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"Saturation mutagenesis"

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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
#1
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-3-hydoxy-5-oxohexanoate ((S)-CHOH). To improve the activity of RtSCR9, 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-, 3.03- and 1...
April 20, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28422960/predicting-the-impact-of-lynch-syndrome-causing-missense-mutations-from-structural-calculations
#2
Sofie V Nielsen, Amelie Stein, Alexander B Dinitzen, Elena Papaleo, Michael H Tatham, Esben G Poulsen, Maher M Kassem, Lene J Rasmussen, Kresten Lindorff-Larsen, Rasmus Hartmann-Petersen
Accurate methods to assess the pathogenicity of mutations are needed to fully leverage the possibilities of genome sequencing in diagnosis. Current data-driven and bioinformatics approaches are, however, limited by the large number of new variations found in each newly sequenced genome, and often do not provide direct mechanistic insight. Here we demonstrate, for the first time, that saturation mutagenesis, biophysical modeling and co-variation analysis, performed in silico, can predict the abundance, metabolic stability, and function of proteins inside living cells...
April 19, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28387245/design-and-application-of-a-lactulose-biosensor
#3
Jieyuan Wu, Peixia Jiang, Wei Chen, Dandan Xiong, Linglan Huang, Junying Jia, Yuanyuan Chen, Jian-Ming Jin, Shuang-Yan Tang
In this study the repressor of Escherichia coli lac operon, LacI, has been engineered for altered effector specificity. A LacI saturation mutagenesis library was subjected to Fluorescence Activated Cell Sorting (FACS) dual screening. Mutant LacI-L5 was selected and it is specifically induced by lactulose but not by other disaccharides tested (lactose, epilactose, maltose, sucrose, cellobiose and melibiose). LacI-L5 has been successfully used to construct a whole-cell lactulose biosensor which was then applied in directed evolution of cellobiose 2-epimerase (C2E) for elevated lactulose production...
April 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28381551/production-of-homogeneous-glycoprotein-with-multi-site-modifications-by-an-engineered-n-glycosyltransferase-mutant
#4
Qitao Song, Zhigang Wu, Yueyuan Fan, Woran Song, Peiru Zhang, Li Wang, Faxing Wang, Yangyang Xu, Peng George Wang, Jiansong Cheng
Naturally occurring N- glycoproteins exhibit glycoform heterogeneity with respect to N-glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). However, access to well-defined glycoproteins is always important for both basic research and therapeutic purposes. As a result, there has been substantial effort to identify and understand the catalytic properties of N-glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study, we found that ApNGT, a newly discovered cytoplasmic N- glycosyltransferase from Actinobacillus pleuropneumoniae, has strict selectivity towards the residues around the Asn of N-glycosylation sequon by screening a small library of synthetic peptides...
April 5, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28284313/one-step-combined-focused-eppcr-and-saturation-mutagenesis-for-thermostability-evolution-of-a-new-cold-active-xylanase
#5
Juan Pablo Acevedo, Manfred T Reetz, Juan A Asenjo, Loreto P Parra
Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry...
May 2017: Enzyme and Microbial Technology
https://www.readbyqxmd.com/read/28255534/the-structure-of-toho1-%C3%AE-lactamase-in-complex-with-penicillin-reveals-the-role-of-tyr105-in-substrate-recognition
#6
Patricia S Langan, Venu Gopal Vandavasi, Kevin L Weiss, Jonathan B Cooper, Stephan L Ginell, Leighton Coates
The role of the conserved residue Tyr105 in class A β-lactamases has been the subject of investigation using both structural studies and saturation mutagenesis. Both have shown that while it does not need to be strictly conserved for activity, it is important for substrate recognition. With this in mind we determined the crystal structure of Toho1 β-lactamase at 15 K to 1.10 Å resolution in complex with penicillin. As expected a ring-opened penicillin molecule bound to Ser70 the catalytic nucleophile, can clearly be seen in electron density in the active site...
December 2016: FEBS Open Bio
https://www.readbyqxmd.com/read/28251763/a-13-week-research-based-biochemistry-laboratory-curriculum
#7
Scott T Lefurgy, Emily C Mundorff
Here, we present a 13-week research-based biochemistry laboratory curriculum designed to provide the students with the experience of engaging in original research while introducing foundational biochemistry laboratory techniques. The laboratory experience has been developed around the directed evolution of an enzyme chosen by the instructor, with mutations designed by the students. Ideal enzymes for this curriculum are able to be structurally modeled, solubly expressed, and monitored for activity by UV/Vis spectroscopy, and an example curriculum for haloalkane dehalogenase is given...
March 2, 2017: Biochemistry and Molecular Biology Education
https://www.readbyqxmd.com/read/28218758/variant-aware-saturating-mutagenesis-using-multiple-cas9-nucleases-identifies-regulatory-elements-at-trait-associated-loci
#8
Matthew C Canver, Samuel Lessard, Luca Pinello, Yuxuan Wu, Yann Ilboudo, Emily N Stern, Austen J Needleman, Frédéric Galactéros, Carlo Brugnara, Abdullah Kutlar, Colin McKenzie, Marvin Reid, Diane D Chen, Partha Pratim Das, Mitchel A Cole, Jing Zeng, Ryo Kurita, Yukio Nakamura, Guo-Cheng Yuan, Guillaume Lettre, Daniel E Bauer, Stuart H Orkin
Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants...
April 2017: Nature Genetics
https://www.readbyqxmd.com/read/28218303/a-combination-of-mutational-and-computational-scanning-guides-the-design-of-an-artificial-ligand-binding-controlled-lipase
#9
Marco Kaschner, Oliver Schillinger, Timo Fettweiss, Christina Nutschel, Frank Krause, Alexander Fulton, Birgit Strodel, Andreas Stadler, Karl-Erich Jaeger, Ulrich Krauss
Allostery, i.e. the control of enzyme activity by a small molecule at a location distant from the enzyme's active site, represents a mechanism essential for sustaining life. The rational design of allostery is a non-trivial task but can be achieved by fusion of a sensory domain, which responds to environmental stimuli with a change in its structure. Hereby, the site of domain fusion is difficult to predict. We here explore the possibility to rationally engineer allostery into the naturally not allosterically regulated Bacillus subtilis lipase A, by fusion of the citrate-binding sensor-domain of the CitA sensory-kinase of Klebsiella pneumoniae...
February 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28170244/structure-activity-relationship-and-molecular-mechanics-reveal-the-importance-of-ring-entropy-in-the-biosynthesis-and-activity-of-a-natural-product
#10
Hai L Tran, Katrina W Lexa, Olivier Julien, Travis S Young, Christopher T Walsh, Matthew P Jacobson, James A Wells
Macrocycles are appealing drug candidates due to their high affinity, specificity, and favorable pharmacological properties. In this study, we explored the effects of chemical modifications to a natural product macrocycle upon its activity, 3D geometry, and conformational entropy. We chose thiocillin as a model system, a thiopeptide in the ribosomally encoded family of natural products that exhibits potent antimicrobial effects against Gram-positive bacteria. Since thiocillin is derived from a genetically encoded peptide scaffold, site-directed mutagenesis allows for rapid generation of analogues...
February 22, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28165493/a-noise-trimming-and-positional-significance-of-transposon-insertion-system-to-identify-essential-genes-in-yersinia-pestis
#11
Zheng Rong Yang, Helen L Bullifent, Karen Moore, Konrad Paszkiewicz, Richard J Saint, Stephanie J Southern, Olivia L Champion, Nicola J Senior, Mitali Sarkar-Tyson, Petra C F Oyston, Timothy P Atkins, Richard W Titball
Massively parallel sequencing technology coupled with saturation mutagenesis has provided new and global insights into gene functions and roles. At a simplistic level, the frequency of mutations within genes can indicate the degree of essentiality. However, this approach neglects to take account of the positional significance of mutations - the function of a gene is less likely to be disrupted by a mutation close to the distal ends. Therefore, a systematic bioinformatics approach to improve the reliability of essential gene identification is desirable...
February 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28163026/engineering-of-cyp106a2-for-steroid-9%C3%AE-and-6%C3%AE-hydroxylation
#12
Julia Nikolaus, Kim Thoa Nguyen, Cornelia Virus, Jan L Riehm, Michael Hutter, Rita Bernhardt
CYP 106A2 from Bacillus megaterium ATCC 13368 has been described as a 15β-hydroxylase showing also minor 11α-, 9α- and 6β-hydroxylase activity for progesterone conversion. Previously, mutant proteins with a changed selectivity towards 11α-OH-progesterone have already been produced. The challenge of this work was to create mutant proteins with a higher regioselectivity towards hydroxylation at positions 9 and 6 of the steroid molecule. 9α-hydroxyprogesterone exhibits pharmaceutical importance, because it is a useful intermediate in the production of physiologically active substances which possess progestational activity...
April 2017: Steroids
https://www.readbyqxmd.com/read/28134951/converting-pasteurella-multocida%C3%AE-2-3-sialyltransferase-1-pmst1-to-a-regioselective-%C3%AE-2-6-sialyltransferase-by-saturation-mutagenesis-and-regioselective-screening
#13
John B McArthur, Hai Yu, Jie Zeng, Xi Chen
A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) for α2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2-6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1...
February 21, 2017: Organic & Biomolecular Chemistry
https://www.readbyqxmd.com/read/28109425/beyond-the-natural-proteome-nondegenerate-saturation-mutagenesis-methodologies-and-advantages
#14
M M Ferreira Amaral, L Frigotto, A V Hine
Beyond the natural proteome, high-throughput mutagenesis offers the protein engineer an opportunity to "tweak" the wild-type activity of a protein to create a recombinant protein with required attributes. Of the various approaches available, saturation mutagenesis is one of the core techniques employed by protein engineers, and in recent times, nondegenerate saturation mutagenesis is emerging as the approach of choice. This review compares the current methodologies available for conducting nondegenerate saturation mutagenesis with traditional, degenerate saturation and briefly outlines the options available for screening the resulting libraries, to discover a novel protein with the required activity and/or specificity...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28096490/comprehensive-essentiality-analysis-of-the-mycobacterium-tuberculosis-genome-via-saturating-transposon-mutagenesis
#15
Michael A DeJesus, Elias R Gerrick, Weizhen Xu, Sae Woong Park, Jarukit E Long, Cara C Boutte, Eric J Rubin, Dirk Schnappinger, Sabine Ehrt, Sarah M Fortune, Christopher M Sassetti, Thomas R Ioerger
For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed...
January 17, 2017: MBio
https://www.readbyqxmd.com/read/28092444/engineering-of-the-bacillus-circulans-%C3%AE-galactosidase-product-specificity
#16
Huifang Yin, Tjaard Pijning, Xiangfeng Meng, Lubbert Dijkhuizen, Sander S van Leeuwen
Microbial β-galactosidase enzymes are widely used as biocatalysts in industry to produce prebiotic galactooligosaccharides (GOS) from lactose. GOS mixtures are used as beneficial additives in infant formula to mimic the prebiotic effects of human milk oligosaccharides (hMOS). The structural variety in GOS mixtures is significantly lower than in hMOS. Since this structural complexity is considered as the basis for the multiple biological functions of hMOS, it is important to broaden the variety of GOS structures...
February 7, 2017: Biochemistry
https://www.readbyqxmd.com/read/28074907/insights-into-an-evolutionary-strategy-leading-to-antibiotic-resistance
#17
Chun-Feng D Hou, Jian-Wei Liu, Charles Collyer, Nataša Mitić, Marcelo Monteiro Pedroso, Gerhard Schenk, David L Ollis
Metallo-β-lactamases (MBLs) with activity towards a broad-spectrum of β-lactam antibiotics have become a major threat to public health, not least due to their ability to rapidly adapt their substrate preference. In this study, the capability of the MBL AIM-1 to evade antibiotic pressure by introducing specific mutations was probed by two alternative methods, i.e. site-saturation mutagenesis (SSM) of active site residues and in vitro evolution. Both approaches demonstrated that a single mutation in AIM-1 can greatly enhance a pathogen's resistance towards broad spectrum antibiotics without significantly compromising the catalytic efficiency of the enzyme...
January 11, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28074221/exploring-the-thermostable-properties-of-halohydrin-dehalogenase-from-agrobacterium-radiobacter-ad1-by-a-combinatorial-directed-evolution-strategy
#18
Zhiyun Wu, Wenfeng Deng, Yapei Tong, Qian Liao, Dongmin Xin, Huashun Yu, Juan Feng, Lixia Tang
As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained...
January 10, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28073929/improving-heterologous-production-of-phenylpropanoids-in-saccharomyces-cerevisiae-by-tackling-an-unwanted-side-reaction-of-tsc13-an-endogenous-double-bond-reductase
#19
Beata Joanna Lehka, Michael Eichenberger, Walden Emil Bjørn-Yoshimoto, Katherina Garcia Vanegas, Nicolaas Buijs, Niels Bjerg Jensen, Jane Dannow Dyekjær, Håvard Jenssen, Ernesto Simon, Michael Naesby
Phenylpropanoids, such as flavonoids and stilbenoids, are of great commercial interest, and their production in Saccharomyces cerevisiae is a very promising strategy. However, to achieve commercially viable production, each step of the process must be optimised. We looked at carbon loss, known to occur in the heterologous flavonoid pathway in yeast, and identified an endogenous enzyme, the enoyl reductase Tsc13, which turned out to be responsible for the accumulation of phloretic acid via reduction of p-coumaroyl-CoA...
January 1, 2017: FEMS Yeast Research
https://www.readbyqxmd.com/read/28064476/chemoselective-coupling-preserves-the-substrate-integrity-of-surface-immobilized-oligonucleotides-for-emulsion-pcr-based-gene-library-construction
#20
Marie L Malone, Valerie J Cavett, Brian M Paegel
Combinatorial bead libraries figure prominently in next-generation sequencing and are also important tools for in vitro evolution. The most common methodology for generating such bead libraries, emulsion PCR (emPCR), enzymatically extends bead-immobilized oligonucleotide PCR primers in emulsion droplets containing a single progenitor library member. Primers are almost always immobilized on beads via noncovalent biotin-streptavidin binding. Here, we describe covalent bead functionalization with primers (∼10(6) primers/2...
January 9, 2017: ACS Combinatorial Science
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