combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.