RESUMO
Corynebacterium glutamicum is an important industrial workhorse for production of amino acids and chemicals. Although recently developed genome editing technologies have advanced the rational genetic engineering of C. glutamicum, continuous genome evolution based on genetic mutators is still unavailable. To address this issue, the DNA replication and repair machinery of C. glutamicum was targeted in this study. DnaQ, the homolog of ϵ subunit of DNA polymerase III responsible for proofreading in Escherichia coli, was proven irrelevant to DNA replication fidelity in C. glutamicum. However, the histidinol phosphatase (PHP) domain of DnaE1, the α subunit of DNA polymerase III, was characterized as the key proofreading element and certain variants with PHP mutations allowed elevated spontaneous mutagenesis. Repression of the NucS-mediated post-replicative mismatch repair pathway or overexpression of newly screened NucS variants also impaired the DNA replication fidelity. Simultaneous interference with the DNA replication and repair machinery generated a binary genetic mutator capable of increasing the mutation rate by up to 2352-fold. The mutators facilitated rapid evolutionary engineering of C. glutamicum to acquire stress tolerance and protein overproduction phenotypes. This study provides efficient tools for evolutionary engineering of C. glutamicum and could inspire the development of mutagenesis strategy for other microbial hosts.
Assuntos
Corynebacterium glutamicum , DNA Polimerase III , DNA Polimerase III/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Replicação do DNA/genética , Mutação , Taxa de Mutação , Engenharia MetabólicaRESUMO
L-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation. The GRAS (generally regarded as safe) industrial workhorse Corynebacterium glutamicum is an attractive chassis for L-threonine production. However, the present L-threonine production in C. glutamicum cannot meet the requirement of industrialization due to the relatively low production level of L-threonine and the accumulation of large amounts of by-products (such as L-lysine, L-isoleucine, and glycine). Herein, to enhance the L-threonine biosynthesis in C. glutamicum, releasing the aspartate kinase (LysC) and homoserine dehydrogenase (Hom) from feedback inhibition by L-lysine and L-threonine, respectively, and overexpressing four flux-control genes were performed. Next, to reduce the formation of by-products L-lysine and L-isoleucine without the cause of an auxotrophic phenotype, the feedback regulation of dihydrodipicolinate synthase (DapA) and threonine dehydratase (IlvA) was strengthened by replacing the native enzymes with heterologous analogues with more sensitive feedback inhibition by L-lysine and L-isoleucine, respectively. The resulting strain maintained the capability of synthesizing enough amounts of L-lysine and L-isoleucine for cell biomass formation but exhibited almost no extracellular accumulation of these two amino acids. To further enhance L-threonine production and reduce the by-product glycine, L-threonine exporter and homoserine kinase were overexpressed. Finally, the rationally engineered non-auxotrophic strain ZcglT9 produced 67.63 g/L (17.2% higher) L-threonine with a productivity of 1.20 g/L/h (108.0% higher) in fed-batch fermentation, along with significantly reduced by-product accumulation, representing the record for L-threonine production in C. glutamicum. In this study, we developed a strategy of reconstructing the feedback regulation of amino acid metabolism and successfully applied this strategy to de novo construct a non-auxotrophic L-threonine producing C. glutamicum. The main end by-products including L-lysine, L-isoleucine, and glycine were almost eliminated in fed-batch fermentation of the engineered C. glutamicum strain. This strategy can also be used for engineering producing strains for other amino acids and derivatives.
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Aquaculture is a rapidly growing food production technology, but there are significant concerns related to its environmental impact and adverse social effects. We examine aquaculture outcomes in a three pillars of sustainability framework by analyzing data collected using the Aquaculture Performance Indicators. Using this approach, comparable data has been collected for 57 aquaculture systems worldwide on 88 metrics that measure social, economic, or environmental outcomes. We first examine the relationships among the three pillars of sustainability and then analyze performance in the three pillars by technology and species. The results show that economic, social, and environmental outcomes are, on average, mutually reinforced in global aquaculture systems. However, the analysis also shows significant variation in the degree of sustainability in different aquaculture systems, and weak performance of some production systems in some dimensions provides opportunity for innovative policy measures and investment to further align sustainability objectives.
Assuntos
Aquicultura , Conservação dos Recursos Naturais , Aquicultura/economia , Aquicultura/métodos , Conservação dos Recursos Naturais/métodos , Conservação dos Recursos Naturais/economia , Humanos , Meio Ambiente , Animais , Desenvolvimento Sustentável/economia , Pesqueiros/economiaRESUMO
Heme is an important tetrapyrrole compound, and has been widely applied in food and medicine industries. Although microbial production of heme has been developed with metabolic engineering strategies during the past 20 years, the production levels are relatively low due to the multistep enzymatic processes and complicated regulatory mechanisms of microbes. Previous studies mainly adopted the strategies of strengthening precursor supply and product transportation to engineer microbes for improving heme biosynthesis. Few studies focused on the engineering and screening of efficient enzymes involved in heme biosynthesis. Herein, a growth-coupled, high-throughput selection platform based on the detoxification of Zinc-protoporphyrin IX (an analogue of heme) was developed and applied to directed evolution of coproporphyrin ferrochelatase, catalyzing the insertion of metal ions into porphyrin ring to generate heme or other tetrapyrrole compounds. A mutant with 3.03-fold increase in k cat/K M was selected. Finally, growth-coupled directed evolution of another three key enzymes involved in heme biosynthesis was tested by using this selection platform. The growth-coupled selection platform developed here can be a simple and effective strategy for directed evolution of the enzymes involved in the biosynthesis of heme or other tetrapyrrole compounds.
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Circulating tumor DNA (ctDNA) was short and rare, making the detection performance of the current targeted sequencing methods unsatisfying. We developed the One-PrimER Amplification (OPERA) system and examined its performance in detecting mutations of low variant allelic frequency (VAF) in various samples with short-sized DNA fragments. In cell line-derived samples containing sonication-sheared DNA fragments with 50-150 bp, OPERA was capable of detecting mutations as low as 0.0025% VAF, while CAPP-Seq only detected mutations of >0.03% VAF. Both single nucleotide variant and insertion/deletion can be detected by OPERA. In synthetic fragments as short as 80 bp with low VAF (0.03%-0.1%), the detection sensitivity of OPERA was significantly higher compared to that of droplet digital polymerase chain reaction. The error rate was 5.9×10-5 errors per base after de-duplication in plasma samples collected from healthy volunteers. By suppressing "single-strand errors", the error rate can be further lowered by >5 folds in EGFR T790M hotspot. In plasma samples collected from lung cancer patients, OPERA detected mutations in 57.1% stage I patients with 100% specificity and achieved a sensitivity of 30.0% in patients with tumor volume of less than 1 cm3. OPERA can effectively detect mutations in rare and highly-fragmented DNA.
Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Receptores ErbB/genética , Mutação , Inibidores de Proteínas Quinases , DNA Tumoral Circulante/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
NAD-independent L-lactate dehydrogenase (l-iLDH) and NAD-independent D-lactate dehydrogenase (D-iLDH) activities are induced coordinately by either enantiomer of lactate in Pseudomonas strains. Inspection of the genomic sequences of different Pseudomonas strains revealed that the lldPDE operon comprises 3 genes, lldP (encoding a lactate permease), lldD (encoding an L-iLDH), and lldE (encoding a D-iLDH). Cotranscription of lldP, lldD, and lldE in Pseudomonas aeruginosa strain XMG starts with the base, C, that is located 138 bp upstream of the lldP ATG start codon. The lldPDE operon is located adjacent to lldR (encoding an FadR-type regulator, LldR). The gel mobility shift assays revealed that the purified His-tagged LldR binds to the upstream region of lldP. An XMG mutant strain that constitutively expresses D-iLDH and L-iLDH was found to contain a mutation in lldR that leads to an Ile23-to-serine substitution in the LldR protein. The mutated protein, LldR(M), lost its DNA-binding activity. A motif with a hyphenated dyad symmetry (TGGTCTTACCA) was identified as essential for the binding of LldR to the upstream region of lldP by using site-directed mutagenesis. L-Lactate and D-lactate interfered with the DNA-binding activity of LldR. Thus, L-iLDH and D-iLDH were expressed when the operon was induced in the presence of L-lactate or D-lactate.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Lactatos/metabolismo , Pseudomonas aeruginosa/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano , Genômica/métodos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligopeptídeos , Óperon , Ligação Proteica , Pseudomonas aeruginosa/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genéticaRESUMO
Metal chalcogenides (MCs) have received widespread attentions in potassium ion storage, due to their high theoretical specific capacity and low cost. However, practical applications are still a challenge because of the slow diffusion rate and large ionic radius, leading to dramatic volume expansion and slow rate performance. Herein, we introduce a simple and large scale solvothermal method to synthesize high-quality two-dimensional (2D) layered CuSbS2 nanosheets with a thickness of about 5 nm. The thin 2D layered structure has a weak van der Waals gap and a large exposed surface area to contact the electrolyte and promotes rapid K+ diffusion kinetics. In addition, the in-situ copper exsolution during potassiation process enhances the rate capability of K+ storage. CuSbS2 half cells exhibited excellent rate performance, delivering specific capacities of 573, 505, 476, 230, 177 mAh g-1 at current densities of 0.1, 0.5, 1, 5, 10 A g-1, respectively. The unique K+ electrochemical storage mechanism and resistance change during reaction process was revealed in detail by operando XRD, XPS and TEM. Finally, potassium ion hybrid capacitors (PIHCs) with CuSbS2 nanosheets as anode and AC as cathode demonstrated excellent performances with the maximum energy density of 127 W h kg-1 and the power density of 2415 W kg-1, providing an example of rationally design a high rate battery-type PIHC anode.
RESUMO
Potassium ion batteries (PIBs) are potential alternative energy storage systems to lithium ion batteries (LIBs), due to elemental abundance of potassium, low cost and similar working principle to LIBs. Recently, metal chalcogenides (MCs) have gained enormous interests, especially antimony (Sb)-, bismuth (Bi)-based chalcogenides because they were able to undergo alloying/conversion dual mechanism, which can provide higher specific capacity and energy density (K3 Sbâ¼660â mA h g-1 , K3 Biâ¼385â mA h g-1 ). However, several challenges hinder the development of Sb-, Bi-based chalcogenide anode materials for PIBs, such as huge volume expansion during potassiation, unstable solid-electrolyte interface (SEI), slow reaction kinetics, and polychalcogenide-induced shuttle effect. In this review, the current state-of-the-art Sb-, Bi-based chalcogenides are comprehensively summarized, including the reaction mechanism, electrochemical performance, ingenious nanostructures, electrolyte systems, and prospects for future development. This review contributes to understanding the K+ storage mechanism and the interaction between active materials and electrolytes, providing guidance and foundation for the design of next-generation high-performance PIBs.
RESUMO
Metal oxides are considered as prospective dual-functional anode candidates for potassium ion batteries (PIBs) and hybrid capacitors (PIHCs) because of their abundance and high theoretic gravimetric capacity; however, due to the inherent insulating property of wide band gaps and deficient ion-transport kinetics, metal oxide anodes exhibit poor K+ electrochemical performance. In this work, we report crystal facet and architecture engineering of metal oxides to achieve significantly enhanced K+ storage performance. A bismuth antimonate (BiSbO4) nanonetwork with an architecture of perpendicularly crossed single crystal nanorods of majorly exposed (001) planes are synthesized via CTAB-mediated growth. (001) is found to be the preferential surface diffusion path for superior adsorption and K+ transport, and in addition, the interconnected nanorods gives rise to a robust matrix to enhance electrical conductivity and ion transport, as well as buffering dramatic volume change during insertion/extraction of K+. Thanks to the synergistic effect of facet and structural engineering of BiSbO4 electrodes, a stable dual conversion-alloying mechanism based on reversible six-electron transfer per formula unit of ternary metal oxides is realized, proceeding by reversible coexistence of potassium peroxide conversion reactions (KO2âK2O) and BixSby alloying reactions (BiSb â KBiSb â K3BiSb). As a result, BiSbO4 nanonetwork anodes show outstanding potassium ion storage in terms of capacity, cycling life, and rate capability. Finally, the implementation of a BiSbO4 nanonetwork anode in the state-of-the-art full cell configuration of both PIBs and PIHCs shows satisfactory performance in a Ragone plot that sheds light on their practical applications for a wide range of K+-based energy storage devices. We believe this study will propose a promising avenue to design advanced hierarchical nanostructures of ternary or binary conversion-type materials for PIBs, PIHCs, or even for extensive energy storage.
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Chemoenzymatic synthesis of N-acetyl-d-neuraminic acid from N-acetyl-d-glucosamine using the spore surface-displayed N-acetyl-d-neuraminic acid aldolase at a high concentration (53.9 g liter(-1)) was achieved in this study. Thus, displaying a target enzyme on the surface of spores might be an alternative for integration of biocatalytic conversion into chemical synthesis.
Assuntos
Acetilglucosamina/metabolismo , Aldeído Liases/metabolismo , Bacillus subtilis/metabolismo , Expressão Gênica , Ácido N-Acetilneuramínico/biossíntese , Esporos Bacterianos/metabolismo , Aldeído Liases/genética , Bacillus subtilis/genética , Vias Biossintéticas/genética , Biotecnologia/métodos , Organismos Geneticamente Modificados , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esporos Bacterianos/genéticaRESUMO
Production of N-acetyl-D-neuraminic acid (Neu5Ac) via biocatalysis is traditionally conducted using isolated enzymes or whole cells. The use of isolated enzymes is restricted by the time-consuming purification process, whereas the application of whole cells is limited by the permeability barrier presented by the microbial cell membrane. In this study, a novel type of biocatalyst, Neu5Ac aldolase presented on the surface of Bacillus subtilis spores, was used for the production of Neu5Ac. Under optimal conditions, Neu5Ac at a high concentration (54.7 g liter⻹) and a high yield (90.2%) was obtained under a 5-fold excess of pyruvate over N-acetyl-D-mannosamine. The novel biocatalyst system, which is able to express and immobilize the target enzyme simultaneously on the surface of B. subtilis spores, represents a suitable alternative for value-added chemical production.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Oxo-Ácido-Liases/genética , Oxo-Ácido-Liases/metabolismo , Esporos Bacterianos/enzimologia , Esporos Bacterianos/metabolismo , Bacillus subtilis/genética , Biotecnologia/métodos , Esporos Bacterianos/genéticaRESUMO
Ebola virus (EBOV) is one of the most pathogenic viruses in humans which can cause a lethal hemorrhagic fever. Understanding the cellular entry mechanisms of EBOV can promote the development of new therapeutic strategies to control virus replication and spread. It has been known that EBOV virions bind to factors expressed at the host cell surface. Subsequently, the virions are internalized by a macropinocytosis-like process, followed by being trafficked through early and late endosomes. Recent researches indicate that the entry of EBOV into cells requires integrated and functional lipid rafts. Whilst lipid rafts have been hypothesized to play a role in virus entry, there is a current lack of supporting data. One major technical hurdle is the lack of effective approaches for observing viral entry. To provide evidence on the involvement of lipid rafts in the entry process of EBOV, we generated the fluorescently labeled Ebola virus like particles (VLPs), and utilized single-particle tracking (SPT) to visualize the entry of fluorescent Ebola VLPs in live cells and the interaction of Ebola VLPs with lipid rafts. In this study, we demonstrate the compartmentalization of Ebola VLPs in lipid rafts during entry process, and inform the essential function of lipid rafts for the entry of Ebola virus. As such, our study provides evidence to show that the raft integrity is critical for Ebola virus pathogenesis and that lipid rafts can serve as potential targets for the development of novel therapeutic strategies.
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The study evaluated GIMMS NDVI based on MODIS NDVI and SPOT NDVI over the same period from 2000 to 2015. We assessed their absolute values, dynamics, trends and cross-relationships between any two of the NDVIs for the national scale, as well as four separate land use types, i.e., paddy field, dry land, forest, and grassland. GIMMS NDVI was numerically greater than MODIS NDVI and SPOT NDVI. The three NDVIs exhibited equal capability of capturing monthly phenological variations. During the study period, the three NDVIs showed increasing trends in most regions, with GIMMS NDVI showing the smallest increment. Pronounced differences were identified in trends between GIMMS NDVI and MODIS NDVI or SPOT NDVI in the northwest, northeast, south-central China, Tibetan Plateau and Yunnan-Guizhou Plateau, implying that GIMMS NDVI trends in these regions should be interpreted with caution. High correlations existed between the three datasets. MODIS NDVI and SPOT NDVI showed stronger correlations at national scale. The GIMMS NDVI and MODIS NDVI were in highest accordance for dry land, while MODIS NDVI and SPOT NDVI were in higher accordance for the paddy field, forest, and grassland than dry lands.
Assuntos
Monitoramento Ambiental , China , FlorestasRESUMO
AIM: The simplified preparative method and formulation for atenolol monolithic osmotic pump tablets were investigated. METHODS: The core tablets with an indentation were compressed by the punch with a needle. Osmotic pump tablets were prepared by coating the indented tablets. Similarity factor was used to evaluate formulation of osmotic pump tablets. RESULTS: The formulation of core tablets and the composition and thickness of coating membrane showed marked effects on drug release. Orifice size of core tablets in the range of 1.00 - 1.14 mm scarcely affected drug release. CONCLUSION: The preparation of osmotic pump tablets was simplified with the exempting of laser drilling. The atenolol monolithic osmotic pump tablets could deliver drug constantly for 24 h.
Assuntos
Antagonistas Adrenérgicos beta/administração & dosagem , Atenolol/administração & dosagem , Polietilenoglicóis/química , Tecnologia Farmacêutica/métodos , Antagonistas Adrenérgicos beta/química , Atenolol/química , Preparações de Ação Retardada , Portadores de Fármacos , Osmose , ComprimidosRESUMO
As the major neurotransmitter in the mammalian central nervous system (CNS), excessive extracellular glutamate (Glu) can activate the Glu receptors and neuronal calcium (Ca2+) overload, then produce neurotoxicity, which is a common pathway for neuronal injury or death, and is associated with acute and chronic neurodegenerative diseases. Therefore, it has been a therapeutic strategy to investigate neuroprotective effects against Glu-induced neurotoxicity for treating both acute and chronic forms of neurodegeneration. Resveratrol (Res), as a naturally occurring polyphenol mainly found in grapes and red wine, has shown a neuroprotective effect in a variety of experimental models for neurodegenerative diseases in vitro and in vivo. This review will focus on the neuroprotective effect of Res against Glu-induced excitotoxicity in neurodegenerative diseases by blocking different Glu receptors and Ca2+ ion channels.
Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/toxicidade , Doenças Neurodegenerativas/prevenção & controle , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estilbenos/farmacologia , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Neurônios/patologia , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , ResveratrolRESUMO
2-Oxobutyrate (2-OBA) is an important intermediate with many applications in the drug and chemical industries. l-Threonine, an industrial fermentation product, could be used as a suitable starting material for the 2-OBA production. In this study, whole cells of Pseudomonas stutzeri SDM were confirmed to possess a good ability to convert l-threonine into 2-OBA. The bioconversion conditions were optimized for the 2-OBA production from l-threonine. Using 9.2g dry cell weight l(-1) of whole cells of strain SDM as biocatalyst, the biocatalytic process produced 2-OBA at a high concentration (25.6gl(-1)) with a high molar conversion rate (99.6%) at 6h from 30g1(-1) of l-threonine.