Base de dados : MEDLINE
Pesquisa : E05.104.500 [Categoria DeCS]
Referências encontradas : 1507 [refinar]
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  1 / 1507 MEDLINE  
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[PMID]:29233696
[Au] Autor:Wang Q; Pu Y; Yang D; Yin X; He Z; Yang Y; Yang Y
[Ad] Endereço:Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China; Plant Germplasm and Genomics Center, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China; Institute of Tibetan Plateau Resear
[Ti] Título:Molecular cloning and characterization of the glutathione reductase gene from Stipa purpurea.
[So] Source:Biochem Biophys Res Commun;495(2):1851-1857, 2018 01 08.
[Is] ISSN:1090-2104
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Reactive oxygen species (ROS) are a key factor in abiotic stresses; excess ROS is harmful to plants. Glutathione reductase (GR) plays an important role in scavenging ROS in plants. Here, a GR gene, named SpGR, was cloned from Stipa purpurea and characterized. The full-length open reading frame was 1497 bp, encoding 498 amino acids. Subcellular localization analysis indicated that SpGR was localized to both the plasma membrane and nucleus. The expression of SpGR was induced by cold, salt, and drought stresses. Functional analysis indicated that ectopic expression of SpGR in Arabidopsis thaliana resulted in greater tolerance to salt stress than that of wild-type plants, but no difference under cold or drought treatments. The results of GR activity and GSSG and GSH content analyses suggested that, under salt stress, transgenic plants produced more GR to reduce GSSG to GSH for scavenging ROS than wild-type plants. Therefore, SpGR may be a candidate gene for plants to resist abiotic stress.
[Mh] Termos MeSH primário: Arabidopsis/fisiologia
Glutationa Redutase/química
Glutationa Redutase/metabolismo
Plantas Geneticamente Modificadas/fisiologia
Poaceae/enzimologia
Espécies Reativas de Oxigênio/metabolismo
Plantas Tolerantes a Sal/genética
[Mh] Termos MeSH secundário: Clonagem Molecular/métodos
Ativação Enzimática
Melhoramento Genético/métodos
Glutationa Redutase/genética
Poaceae/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Reactive Oxygen Species); EC 1.8.1.7 (Glutathione Reductase)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180214
[Lr] Data última revisão:
180214
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171214
[St] Status:MEDLINE


  2 / 1507 MEDLINE  
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[PMID]:28467058
[Au] Autor:Bouzon M; Perret A; Loreau O; Delmas V; Perchat N; Weissenbach J; Taran F; Marlière P
[Ad] Endereço:CEA, Genoscope , 2 rue Gaston Crémieux, 91000 Evry, France.
[Ti] Título:A Synthetic Alternative to Canonical One-Carbon Metabolism.
[So] Source:ACS Synth Biol;6(8):1520-1533, 2017 Aug 18.
[Is] ISSN:2161-5063
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:One-carbon metabolism is an ubiquitous metabolic pathway that encompasses the reactions transferring formyl-, hydroxymethyl- and methyl-groups bound to tetrahydrofolate for the synthesis of purine nucleotides, thymidylate, methionine and dehydropantoate, the precursor of coenzyme A. An alternative cyclic pathway was designed that substitutes 4-hydroxy-2-oxobutanoic acid (HOB), a compound absent from known metabolism, for the amino acids serine and glycine as one-carbon donors. It involves two novel reactions, the transamination of l-homoserine and the transfer of a one-carbon unit from HOB to tetrahydrofolate releasing pyruvate as coproduct. Since canonical reactions regenerate l-homoserine from pyruvate by carboxylation and subsequent reduction, every one-carbon moiety made available for anabolic reactions originates from CO . The HOB-dependent pathway was established in an Escherichia coli auxotroph selected for prototrophy using long-term cultivation protocols. Genetic, metabolic and biochemical evidence support the emergence of a functional HOB-dependent one-carbon pathway achieved with the recruitment of the two enzymes l-homoserine transaminase and HOB-hydroxymethyltransferase and of HOB as an essential metabolic intermediate. Escherichia coli biochemical reprogramming was achieved by minimally altering canonical metabolism and leveraging on natural selection mechanisms, thereby launching the resulting strain on an evolutionary trajectory diverging from all known extant species.
[Mh] Termos MeSH primário: Acetoacetatos/metabolismo
Carbono/metabolismo
Proteínas de Escherichia coli/metabolismo
Escherichia coli/metabolismo
Melhoramento Genético/métodos
Engenharia Metabólica/métodos
Redes e Vias Metabólicas/genética
[Mh] Termos MeSH secundário: Escherichia coli/genética
Proteínas de Escherichia coli/genética
Glicina/genética
Glicina/metabolismo
Ácido Pirúvico/metabolismo
Serina/genética
Serina/metabolismo
Biologia Sintética/métodos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Acetoacetates); 0 (Escherichia coli Proteins); 452VLY9402 (Serine); 4ZI204Y1MC (acetoacetic acid); 7440-44-0 (Carbon); 8558G7RUTR (Pyruvic Acid); TE7660XO1C (Glycine)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180201
[Lr] Data última revisão:
180201
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170504
[St] Status:MEDLINE
[do] DOI:10.1021/acssynbio.7b00029


  3 / 1507 MEDLINE  
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[PMID]:29175328
[Au] Autor:Phanthanawiboon S; Pambudi S; Omokoko MD; Hanabara K; A-Nuegoonpipat A; Kamitani W; Ikuta K; Kurosu T
[Ad] Endereço:Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
[Ti] Título:Construction of a high-yield dengue virus by replacing nonstructural proteins 3-4B without increasing virulence.
[So] Source:Biochem Biophys Res Commun;495(1):1221-1226, 2018 01 01.
[Is] ISSN:1090-2104
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Producing virus at high yield is critically important for development of whole virion inactivated vaccines or live attenuated vaccines. Most dengue virus (DENV) clinical isolates, however, replicate at low levels in cultured cells, which limits their use for vaccine development. The present study examined differences between low-replicating DENV clinical isolates and high-replicating laboratory strains with the aim of engineering high-yield DENV clinical isolates. Construction of a series of recombinant chimeric viruses derived from a high-replicating laboratory DENV type 4 (DENV-4) H241 strain and a clinical isolate revealed that the NS3-NS4B region of H241 conferred a replication advantage in cultured cells. Furthermore, northern blot analysis revealed that this advantage was due to more efficient synthesis of viral RNA. Importantly, replacement of the NS3-NS4B region of H241 did not increase virulence in mice, suggesting that viral production can be increased safely. This study provided information that will facilitate engineering of safe and high-yield viruses that can be used for vaccine development.
[Mh] Termos MeSH primário: Técnicas de Cultura Celular por Lotes/métodos
Vírus da Dengue/crescimento & desenvolvimento
Vírus da Dengue/genética
Melhoramento Genético/métodos
Carga Viral/genética
Proteínas não Estruturais Virais/metabolismo
Virulência/fisiologia
[Mh] Termos MeSH secundário: Recombinação Genética/genética
Carga Viral/fisiologia
Proteínas não Estruturais Virais/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Viral Nonstructural Proteins)
[Em] Mês de entrada:1712
[Cu] Atualização por classe:180105
[Lr] Data última revisão:
180105
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171128
[St] Status:MEDLINE


  4 / 1507 MEDLINE  
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[PMID]:28464284
[Au] Autor:Su H; Lin J; Wang Y; Chen Q; Wang G; Tan F
[Ad] Endereço:Chongqing Institute of Green and Interligent Technology, Chinese Academy of Science, 266, Fangzheng Avenue, Shuitu High-Tech Park, Beibei, Chongqing 400714, P. R. China.
[Ti] Título:Engineering Brevibacterium flavum for the production of renewable bioenergy: C4-C5 advanced alcohols.
[So] Source:Biotechnol Bioeng;114(9):1946-1958, 2017 09.
[Is] ISSN:1097-0290
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Biosynthesis of advanced biofuels by engineered non-natural microorganisms has been proposed to be the most promising approach for the replacement of dwindling fossil fuel resources. Brevibacterium flavum (Bf) is a model brevibacterium aerobe which lacks basic and applied research that could enable this species to produce biofuels. There are no reports regarding engineering this microorganism to produce advanced alcohols before. Here, for the first time, we developed the bacterium as a novel biosynthetic platform for advanced alcohols production via the mutagenesis and engineering to produce 2-ketoacids derived alcohols. In order to enhance the strain's capability of producing advanced alcohols, we preferentially improved intrinsic metabolism ability of the strain to obtain improved expression host (IEH) via generating mutagenesis libraries by whole cell mutagenesis (WCM). The IEH was determined via screening out the mutant strain with the highest production of branched-chain organic acids (BCOA) using high throughput screening method.. Subsequently, a novel vector system for Bf was established, and the corresponding biosynthetic pathway of directing carbon flux into the target advanced alcohols was recruited to make the bacterium possess the capability of producing advanced alcohols and further enhance the production using the IEH. Specifically, we generated bioengineered strains that were able to synthesize up to the highest 5362 and 4976 mg/L isobutanol, 1945 and 1747 mg/L 2-methyl-1-butanol (2 MB), and 785.34 and 781 mg/L 3-methyl-1-butanol (3 MB) from pure glucose and duckweed substrates, respectively. Our findings confirmed the feasibility and potential of using Bf as a novel biosynthetic platform to generate advanced biofuels with glucose and inexpensive renewable feedstock-duckweed as a fermentation substrate. Biotechnol. Bioeng. 2017;114: 1946-1958. © 2017 Wiley Periodicals, Inc.
[Mh] Termos MeSH primário: Álcoois/metabolismo
Biocombustíveis/microbiologia
Vias Biossintéticas/fisiologia
Brevibacterium flavum/fisiologia
Cetoácidos/metabolismo
Engenharia Metabólica/métodos
Energia Renovável
[Mh] Termos MeSH secundário: Álcoois/isolamento & purificação
Melhoramento Genético/métodos
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Alcohols); 0 (Biofuels); 0 (Keto Acids)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171226
[Lr] Data última revisão:
171226
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170503
[St] Status:MEDLINE
[do] DOI:10.1002/bit.26324


  5 / 1507 MEDLINE  
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[PMID]:28464256
[Au] Autor:Shin HY; Nijland JG; de Waal PP; Driessen AJM
[Ad] Endereço:Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials and Kluyver Centre for Genomics of Industrial Fermentation, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
[Ti] Título:The amino-terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid.
[So] Source:Biotechnol Bioeng;114(9):1937-1945, 2017 09.
[Is] ISSN:1097-0290
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N-terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% d-glucose and 4% d-xylose. Mutation of N361 of Hxt11/2 into threonine reversed the specificity for d-xylose over d-glucose with high d-xylose transport rates. This mutant supported efficient sugar fermentation of both d-glucose and d-xylose at industrially relevant sugar concentrations even in the presence of the inhibitor acetic acid which is normally present in lignocellulosic hydrolysates. Biotechnol. Bioeng. 2017;114: 1937-1945. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
[Mh] Termos MeSH primário: Ácido Acético/metabolismo
Membrana Celular/metabolismo
Melhoramento Genético/métodos
Proteínas Facilitadoras de Transporte de Glucose/metabolismo
Proteínas de Transporte de Monossacarídeos/metabolismo
Proteínas de Saccharomyces cerevisiae/metabolismo
Saccharomyces cerevisiae/fisiologia
Xilose/metabolismo
[Mh] Termos MeSH secundário: Aminoácidos/genética
Glucose
Proteínas Facilitadoras de Transporte de Glucose/genética
Engenharia Metabólica/métodos
Proteínas de Transporte de Monossacarídeos/genética
Proteínas de Saccharomyces cerevisiae/genética
Relação Estrutura-Atividade
Xilose/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Amino Acids); 0 (Glucose Transport Proteins, Facilitative); 0 (HXT11 protein, S cerevisiae); 0 (HXT2 protein, S cerevisiae); 0 (Monosaccharide Transport Proteins); 0 (Saccharomyces cerevisiae Proteins); A1TA934AKO (Xylose); IY9XDZ35W2 (Glucose); Q40Q9N063P (Acetic Acid)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171226
[Lr] Data última revisão:
171226
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170503
[St] Status:MEDLINE
[do] DOI:10.1002/bit.26322


  6 / 1507 MEDLINE  
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[PMID]:28926680
[Au] Autor:Ahn JH; Bang J; Kim WJ; Lee SY
[Ad] Endereço:Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus program), BioProcess Engineering Research Center, Institute for the BioCentury, Yuseong-gu, Daejeon, Republic of Korea.
[Ti] Título:Formic acid as a secondary substrate for succinic acid production by metabolically engineered Mannheimia succiniciproducens.
[So] Source:Biotechnol Bioeng;114(12):2837-2847, 2017 Dec.
[Is] ISSN:1097-0290
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:There has been much effort exerted to reduce one carbon (C1) gas emission to address climate change. As one promising way to more conveniently utilize C1 gas, several technologies have been developed to convert C1 gas into useful chemicals such as formic acid (FA). In this study, systems metabolic engineering was utilized to engineer Mannheimia succiniciproducens to efficiently utilize FA. C isotope analysis of M. succiniciproducens showed that FA could be utilized through formate dehydrogenase (FDH) reaction and/or the reverse reaction of pyruvate formate lyase (PFL). However, the naturally favored forward reaction of PFL was found to lower the SA yield from FA. In addition, FA assimilation via FDH was found to be more efficient than the reverse reaction of PFL. Thus, the M. succiniciproducens LPK7 strain, which lacks in pfl, ldh, pta, and ack genes, was selected as a base strain. In silico metabolic analysis confirmed that utilization of FA would be beneficial for the enhanced production of SA and suggested FDH as an amplification target. To find a suitable FDH, four different FDHs from M. succiniciproducens, Methylobacterium extorquens, and Candida boidinii were amplified in LPK7 strain to enhance FA assimilation. High-inoculum density cultivation using C labeled sodium formate was performed to evaluate FA assimilation efficiency. Fed-batch fermentations of the LPK7 (pMS3-fdh2 meq) strain was carried out using glucose, sucrose, or glycerol as a primary carbon source and FA as a secondary carbon source. As a result, this strain produced 76.11 g/L SA with the yield and productivity of 1.28 mol/mol and 4.08 g/L/h, respectively, using sucrose and FA as dual carbon sources. The strategy employed here will be similarly applicable in developing microorganisms to utilize FA and to produce valuable chemicals and materials from FA.
[Mh] Termos MeSH primário: Formiato Desidrogenases/genética
Formiatos/metabolismo
Melhoramento Genético/métodos
Mannheimia/fisiologia
Engenharia Metabólica/métodos
Análise do Fluxo Metabólico/métodos
Ácido Succínico/metabolismo
[Mh] Termos MeSH secundário: Simulação por Computador
Mannheimia/classificação
Modelos Biológicos
Especificidade da Espécie
Especificidade por Substrato
Ácido Succínico/isolamento & purificação
Regulação para Cima/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Formates); 0YIW783RG1 (formic acid); AB6MNQ6J6L (Succinic Acid); EC 1.2.1.2 (Formate Dehydrogenases)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171113
[Lr] Data última revisão:
171113
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170920
[St] Status:MEDLINE
[do] DOI:10.1002/bit.26435


  7 / 1507 MEDLINE  
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[PMID]:28926673
[Au] Autor:Chung CY; Wang Q; Yang S; Ponce SA; Kirsch BJ; Zhang H; Betenbaugh MJ
[Ad] Endereço:Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland.
[Ti] Título:Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells.
[So] Source:Biotechnol Bioeng;114(12):2848-2856, 2017 Dec.
[Is] ISSN:1097-0290
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities. In this study, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms standards for specific biomedical and biotechnology applications.
[Mh] Termos MeSH primário: Anticorpos Monoclonais/biossíntese
Anticorpos Monoclonais/genética
Técnicas de Química Combinatória/métodos
Galactose/metabolismo
Imunoglobulina G/metabolismo
Engenharia de Proteínas/métodos
[Mh] Termos MeSH secundário: Animais
Células CHO
Mapeamento Cromossômico/métodos
Cricetulus
Melhoramento Genético/métodos
Glicosilação
Imunoglobulina G/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antibodies, Monoclonal); 0 (Immunoglobulin G); X2RN3Q8DNE (Galactose)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171113
[Lr] Data última revisão:
171113
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170920
[St] Status:MEDLINE
[do] DOI:10.1002/bit.26375


  8 / 1507 MEDLINE  
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[PMID]:28842992
[Au] Autor:Kinfu BM; Jahnke M; Janus M; Besirlioglu V; Roggenbuck M; Meurer R; Vojcic L; Borchert M; Schwaneberg U; Chow J; Streit WR
[Ad] Endereço:Microbiology and Biotechnology, Biocenter Klein Flottbek, University of Hamburg, Ohnhorststr, Hamburg, Germany.
[Ti] Título:Recombinant RNA Polymerase from Geobacillus sp. GHH01 as tool for rapid generation of metagenomic RNAs using in vitro technologies.
[So] Source:Biotechnol Bioeng;114(12):2739-2752, 2017 Dec.
[Is] ISSN:1097-0290
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The exciting promises of functional metagenomics for the efficient discovery of novel biomolecules from nature are often hindered by factors associated with expression hosts. Aiming to shift functional metagenomics to a host independent innovative system, we here report on the cloning, heterologous expression, and reconstitution of an RNA polymerase (RNAP) from the thermophilic Geobacillus sp. GHH01 and in vitro transcription thereafter. The five genes coding for RNAP subunits, a house keeping sigma factor and two transcription elongation factors were cloned and over expressed as His -tagged and/ or tag-free proteins. Purified subunits were reconstituted into a functional polymerase through either the classical method of denaturation and subsequent renaturation or through a new resource and time efficient thermo-reconstitution method which takes advantage of the subunits' temperature stability. Additionally, all subunits were cloned into a single vector system for a co-expression and in vivo reconstitution to the RNAP core enzyme. Both the core and holoenzyme form of the RNAP exhibited a robust transcription activity and were stable up to a temperature of 55°C close to their fullest activity. The Geobacillus RNAP showed a remarkable in vitro transcription profile recognizing DNA template sequences of diverse bacteria and archaea as well as metagenomic samples. Coupled with a subsequent in vitro translation step, this recombinant transcription system could allow a new, clone-free, and functional metagenomic screening approach.
[Mh] Termos MeSH primário: RNA Polimerases Dirigidas por DNA/genética
Melhoramento Genético/métodos
Geobacillus/genética
Metagenoma/genética
RNA/biossíntese
Proteínas Recombinantes/genética
[Mh] Termos MeSH secundário: Regulação Bacteriana da Expressão Gênica/genética
Regulação Enzimológica da Expressão Gênica/genética
RNA/genética
Proteínas Recombinantes/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Recombinant Proteins); 63231-63-0 (RNA); EC 2.7.7.6 (DNA-Directed RNA Polymerases)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171113
[Lr] Data última revisão:
171113
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170827
[St] Status:MEDLINE
[do] DOI:10.1002/bit.26436


  9 / 1507 MEDLINE  
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[PMID]:28842980
[Au] Autor:Browning DF; Richards KL; Peswani AR; Roobol J; Busby SJW; Robinson C
[Ad] Endereço:Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.
[Ti] Título:Escherichia coli "TatExpress" strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway.
[So] Source:Biotechnol Bioeng;114(12):2828-2836, 2017 Dec.
[Is] ISSN:1097-0290
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat-dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.
[Mh] Termos MeSH primário: Escherichia coli/genética
Escherichia coli/metabolismo
Produtos do Gene tat/genética
Melhoramento Genético/métodos
Hormônio do Crescimento/biossíntese
Periplasma/metabolismo
Via Secretória/genética
[Mh] Termos MeSH secundário: Hormônio do Crescimento/genética
Hormônio do Crescimento/isolamento & purificação
Seres Humanos
Redes e Vias Metabólicas/genética
Regiões Promotoras Genéticas/genética
Proteínas Recombinantes/biossíntese
Proteínas Recombinantes/genética
Proteínas Recombinantes/isolamento & purificação
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Gene Products, tat); 0 (Recombinant Proteins); 9002-72-6 (Growth Hormone)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171113
[Lr] Data última revisão:
171113
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170827
[St] Status:MEDLINE
[do] DOI:10.1002/bit.26434


  10 / 1507 MEDLINE  
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[PMID]:28832943
[Au] Autor:Schwartz C; Frogue K; Ramesh A; Misa J; Wheeldon I
[Ad] Endereço:Chemical and Environmental Engineering, University of California Riverside, Riverside, California.
[Ti] Título:CRISPRi repression of nonhomologous end-joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica.
[So] Source:Biotechnol Bioeng;114(12):2896-2906, 2017 Dec.
[Is] ISSN:1097-0290
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical-producing yeast Yarrowia lipolytica. By using a multiplexed sgRNA targeting strategy, we demonstrate efficient repression of eight out of nine targeted genes to enhance HR. Strains with nonhomologous end-joining repressed were shown to have increased rates of HR when transformed with a linear DNA fragment with homology to a genomic locus. With multiplexed targeting of KU70 and KU80, and enhanced repression with Mxi1 fused to deactivated Cas9 (dCas9), rates of HR as high as 90% were achieved. The developed CRISPRi system enables enhanced HR in Y. lipolytica without permanent genetic knockouts and promises to be a potent tool for other metabolic engineering, synthetic biology, and functional genomics studies.
[Mh] Termos MeSH primário: Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética
Reparo do DNA por Junção de Extremidades/genética
Edição de Genes/métodos
Melhoramento Genético/métodos
Genoma Fúngico/genética
Proteínas Recombinantes/biossíntese
Yarrowia/genética
[Mh] Termos MeSH secundário: Regulação Fúngica da Expressão Gênica/genética
Recombinação Homóloga/genética
Proteínas Recombinantes/genética
Proteínas Repressoras/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Recombinant Proteins); 0 (Repressor Proteins)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171113
[Lr] Data última revisão:
171113
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170824
[St] Status:MEDLINE
[do] DOI:10.1002/bit.26404



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