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"Saturation mutagenesis"

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https://www.readbyqxmd.com/read/28109425/beyond-the-natural-proteome-nondegenerate-saturation-mutagenesis-methodologies-and-advantages
#1
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
#2
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
#3
Huifang Yin, Tjaard Pijning, Xiangfeng Meng, Lubbert Dijkhuizen, Sander Sebastiaan 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...
January 16, 2017: Biochemistry
https://www.readbyqxmd.com/read/28074907/insights-into-an-evolutionary-strategy-leading-to-antibiotic-resistance
#4
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
#5
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
#6
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 S. cerevisiae is a very promising strategy. However, to achieve commercially viable production, each step of the process must be optimized. 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 10, 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
#7
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
https://www.readbyqxmd.com/read/28050632/amino-acid-substitutions-in-random-mutagenesis-libraries-lessons-from-analyzing-3000-mutations
#8
Jing Zhao, Victorine Josiane Frauenkron-Machedjou, Tsvetan Kardashliev, Anna Joëlle Ruff, Leilei Zhu, Marco Bocola, Ulrich Schwaneberg
The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis...
January 3, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28049717/a-novel-hect-ubiquitin-ligase-regulating-chemotaxis-and-development-in-dictyostelium-discoideum
#9
Barbara Pergolizzi, Enrico Bracco, Salvatore Bozzaro
Cyclic AMP binding to G protein-coupled receptors orchestrates chemotaxis and development in Dictyostelium. By activating the RasC-TORC2-AKT/PKB module, cAMP regulates cell polarization during chemotaxis. TORC2 also mediates GPCR-dependent stimulation of adenylyl cyclase A (ACA), enhancing cAMP relay and developmental gene expression. Thus, mutants defective in the TORC2 Pia/Rictor subunit are impaired in chemotaxis and development. Near-saturation mutagenesis of a Pia/Rictor mutant by random gene disruption led to selection of two suppressor mutants, in which spontaneous chemotaxis and development were restored...
January 3, 2017: Journal of Cell Science
https://www.readbyqxmd.com/read/28044225/simple-mssm-a-simple-and-efficient-method-for-simultaneous-multi-site-saturation-mutagenesis
#10
Feng Cheng, Jian-Miao Xu, Chao Xiang, Zhi-Qiang Liu, Li-Qing Zhao, Yu-Guo Zheng
OBJECTIVE: To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. RESULTS: A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons...
January 2, 2017: Biotechnology Letters
https://www.readbyqxmd.com/read/28044007/modification-of-the-peroxygenative-peroxidative-activity-ratio-in-the-unspecific-peroxygenase-from-agrocybe-aegerita-by-structure-guided-evolution
#11
Diana M Mate, Miguel A Palomino, Patricia Molina-Espeja, Javier Martin-Diaz, Miguel Alcalde
Unspecific peroxygenase (UPO) is a heme-thiolate peroxidase capable of performing with high-selectivity C-H oxyfunctionalizations of great interest in organic synthesis through its peroxygenative activity. However, the convergence of such activity with an unwanted peroxidative activity encumbers practical applications. In this study, we have modified the peroxygenative:peroxidative activity ratio (P:p ratio) of UPO from Agrocybe aegerita by structure-guided evolution. Several flexible loops (Glu1-Pro35, Gly103-Asp131, Ser226-Gly243, Gln254-Thr276 and Ty293-Arg327) were selected on the basis on their B-factors and ΔΔG values...
January 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28007937/directed-evolution-of-p450cin-for-mediated-electron-transfer
#12
Ketaki D Belsare, Thomas Horn, Anna Joëlle Ruff, Ronny Martinez, Anders Magnusson, Dirk Holtmann, Jens Schrader, Ulrich Schwaneberg
Directed evolution is a powerful method to optimize enzyme properties for application demands. Interesting targets are P450 monooxygenases which catalyze the stereo- and regiospecific hydroxylation of chemically inert C-H bonds. Synthesis employing P450s under cell-free reaction conditions is limited by low total turnover numbers, enzyme instability, low product yields and the requirement of the expensive co-factor NADPH. Bioelectrocatalysis is an alternative to replace NADPH in cell-free P450-catalyzed reactions...
December 22, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28005308/second-generation-engineering-of-thermostable-transketolase-tkgst-for-aliphatic-aldehyde-acceptors-with-improved-and-reversed-stereoselectivity
#13
Chaoqiang Zhou, Thangavelu Saravanan, Marion Lorillière, Dongzhi Wei, Franck Charmantray, Laurence Hecquet, Wolf-Dieter Fessner, Dong Yi
Transketolase from Geobacillus stearothermophilus (TKgst) is a novel thermostable enzyme with notable high activity and stability at elevated temperatures, but accepts non-α-hydroxylated aldehydes only with low efficiency. Here, we report on a protein engineering study by double-site saturation mutagenesis at either Leu191 or Phe435 in combination with Asp470 of TKgst, which are responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with up to 7...
December 22, 2016: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28005186/reengineering-of-the-feedback-inhibition-enzyme-n-acetyl-l-glutamate-kinase-to-enhance-l-arginine-production-in-corynebacterium-crenatum
#14
Jingjing Zhang, Meijuan Xu, Xiaoxun Ge, Xian Zhang, Taowei Yang, Zhenghong Xu, Zhiming Rao
N-acetyl-L-glutamate kinase (NAGK) catalyzes the second step of L-arginine biosynthesis and is inhibited by L-arginine in Corynebacterium crenatum. To ascertain the basis for the arginine sensitivity of CcNAGK, residue E19 which located at the entrance of the Arginine-ring was subjected to site-saturated mutagenesis and we successfully illustrated the inhibition-resistant mechanism. Typically, the E19Y mutant displayed the greatest deregulation of L-arginine feedback inhibition. An equally important strategy is to improve the catalytic activity and thermostability of CcNAGK...
December 22, 2016: Journal of Industrial Microbiology & Biotechnology
https://www.readbyqxmd.com/read/28000046/semi-rational-engineering-of-leucine-dehydrogenase-for-l-2-aminobutyric-acid-production
#15
Jian-Miao Xu, Feng Cheng, Fang-Tian Fu, Hai-Feng Hu, Yu-Guo Zheng
L-2-aminobutyric acid (L-ABA) as a precursor for the anticonvulsant and the antituberculotic is a key intermediate in the chemical and pharmaceutical industries. Recently, leucine dehydrogenase (LeuDH) with NAD(+) regeneration was developed for L-ABA production on a large scale. Previously, the L-ABA yield was improved by optimizing conversion conditions, including cofactor regeneration and enzyme immobilization but not protein engineering on LeuDH due to lacking an applicable high-throughput screening (HTS) method...
December 20, 2016: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/27978710/interrogating-key-positions-of-size-reduced-tale-repeats-reveals-a-programmable-sensor-of-5-carboxylcytosine
#16
Sara Maurer, Mario Giess, Oliver Koch, Daniel Summerer
Transcription-activator-like effector (TALE) proteins consist of concatenated repeats that recognize consecutive canonical nucleobases of DNA via the major groove in a programmable fashion. Since this groove displays unique chemical information for the four human epigenetic cytosine nucleobases, TALE repeats with epigenetic selectivity can be engineered, with potential to establish receptors for the programmable decoding of all human nucleobases. TALE repeats recognize nucleobases via key amino acids in a structurally conserved loop whose backbone is positioned very close to the cytosine 5-carbon...
December 16, 2016: ACS Chemical Biology
https://www.readbyqxmd.com/read/27941803/genome-wide-mapping-of-mutations-at-single-nucleotide-resolution-for-protein-metabolic-and-genome-engineering
#17
Andrew D Garst, Marcelo C Bassalo, Gur Pines, Sean A Lynch, Andrea L Halweg-Edwards, Rongming Liu, Liya Liang, Zhiwen Wang, Ramsey Zeitoun, William G Alexander, Ryan T Gill
Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale...
January 2017: Nature Biotechnology
https://www.readbyqxmd.com/read/27935281/engineering-cyclohexanone-monooxygenase-for-the-production-of-methyl-propanoate
#18
Hugo L van Beek, Elvira Romero, Marco W Fraaije
A previous study showed that cyclohexanone monooxygenase from Acinetobacter calcoaceticus (AcCHMO) catalyzes the Baeyer-Villiger oxidation of 2-butanone, yielding ethyl acetate and methyl propanoate as products. Methyl propanoate is of industrial interest as a precursor of acrylic plastic. Here, various residues near the substrate and NADP(+) binding sites in AcCHMO were subjected to saturation mutagenesis to enhance both the activity on 2-butanone and the regioselectivity toward methyl propanoate. The resulting libraries were screened using whole cell biotransformations, and headspace gas chromatography-mass spectrometry was used to identify improved AcCHMO variants...
December 20, 2016: ACS Chemical Biology
https://www.readbyqxmd.com/read/27923373/microbial-production-of-next-generation-stevia-sweeteners
#19
Kim Olsson, Simon Carlsen, Angelika Semmler, Ernesto Simón, Michael Dalgaard Mikkelsen, Birger Lindberg Møller
BACKGROUND: The glucosyltransferase UGT76G1 from Stevia rebaudiana is a chameleon enzyme in the targeted biosynthesis of the next-generation premium stevia sweeteners, rebaudioside D (Reb D) and rebaudioside M (Reb M). These steviol glucosides carry five and six glucose units, respectively, and have low sweetness thresholds, high maximum sweet intensities and exhibit a greatly reduced lingering bitter taste compared to stevioside and rebaudioside A, the most abundant steviol glucosides in the leaves of Stevia rebaudiana...
December 7, 2016: Microbial Cell Factories
https://www.readbyqxmd.com/read/27887027/expansion-of-the-substrate-range-of-the-gentisate-1-2-dioxygenase-from-corynebacterium-glutamicum-for-the-conversion-of-monohydroxylated-benzoates
#20
Erik Eppinger, Andreas Stolz
The gentisate 1,2-dioxygenases (GDOs) from Corynebacterium glutamicum and various other organisms oxidatively cleave the aromatic nucleus of gentisate (2,5-dihydroxybenzoate), but are not able to convert salicylate (2-hydroxybenzoate). In contrast, the α-proteobacterium Pseudaminobacter salicylatoxidans synthesises an enzyme ('salicylate dioxygenase', SDO) which cleaves gentisate, but also (substituted) salicylate(s). Sequence comparisons showed that the SDO belongs to a group of GDOs mainly originating from Gram-positive bacteria which also include the GDO from C...
January 2017: Protein Engineering, Design & Selection: PEDS
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