RESUMO
Glycerol is an eco-friendly solvent that enhances plant biomass decomposition via glycerolysis in many pretreatment methods. Nonetheless, inefficient conversion of glycerol to ethanol by natural Saccharomyces cerevisiae limits its use in these processes. In this study, we have developed an efficient glycerol-converting yeast strain by genetically modifying the oxidation of cytosolic NAD (NADH) by an O2-dependent dynamic shuttle and abolishing both glycerol phosphorylation and biosynthesis in S. cerevisiae strain D452-2, as well as by vigorous expression of whole genes in the dihydroxyacetone (DHA) pathway (Candida utilis glycerol facilitator, Ogataea polymorpha glycerol dehydrogenase, endogenous dihydroxyacetone kinase, and triosephosphate isomerase). The engineered strain showed conversion efficiencies (CE) up to 0.49 g ethanol/g glycerol (98% of theoretical CE), with a production rate of >1 g liter-1 h-1 when glycerol was supplemented in a single fed-batch fermentation in a rich medium. Furthermore, the engineered strain converted a mixture of glycerol and glucose into bioethanol (>86 g/liter) with 92.8% CE. To the best of our knowledge, this is the highest reported titer of bioethanol produced from glycerol and glucose. Notably, we developed a glycerol-utilizing transformant from a parent strain which cannot utilize glycerol as a sole carbon source. The developed strain converted glycerol to ethanol with a productivity of 0.44 g liter-1 h-1 on minimal medium under semiaerobic conditions. Our findings will promote the utilization of glycerol in eco-friendly biorefineries and integrate bioethanol and plant oil industries. IMPORTANCE With the development of efficient lignocellulosic biorefineries, glycerol has attracted attention as an eco-friendly biomass-derived solvent that can enhance the dissociation of lignin and cell wall polysaccharides during the pretreatment process. Coconversion of glycerol with the sugars released from biomass after glycerolysis increases the resources for ethanol production and lowers the burden of component separation. However, low conversion efficiency from glycerol and sugars limits the industrial application of this process. Therefore, the generation of an efficient glycerol-fermenting yeast will promote the applicability of integrated biorefineries. Hence, metabolic flux control in yeast grown on glycerol will lead to the generation of cell factories that produce chemicals, which will boost biodiesel and bioethanol industries. Additionally, the use of glycerol-fermenting yeast will reduce global warming and generation of agricultural waste, leading to the establishment of a sustainable society.
Assuntos
Etanol , Glicerol , Saccharomyces cerevisiae , Etanol/metabolismo , Glucose , Glicerol/metabolismo , Microbiologia Industrial , Microrganismos Geneticamente Modificados , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Solventes , AçúcaresRESUMO
Tunicosaponins are natural products extracted from Psammosilene tunicoides, which is an important ingredient of Yunnan Baiyao Powder, an ancient and famous Asian herbal medicine. The representative aglycones of tunicosaponins are the oleanane-type triterpenoids of gypsogenin and quillaic acid, which were found to manipulate a broad range of virus-host fusion via wrapping the heptad repeat-2 (HR2) domain prevalent in viral envelopes. However, the unknown biosynthetic pathway and difficulty in chemical synthesis hinder the therapeutic use of tunicosaponins. Here, two novel cytochrome P450-dependent monooxygenases that take part in the biosynthesis of tunicosaponins, CYP716A262 (CYP091) and CYP72A567 (CYP099), were identified from P. tunicoides. In addition, the whole biosynthesis pathway of the tunicosaponin aglycones was reconstituted in yeast by transforming the platform strain BY-bAS with the CYP716A262 and CYP716A567 genes, the resulting strain could produce 146.84 and 314.01 mg/L of gypsogenin and quillaic acid, respectively. This synthetic biology platform for complicated metabolic pathways elucidation and microbial cell factories construction can provide alternative sources of important natural products, helping conserve natural plant resources.
Assuntos
Caryophyllaceae/genética , Sistema Enzimático do Citocromo P-450 , Ácido Oleanólico , Proteínas de Plantas , Plantas Medicinais/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Ácido Oleanólico/biossíntese , Ácido Oleanólico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saponinas/biossíntese , Saponinas/genéticaRESUMO
Synthetic microbial cocultures carry enormous potential for applied biotechnology and are increasingly the subject of fundamental research. So far, most cocultures have been designed and characterized based on bulk cultivations without considering the potentially highly heterogeneous and diverse single-cell behavior. However, an in-depth understanding of cocultures including their interacting single cells is indispensable for the development of novel cultivation approaches and control of cocultures. We present the development, validation, and experimental characterization of an optochemically controllable bacterial coculture on a microcolony level consisting of two Corynebacterium glutamicum strains. Our coculture combines an l-lysine auxotrophic strain together with a l-lysine-producing variant carrying the genetically IPTG-mediated induction of l-lysine production. We implemented two control approaches utilizing IPTG as inducer molecule. First, unmodified IPTG was supplemented to the culture enabling a medium-based control of the production of l-lysine, which serves as the main interacting component. Second, optochemical control was successfully performed by utilizing photocaged IPTG activated by appropriate illumination. Both control strategies were validated studying cellular growth on a microcolony level. The novel microfluidic single-cell cultivation strategies applied in this work can serve as a blueprint to validate cellular control strategies of synthetic mono- and cocultures with single-cell resolution at defined environmental conditions.
Assuntos
Proliferação de Células/efeitos da radiação , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Engenharia Metabólica/métodos , Interações Microbianas/efeitos da radiação , Raios Ultravioleta , Biotecnologia/métodos , Proliferação de Células/genética , Técnicas de Cocultura/métodos , Corynebacterium glutamicum/classificação , Meios de Cultura/química , Fluorescência , Isopropiltiogalactosídeo/genética , Isopropiltiogalactosídeo/metabolismo , Lisina/biossíntese , Interações Microbianas/genética , Técnicas Analíticas Microfluídicas/métodos , Microrganismos Geneticamente ModificadosRESUMO
With the aid of a chain transfer (CT) reaction, hydroxyalkanoate (HA) oligomers can be secreted by recombinant Escherichia coli carrying the gene encoding a lactate-polymerizing enzyme (PhaC1PsSTQK) in Luria-Bertani (LB) medium supplemented with a carbon source and CT agent. In this study, HA oligomers were produced through microbial secretion using a mineral-based medium instead of LB medium, and the impact of medium composition on HA oligomer secretion was investigated. The focused targets were medium composition and NaCl concentration related to osmotic conditions. It was observed that 4.21 g/L HA oligomer was secreted by recombinant E. coli in LB medium, but the amount secreted in the mineral-based modified R (MR) medium was negligible. However, when the MR medium was supplemented with 5 g/L yeast extract, 3.75 g/L HA oligomer was secreted. This can be accounted for by the enhanced expression and activity of PhaC1PsSTQK upon supplementation with growth-activated nutrients as supplementation with yeast extract also promoted cell growth and intracellular growth-associated polymer accumulation. Furthermore, upon adding 10 g/L NaCl to the yeast extract-supplemented MR medium, HA oligomer secretion increased to 6.86 g/L, implying that NaCl-induced osmotic pressure promotes HA oligomer secretion. These findings may facilitate the secretory production of HA oligomers using an inexpensive medium.
Assuntos
Meios de Cultura/análise , Escherichia coli/metabolismo , Poli-Hidroxialcanoatos/biossíntese , Polimerização , Escherichia coli/química , Microrganismos Geneticamente Modificados/química , Microrganismos Geneticamente Modificados/metabolismoRESUMO
Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met-bottromycin A2 up to 109 mg L-1 , the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L-1 talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re-balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of non-cyclized shunt by-products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13-fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline-type oxytetracycline, and the anthramycin-analogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone-type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and synthetic control. Given its simplicity and broad benefits, microparticle-supplementation appears as an enabling technology in natural product research of these most important microbes.
Assuntos
Antibacterianos/biossíntese , Engenharia Metabólica , Microrganismos Geneticamente Modificados , Peptídeos Cíclicos , Streptomyces lividans , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , Streptomyces lividans/genética , Streptomyces lividans/metabolismoRESUMO
Baicalein and scutellarein are bioactive flavonoids isolated from the traditional Chinese medicine Scutellaria baicalensis Georgi; however, there is a lack of effective strategies for producing baicalein and scutellarein. In this study, we developed a sequential self-assembly enzyme reactor involving two enzymes in the baicalein pathway with a pair of protein-peptide interactions in E. coli. These domains enabled us to optimize the stoichiometry of two baicalein biosynthetic enzymes recruited to be an enzymes complex. This strategy reduces the accumulation of intermediates and removes the pathway bottleneck. With this strategy, we successfully promoted the titer of baicalein by 6.6-fold (from 21.6 to 143.5 mg/L) and that of scutellarein by 1.4-fold (from 84.3 to 120.4 mg/L) in a flask fermentation, respectively. Furthermore, we first achieved the de novo biosynthesis of baicalein directly from glucose, and the strain was capable of producing 214.1 mg/L baicalein by fed-batch fermentation. This work provides novel insights for future optimization and large-scale fermentation of baicalein and scutellarein.
Assuntos
Apigenina/biossíntese , Reatores Biológicos , Medicamentos de Ervas Chinesas/metabolismo , Escherichia coli/metabolismo , Flavanonas/biossíntese , Engenharia Metabólica/métodos , Extratos Vegetais/biossíntese , Técnicas de Cultura Celular por Lotes/métodos , Escherichia coli/genética , Fermentação , Glucose/metabolismo , Malonil Coenzima A/metabolismo , Microrganismos Geneticamente Modificados , Domínios PDZ , Fenilalanina Amônia-Liase/química , Fenilalanina Amônia-Liase/metabolismo , Scutellaria baicalensis , Sirolimo/metabolismoRESUMO
Recombinant viruses expressing fluorescent or luminescent reporter proteins are used to quantitate and visualize viral replication and transmission. Here, we used a split NanoLuc luciferase (NLuc) system comprising large LgBiT and small HiBiT peptide fragments to generate stable reporter rotaviruses (RVs). Reporter RVs expressing NSP1-HiBiT fusion protein were generated by placing an 11 amino acid HiBiT peptide tag at the C-terminus of the intact simian RV NSP1 open reading frame or truncated human RV NSP1 open reading frame. Virus-infected cell lysates exhibited NLuc activity that paralleled virus replication. The antiviral activity of neutralizing antibodies and antiviral reagents against the recombinant HiBiT reporter viruses were monitored by measuring reductions in NLuc expression. These findings demonstrate that the HiBiT reporter RV systems are powerful tools for studying the viral life cycle and pathogenesis, and a robust platform for developing novel antiviral drugs.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Luciferases/genética , Peptídeos/genética , Rotavirus/genética , Animais , Antivirais/farmacologia , Cricetinae , Humanos , Camundongos , Microrganismos Geneticamente Modificados , Testes de Neutralização , Ribavirina/farmacologia , Rotavirus/fisiologia , Infecções por Rotavirus/tratamento farmacológico , Infecções por Rotavirus/virologia , Proteínas não Estruturais Virais/genética , Replicação Viral/genéticaRESUMO
Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that encode pectinolytic enzymes. Pectate lyases that cleave the α-1,4-galacturonosidic linkage of pectin are widely used in industries such as papermaking and fruit softening. However, there are few reports on pectate lyases with good thermostability. Here, two pectate lyases (CbPL3 and CbPL9) from a hyperthermophilic bacterium, Caldicellulosiruptor bescii, belonging to family 3 and family 9 polysaccharide lyases, respectively, were investigated. The biochemical properties of the two CbPLs were shown to be similar under optimized conditions of 80°C to 85°C and pH 8 to 9. However, the degradation products from pectin and polygalacturonic acids (pGAs) were different. A family 66 carbohydrate-binding module (CbCBM66) located in the N terminus of the two CbPLs shares 100% amino acid identity. A CbCBM66-truncated mutant of CbPL9 showed lower activities than the wild type, whereas CbPL3 with a CbCBM66 knockout portion was reported to have enhanced activities, thereby revealing the different effect of CbCBM66. Prediction by the I-TASSER server revealed that CbCBM66 is structurally close to BsCBM66 from Bacillus subtilis; however, the COFACTOR and COACH programs indicated that the substrate-binding sites between CbCBM66 and BsCBM66 are different. Furthermore, a substrate-binding assay indicated that the catalytic domains in the two CbPLs had strong affinities for pectate-related substrates, but CbCBM66 showed a weak interaction with a number of lignocellulosic carbohydrates. Finally, scanning electron microscopy (SEM) analysis and a total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two CbPLs are impressive sources for the degradation of plant biomass.IMPORTANCE Thermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium, Caldicellulosiruptor bescii The two enzymes share a high optimum temperature, a low optimum pH, and good thermostability at the evaluated temperature. A family 66 carbohydrate-binding module (CbCBM66) was identified in the two CbPLs, sharing 100% amino acid identity. The deletion of CbCBM66 dramatically decreased the activity of CbPL9 but increased the activity and thermostability of CbPL3, suggesting different roles of CbCBM66 in the two enzymes. Moreover, the degradation products of the two CbPLs were different. These results revealed that these enzymes could represent potential pectate lyases for applications in the paper and textile industries.
Assuntos
Proteínas de Bactérias/genética , Firmicutes/genética , Pectinas/metabolismo , Polissacarídeo-Liases/genética , Proteínas de Bactérias/metabolismo , Biomassa , Caldicellulosiruptor , Escherichia coli/enzimologia , Escherichia coli/genética , Firmicutes/enzimologia , Microrganismos Geneticamente Modificados/enzimologia , Microrganismos Geneticamente Modificados/genética , Polissacarídeo-Liases/metabolismoRESUMO
The root-knot nematode Meloidogyne chitwoodi is a pest that affects potato production in the Pacific Northwest of the United States. Here, to develop new strategies against M. chitwoodi infection of potato, we engineered Bacillus subtilis to secrete the plant-defence elicitor peptide StPep1. Pre-treatment of potato roots with the bacteria secreting StPep1 substantially reduced root galling, indicating that a bacterial secretion of a plant elicitor is an effective strategy for plant protection.
Assuntos
Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Solanum tuberosum/imunologia , Tylenchoidea/fisiologia , Animais , Bacillus subtilis/genética , Microrganismos Geneticamente Modificados/genética , Doenças das Plantas/parasitologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/microbiologia , Solanum tuberosum/genética , Solanum tuberosum/microbiologia , Solanum tuberosum/parasitologiaRESUMO
Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiaeBIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiaeIMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.
Assuntos
Biotina/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Ascomicetos/enzimologia , Ascomicetos/genética , Engenharia Metabólica , Microrganismos Geneticamente Modificados/enzimologia , Microrganismos Geneticamente Modificados/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Leveduras/enzimologia , Leveduras/genéticaRESUMO
BACKGROUND: Acridone alkaloids are heterocyclic compounds that exhibit a broad-range of pharmaceutical and chemotherapeutic activities, including anticancer, antiviral, anti-inflammatory, antimalarial, and antimicrobial effects. Certain plant species such as Citrus microcarpa, Ruta graveolens, and Toddaliopsis bremekampii synthesize acridone alkaloids from anthranilate and malonyl-CoA. RESULTS: We synthesized two acridones in Escherichia coli. Acridone synthase (ACS) and anthraniloyl-CoA ligase genes were transformed into E. coli, and the synthesis of acridone was examined. To increase the levels of endogenous anthranilate, we tested several constructs expressing proteins involved in the shikimate pathway and selected the best construct. To boost the supply of malonyl-CoA, genes coding for acetyl-coenzyme A carboxylase (ACC) from Photorhabdus luminescens were overexpressed in E. coli. For the synthesis of 1,3-dihydroxy-10-methylacridone, we utilized an N-methyltransferase gene (NMT) to supply N-methylanthranilate and a new N-methylanthraniloyl-CoA ligase. After selecting the best combination of genes, approximately 17.3 mg/L of 1,3-dihydroxy-9(10H)-acridone (DHA) and 26.0 mg/L of 1,3-dihydroxy-10-methylacridone (NMA) were synthesized. CONCLUSIONS: Two bioactive acridone derivatives were synthesized by expressing type III plant polyketide synthases and other genes in E. coli, which increased the supplement of substrates. This study showed that is possible to synthesize diverse polyketides in E. coli using plant polyketide synthases.
Assuntos
Acridonas/metabolismo , Escherichia coli , Aciltransferases/genética , Proteínas de Bactérias/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Microrganismos Geneticamente Modificados/metabolismo , Photorhabdus/enzimologia , Proteínas de Plantas/genética , Policetídeo Sintases/genética , Proteínas Recombinantes/genéticaRESUMO
The mevalonate pathway is a well-known metabolic route that provides biosynthetic precursors for myriad isoprenoids. An unexpected variety of the pathway has been discovered from recent studies on microorganisms, mainly on archaea. The most recently discovered example, called the "archaeal" mevalonate pathway, is a modified version of the canonical eukaryotic mevalonate pathway and was elucidated in our previous study using the hyperthermophilic archaeon Aeropyrum pernix This pathway comprises four known enzymes that can produce mevalonate 5-phosphate from acetyl coenzyme A, two recently discovered enzymes designated phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, and two more known enzymes, i.e., isopentenyl phosphate kinase and isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase. To show its wide distribution in archaea and to confirm if its enzyme configuration is identical among species, the putative genes of a lower portion of the pathway-from mevalonate to isopentenyl pyrophosphate-were isolated from the methanogenic archaeon Methanosarcina mazei, which is taxonomically distant from A. pernix, and were introduced into an engineered Escherichia coli strain that produces lycopene, a red carotenoid pigment. Lycopene production, as a measure of isoprenoid productivity, was enhanced when the cells were grown semianaerobically with the supplementation of mevalonolactone, which demonstrates that the archaeal pathway can function in bacterial cells to convert mevalonate into isopentenyl pyrophosphate. Gene deletion and complementation analysis using the carotenogenic E. coli strain suggests that both phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase from M. mazei are required for the enhancement of lycopene production.IMPORTANCE Two enzymes that have recently been identified from the hyperthermophilic archaeon A. pernix as components of the archaeal mevalonate pathway do not require ATP for their reactions. This pathway, therefore, might consume less energy than other mevalonate pathways to produce precursors for isoprenoids. Thus, the pathway might be applicable to metabolic engineering and production of valuable isoprenoids that have application as pharmaceuticals. The archaeal mevalonate pathway was successfully reconstructed in E. coli cells by introducing several genes from the methanogenic or hyperthermophilic archaeon, which demonstrated that the pathway requires the same components even in distantly related archaeal species and can function in bacterial cells.
Assuntos
Escherichia coli/metabolismo , Methanosarcina/metabolismo , Ácido Mevalônico/metabolismo , Escherichia coli/genética , Redes e Vias Metabólicas , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismoRESUMO
Recombinant viral vaccines expressing antigens of pathogenic microbes (e.g., HIV, Ebola virus, and malaria) have been designed to overcome the insufficient immune responses induced by the conventional vaccines. Our knowledge of and clinical experience with the new recombinant viral vaccines are insufficient, and a clear regulatory pathway is needed for the further development and evaluation of recombinant viral vaccines. In 2018, the research group supported by the Ministry of Health, Labour and Welfare, Japan (MHLW) published a concept paper to address the development of recombinant viral vaccines against infectious diseases. Herein we summarize the concept paper-which explains the Japanese regulatory concerns about recombinant viral vaccines-and provide a focus of discussion about the development of recombinant viral vaccines.
Assuntos
Controle de Medicamentos e Entorpecentes/legislação & jurisprudência , Vacinas Sintéticas/normas , Vacinas Virais/normas , Animais , Anticoncepcionais Masculinos/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Hospedeiro Imunocomprometido , Japão , Microrganismos Geneticamente Modificados , Controle de Qualidade , Distribuição Tecidual , Vacinas Sintéticas/farmacologia , Vacinas Virais/farmacocinética , Replicação Viral/fisiologia , Eliminação de Partículas ViraisRESUMO
It has been challenging to produce ex vivo models of the inclusion pathologies that are hallmark pathologies of many neurodegenerative diseases. Using three-dimensional mouse brain slice cultures (BSCs), we have developed a paradigm that rapidly and robustly recapitulates mature neurofibrillary inclusion and Lewy body formation found in Alzheimer's and Parkinson's disease, respectively. This was achieved by transducing the BSCs with recombinant adeno-associated viruses (rAAVs) that express α-synuclein or variants of tau. Notably, the tauopathy BSC model enables screening of small molecule therapeutics and tracking of neurodegeneration. More generally, the rAAV BSC "toolkit" enables efficient transduction and transgene expression from neurons, microglia, astrocytes, and oligodendrocytes, alone or in combination, with transgene expression lasting for many months. These rAAV-based BSC models provide a cost-effective and facile alternative to in vivo studies, and in the future can become a widely adopted methodology to explore physiological and pathological mechanisms related to brain function and dysfunction.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Dependovirus/genética , Doença de Parkinson/patologia , Doença de Alzheimer/virologia , Animais , Encéfalo/metabolismo , Encéfalo/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Expressão Gênica , Humanos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Microrganismos Geneticamente Modificados , Mutação , Neurônios/patologia , Técnicas de Cultura de Órgãos , Doença de Parkinson/virologia , Transdução Genética , Transgenes , alfa-Sinucleína/genética , Proteínas tau/genéticaRESUMO
Cadmium (Cd) is a major heavy metal pollutant, and Cd toxicity is a serious cause of abiotic stress in the environment. Plants protect themselves against Cd stress through a variety of pathways. In a recent study, we found that mitochondrial pyruvate carriers (MPCs) are involved in Cd tolerance in Arabidopsis (Arabidopsis thaliana). Following the identification of MPCs in yeast (Saccharomyces cerevisiae) in 2012, most studies have focused on the function of MPCs in animals, as a possible approach to reduce the risk of cancer developing. The results of this study show that AtMPC protein complexes are required for Cd tolerance and prevention of Cd accumulation in Arabidopsis. AtMPC complexes are composed of two elements, AtMPC1 and AtMPC2 (AtNRGA1 or AtMPC3). When the formation of AtMPCs was interrupted by the loss of AtMPC1, glutamate could supplement the synthesis of acetyl-coenzyme A and sustain the TCA cycle. With the up-regulation of glutathione synthesis following exposure to Cd stress, the supplementary pathway could not efficiently drive the tricarboxylic acid cycle without AtMPC. The ATP content decreased concomitantly with the deletion of tricarboxylic acid activity, which led to Cd accumulation in Arabidopsis. More importantly, ScMPCs were also required for Cd tolerance in yeast. Our results suggest that the mechanism of Cd tolerance may be similar in other species.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Cádmio/toxicidade , Glutationa/biossíntese , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte de Ânions/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Cádmio/farmacocinética , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/genética , Ácido Glutâmico/metabolismo , Proteínas de Membrana/genética , Microrganismos Geneticamente Modificados , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/genética , Transportadores de Ácidos Monocarboxílicos/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plantas Geneticamente Modificadas , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico/efeitos dos fármacos , Nicotiana/genéticaRESUMO
BACKGROUND: Electrochemical energy is a key factor of biosynthesis, and is necessary for the reduction or assimilation of substrates such as CO2. Previous microbial electrosynthesis (MES) research mainly utilized naturally electroactive microbes to generate non-specific products. RESULTS: In this research, an electroactive succinate-producing cell factory was engineered in E. coli T110(pMtrABC, pFccA-CymA) by expressing mtrABC, fccA and cymA from Shewanella oneidensis MR-1, which can utilize electricity to reduce fumarate. The electroactive T110 strain was further improved by incorporating a carbon concentration mechanism (CCM). This strain was fermented in an MES system with neutral red as the electron carrier and supplemented with HCO3+, which produced a succinate yield of 1.10 mol/mol glucose-a 1.6-fold improvement over the parent strain T110. CONCLUSIONS: The strain T110(pMtrABC, pFccA-CymA, pBTCA) is to our best knowledge the first electroactive microbial cell factory engineered to directly utilize electricity for the production of a specific product. Due to the versatility of the E. coli platform, this pioneering research opens the possibility of engineering various other cell factories to utilize electricity for bioproduction.
Assuntos
Dióxido de Carbono/metabolismo , Técnicas Eletroquímicas , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Ácido Succínico/metabolismo , Reatores Biológicos , Ciclo do Carbono , Microbiologia Industrial , Engenharia Metabólica , Microrganismos Geneticamente ModificadosRESUMO
Allergic poly-sensitization affects a large number of allergic patients and poses a great challenge for their treatment. In this study we evaluated the effects of the probiotic Escherichia coli Nissle 1917 (EcN) expressing a birch and grass pollen allergen chimera 'Bet v 1, Phl p 1 and Phl p 5' (EcN-Chim) on allergy prevention after oral or intranasal application in poly-sensitized mice. In contrast to oral application, intranasal pretreatment with EcN-Chim prior to poly-sensitization led to a significant reduction of lung inflammation (eosinophils, IL-5, and IL-13 in bronchoalveolar lavage) along with suppressed levels of allergen-specific serum IgE. The suppression was associated with increased levels of allergen-specific IgA in lungs and serum IgG2a along with increased Foxp3, TGF-ß, and IL-10 mRNA in bronchial lymph nodes. In vitro EcN induced high levels of IL-10 and IL-6 in both lung and intestinal epithelial cells. Importantly, using in vivo imaging techniques we demonstrated that intranasally applied EcN do not permanently colonize nose, lung, and gut and this strain might therefore be a safe delivery vector against allergy in humans. In conclusion, our data show that intranasal application of recombinant EcN expressing a multiallergen chimera presents a novel and promising treatment strategy for prevention of allergic poly-sensitization.
Assuntos
Escherichia coli/genética , Vetores Genéticos/genética , Hipersensibilidade/imunologia , Mucosa/fisiologia , Alérgenos/genética , Alérgenos/imunologia , Animais , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Betula/imunologia , Reações Cruzadas , Citocinas/genética , Citocinas/metabolismo , Feminino , Humanos , Imunização , Imunoglobulina E/sangue , Camundongos , Camundongos Endogâmicos BALB C , Microrganismos Geneticamente Modificados , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Poaceae/imunologia , Pólen/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , VacinaçãoRESUMO
Usnic acid, a lichen secondary metabolite produced by a whole number of lichens, has attracted the interest of researchers owing to its broad range of biological activity, including antiviral, antibiotic, anticancer properties, and it possessing a certain toxicity. The synthesis of new usnic acid derivatives and the investigation of their biological activity may lead to the discovery of compounds with better pharmacological and toxicity profiles. In this context, a series of new usnic acid derivatives comprising a terpenoid moiety were synthesized, and their ability to inhibit the catalytic activity of the human DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 was investigated. The most potent compounds (15A, 15B, 15G: , and 16A, 16B, 16G: ) had IC50 values in the range of 0.33â-â2.7 µM. The inhibitory properties were mainly dependent on the flexibility and length of the terpenoid moiety, but not strongly dependent on the configuration of the asymmetric centers. The synthesized derivatives showed low cytotoxicity against human cell lines in an MTT assay. They could be used as a basis for the development of more effective anticancer therapies when combined with topoisomerase 1 inhibitors.
Assuntos
Benzofuranos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/efeitos dos fármacos , Benzofuranos/síntese química , Benzofuranos/química , Linhagem Celular Tumoral/efeitos dos fármacos , Escherichia coli , Células HEK293/efeitos dos fármacos , Humanos , Células MCF-7/efeitos dos fármacos , Microrganismos Geneticamente Modificados , Simulação de Acoplamento Molecular , Inibidores de Fosfodiesterase/químicaRESUMO
Flavonols are a flavonoid subfamily widely distributed in plants, including several ones of great importance in human and animal diet (apple, tomato, broccoli, onion, beans, tea). These polyphenolic nutraceuticals exert potent antimicrobial (membrane potential disruptors), antioxidant (free-radical scavengers), pharmacokinetic (CYP450 modulators), anti-inflammatory (lipoxygenase inhibitors), antiangiogenic (VEGF inhibitors) and antitumor (cyclin inhibitors) activities. Biotechnological production of these nutraceuticals, for example via heterologous biosynthesis in industrial actinomycetes, is favored since in plants these polyphenols appear as inactive glycosylated derivatives, in low concentrations or as part of complex mixtures with other polyphenolic compounds. In this work, we describe the de novo biosynthesis of three important flavonols, myricetin, kaempferol and quercetin, in the industrially relevant actinomycetes Streptomyces coelicolor and S. albus. De novo biosynthesis of kaempferol, myricetin and quercetin in actinomycetes has not been described before.
Assuntos
Suplementos Nutricionais , Flavonoides , Quempferóis , Microrganismos Geneticamente Modificados , Quercetina , Streptomyces coelicolor , Flavonoides/biossíntese , Flavonoides/genética , Quempferóis/biossíntese , Quempferóis/genética , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Quercetina/biossíntese , Quercetina/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismoRESUMO
In previous work, a series of bioactive natural products had been isolated from the plant endophytic Streptomyces sp. CS, which was isolated from Maytenus hookeri. To mine new active metabolites, we describe introducing an alien carbamoyltransferase (asm21) gene into the strain CS by conjugal transfer. As a result, three recombinatorial mutants named CS/asm21-1, CS/asm21-2 and CS/asm21-4 were successfully constructed. Three mutants and wild type CS were cultured on solid medium, and the extracts were detected and analyzed by liquid chromatography-mass spectrometry (LC-MS). The LC-MS profiles showed several unknown peaks that were present in the spectra of extracts of the CS/asm21-4 cultured on oatmeal solid medium. Then, three new naphthomycins O-Q (1-3), a new macrolide hookerolide (4) as well as nine known compounds were obtained from the solid cultured medium. Their structures were identified by spectra data. These new compounds showed moderate antimicrobial activities.