Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.026
Filtrar
1.
Biochem Biophys Res Commun ; 497(1): 25-31, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29407173

RESUMO

Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6ß-hydroxylase genes of Hyoscyamus niger (HnH6H) and S. lurida (SlH6H) were cloned and respectively overexpressed in hairy root cultures of S. lurida, to compare their effects on promoting the production of TAs, especially the high-value scopolamine. Root cultures with SlH6H/HnH6H overexpression were confirmed by PCR and real-time quantitative PCR, suggesting that the enzymatic steps defined by H6H were strongly elevated at the transcriptional level. Tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were analyzed by HPLC. Scopolamine and anisodamine contents were remarkably elevated in the root cultures overexpressing SlH6H/HnH6H, whereas that of hyoscyamine was more or less reduced, when compared with those of the control. These results also indicated that SlH6H and HnH6H promoted anisodamine production at similar levels in S. lurida root cultures. More importantly, HnH6H-overexpressing root cultures had more scopolamine in them that did SlH6H-overexpressing root cultures. This study not only provides a feasible way of overexpressing H6H to produce high-value scopolamine in engineered root cultures of S. lurida but also found that HnH6H was better than SlH6H for engineering scopolamine production.


Assuntos
Engenharia Metabólica/métodos , Oxigenases de Função Mista/genética , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas/fisiologia , Escopolamina/metabolismo , Scopolia/fisiologia , Ativação Enzimática , Estabilidade Enzimática , Melhoramento Genético/métodos , Oxigenases de Função Mista/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Escopolamina/isolamento & purificação
2.
Biochem Biophys Res Commun ; 497(1): 160-166, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29428735

RESUMO

Germin-like proteins (GLPs) are ubiquitous water-soluble glycoproteins that are located in the extracellular matrix. These proteins have been reported to play vital roles in diverse biological processes. In the present study, a GLP in soybean (Glycine max L. Merr.), GmGLP10, was characterized. Sequence analysis revealed that the GmGLP10 gene (GenBank Accession Number EU916258) encodes a 213-amino acid (aa) protein, which contains a N-terminal signal peptide at 1-22 aa and is highly homologous to the members of the GER2 subfamily. GmGLP10 was highly expressed in the leaves, but very faint in the roots. The expression of GmGLP10 was induced by methyl jasmonate (MeJA), ethylene (ET), salicylic acid (SA), oxalate acid (OA), and the infection of Sclerotinia sclerotiorum. Overexpression of GmGLP10 in transgenic tobacco significantly enhanced tolerance to OA and S. sclerotiorum infection. Moreover, higher levels of H2O2 and the upregulated expression of a set of plant defense-related genes and HR (hypersensitive response)-associated genes were detected in the transgenic plants. These results suggest that GmGLP10 functions as a positive regulator of resistance to S. sclerotiorum.


Assuntos
Ascomicetos/fisiologia , Glicoproteínas/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Plantas Geneticamente Modificadas/fisiologia , Tabaco/metabolismo , Tabaco/microbiologia , Resistência à Doença/fisiologia , Melhoramento Genético/métodos , Glicoproteínas/genética , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/genética , Regulação para Cima
3.
Biochem Biophys Res Commun ; 495(1): 686-692, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29113801

RESUMO

Although Escherichia coli has been engineered to perform N-glycosylation of recombinant proteins, an optimal glycosylating strain has not been created. By inserting a codon optimised Campylobacter oligosaccharyltransferase onto the E. coli chromosome, we created a glycoprotein platform strain, where the target glycoprotein, sugar synthesis and glycosyltransferase enzymes, can be inserted using expression vectors to produce the desired homogenous glycoform. To assess the functionality and glycoprotein producing capacity of the chromosomally based OST, a combined Western blot and parallel reaction monitoring mass spectrometry approach was applied, with absolute quantification of glycoprotein. We demonstrated that chromosomal oligosaccharyltransferase remained functional and facilitated N-glycosylation. Although the engineered strain produced less total recombinant protein, the glycosylation efficiency increased by 85%, and total glycoprotein production was enhanced by 17%.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/fisiologia , Edição de Genes/métodos , Genoma Bacteriano/genética , Glicoproteínas/biossíntese , Hexosiltransferases/genética , Proteínas de Membrana/genética , Engenharia Metabólica/métodos , Proteínas de Bactérias/metabolismo , Melhoramento Genético/métodos , Glicoproteínas/genética , Glicosilação , Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo
4.
Biochem Biophys Res Commun ; 495(2): 1851-1857, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29233696

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.


Assuntos
Arabidopsis/fisiologia , Glutationa Redutase/química , Glutationa Redutase/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Poaceae/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Plantas Tolerantes a Sal/genética , Clonagem Molecular/métodos , Ativação Enzimática , Melhoramento Genético/métodos , Glutationa Redutase/genética , Poaceae/genética
5.
Biochem Biophys Res Commun ; 495(1): 1221-1226, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29175328

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.


Assuntos
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 , Recombinação Genética/genética , Carga Viral/fisiologia , Proteínas não Estruturais Virais/genética
6.
Biotechnol Bioeng ; 114(12): 2848-2856, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28926673

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.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Técnicas de Química Combinatória/métodos , Galactose/metabolismo , Imunoglobulina G/metabolismo , Engenharia de Proteínas/métodos , Animais , Células CHO , Mapeamento Cromossômico/métodos , Cricetulus , Melhoramento Genético/métodos , Glicosilação , Imunoglobulina G/genética
7.
Biotechnol Bioeng ; 114(12): 2837-2847, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28926680

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. 13 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 13 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.


Assuntos
Formiato Desidrogenases/genética , Formiatos/metabolismo , Melhoramento Genético/métodos , Mannheimia/fisiologia , Engenharia Metabólica/métodos , Análise do Fluxo Metabólico/métodos , Ácido Succínico/metabolismo , Simulação por Computador , Mannheimia/classificação , Modelos Biológicos , Especificidade da Espécie , Especificidade por Substrato , Ácido Succínico/isolamento & purificação , Regulação para Cima/genética
8.
Biochem Biophys Res Commun ; 491(3): 642-648, 2017 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-28757414

RESUMO

MYB transcription factors are a large family of proteins involved in plant development and responses to stress. In this study, the wheat salinity-induced R2R3-MYB transcription factor TaSIM was functionally characterized, with a focus on its role in salt stress tolerance. TaSIM protein enters the nucleus and binds to the MYB-binding site II motif. Expression analysis revealed that TaSIM was induced by drought, high salinity, low temperature, and abscisic acid treatment. Overexpression of TaSIM improved salt stress tolerance in transgenic plants. Furthermore, the transcript levels of genes involved in abscisic acid (ABA)-dependent (RD22) and ABA-independent (RD29A) signaling were higher in TaSIM-overexpressing plants than in the wild type. These results suggest that TaSIM positively modulates salt stress tolerance and has potential applications in molecular breeding to enhance salt tolerance in crops.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Melhoramento Genético/métodos , Tolerância ao Sal/fisiologia , Estresse Fisiológico/fisiologia , Fatores de Transcrição/genética , Triticum/genética , Plantas Geneticamente Modificadas/fisiologia
9.
Biotechnol Bioeng ; 114(12): 2896-2906, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28832943

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.


Assuntos
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 , Regulação Fúngica da Expressão Gênica/genética , Recombinação Homóloga/genética , Proteínas Recombinantes/genética , Proteínas Repressoras/genética
10.
Biotechnol Bioeng ; 114(12): 2828-2836, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28842980

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-1 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.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Produtos do Gene tat/genética , Melhoramento Genético/métodos , Hormônio do Crescimento/biossíntese , Periplasma/metabolismo , Via Secretória/genética , Hormônio do Crescimento/genética , Hormônio do Crescimento/isolamento & purificação , Humanos , Redes e Vias Metabólicas/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
11.
Biotechnol Bioeng ; 114(12): 2739-2752, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28842992

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 His6 -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.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Melhoramento Genético/métodos , Geobacillus/genética , Metagenoma/genética , RNA/biossíntese , Proteínas Recombinantes/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , RNA/genética , Proteínas Recombinantes/metabolismo
12.
Biotechnol Bioeng ; 114(12): 2794-2806, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28782796

RESUMO

Ansamitocin P-3 (AP-3) is a maytansinoid with its most compelling antitumor activity, however, the low production titer of AP-3 greatly restricts its wide commercial application. In this work, a combinatorial approach including random mutation and metabolic engineering was conducted to enhance AP-3 biosynthesis in Actinosynnema pretiosum. First, a mutant strain M was isolated by N-methyl-N'-nitro-N-nitrosoguanidine mutation, which could produce AP-3 almost threefold that of wild type (WT) in 48 deep-well plates. Then, by overexpressing key biosynthetic genes asmUdpg and asm13-17 in the M strain, a further 60% increase of AP-3 production in 250-ml shake flasks was achieved in the engineered strain M-asmUdpg:asm13-17 compared to the M strain, and its maximum AP-3 production reached 582.7 mg/L, which is the highest as ever reported. Both the gene transcription levels and intracellular intermediate concentrations in AP-3 biosynthesis pathway were significantly increased in the M and M-asmUdpg:asm13-17 during fermentation compared to the WT. The good fermentation performance of the engineered strain was also confirmed in a lab-scale bioreactor. This work demonstrated that combination of random mutation and metabolic engineering could promote AP-3 biosynthesis and might be helpful for increasing the production of other industrially important secondary metabolites.


Assuntos
Actinobacteria/fisiologia , Vias Biossintéticas/genética , Melhoramento Genético/métodos , Maitansina/análogos & derivados , Engenharia Metabólica/métodos , Mutação/genética , Actinobacteria/classificação , Maitansina/biossíntese , Especificidade da Espécie , Regulação para Cima/genética
13.
Biotechnol Bioeng ; 114(12): 2807-2817, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28802003

RESUMO

Lactic acid bacteria (LAB) are among the most interesting organisms for industrial processes with a long history of application as food starters and biocontrol agents, and an underexploited potential for biorefineries converting biomass into high-value compounds. Lactic acid (LA), their main fermentation product, is among the most requested chemicals owing to its broad range of applications. Notably, LA polymers, that is, polylactides, have high potential as biodegradable substitutes of fossil-derived plastics. However, LA production by LAB fermentation is currently too expensive for polylactide to be cost-competitive with traditional plastics. LAB have complex nutritional requirements and cannot ferment inexpensive substrates such as cellulose. Metabolic engineering could help reduce such nutritional requirements and enable LAB to directly ferment low-cost polysaccharides. Here, we engineered a Lactococcus lactis strain which constitutively secretes a ß-glucosidase and an endoglucanase. The recombinant strain can grow on cellooligosaccharides up to at least cellooctaose and efficiently metabolizes them to L-LA in single-step fermentation. This is the first report of a LAB able to directly metabolize cellooligosaccharides longer that cellohexaose and a significant step toward cost-sustainable consolidated bioprocessing of cellulose into optically pure LA.


Assuntos
Celulose/análogos & derivados , Dextrinas/metabolismo , Melhoramento Genético/métodos , Ácido Láctico/biossíntese , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas Recombinantes/metabolismo , Recombinação Genética/genética , Celulose/genética , Celulose/metabolismo , Dextrinas/genética , Ácido Láctico/isolamento & purificação
14.
Biotechnol Bioeng ; 114(11): 2432-2444, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28688187

RESUMO

Antibodies are an important class of therapeutics and are predominantly produced in Chinese Hamster Ovary (CHO) cell lines. While this manufacturing platform is sufficiently productive to supply patient populations of currently approved therapies, it is unclear whether or not the current CHO platform can address two significant areas of need: affordable access to biologics for patients around the globe and production of unprecedented quantities needed for very large populations of patients. Novel approaches to recombinant protein production for therapeutic biologic products may be needed, and might be enabled by non-mammalian expression systems and recent advances in bioengineering. Eukaryotic microorganisms such as fungi, microalgae, and protozoa offer the potential to produce high-quality antibodies in large quantities. In this review, we lay out the current understanding of a wide range of species and evaluate based on theoretical considerations which are best poised to deliver a step change in cost of manufacturing and volumetric productivity within the next decade.Related article: http://onlinelibrary.wiley.com/doi/10.1002/bit.26383/full.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/uso terapêutico , Eucariotos/genética , Eucariotos/metabolismo , Engenharia de Proteínas/métodos , Anticorpos Monoclonais/genética , Desenho de Drogas , Eucariotos/classificação , Melhoramento Genético/métodos , Especificidade da Espécie
15.
Biotechnol Bioeng ; 114(11): 2581-2591, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28667762

RESUMO

Saccharomyces cerevisiae has limited capabilities for producing fuels and chemicals derived from acetyl-CoA, such as isoprenoids, due to a rigid flux partition toward ethanol during glucose metabolism. Despite numerous efforts, xylose fermentation by engineered yeast harboring heterologous xylose metabolic pathways was not as efficient as glucose fermentation for producing ethanol. Therefore, we hypothesized that xylose metabolism by engineered yeast might be a better fit for producing non-ethanol metabolites. We indeed found that engineered S. cerevisiae on xylose showed higher expression levels of the enzymes involved in ethanol assimilation and cytosolic acetyl-CoA synthesis than on glucose. When genetic perturbations necessary for overproducing squalene and amorphadiene were introduced into engineered S. cerevisiae capable of fermenting xylose, we observed higher titers and yields of isoprenoids under xylose than glucose conditions. Specifically, co-overexpression of a truncated HMG1 (tHMG1) and ERG10 led to substantially higher squalene accumulation under xylose than glucose conditions. In contrast to glucose utilization producing massive amounts of ethanol regardless of aeration, xylose utilization allowed much less amounts of ethanol accumulation, indicating ethanol is simultaneously re-assimilated with xylose consumption and utilized for the biosynthesis of cytosolic acetyl-CoA. In addition, xylose utilization by engineered yeast with overexpression of tHMG1, ERG10, and ADS coding for amorphadiene synthase, and the down-regulation of ERG9 resulted in enhanced amorphadiene production as compared to glucose utilization. These results suggest that the problem of the rigid flux partition toward ethanol production in yeast during the production of isoprenoids and other acetyl-CoA derived chemicals can be bypassed by using xylose instead of glucose as a carbon source. Biotechnol. Bioeng. 2017;114: 2581-2591. © 2017 Wiley Periodicals, Inc.


Assuntos
Etanol/metabolismo , Melhoramento Genético/métodos , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/fisiologia , Terpenos/metabolismo , Xilose/metabolismo , Terpenos/isolamento & purificação , Regulação para Cima/genética , Xilose/genética
16.
Biotechnol Bioeng ; 114(11): 2539-2549, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28710851

RESUMO

Apoptosis has important functions during pathophysiologic processes. However, from a biopharmaceutical point of view, active apoptosis of host cells is undesirable during viral packaging or protein expression, because it decreases the efficiency of viral or protein production. Here we used the CRISPR/Cas technique to knock out four pro-apoptotic genes, Caspase3, Caspase6, Caspase7 and AIF1, in HEK293 cells, and successfully produced an apoptosis-resistant cell line. Furthermore, this cell line showed higher expression levels of pro-apoptotic proteins and higher packaging efficiency for the virus carrying these proteins than control HEK293 cells. This study not only produced an apoptosis-resistant cell line that is useful in producing apoptosis-inducing proteins or viruses expressing these proteins, but also provides a methodology to build other apoptosis-resistant cell lines.


Assuntos
Apoptose/genética , Sistemas CRISPR-Cas/genética , Melhoramento Genético/métodos , Células HEK293/fisiologia , Células HEK293/virologia , Lentivirus/crescimento & desenvolvimento , Proteínas Recombinantes/biossíntese , Técnicas de Inativação de Genes/métodos , Células HEK293/citologia , Humanos , Lentivirus/isolamento & purificação , Engenharia de Proteínas/métodos , Proteínas Recombinantes/isolamento & purificação
17.
Biotechnol Bioeng ; 114(11): 2685-2689, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28710857

RESUMO

The economical production of chemicals and fuels by microbial processes remains an intense area of interest in biotechnology. A key limitation in such efforts concerns the availability of key co-factors, in this case NADPH, required for target pathways. Many of the strategies pursued for increasing NADPH availability in Escherichia coli involve manipulations to the central metabolism, which can create redox imbalances and overall growth defects. In this study we used a reactive oxygen species based selection to search for novel methods of increasing NADPH availability. We report a loss of function mutation in the gene hdfR appears to increase NADPH availability in E. coli. Additionally, we show this excess NADPH can be used to improve the production of 3HP in E. coli.


Assuntos
Escherichia coli/fisiologia , Melhoramento Genético/métodos , Ácido Láctico/análogos & derivados , Engenharia Metabólica/métodos , NADP/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Disponibilidade Biológica , Ciclo do Ácido Cítrico/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Ácido Láctico/isolamento & purificação , Ácido Láctico/metabolismo , Via de Pentose Fosfato/fisiologia
18.
Biotechnol Bioeng ; 114(12): 2883-2895, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28755474

RESUMO

Probiotics, whether taken as capsules or consumed in foods, have been regarded as safe for human use by regulatory agencies. Being living cells, they serve as "tunable" factories for the synthesis of a vast array of beneficial molecules. The idea of reprogramming probiotics to act as controllable factories, producing potential therapeutic molecules under user-specified conditions, represents a new and powerful concept in drug synthesis and delivery. Probiotics that serve as drug delivery vehicles pose several challenges, one being targeting (as seen with nanoparticle approaches). Here, we employ synthetic biology to control swimming directionality in a process referred to as "pseudotaxis." Escherichia coli, absent the motility regulator cheZ, swim sporadically, missing the traditional "run" in the run:tumble swimming paradigm. Upon introduction of cheZ in trans and its signal-generated upregulation, engineered bacteria can be "programmed" to swim toward the source of the chemical cue. Here, engineered cells that encounter sufficient levels of the small signal molecule pyocyanin, produce an engineered CheZ and swim with programmed directionality. By incorporating a degradation tag at the C-terminus of CheZ, the cells stop running when they exit spaces containing pyocyanin. That is, the engineered CheZ modified with a C-terminal extension derived from the putative DNA-binding transcriptional regulator YbaQ (RREERAAKKVA) is consumed by the ClpXP protease machine at a rate sufficient to "brake" the cells when pyocyanin levels are too low. Through this process, we demonstrate that over time, these engineered E. coli accumulate in pyocyanin-rich locales. We suggest that such approaches may find utility in engineering probiotics so that their beneficial functions can be focused in areas of principal benefit.


Assuntos
Quimiotaxia/fisiologia , Proteínas de Escherichia coli/genética , Escherichia coli/fisiologia , Redes Reguladoras de Genes/genética , Melhoramento Genético/métodos , Proteínas Quimiotáticas Aceptoras de Metil/genética , Transativadores/genética , Quimiotaxia/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Piocianina/administração & dosagem , Biologia Sintética/métodos
19.
Biotechnol Bioeng ; 114(12): 2782-2793, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28755490

RESUMO

Metabolic engineering of isopropyl alcohol (IPA)-producing Escherichia coli strains was conducted along with 13 C-metabolic flux analysis (MFA). A metabolically engineered E. coli strain expressing the adc gene derived from Clostridium acetobutylicum and the IPADH gene from C. beijerinckii did not produce IPA during its exponential growth phase in the aerobic batch culture. 13 C-MFA was carried out, and revealed a deficiency in NADPH regeneration for IPA production in growth phase. Based on these findings, we used nitrogen-starved culture conditions to reduce NADPH consumption for biomass synthesis. As a result, IPA yield was increased to 20% mol/mol glucose. 13 C-MFA revealed that the relative flux levels through the oxidative pentose phosphate (PP) pathway and the TCA cycle were elevated in nitrogen-starved condition relative to glucose uptake rate. To prevent CO2 release in the 6-phosphogluconate dehydrogenase (6PGDH) reaction, metabolism of this E. coli strain was further engineered to redirect glycolytic flux to the glucose 6-phosphate dehydrogenase (G6PDH) and Entner-Doudoroff (ED) pathway. IPA yield of 55% mol/mol glucose was achieved by combining the nitrogen-starved culture condition with the metabolic redirection. The 13 C-MFA data and intracellular NADPH levels obtained under these IPA production conditions revealed linear correlations between the specific IPA production rate and NADPH concentration, as well as between IPA yield and the pyruvate dehydrogenase (PDH) flux. Our results showed that 13 C-MFA is a helpful tool for metabolic engineering studies, and that further improvement in IPA production by E. coli may be achieved by fine-tuning the cofactor ratio and concentrations, as well as optimizing the metabolic pathways and culture conditions.


Assuntos
2-Propanol/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Análise do Fluxo Metabólico/métodos , Redes e Vias Metabólicas/fisiologia , 2-Propanol/isolamento & purificação , Proteínas de Bactérias/genética , Isótopos de Carbono/farmacocinética , Escherichia coli/classificação , Escherichia coli/citologia , Melhoramento Genético/métodos , Especificidade da Espécie
20.
BMC Biotechnol ; 17(1): 57, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28673305

RESUMO

BACKGROUND: Effective and simple methods that lead to higher enzymatic efficiencies are highly sough. Here we proposed a foldon-triggered trimerization of the target enzymes with significantly improved catalytic performances by fusing a foldon domain at the C-terminus of the enzymes via elastin-like polypeptides (ELPs). The foldon domain comprises 27 residues and can forms trimers with high stability. RESULTS: Lichenase and xylanase can hydrolyze lichenan and xylan to produce value added products and biofuels, and they have great potentials as biotechnological tools in various industrial applications. We took them as the examples and compared the kinetic parameters of the engineered trimeric enzymes to those of the monomeric and wild type ones. When compared with the monomeric ones, the catalytic efficiency (k cat /K m ) of the trimeric lichenase and xylanase increased 4.2- and 3.9- fold. The catalytic constant (k cat ) of the trimeric lichenase and xylanase increased 1.8- fold and 5.0- fold than their corresponding wild-type counterparts. Also, the specific activities of trimeric lichenase and xylanase increased by 149% and 94% than those of the monomeric ones. Besides, the recovery of the lichenase and xylanase activities increased by 12.4% and 6.1% during the purification process using ELPs as the non-chromatographic tag. The possible reason is the foldon domain can reduce the transition temperature of the ELPs. CONCLUSION: The trimeric lichenase and xylanase induced by foldon have advantages in the catalytic performances. Besides, they were easier to purify with increased purification fold and decreased the loss of activities compared to their corresponding monomeric ones. Trimerizing of the target enzymes triggered by the foldon domain could improve their activities and facilitate the purification, which represents a simple and effective enzyme-engineering tool. It should have exciting potentials both in industrial and laboratory scales.


Assuntos
Bacillus subtilis/fisiologia , Endo-1,4-beta-Xilanases/química , Escherichia coli/fisiologia , Melhoramento Genético/métodos , Glicosídeo Hidrolases/química , Engenharia de Proteínas/métodos , Catálise , Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/genética , Ativação Enzimática , Evolução Molecular , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , Glicosídeo Hidrolases/biossíntese , Glicosídeo Hidrolases/genética , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica/genética , Especificidade por Substrato
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA