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https://www.readbyqxmd.com/read/28922354/reconstructing-promoter-activity-from-lux-bioluminescent-reporters
#1
Mudassar Iqbal, Neil Doherty, Anna M L Page, Saara N A Qazi, Ishan Ajmera, Peter A Lund, Theodore Kypraios, David J Scott, Philip J Hill, Dov J Stekel
The bacterial Lux system is used as a gene expression reporter. It is fast, sensitive and non-destructive, enabling high frequency measurements. Originally developed for bacterial cells, it has also been adapted for eukaryotic cells, and can be used for whole cell biosensors, or in real time with live animals without the need for euthanasia. However, correct interpretation of bioluminescent data is limited: the bioluminescence is different from gene expression because of nonlinear molecular and enzyme dynamics of the Lux system...
September 18, 2017: PLoS Computational Biology
https://www.readbyqxmd.com/read/28921549/%C3%AE-flucs-brighter-photinus-pyralis-firefly-luciferases-identified-by-surveying-consecutive-single-amino-acid-deletion-mutations-in-a-thermostable-variant
#2
Lisa M Halliwell, Amit P Jathoul, Jack P Bate, Harley L Worthy, James C Anderson, Dafydd D Jones, James A H Murray
The bright bioluminescence catalysed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bioimaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within 6 loop structures of the bright and thermostable x11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes...
September 16, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28921510/development-of-a-formaldehyde-biosensor-with-application-to-synthetic-methylotrophy
#3
Benjamin M Woolston, Timothy Roth, Ishwar Kohale, David Liu, Gregory Stephanopoulos
Formaldehyde is a prevalent environmental toxin and a key intermediate in single carbon metabolism. The ability to monitor formaldehyde concentration is therefore of interest for both environmental monitoring and for metabolic engineering of native and synthetic methylotrophs, but current methods suffer from low sensitivity, complex workflows, or require expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of E. coli. Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 1 µM...
September 16, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28921443/polyester-based-nanoparticles-for-the-encapsulation-of-monoclonal-antibodies
#4
Flávia Sousa, Pedro Fonte, Andreia Cruz, Patrick J Kennedy, Inês Mendes Pinto, Bruno Sarmento
Aliphatic polyesters have been widely explored for biomedical applications (e.g., drug delivery systems, biomedical devices, and tissue engineering). Recently, polyesters have been used in nanoparticle formulations for the controlled release of monoclonal antibodies (mAbs) for the enhanced efficacy of antibody-based therapy. Polyester-based nanoparticles for mAb delivery provide decreased antibody dosage, increased antibody stability and protection and longer therapeutic action, ultimately translating to an increased therapeutic index...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28920402/-preparation-and-transformation-optimization-for-supercompetent-b-subtilis-sck6-cells
#5
Xinzhi Li, Zhenghui Lu, Yuling Zhou, Shiyu Li, Guimin Zhang
Bacillus subtilis is Gram-positive aerobic bacterium and widely used as a heterologous protein expression host because of its safety and high protein secretion property. However, comparing to Escherichia coli, the low transformation efficiency limits the application of B. subtilis as a host cell for directed evolution of heterologous enzymes. Therefore, we optimized the competent cell preparation conditions for conventional plasmid, including the alteration of the medium, the concentration of inducer, the plasmid type, and other parameters...
April 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/28920398/-fermentation-purification-and-immunogenicity-evaluation-of-hepatitis-e-virus-like-particles-expressed-in-hansenula-polymorpha
#6
Caixia Su, Li Li, Zhenji Jin, Xudong Han, Ping Zhao, Lin Wang, Chunhu Jiang, Yueli Wang, Wenwen Wang, Deqi Xu, Naishuo Zhu
To develop a new recombinant hepatitis E vaccine, we used Hansenula polymorpha expression system to express recombinant hepatitis E virus-like particles (HEV VLPs), to construct a recombinant engineered strain HP/HEV2.3. The fermentation conditions and purification process were studied next. The first working seed lots were fermented in liquid culture, and the fermentation products were collected, then crushed, clarified, purified by ultrafiltration, silica gel adsorbed and desorbed, concentrated by ultrafiltration, purified by liquid chromatography and sterilized by filtration...
April 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/28920389/-research-progress-in-multi-enzyme-regulation-of-genetically-engineered-bacteria-producing-lycopene
#7
Jiali Xu, Siyi Zuo, Chengjia Xie, Ling Jiang, Shuang Li, He Huang, Xian Xu
Lycopene plays a crucial role in the biosynthesis pathway of 2-methyl-derythritol-4-phosphate (MEP) and mevalonic acid (MVA). It is a representative product of isoprenoid family, and a typical product of multi-enzyme catalytic reaction in organism. In this paper, we first introduced the general regulation methods in multi-enzyme synthesis reaction, including the construction of multi gene co-expression plasmid, gene order regulation, promoter and ribosome binding site regulation, gene knockout and replacement, aiming at the optimization strategies of multi-enzyme catalytic reaction in lycopene synthesis pathway...
April 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/28920010/exploring-step-by-step-assembly-of-nanoparticle-cytochrome-biohybrid-photoanodes
#8
Ee Taek Hwang, Katherine L Orchard, Daisuke Hojo, Joseph Beton, Colin W J Lockwood, Tadafumi Adschiri, Julea N Butt, Erwin Reisner, Lars J C Jeuken
Coupling light-harvesting semiconducting nanoparticles (NPs) with redox enzymes has been shown to create artificial photosynthetic systems that hold promise for the synthesis of solar fuels. High quantum yields require efficient electron transfer from the nanoparticle to the redox protein, a property that can be difficult to control. Here, we have compared binding and electron transfer between dye-sensitized TiO2 nanocrystals or CdS quantum dots and two decaheme cytochromes on photoanodes. The effect of NP surface chemistry was assessed by preparing NPs capped with amine or carboxylic acid functionalities...
August 2017: ChemElectroChem
https://www.readbyqxmd.com/read/28919509/molecular-and-macro-scale-analysis-of-enzyme-crosslinked-silk-hydrogels-for-rational-biomaterial-design
#9
Meghan McGill, Jeannine M Coburn, Benjamin P Partlow, Xuan Mu, David L Kaplan
Silk fibroin-based hydrogels have exciting applications in tissue engineering and therapeutic molecule delivery; however, their utility is dependent on their diffusive properties. The present study describes a molecular and macro-scale investigation of enzymatically-crosslinked silk fibroin hydrogels, and demonstrates that these systems have tunable crosslink density and diffusivity. We developed a liquid chromatography tandem mass spectroscopy (LC-MS/MS) method to assess the quantity and order of covalent tyrosine crosslinks in the hydrogels...
September 14, 2017: Acta Biomaterialia
https://www.readbyqxmd.com/read/28917970/how-do-enzymes-meet-nanoparticles-and-nanomaterials
#10
REVIEW
Ming Chen, Guangming Zeng, Piao Xu, Cui Lai, Lin Tang
Enzymes are fundamental biological catalysts responsible for biological regulation and metabolism. The opportunity for enzymes to 'meet' nanoparticles and nanomaterials is rapidly increasing due to growing demands for applications in nanomaterial design, environmental monitoring, biochemical engineering, and biomedicine. Therefore, understanding the nature of nanomaterial-enzyme interactions is becoming important. Since 2014, enzymes have been used to modify, degrade, or make nanoparticles/nanomaterials, while numerous nanoparticles/nanomaterials have been used as materials for enzymatic immobilization and biosensors and as enzyme mimicry...
September 13, 2017: Trends in Biochemical Sciences
https://www.readbyqxmd.com/read/28917628/an-engineered-biomarker-system-to-monitor-and-modulate-immune-clearance-of-cell-therapies
#11
Amy Singleton, Danika Khong, Ling-Yee Chin, Shilpaa Mukundan, Matthew Li, Biju Parekkadan
BACKGROUND AIMS: Cell transplants offer a new opportunity to deliver therapies with novel and complex mechanisms of action. Understanding the pharmacology of cell transplants is important to deliver this new therapy effectively. Currently, however, there are limited techniques to easily track cells after intravenous administration due to the dispersion of the graft throughout the entire body. METHODS: We herein developed an engineered cell system that secretes a luciferase enzyme to sensitively detect cell transplants independent of their locale by a simple blood test...
September 13, 2017: Cytotherapy
https://www.readbyqxmd.com/read/28916461/metabolic-engineering-of-escherichia-coli-for-the-synthesis-of-the-quadripolymer-poly-glycolate-co-lactate-co-3-hydroxybutyrate-co-4-hydroxybutyrate-from-glucose
#12
Zheng-Jun Li, Kangjian Qiao, Xue-Mei Che, Gregory Stephanopoulos
Escherichia coli was metabolically engineered to effectively produce a series of biopolymers consisted of four types of monomers including glycolate, lactate, 3-hydroxybutyrate and 4-hydroxybutyrate from glucose as the carbon source. The biosynthetic route of novel quadripolymers was achieved by the overexpression of a range of homologous and heterologous enzymes including isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, glyoxylate/hydroxypyruvate reductase, propionyl-CoA transferase, β-ketothiolase, acetoacetyl-CoA reductase, succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, CoA transferase and PHA synthase...
September 12, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28915575/hexokinase-2-promotes-tumor-growth-and-metastasis-by-regulating-lactate-production-in-pancreatic-cancer
#13
Marybeth Anderson, Raoud Marayati, Richard Moffitt, Jen Jen Yeh
Pancreatic ductal adenocarcinoma (PDAC) is a KRAS-driven cancer with a high incidence of metastasis and an overall poor prognosis. Previous work in a genetically engineered mouse model of PDAC showed glucose metabolism to be important for maintaining tumor growth. Multiple glycolytic enzymes, including hexokinase 2 (HK2), were upregulated in primary PDAC patient tumors, supporting a role for glycolysis in promoting human disease. HK2 was most highly expressed in PDAC metastases, suggesting a link between HK2 and aggressive tumor biology...
August 22, 2017: Oncotarget
https://www.readbyqxmd.com/read/28915025/structural-evidence-for-the-dopamine-first-mechanism-of-norcoclaurine-synthase
#14
Benjamin R Lichman, Altin Sula, Thomas Pesnot, Helen C Hailes, John Ward, Nicholas H Keep
Norcoclaurine synthase (NCS) is a Pictet-Spenglerase that catalyzes the first key step in plant benzylisoquinoline alkaloid metabolism, a compound family that includes bioactive natural products such as morphine. The enzyme has also shown great potential as a biocata-lyst for the formation of chiral isoquinolines. Here we present new high-resolution X-ray crystallography data describing Thalictrum flavum NCS bound to a mechanism inspired ligand. The structure supports two key features of the NCS 'dopamine-first' mechanism: the binding of dopamine catechol to Lys-122 and the position of the carbonyl substrate binding site at the active site entrance...
September 15, 2017: Biochemistry
https://www.readbyqxmd.com/read/28911121/engineering-human-primpol-into-an-efficient-rna-dependent-dna-primase-polymerase
#15
Rubén Agudo, Patricia A Calvo, María I Martínez-Jiménez, Luis Blanco
We have developed a straightforward fluorometric assay to measure primase-polymerase activity of human PrimPol (HsPrimPol). The sensitivity of this procedure uncovered a novel RNA-dependent DNA priming-polymerization activity (RdDP) of this enzyme. In an attempt to enhance HsPrimPol RdDP activity, we constructed a smart mutant library guided by prior sequence-function analysis, and tested this library in an adapted screening platform of our fluorometric assay. After screening less than 500 variants, we found a specific HsPrimPol mutant, Y89R, which displays 10-fold higher RdDP activity than the wild-type enzyme...
September 6, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28910986/elimination-of-the-last-reactions-in-ergosterol-biosynthesis-alters-the-resistance-of-saccharomyces-cerevisiae-to-multiple-stresses
#16
Guodong Liu, Yun Chen, Nils J Færgeman, Jens Nielsen
The sterol composition of membranes is known to influence many phenotypes of yeast. However, a systematic study of the relationship between sterol composition and stress resistances has not been conducted. Here, we therefore constructed single or double gene deletion mutants of the last four enzymes in ergosterol biosynthesis in a prototrophic genetic background of Saccharomyces cerevisiae. Identification of the sterol composition of these mutants revealed a high flexibility of the sterol-processing steps instead of the previously proposed sequential conversion...
September 1, 2017: FEMS Yeast Research
https://www.readbyqxmd.com/read/28904563/bacillus-coagulans-ma-13-a-promising-thermophilic-and-cellulolytic-strain-for-the-production-of-lactic-acid-from-lignocellulosic-hydrolysate
#17
Martina Aulitto, Salvatore Fusco, Simonetta Bartolucci, Carl Johan Franzén, Patrizia Contursi
BACKGROUND: The transition from a petroleum-based economy towards more sustainable bioprocesses for the production of fuels and chemicals (circular economy) is necessary to alleviate the impact of anthropic activities on the global ecosystem. Lignocellulosic biomass-derived sugars are suitable alternative feedstocks that can be fermented or biochemically converted to value-added products. An example is lactic acid, which is an essential chemical for the production of polylactic acid, a biodegradable bioplastic...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28903556/4-hydroxyphenylpyruvate-dioxygenase-inhibitors-from-chemical-biology-to-agrochemicals
#18
Ferdinand Ndikuryayo, Behrooz Moosavi, Wen-Chao Yang, Guang-Fu Yang
The development of new herbicides is receiving considerable attention to control weed biotypes resistant to current herbicides. Consequently, new enzymes are always desired as targets for herbicide discovery. 4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is an enzyme engaged in photosynthetic activity and catalyzes the transformation of 4-hydroxyphenylpyruvic acid (HPPA) into homogentisic acid (HGA). HPPD inhibitors constitute a promising area of discovery and development of innovative herbicides with some advantages, including excellent crop selectivity, low application rates, and broad-spectrum weed control...
September 14, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28902999/self-assembled-2d-free-standing-janus-nanosheets-with-single-layer-thickness
#19
Yiyang Lin, Michael R Thomas, Amy Gelmi, Vincent Leonardo, E Thomas Pashuck, Stephanie A Maynard, Ye Wang, Molly M Stevens
We report the thermodynamically controlled growth of solution-processable and free-standing nanosheets via peptide assembly in two dimensions. By taking advantage of self-sorting between peptide β-strands and hydrocarbon chains, we have demonstrated the formation of Janus 2D structures with single-layer thickness, which enable a predetermined surface heterofunctionalization. A controlled 2D-to-1D morphological transition was achieved by subtly adjusting the intermolecular forces. These nanosheets provide an ideal substrate for the engineering of guest components (e...
September 13, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28902886/metabolic-engineering-to-simultaneously-activate-anthocyanin-and-proanthocyanidin-biosynthetic-pathways-in-nicotiana-spp
#20
Sandra Fresquet-Corrales, Edelín Roque, Alejandro Sarrión-Perdigones, Maricruz Rochina, María P López-Gresa, Huertas M Díaz-Mula, José M Bellés, Francisco Tomás-Barberán, José P Beltrán, Luis A Cañas
Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production...
2017: PloS One
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