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"Saturation mutagenesis"

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https://www.readbyqxmd.com/read/28764860/crispring-the-regulatory-genome-the-challenge-ahead
#1
Stephanie E Sansbury, Katelyn M Sweeney, Ophir Shalem
CRISPR saturation mutagenesis has the potential to dissect the functional landscape of noncoding regions, but is highly susceptible to false discovery and misinterpretation. As recently published, Canver et al. have now taken the first steps towards addressing these issues by increasing screening resolution and analyzing the effects of off targets on hit calling.
July 29, 2017: Trends in Genetics: TIG
https://www.readbyqxmd.com/read/28757433/technical-considerations-for-the-use-of-crispr-cas9-in-hematology-research
#2
REVIEW
Michael C Gundry, Daniel P Dever, David Yudovich, Daniel E Bauer, Simon Haas, Adam C Wilkinson, Sofie Singbrant
The hematopoietic system is responsible for transporting oxygen and nutrients, fighting infections, and repairing tissue damage. Hematopoietic system dysfunction therefore causes a range of serious health consequences. Lifelong hematopoiesis is maintained by repopulating multipotent hematopoietic stem cells (HSCs) that replenish shorter-lived, mature blood cell types. A prokaryotic mechanism of immunity, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system, has recently been "repurposed" to efficiently mutate mammalian genomes in a sequence-specific manner...
July 27, 2017: Experimental Hematology
https://www.readbyqxmd.com/read/28754922/shadowy-a-dark-yellow-fluorescent-protein-for-flim-based-fret-measurement
#3
Hideji Murakoshi, Akihiro C E Shibata
Fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurement (FLIM-FRET) is one of the powerful methods for imaging of intracellular protein activities such as protein-protein interactions and conformational changes. Here, using saturation mutagenesis, we developed a dark yellow fluorescent protein named ShadowY that can serve as an acceptor for FLIM-FRET. ShadowY is spectrally similar to the previously reported dark YFP but has a much smaller quantum yield, greater extinction coefficient, and superior folding property...
July 28, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28715793/monitoring-in-vivo-metabolic-flux-with-a-designed-whole-cell-metabolite-biosensor-of-shikimic-acid
#4
Heng Li, Chaoning Liang, Wei Chen, Jian-Ming Jin, Shuang-Yan Tang, Yong Tao
Knowledge of intracellular metabolite levels is important for the understanding of metabolic flux distributions. Whole-cell biosensors of key metabolites are ideal for the monitoring of carbon flow in important metabolic pathways, thus guiding metabolic engineering for microbial improvement. However, lack of biosensors for metabolites of interests has limited their applications. In this study, a genetically encoded whole-cell biosensor specifically responding to shikimic acid has been developed by screening a site-saturation mutagenesis library of the binding pocket of a uric acid-responsive regulatory protein...
July 11, 2017: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/28693917/biocatalysts-for-the-pharmaceutical-industry-created-by-structure-guided-directed-evolution-of-stereoselective-enzymes
#5
REVIEW
Guangyue Li, Jian-Bo Wang, Manfred T Reetz
Enzymes have been used for a long time as catalysts in the asymmetric synthesis of chiral intermediates needed in the production of therapeutic drugs. However, this alternative to man-made catalysts has suffered traditionally from distinct limitations, namely the often observed wrong or insufficient enantio- and/or regioselectivity, low activity, narrow substrate range, and insufficient thermostability. With the advent of directed evolution, these problems can be generally solved. The challenge is to develop and apply the most efficient mutagenesis methods which lead to highest-quality mutant libraries requiring minimal screening...
May 11, 2017: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/28686159/deconstruction-of-the-ras-switching-cycle-through-saturation-mutagenesis
#6
Pradeep Bandaru, Neel H Shah, Moitrayee Bhattacharyya, John P Barton, Yasushi Kondo, Joshua C Cofsky, Christine L Gee, Arup K Chakraborty, Tanja Kortemme, Rama Ranganathan, John Kuriyan
Ras proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between active and inactive states. The high conservation of Ras requires mechanistic explanation, especially given the general mutational tolerance of proteins. Here, we use deep mutational scanning, biochemical analysis and molecular simulations to understand constraints on Ras sequence. Ras exhibits global sensitivity to mutation when regulated by a GTPase activating protein and a nucleotide exchange factor...
July 7, 2017: ELife
https://www.readbyqxmd.com/read/28674381/engineered-botulinum-neurotoxin-b-with-improved-efficacy-for-targeting-human-receptors
#7
Liang Tao, Lisheng Peng, Ronnie P-A Berntsson, Sai Man Liu, SunHyun Park, Feifan Yu, Christopher Boone, Shilpa Palan, Matthew Beard, Pierre-Etienne Chabrier, Pål Stenmark, Johannes Krupp, Min Dong
Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However, it has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B's therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B...
July 3, 2017: Nature Communications
https://www.readbyqxmd.com/read/28659942/identification-of-two-new-mechanisms-that-regulate-fruit-growth-by-cell-expansion-in-tomato
#8
Constance Musseau, Daniel Just, Joana Jorly, Frédéric Gévaudant, Annick Moing, Christian Chevalier, Martine Lemaire-Chamley, Christophe Rothan, Lucie Fernandez
Key mechanisms controlling fruit weight and shape at the levels of meristem, ovary or very young fruit have already been identified using natural tomato diversity. We reasoned that new developmental modules prominent at later stages of fruit growth could be discovered by using new genetic and phenotypic diversity generated by saturated mutagenesis. Twelve fruit weight and tissue morphology mutants likely affected in late fruit growth were selected among thousands of fruit size and shape EMS mutants available in our tomato EMS mutant collection...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28638047/semi-rational-engineering-of-a-thermostable-aldo-keto-reductase-from-thermotoga-maritima-for-synthesis-of-enantiopure-ethyl-2-hydroxy-4-phenylbutyrate-ehpb
#9
Zhiguo Wang, Shuo Zhou, Shuangling Zhang, Sa Zhang, Fangmeng Zhu, Xiaolu Jin, Zhenming Chen, Xiaoling Xu
A novel aldo-keto reductase Tm1743 characterized from Thermotoga maritima was explored as an effective biocatalyst in chiral alcohol production. Natural Tm1743 catalyzes asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) at high efficiency, but the production of, ethyl (S)-2-hydroxy-4-phenylbutyrate ((S)-EHPB), which is less desirable, is preferred with an enantiomeric excess (ee) value of 76.5%. Thus, altering the enantioselectivity of Tm1743 to obtain the more valuable product (R)-EHPB for angiotensin drug synthesis is highly desired...
June 21, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28637270/in-planta-recapitulation-of-isoprene-synthases-evolution-from-ocimene-synthases
#10
Mingai Li, Jia Xu, Alberto Algarra Alarcon, Silvia Carlin, Enrico Barbaro, Luca Cappellin, Violeta Velikova, Urska Vrhovsek, Francesco Loreto, Claudio Varotto
Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated.We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases...
June 16, 2017: Molecular Biology and Evolution
https://www.readbyqxmd.com/read/28601895/site-saturation-mutagenesis-library-construction-and-screening-for-specific-broad-spectrum-single-domain-antibodies-against-multiple-cry1-toxins
#11
Lingxia Jiao, Yuan Liu, Xiao Zhang, Beibei Liu, Cunzheng Zhang, Xianjin Liu
Potential ecological environmental and food safety risks of various Cry toxins of Bacillus thuringiensis (Bt) in transgenic food have received gradually increasing attention, which urged to establish an efficient and broad-spectrum detection technology for Cry toxins. Based on the single-domain antibody (sdAb) A8 against Bt Cry1Ab toxin screened from the humanized domain antibody library, the key amino acids of sdAb (A8) binding five kinds of Cry1 toxins were predicted using homology modeling and molecular docking technology, and the results showed that 105th asparagine, 106th arginine, 107th valine, and 114th arginine, respectively, located in heavy-chain complementarity-determining region 3 were common key amino acid sites...
June 11, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28571627/directed-evolution-of-thermomyces-lanuginosus-lipase-to-enhance-methanol-tolerance-for-efficient-production-of-biodiesel-from-waste-grease
#12
Kaiyuan Tian, Kee Tai, Bryan Jian Wei Chua, Zhi Li
Engineering a methanol tolerant lipase is of great importance in biodiesel production. Here, the first semi-rational method for directed enzyme evolution to enhance methanol tolerance by targeting high B-factor residues for iterative saturation mutagenesis (ISM) is reported. The best double mutant, TLL-S105C/D27R, retained 71% of its original activity after incubation in methanol, showing 30% greater methanol tolerance than TLL. TLL-S105C/D27R also displayed 27% higher activity over TLL. Structure modelling suggested that the increased stability of TLL-S105C/D27R was caused by the formation of a new hydrogen bond which stabilized the protein structure...
May 19, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28530095/rational-design-of-bacillus-coagulans-nl01-l-arabinose-isomerase-and-use-of-its-f279i-variant-in-d-tagatose-production
#13
Zhaojuan Zheng, Wending Mei, Meijuan Xia, Qin He, Jia Ouyang
d-Tagatose is a prospective functional sweetener that can be produced by l-arabinose isomerase (AI) from d-galactose. To improve the activity of AI toward d-galactose, the AI of Bacillus coagulans was rationally designed on the basis of molecular modeling and docking. After alanine scanning and site-saturation mutagenesis, variant F279I that exhibited improved activity toward d-galactose was obtained. The optimal temperature and pH of F279I were determined to be 50 °C and 8.0, respectively. This variant possessed 1...
June 14, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28526714/constans-imparts-dna-sequence-specificity-to-the-histone-fold-nf-yb-nf-yc-dimer
#14
Nerina Gnesutta, Roderick W Kumimoto, Swadhin Swain, Matteo Chiara, Chamindika Siriwardana, David S Horner, Ben F Holt, Roberto Mantovani
Nuclear Factor Y (NF-Y) is a heterotrimeric transcription factor that binds CCAAT elements. The NF-Y trimer is composed of a Histone Fold Domain (HFD) dimer (NF-YB/NF-YC) and NF-YA, which confers DNA sequence specificity. NF-YA shares a conserved domain with the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) proteins. We show that CONSTANS (CO/B-BOX PROTEIN1 BBX1), a master flowering regulator, forms a trimer with Arabidopsis thaliana NF-YB2/NF-YC3 to efficiently bind the CORE element of the FLOWERING LOCUS T promoter...
June 2017: Plant Cell
https://www.readbyqxmd.com/read/28508385/improving-the-temperature-characteristics-and-catalytic-efficiency-of-a-mesophilic-xylanase-from-aspergillus-oryzae-aoxyn11a-by-iterative-mutagenesis-based-on-in-silico-design
#15
Xue-Qing Li, Qin Wu, Die Hu, Rui Wang, Yan Liu, Min-Chen Wu, Jian-Fang Li
To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly(21) with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11A(G21I), was selected from the mutagenesis library, E...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28504265/in-vitro-evolution-of-an-influenza-broadly-neutralizing-antibody-is-modulated-by-hemagglutinin-receptor-specificity
#16
Nicholas C Wu, Geramie Grande, Hannah L Turner, Andrew B Ward, Jia Xie, Richard A Lerner, Ian A Wilson
The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. However, the factors that influence the evolution of high-affinity bnAbs remain elusive. We therefore explore the functional sequence space of bnAb C05, which targets the receptor-binding site (RBS) of influenza haemagglutinin (HA) via a long CDR H3. We combine saturation mutagenesis with yeast display to enrich for C05 variants of CDR H3 that bind to H1 and H3 HAs...
May 15, 2017: Nature Communications
https://www.readbyqxmd.com/read/28469274/crispr-cpf1-assisted-genome-editing-of-corynebacterium-glutamicum
#17
Yu Jiang, Fenghui Qian, Junjie Yang, Yingmiao Liu, Feng Dong, Chongmao Xu, Bingbing Sun, Biao Chen, Xiaoshu Xu, Yan Li, Renxiao Wang, Sheng Yang
Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86-100%...
May 4, 2017: Nature Communications
https://www.readbyqxmd.com/read/28464395/specificity-effects-of-amino-acid-substitutions-in-promiscuous-hydrolases-context-dependence-of-catalytic-residue-contributions-to-local-fitness-landscapes-in-nearby-sequence-space
#18
Christopher D Bayer, Bert van Loo, Florian Hollfelder
Catalytic promiscuity can facilitate evolution of enzyme functions-a multifunctional catalyst may act as a springboard for efficient functional adaptation. We test the effect of single mutations on multiple activities in two groups of promiscuous AP superfamily members to probe this hypothesis. We quantify the effect of site-saturating mutagenesis of an analogous, nucleophile-flanking residue in two superfamily members: an arylsulfatase (AS) and a phosphonate monoester hydrolase (PMH). Statistical analysis suggests that no one physicochemical characteristic alone explains the mutational effects...
June 1, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28464223/casting-eppcr-ceppcr-a-simple-random-mutagenesis-method-to-generate-high-quality-mutant-libraries
#19
Jianhua Yang, Anna J Ruff, Marcus Arlt, Ulrich Schwaneberg
During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g., high temperature, extreme pH, ionic liquids, or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is random mutagenesis by error-prone polymerase chain reaction (epPCR). However, recent reports show that epPCR often fails to produce >70% of beneficial positions/amino acid exchanges which improve enzyme properties such as organic solvent or ionic liquid resistance...
September 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28425285/directed-evolution-of-carbonyl-reductase-from-rhodosporidium-toruloides-and-its-application-in-stereoselective-synthesis-of-tert-butyl-3r-5s-6-chloro-3-5-dihydroxyhexanoate
#20
Zhi-Qiang Liu, Lin Wu, Xiao-Jian Zhang, Ya-Ping Xue, Yu-Guo Zheng
tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl reductase RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH). For the activity of RtSCR9 to be improved, random mutagenesis and site-saturation mutagenesis were performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys, and mut-Phe156Gln). These mutants exhibited 1.94-, 3...
May 10, 2017: Journal of Agricultural and Food Chemistry
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