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https://www.readbyqxmd.com/read/28641402/ec-tagging-allows-cell-type-specific-rna-analysis
#1
Naoki Hida, Mohamed Y Aboukilila, Dana A Burow, Rakesh Paul, Marc M Greenberg, Michael Fazio, Samantha Beasley, Robert C Spitale, Michael D Cleary
Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT...
June 21, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28637248/expression-and-comparison-of-codon-optimised-aspergillus-tubingensis-amylase-variants-in-saccharomyces-cerevisiae
#2
Rosemary A Cripwell, Shaunita H Rose, Willem H van Zyl
The expression of codon optimised genes is a popular genetic engineering approach for the production of industrially relevant proteins. This study investigates and compares the expression of codon optimised and codon adapted amylase variants. The Aspergillus tubingensis raw starch hydrolysing α-amylase (amyA) and glucoamylase (glaA) encoding genes were redesigned using synonymous codons and expressed in S. cerevisiae Y294. Codon optimisation to favour S. cerevisiae codon bias resulted in a decrease in extracellular enzyme activity of 72% (30...
June 15, 2017: FEMS Yeast Research
https://www.readbyqxmd.com/read/28634826/tai-vacuolar-invertase-orthologs-the-interspecific-variability-in-tomato-plants-solanum-section-lycopersicon
#3
M A Slugina, A V Shchennikova, E Z Kochieva
Understanding the genetic mechanisms underlying carbohydrate metabolism can promote the development of biotechnological advances in fruit plants. The flesh tomato fruit represents an ideal system for examining the role of sucrose cleavage enzymes in fruit development, and wild tomato species differing in storage sugars serve as an excellent research material for this purpose. Plant vacuolar invertase is a key enzyme of sucrose metabolism in the sink organs. In the present study, we identified complete gene sequences encoding the TAI vacuolar invertase in 11 wild and one cultivated tomato accessions of the Solanum section Lycopersicon...
June 20, 2017: Molecular Genetics and Genomics: MGG
https://www.readbyqxmd.com/read/28634712/site-directed-mutagenesis-under-the-direction-of-in-silico-protein-docking-modeling-reveals-the-active-site-residues-of-3-ketosteroid-%C3%AE-1-dehydrogenase-from-mycobacterium-neoaurum
#4
Ning Qin, Yanbing Shen, Xu Yang, Liqiu Su, Rui Tang, Wei Li, Min Wang
3-Ketosteroid-Δ(1)-dehydrogenases (KsdD) from Mycobacterium neoaurum could transform androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione. This reaction has a significant effect on the product of pharmaceutical steroid. The crystal structure and active site residues information of KsdD from Mycobacterium is not yet available, which result in the engineering of KsdD is tedious. In this study, by the way of protein modeling and site-directed mutagenesis, we find that, Y122, Y125, S138, E140 and Y541 from the FAD-binding domain and Y365 from the catalytic domain play a key role in this transformation...
July 2017: World Journal of Microbiology & Biotechnology
https://www.readbyqxmd.com/read/28634692/crispr-cas-orthologues-and-variants-optimizing-the-repertoire-specificity-and-delivery-of-genome-engineering-tools
#5
Alberto Cebrian-Serrano, Benjamin Davies
Robust and cost-effective genome editing in a diverse array of cells and model organisms is now possible thanks to the discovery of the RNA-guided endonucleases of the CRISPR-Cas system. The commonly used Cas9 of Streptococcus pyogenes shows high levels of activity but, depending on the application, has been associated with some shortcomings. Firstly, the enzyme has been shown to cause mutagenesis at genomic sequences resembling the target sequence. Secondly, the stringent requirement for a specific motif adjacent to the selected target site can limit the target range of this enzyme...
June 20, 2017: Mammalian Genome: Official Journal of the International Mammalian Genome Society
https://www.readbyqxmd.com/read/28633594/targeting-and-delivery-of-therapeutic-enzymes
#6
Scott N Dean, Kendrick B Turner, Igor L Medintz, Scott A Walper
Biotechnology has revolutionized therapeutics for the treatment of a wide range of diseases. Recent advances in protein engineering and material science have made the targeted delivery of enzyme therapeutics using nanocarriers (NCs) a new model of treatment. Several NCs have been approved for clinical use in drug delivery. Despite their advantages, few NCs have been approved to deliver enzyme cargo in a targeted manner. This review details the current arsenal of platforms developed to deliver enzyme therapeutics as well as the advantages and challenges of using enzymes as drugs, with examples from the literature, and discusses the benefits and liabilities of a given approach...
July 2017: Therapeutic Delivery
https://www.readbyqxmd.com/read/28633311/cofactor-specificity-switch-in-shikimate-dehydrogenase-by-rational-design-and-consensus-engineering
#7
Fernando García-Guevara, Iris Bravo, Claudia Martínez-Anaya, Lorenzo Segovia
Consensus engineering has been used to design more stable variants using the most frequent amino acid at each site of a multiple sequence alignment; sometimes consensus engineering modifies function, but efforts have mainly been focused on studying stability. Here we constructed a consensus Rossmann domain for the Shikimate dehydrogenase enzyme; separately we decided to switch the cofactor specificity through rational design in the Escherichia coli Shikimate dehydrogenase enzyme and then analyzed the effect of consensus mutations on top of our design...
June 13, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28632991/uncovering-the-repertoire-of-fungal-secondary-metabolites-from-fleming-s-lab-to-the-international-space-station
#8
Tomasz Boruta
Fungi produce a variety of secondary metabolites (SMs), low-molecular weight compounds associated with many potentially useful biological activities. The examples of biotechnologically relevant fungal metabolites include penicillin, a beta-lactam antibiotic, and lovastatin, a cholesterol-lowering drug. The discovery of pharmaceutical lead compounds within the microbial metabolic pools relies on the selection and biochemical characterization of promising strains. Not all SMs are produced under standard cultivation conditions, hence the uncovering of chemical potential of investigated strains often requires the use of induction strategies to awake the associated biosynthetic genes...
June 20, 2017: Bioengineered
https://www.readbyqxmd.com/read/28631755/construction-of-a-synthetic-metabolic-pathway-for-biosynthesis-of-the-non-natural-methionine-precursor-2-4-dihydroxybutyric-acid
#9
Thomas Walther, Christopher M Topham, Romain Irague, Clément Auriol, Audrey Baylac, Hélène Cordier, Clémentine Dressaire, Luce Lozano-Huguet, Nathalie Tarrat, Nelly Martineau, Marion Stodel, Yannick Malbert, Marc Maestracci, Robert Huet, Isabelle André, Magali Remaud-Siméon, Jean Marie François
2,4-Dihydroxybutyric acid (DHB) is a molecule with considerable potential as a versatile chemical synthon. Notably, it may serve as a precursor for chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate, thus, targeting a considerable market in animal nutrition. However, no natural metabolic pathway exists for the biosynthesis of DHB. Here we have therefore conceived a three-step metabolic pathway for the synthesis of DHB starting from the natural metabolite malate. The pathway employs previously unreported malate kinase, malate semialdehyde dehydrogenase and malate semialdehyde reductase activities...
June 20, 2017: Nature Communications
https://www.readbyqxmd.com/read/28630410/investigating-the-life-expectancy-and-proteolytic-degradation-of-engineered-skeletal-muscle-biological-machines
#10
Caroline Cvetkovic, Meghan C Ferrall-Fairbanks, Eunkyung Ko, Lauren Grant, Hyunjoon Kong, Manu O Platt, Rashid Bashir
A combination of techniques from 3D printing, tissue engineering and biomaterials has yielded a new class of engineered biological robots that could be reliably controlled via applied signals. These machines are powered by a muscle strip composed of differentiated skeletal myofibers in a matrix of natural proteins, including fibrin, that provide physical support and cues to the cells as an engineered basement membrane. However, maintaining consistent results becomes challenging when sustaining a living system in vitro...
June 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28630108/inducing-mucosal-iga-a-challenge-for-vaccine-adjuvants-and-delivery-systems
#11
REVIEW
Prosper N Boyaka
Mucosal IgA or secretory IgA (SIgA) are structurally equipped to resist chemical degradation in the harsh environment of mucosal surfaces and enzymes of host or microbial origin. Production of SIgA is finely regulated, and distinct T-independent and T-dependent mechanisms orchestrate Ig α class switching and SIgA responses against commensal and pathogenic microbes. Most infectious pathogens enter the host via mucosal surfaces. To provide a first line of protection at these entry ports, vaccines are being developed to induce pathogen-specific SIgA in addition to systemic immunity achieved by injected vaccines...
July 1, 2017: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/28629697/increase-in-furfural-tolerance-by-combinatorial-overexpression-of-nad-salvage-pathway-enzymes-in-engineered-isobutanol-producing-e-coli
#12
Hun-Suk Song, Jong-Min Jeon, Hyun-Joong Kim, Shashi Kant Bhatia, Ganesan Sathiyanarayanan, Junyoung Kim, Ju Won Hong, Yoon Gi Hong, Kwon Young Choi, Yun-Gon Kim, Wooseong Kim, Yung-Hun Yang
To reduce the furfural toxicity for biochemical production in E. coli, a new strategy was successfully applied by supplying NAD(P)H through the nicotine amide salvage pathway. To alleviate the toxicity, nicotinamide salvage pathway genes were overexpressed in recombinant, isobutanol-producing E. coli. Gene expression of pncB and nadE respectively showed increased tolerance to furfural among these pathways. The combined expression of pncB and nadE was the most effective in increasing the tolerance of the cells to toxic aldehydes...
June 1, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28628207/engineered-polymerases-with-altered-substrate-specificity-expression-and-purification
#13
Ali Nikoomanzar, Matthew R Dunn, John C Chaput
Polymerase engineering is making it possible to synthesize xeno-nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which aims to study the genetic concepts of heredity and evolution in artificial genetic polymers. Since many of the polymerases needed to synthesize XNA polymers are not available commercially, researchers must express and purify these enzymes as recombinant proteins from E...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28627478/a-glimpse-into-the-specialization-history-of-the-lipases-acyltransferases-family-of-cplip2
#14
Anne-Hélène Jan, Eric Dubreucq, Jullien Drone, Maeva Subileau
Lipases/acyltransferases homologous to CpLIP2 from Candida parapsilosis belong to the α/β hydrolase superfamily as lipase A from Moesziomyces antarcticus (Candida antarctica), and constitute a consistent phylogenetic subgroup with at least 56% identity. Lipases/acyltransferases share the phenotypic characteristic of a high acyltransfer activity even in aqueous media with very high water thermodynamic activity. Previous mutagenesis and evolution strategies have given insights into the role of key residues and protein subdomains in the reaction and substrate specificities of these enzymes...
June 13, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28627452/engineering-of-unconventional-yeast-yarrowia-lipolytica-for-efficient-succinic-acid-production-from-glycerol-at-low-ph
#15
Zhiyong Cui, Cuijuan Gao, Jiaojiao Li, Jin Hou, Carol Sze Ki Lin, Qingsheng Qi
Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application...
June 13, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28627424/channeling-in-native-microbial-pathways-implications-and-challenges-for-metabolic-engineering
#16
REVIEW
Mary H Abernathy, Lian He, Yinjie J Tang
Intracellular enzymes can be organized into a variety of assemblies, shuttling intermediates from one active site to the next. Eukaryotic compartmentalization within mitochondrion and peroxisomes and substrate tunneling within multi-enzyme complexes have been well recognized. Intriguingly, the central pathways in prokaryotes may also form extensive channels, including the heavily branched glycolysis pathway. In vivo channeling through cascade enzymes is difficult to directly measure, but can be inferred from in vitro tests, reaction thermodynamics, transport/reaction modeling, analysis of molecular diffusion and protein interactions, or steady state/dynamic isotopic labeling...
June 13, 2017: Biotechnology Advances
https://www.readbyqxmd.com/read/28626501/nadph-oxidases-insights-into-selected-functions-and-mechanisms-of-action-in-cancer-and-stem-cells
#17
REVIEW
Magdalena Skonieczna, Tomasz Hejmo, Aleksandra Poterala-Hejmo, Artur Cieslar-Pobuda, Rafal J Buldak
NADPH oxidases (NOX) are reactive oxygen species- (ROS-) generating enzymes regulating numerous redox-dependent signaling pathways. NOX are important regulators of cell differentiation, growth, and proliferation and of mechanisms, important for a wide range of processes from embryonic development, through tissue regeneration to the development and spread of cancer. In this review, we discuss the roles of NOX and NOX-derived ROS in the functioning of stem cells and cancer stem cells and in selected aspects of cancer cell physiology...
2017: Oxidative Medicine and Cellular Longevity
https://www.readbyqxmd.com/read/28625987/metabolic-engineering-of-actinobacillus-succinogenes-provides-insights-into-succinic-acid-biosynthesis
#18
Michael T Guarnieri, Yat-Chen Chou, Davinia Salvachúa, Ali Mohagheghi, Peter C St John, Darren J Peterson, Yannick J Bomble, Gregg T Beckham
Actinobacillus succinogenes, a gram-negative, facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A...
June 16, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28625981/heterologous-expression-of-the-clostridium-carboxidivorans-co-dehydrogenase-alone-or-together-with-the-acetyl-coa-synthase-enables-clostridium-acetobutylicum-to-both-reduce-co2-and-oxidize-co
#19
Ellinor D Carlson, Eleftherios T Papoutsakis
With recent advances in synthetic biology, CO2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO2 fixation are the CO dehydrogenase (CODH) and acetyl-CoA synthase (ACS), which form a bifunctional heterotetrameric complex. CODH/ACS can reversibly catalyze CO2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO2 and has been well characterized in native hosts...
June 16, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28625755/multilevel-engineering-of-the-upstream-module-of-aromatic-amino-acid-biosynthesis-in-saccharomyces-cerevisiae-for-high-production-of-polymer-and-drug-precursors
#20
Miguel Suástegui, Chiam Yu Ng, Anupam Chowdhury, Wan Sun, Mingfeng Cao, Emma House, Costas D Maranas, Zengyi Shao
A multilevel approach was implemented in Saccharomyces cerevisiae to optimize the precursor module of the aromatic amino acid biosynthesis pathway, which is a rich resource for synthesizing a great variety of chemicals ranging from polymer precursor, to nutraceuticals and pain-relief drugs. To facilitate the discovery of novel targets to enhance the pathway flux, we incorporated the computational tool YEASTRACT for predicting novel transcriptional repressors and OptForce strain-design for identifying non-intuitive pathway interventions...
June 15, 2017: Metabolic Engineering
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