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Promoter-centric genetic tools play a crucial role in controlling gene expression for various applications, such as strain engineering and synthetic biology studies. Hence, a critical need persists for the development of robust gene expression tools. Streptomyces are well-known prolific producers of natural products and exceptional surrogate hosts for the production of high-value chemical compounds and enzymes. In this study, we reported a straightforward and effective strategy for the creation of potent gene expression tools. This was primarily achieved by introducing an additional -35-like motif upstream of the original -35 region of the promoter, coupled with the integration of a palindromic cis-element into the 5'-UTR region. This approach has generated a collection of robust constitutive and inducible gene expression tools tailored for Streptomyces. Of particular note, the fully activated oxytetracycline-inducible gene expression system containing an engineered kasOp* promoter (OK) exhibited nearly an order of magnitude greater activity compared to the well-established high-strength promoter kasOp* under the tested conditions, establishing itself as a powerful gene expression system for Streptomyces. This strategy is expected to be applicable in modifying various other promoters to acquire robust gene expression tools, as evidenced by the enhancement observed in the other two promoters, PL and P21 in this study. Moreover, the effectiveness of these tools has been demonstrated through the augmented production of transglutaminase and daptomycin. The gene expression tools established in this study, alongside those anticipated in forthcoming research, are positioned to markedly advance pathway engineering and synthetic biology investigations in Streptomyces and other microbial strains.
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Transcription factor ELONGATED HYPOCOTYL5 (HY5) is the central hub for seedling photomorphogenesis. E3 ubiquitin (Ub) ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) inhibits HY5 protein accumulation through ubiquitination. However, the process of HY5 deubiquitination, which antagonizes E3 ligase-mediated ubiquitination to maintain HY5 homeostasis has never been studied. Here, we identified that Arabidopsis thaliana deubiquitinating enzyme, Ub-SPECIFIC PROTEASE 14 (UBP14) physically interacts with HY5 and enhances its protein stability by deubiquitination. The da3-1 mutant lacking UBP14 function exhibited a long hypocotyl phenotype, and UBP14 deficiency led to the failure of rapid accumulation of HY5 during dark to light. In addition, UBP14 preferred to stabilize nonphosphorylated form of HY5 which is more readily bound to downstream target genes. HY5 promoted the expression and protein accumulation of UBP14 for positive feedback to facilitate photomorphogenesis. Our findings thus established a mechanism by which UBP14 stabilizes HY5 protein by deubiquitination to promote photomorphogenesis in A. thaliana.
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Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição de Zíper de Leucina Básica , Regulação da Expressão Gênica de Plantas , Ubiquitinação , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Estabilidade Proteica/efeitos da radiação , Luz , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Hipocótilo/genéticaRESUMO
Biosynthesis of atypical angucyclines involves unique oxidative B-ring cleavage and rearrangement reactions, which are catalyzed by AlpJ-family oxygenases, including AlpJ, JadG, and GilOII. Prior investigations established the essential requirement for FADH2/FMNH2 as cofactors when utilizing the quinone intermediate dehydrorabelomycin as a substrate. In this study, we unveil a previously unrecognized facet of these enzymes as cofactor-independent oxygenases when employing the hydroquinone intermediate CR1 as a substrate. The enzymes autonomously drive oxidative ring cleavage and rearrangement reactions of CR1, yielding products identical to those observed in cofactor-dependent reactions of AlpJ-family oxygenases. Furthermore, the AlpJ- and JadG-catalyzed reactions of CR1 could be quenched by superoxide dismutase, supporting a catalytic mechanism wherein the substrate CR1 reductively activates molecular oxygen, generating a substrate radical and the superoxide anion O2 â¢-. Our findings illuminate a substrate-controlled catalytic mechanism of AlpJ-family oxygenases, expanding the realm of cofactor-independent oxygenases. Notably, AlpJ-family oxygenases stand as a pioneering example of enzymes capable of catalyzing oxidative reactions in either an FADH2/FMNH2-dependent or cofactor-independent manner.
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We confirm the previously revised stereochemistry of spiroviolene by X-ray crystallographically characterizing a hydrazone derivative of 9-oxospiroviolane, which is synthesized by hydroboration/oxidation of spiroviolene followed by oxidation of the resultant hydroxy group. An unexpected thermal boron migration occurred during the hydroboration process of spiroviolene that resulted in the production of a mixture of 1α-hydroxyspiroviolane, 9α- and 9ß-hydroxyspiroviolane after oxidation. The assertion of the cis-orientation of the 19- and 20-methyl groups provided further support for the revised cyclization mechanism of spiroviolene.
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Iso-Migrastatin (iso-MGS) and lactimidomycin (LTM) are glutarimide-containing polyketide natural products (NPs) that are biosynthesized by homologous acyltransferase (AT)-less type I polyketide synthase (PKS) assembly lines. The biological activities of iso-MGS and LTM have inspired numerous efforts to generate analogues via genetic manipulation of their biosynthetic machinery in both native producers and model heterologous hosts. A detailed understanding of the MGS and LTM AT-less type I PKSs would serve to inspire future engineering efforts while advancing the fundamental knowledge of AT-less type I PKS enzymology. The mgs and ltm biosynthetic gene clusters (BGCs) encode for two discrete ATs of the architecture AT-enoylreductase (AT-ER) and AT-type II thioesterase (AT-TE). Herein, we report the functional characterization of the mgsB and ltmB and the mgsH and ltmH gene products, revealing that MgsB and LtmB function as type II thioesterases (TEs) and MgsH and LtmH are the dedicated trans-ATs for the MGS and LTM AT-less type I PKSs. In vivo and in vitro experiments demonstrated that MgsB was devoid of any AT activity, despite the presence of the conserved catalytic triad of canonical ATs. Cross-complementation experiments demonstrated that MgsH and LtmH are functionally interchangeable between the MGS and LTM AT-less type I PKSs. This work sets the stage for future mechanistic studies of AT-less type I PKSs and efforts to engineer the MGS and LTM AT-less type I PKS assembly lines for novel glutarimide-containing polyketides.
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Glycerol is a readily available and low-cost simple polyol compound, which can be used as a carbon source for microorganisms to produce various value-added products. Understanding the underlying regulatory mechanism in glycerol metabolism is critical for making better use of glycerol for diverse applications. In a few reported Streptomyces strains, the glycerol utilization gene cluster (glp operon) was shown to be regulated by the IclR family transcriptional regulator GylR. However, the molecular regulatory mechanism mediated by GylR has not been fully elucidated. In this study, we first analyzed the available Actinobacteria genomes in the NCBI Genome database, and found that the glp operon-like gene clusters are conserved in Streptomyces and several other genera of Actinobacteria. By taking Streptomyces clavuligerus NRRL 3585 as a model system, we identified that GylR represses the expressions of glp operon and gylR by directly binding to their promoter regions. Both glycerol-3-phosphate and dihydroxyacetone phosphate can induce the dissociation of GylR from its binding sequences. Furthermore, we identified a minimal essential operator site (a palindromic 18-bp sequence) of GylR-like regulators in Streptomyces. Our study for the first time reported the binding sequences and effector molecules of GylR-like proteins in Streptomyces. The molecular regulatory mechanism mediated by GylR presumably exists widely in Streptomyces. Our findings would facilitate the design of glycerol utilization pathways for producing valuable products. Moreover, our study provided new basic elements for the development of glycerol-inducible regulatory tools for synthetic biology research in the future.
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Tetracycline (TC) natural products possess a variety of remarkable bioactivities and diverse structures. They are an important and fertile source for developing novel drugs. As one of the most successful drug families, TC antibiotics have been in clinical use for over seven decades, and continue to make an important contribution to human health nowadays. To date, studies on TC natural products and their biosynthesis have revealed numerous novel biochemical mechanisms and regulatory elements, which facilitates the rational metabolic engineering studies for generating novel bioactive TC analogs and inspires the development of new synthetic biology tools. In this review, we provide a comprehensive overview on the discovery, biosynthesis, and engineering of the existing TC natural products. These analyses will be of great value for the discovery, design and development of novel TC drugs in the future.
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Produtos Biológicos , Humanos , Produtos Biológicos/farmacologia , Produtos Biológicos/metabolismo , Antibacterianos , Engenharia Metabólica , Biologia Sintética , TetraciclinaRESUMO
Efficient utilization of both glucose and xylose, the two most abundant sugars in biomass hydrolysates, is one of the main objectives of biofermentation with lignocellulosic materials. The utilization of xylose is commonly inhibited by glucose, which is known as glucose catabolite repression (GCR). Here, we report a GCR-based dynamic control (GCR-DC) strategy aiming at better co-utilization of glucose and xylose, by decoupling the cell growth and biosynthesis of riboflavin as a product. Using the thermophilic strain Geobacillus thermoglucosidasius DSM 2542 as a host, we constructed additional riboflavin biosynthetic pathways that were activated by xylose but not glucose. The engineered strains showed a two-stage fermentation process. In the first stage, glucose was preferentially used for cell growth and no production of riboflavin was observed, while in the second stage where glucose was nearly depleted, xylose was effectively utilized for riboflavin biosynthesis. Using corn cob hydrolysate as a carbon source, the optimized riboflavin yields of strains DSM2542-DCall-MSS (full pathway dynamic control strategy) and DSM2542-DCrib (single-module dynamic control strategy) were 5.3- and 2.3-fold higher than that of the control strain DSM 2542 Rib-Gtg constitutively producing riboflavin, respectively. This GCR-DC strategy should also be applicable to the construction of cell factories that can efficiently use natural carbon sources with multiple sugar components for the production of high-value chemicals in future.
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Glucose , Xilose , Bacillaceae , Carbono , Fermentação , Glucose/metabolismo , Lignina , Riboflavina/metabolismo , Açúcares , Xilose/metabolismoRESUMO
Broadband near-infrared (NIR) phosphor-converted light-emitting diodes (pc-LEDs) are essential to integrate near-infrared spectrometers into mobile devices for the rapid and noninvasive detection of biological components. However, efficient broadband NIR phosphors with a peak emission wavelength longer than 800 nm are deficient. In this study, CaMgGe2O6:Cr3+ phosphor was prepared by a high-temperature solid-state reaction. The phosphor doped with 0.02Cr3+ showed an emission band at 845 nm with a broad bandwidth of 160 nm and a high quantum yield of 84% under 450 nm excitation. The broadband NIR pc-LED was fabricated using CaMgGe2O6:0.02Cr3+ phosphor based on a blue light-emitting diode (LED) chip. A photoelectric efficiency of 27.2% @ 10 mA and an NIR output power of 57.98 mW @ 100 mA were achieved, which are the highest values reported yet for broadband NIR pc-LEDs with a peak wavelength longer than 800 nm. Using the fabricated NIR pc-LED as the light source, the characteristic absorption spectra of some substances were obtained. All of the results indicated that the CaMgGe2O6:Cr3+ phosphor has considerable potential in near-infrared spectroscopic applications.
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The biosynthetic investigations of microbial natural products continuously provide powerful biocatalysts for the preparation of valuable chemicals. Practical methods for preparing (S)-3-aminopiperidine-2,6-dione (2), the pharmacophore of thalidomide (1) and its analog drugs, are highly desired. To develop a biocatalyst for producing (S)-2, we dissected the domain functions of IdgS, which is responsible for the biosynthesis of indigoidine (3), a microbial blue pigment that consists of two 2-like moieties. Our data supported that the L-glutamine tethered to the indigoidine assembly line is first offloaded and cyclized by the thioesterase domain to form (S)-2, which is then dehydrogenated by the oxidation (Ox) domain and finally dimerized to yield 3. Based on this, we developed an IdgS-derived enzyme biocatalyst, IdgS-Ox* R539A, for preparing enantiomerically pure (S)-2. As a proof of concept, one-pot chemoenzymatic synthesis of 1 was achieved by combining the biocatalytic and chemical approaches.
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Nature forms S-S bonds by oxidizing two sulfhydryl groups, and no enzyme installing an intact hydropersulfide (-SSH) group into a natural product has been identified to date. The leinamycin (LNM) family of natural products features intact S-S bonds, and previously we reported an SH domain (LnmJ-SH) within the LNM hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line as a cysteine lyase that plays a role in sulfur incorporation. Here we report the characterization of an S-adenosyl methionine (SAM)-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin (GNM) biosynthesis, discovery of hydropersulfides as the nascent products of the GNM and LNM hybrid NRPS-PKS assembly lines, and revelation of three SH domains (GnmT-SH, LnmJ-SH, and WsmR-SH) within the GNM, LNM, and weishanmycin (WSM) hybrid NRPS-PKS assembly lines as thiocysteine lyases. Based on these findings, we propose a biosynthetic model for the LNM family of natural products, featuring thiocysteine lyases as PKS domains that directly install a -SSH group into the GNM, LNM, or WSM polyketide scaffold. Genome mining reveals that SH domains are widespread in Nature, extending beyond the LNM family of natural products. The SH domains could also be leveraged as biocatalysts to install an -SSH group into other biologically relevant scaffolds.
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Produtos Biológicos/metabolismo , Liases de Carbono-Enxofre/metabolismo , Cisteína/análogos & derivados , Metiltransferases/metabolismo , Policetídeo Sintases/metabolismo , Sulfetos/metabolismo , Animais , Produtos Biológicos/química , Cisteína/metabolismo , Cistina/química , Cistina/metabolismo , Humanos , Lactamas/síntese química , Lactamas/química , Lactamas/metabolismo , Macrolídeos/síntese química , Macrolídeos/química , Macrolídeos/metabolismo , Modelos Químicos , Estrutura Molecular , Peptídeo Sintases/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Especificidade por Substrato , Sulfetos/química , Tiazóis/síntese química , Tiazóis/química , Tiazóis/metabolismo , Tionas/síntese química , Tionas/química , Tionas/metabolismo , Domínios de Homologia de srcRESUMO
Catalases play a key role in the defense against oxidative stress in bacteria by catalyzing the decomposition of H2O2. In addition, catalases are also involved in multiple cellular processes, such as cell development and differentiation, as well as metabolite production. However, little is known about the abundance, diversity, and distribution of catalases in bacteria. In this study, we systematically surveyed and classified the homologs of three catalase families from 2,634 bacterial genomes. It was found that both of the typical catalase and Mn-catalase families could be divided into distinct groups, while the catalase-peroxidase homologs formed a tight family. The typical catalases are rich in all the analyzed bacterial phyla except Chlorobi, in which the catalase-peroxidases are dominant. Catalase-peroxidases are rich in many phyla, but lacking in Deinococcus-Thermus, Spirochetes, and Firmicutes. Mn-catalases are found mainly in Firmicutes and Deinococcus-Thermus, but are rare in many other phyla. Given the fact that catalases were reported to be involved in secondary metabolite biosynthesis in several Streptomyces strains, the distribution of catalases in the genus Streptomyces was given more attention herein. On average, there are 2.99 typical catalases and 0.99 catalase-peroxidases in each Streptomyces genome, while no Mn-catalases were identified. To understand detailed properties of catalases in Streptomyces, we characterized all the five typical catalases from S. rimosus ATCC 10970, the oxytetracycline-producing strain. The five catalases showed typical catalase activity, but possessed different catalytic properties. Our findings contribute to the more detailed classification of catalases and facilitate further studies about their physiological roles in secondary metabolite biosynthesis and other cellular processes, which might facilitate the yield improvement of valuable secondary metabolites in engineered bacteria.
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The ever-growing drug resistance problem worldwide highlights the urgency to discover and develop new drugs. Microbial natural products are a prolific source of drugs. Genome sequencing has revealed a tremendous amount of uncharacterized natural product biosynthetic gene clusters (BGCs) encoded within microbial genomes, most of which are cryptic or express at very low levels under standard culture conditions. Therefore, developing effective strategies to awaken these cryptic BGCs is of great interest for natural product discovery. In this study, we designed and validated a Transcription-Translation in One (TTO) approach for activation of cryptic BGCs. This approach aims to alter the metabolite profiles of target strains by directly overexpressing exogenous rpsL (encoding ribosomal protein S12) and rpoB (encoding RNA polymerase ß subunit) genes containing beneficial mutations for natural product production using a plug-and-play plasmid system. As a result, this approach bypasses the tedious screening work and overcomes the false positive problem in the traditional ribosome engineering approach. In this work, the TTO approach was successfully applied to activating cryptic BGCs in three Streptomyces strains, leading to the discovery of two aromatic polyketide antibiotics, piloquinone and homopiloquinone. We further identified a single BGC responsible for the biosynthesis of both piloquinone and homopiloquinone, which features an unusual starter unit incorporation step. This powerful strategy can be further exploited for BGC activation in strains even beyond streptomycetes, thus facilitating natural product discovery research in the future.
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RNA Polimerases Dirigidas por DNA/metabolismo , Metaboloma/genética , Família Multigênica , Proteínas Ribossômicas/metabolismo , Antraquinonas/metabolismo , Produtos Biológicos/metabolismo , Técnicas Genéticas , Biossíntese de Proteínas , Streptomyces lividans/genética , Transcrição GênicaRESUMO
Bacterial natural products (NPs) and their analogs constitute more than half of the new small molecule drugs developed over the past few decades. Despite this success, interest in natural products from major pharmaceutical companies has decreased even as genomics has uncovered the large number of biosynthetic gene clusters (BGCs) that encode for novel natural products. To date, there is still a lack of universal strategies and enabling technologies to discover natural products at scale and speed. This review highlights several of the opportunities provided by genome sequencing and bioinformatics, challenges associated with translating genomes into natural products, and examples of successful strain prioritization and BGC activation strategies that have been used in the genomic era for natural product discovery from cultivatable bacteria.
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Bactérias/genética , Produtos Biológicos/isolamento & purificação , Bactérias/química , Bactérias/metabolismo , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Genoma Bacteriano , Humanos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
In Ce3+ activated SrLn2O4 type phosphors (Ln = Y, Lu, Sc, etc.) only one Ce3+ center was previously reported to show a blue emission band. In this paper, we report the observation of a second Ce3+ center in SrLu2O4:Ce3+. The new center shows a red emission band peaking at 600 nm with an excitation band at 485 nm. We attributed the new center (Ce(ii)) to the substitution of the Lu3+ site and the original blue center (Ce(i)) to the substitution of the Sr2+ site. Spectroscopy studies indicate that Ce(i) centers are preferentially formed at a low doping concentration and the number ratio of Ce(i)/Ce(ii) decreases with increasing Ce3+ concentration until beyond 0.002. The fluorescence lifetimes of the two centers were measured for various doping concentrations. Energy transfer from Ce(i) to Ce(ii) was observed. It was found that the emission intensity of Ce(ii) centers reduces much faster than that of Ce(i) with increasing temperature from 83 K up to 350 K, implying their potential application in temperature sensing based on their temperature dependent intensity ratios. A relative sensing sensitivity as high as 2.28% K-1 at 283 K was achieved.
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Covering: up to 2018With contributions from the global natural product (NP) research community, and continuing the Raw Data Initiative, this review collects a comprehensive demonstration of the immense scientific value of disseminating raw nuclear magnetic resonance (NMR) data, independently of, and in parallel with, classical publishing outlets. A comprehensive compilation of historic to present-day cases as well as contemporary and future applications show that addressing the urgent need for a repository of publicly accessible raw NMR data has the potential to transform natural products (NPs) and associated fields of chemical and biomedical research. The call for advancing open sharing mechanisms for raw data is intended to enhance the transparency of experimental protocols, augment the reproducibility of reported outcomes, including biological studies, become a regular component of responsible research, and thereby enrich the integrity of NP research and related fields.
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Produtos Biológicos/química , Espectroscopia de Ressonância Magnética/métodos , Conformação Molecular , Reprodutibilidade dos TestesRESUMO
Correction for 'The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research' by James B. McAlpine et al., Nat. Prod. Rep., 2018, DOI: .
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Correction for 'Dye-embedded YAG:Ce3+@SiO2 composite phosphors toward warm wLEDs through radiative energy transfer: preparation, characterization and luminescence properties' by Guo-Hui Pan, Jiahua Zhang et al., Nanoscale, 2018, DOI: 10.1039/c8nr07360k.
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The most common yellow phosphor for wLEDs, Y3Al5O12:Ce3+ (YAG:Ce3+), suffers from a deficiency of red in its spectral content of light. In this paper, a new strategy is provided to tailor the Ce3+ spectral profile through surface-located dye molecules of ATTO-Rho101, which feature intense, broad absorption in the green-yellow spectral region of Ce3+ emission as well as bright red emission. Sphere-shaped and highly dispersed micrometer and nanometer-sized YAG:Ce3+ (micro/nano-YAG:Ce3+) was synthesized through a modified solvothermal method. Surface SiO2 coating and simultaneous dye embedding were performed on the solvothermally derived YAG:Ce3+, heat-treated micro-YAG:Ce3+ and commercial phosphors. Efficient radiative transfer/reabsorption from Ce3+ in the inner core of YAG to the dye molecules in the SiO2 outer shell, irrespective of the size of the phosphors, was demonstrated in the accumulated YAG:Ce3+@SiO2 + dye powder upon blue light excitation; this enhanced its red emission. Fluorescence microscopy was demonstrated to be a powerful tool to identify the reabsorption phenomenon of the powdered materials. Packaging the heat-treated micro-YAG:Ce3+@SiO2 + dye phosphors on blue LED chips yielded a warm wLED (Raâ¼ 93), but an Ra of only â¼79 was obtained for the wLED with commercial YAG:Ce3+@(SiO2 + dye)5 due to the low concentration of phosphors dispersed in the epoxy resin and the resulting decreased reabsorption by dye molecules. Surface-protonated amine species were found to induce Ce3+â Ce4+ oxidation upon activation by heating or photoirradiation and then quench the photoluminescence (PL) of micro-YAG:Ce3+ even after surface modification by SiO2, YAG or being embedded in an epoxy resin matrix. High calcination temperatures greatly improved the PL stability of micro-YAG:Ce3+ through the removal of surface-capped species. The dye in the silica matrix showed high stability against heating and irradiation due to the so-called "caging effects"; however, decreased photo-stability was found in commercial YAG:Ce3+@(SiO2 + dye)5 due to the incomplete and/or loose SiO2 layer grown during multiple surface modifications.