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"Saturation mutagenesis"

Gur Pines, James D Winkler, Assaf Pines, Ryan T Gill
The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations...
November 14, 2017: MBio
Guangyue Li, Maximilian J L J Fürst, Hamid Reza Mansouri, Anna K Ressmann, Adriana Ilie, Florian Rudroff, Marko D Mihovilovic, Marco W Fraaije, Manfred T Reetz
Baeyer-Villiger monooxygenases (BVMOs) and evolved mutants have been shown to be excellent biocatalysts in many stereoselective Baeyer-Villiger transformations, but industrial applications are rare which is partly due to the insufficient thermostability of BVMOs under operating conditions. In the present study, the substrate scope of the recently discovered thermally stable BVMO, TmCHMO from Thermocrispum municipale, was studied. This revealed that the wild-type (WT) enzyme catalyzes the oxidation of a variety of structurally different ketones with notable activity and enantioselectivity, including the desymmetrization of 4-methylcyclohexanone (99% ee, S)...
November 13, 2017: Organic & Biomolecular Chemistry
Eunok Jung, Beom Gi Park, Hee-Wang Yoo, Joonwon Kim, Kwon-Young Choi, Byung-Gee Kim
CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed saturation mutagenesis was attempted using a semi-rational approach that combined structure-based computational analysis and subsequent saturation mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover number (TTN) and catalytic efficiency (k cat/K M) toward palmitic acid compared to wild-type, respectively...
November 9, 2017: Applied Microbiology and Biotechnology
Yanbing Zhu, Chaochao Qiao, Hebin Li, Lijun Li, Anfeng Xiao, Hui Ni, Zedong Jiang
This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A total of 10 single-site mutants were chosen using the PoPMuSiC program, and two mutants of K253N and P314T showed enhanced thermal stability. By saturation mutagenesis and thermostability analysis, K253H and P314T were the best mutants at the two sites. Combinational mutations of K253H, P314T and H260L were subsequently introduced, and the best mutant of K253H/H260L was selected. Thermal inactivation analysis showed the half-life (t1/2) value at 55°C for K253H/H260L was 7...
November 4, 2017: International Journal of Biological Macromolecules
Belén Infanzón, Pablo H Sotelo, Josefina Martínez, Pilar Diaz
Rhodococcus sp CR-53 lipase LipR was the first characterized member of bacterial lipase family X. Interestingly, LipR displays some similarity with α/β-hydrolases of the C. antartica lipase A (CAL-A)-like superfamily (abH38), bearing a Y-type oxyanion hole, never found before among bacterial lipases. In order to explore this unusual Y-type oxyanion hole, and to improve LipR performance, two modification strategies based on site directed or saturation mutagenesis were addressed. Initially, a small library of mutants was designed to convert LipR Y-type oxyanion hole (YDS) into one closer to those most frequently found in bacteria (GGG(X))...
January 2018: Enzyme and Microbial Technology
Thomas M Schmitt, David H Aggen, Kumiko Ishida-Tsubota, Sebastian Ochsenreither, David M Kranz, Philip D Greenberg
Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells on antigen-expressing feeder cells to develop higher-affinity TCRs...
November 6, 2017: Nature Biotechnology
Yuta Katano, Tongyang Li, Misato Baba, Miyo Nakamura, Masaaki Ito, Kenji Kojima, Teisuke Takita, Kiyoshi Yasukawa
We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity...
November 6, 2017: Bioscience, Biotechnology, and Biochemistry
Arong Jung, Dhanarajan Rajakumar, Bong-June Yoon, Bradley J Baker
Saturation mutagenesis was performed on a single position in the voltage-sensing domain (VSD) of a genetically encoded voltage indicator (GEVI). The VSD consists of four transmembrane helixes designated S1-S4. The V220 position located near the plasma membrane/extracellular interface had previously been shown to affect the voltage range of the optical signal. Introduction of polar amino acids at this position reduced the voltage-dependent optical signal of the GEVI. Negatively charged amino acids slightly reduced the optical signal by 33 percent while positively charge amino acids at this position reduced the optical signal by 80%...
October 2017: Experimental Neurobiology
Xueying Wang, Lei Wang, Xinping Lin, Xiaobing Yang, Wujun Liu, Zongbao K Zhao
Directed evolution-based protein engineering usually generates large library contained insoluble mutants because of structural disturbance by mutation. To reduce the workload and costs, it is crucial to identify and eliminate those insoluble variants prior to dedicated analysis. Here, we demonstrate a method to visualize soluble protein mutants by using monomeric red fluorescent protein (mRFP) as a fusion tag. A plasmid was devised to express nicotinic acid mononucleotide adenylyltransferase (NadD) fused with a GGGS-linked mRFP tag at the C-terminus...
October 30, 2017: Applied Biochemistry and Biotechnology
Zhongyi Cheng, Lukasz Peplowski, Wenjing Cui, Yuanyuan Xia, Zhongmei Liu, Jialei Zhang, Michihiko Kobayashi, Zhemin Zhou
Optically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high-value chiral chemicals is often time-, money- and resource-intensive; traditional semi-rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot-spot residues. In this study, through ligand-protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity towards rac-mandelonitrile (MAN)...
October 28, 2017: Biotechnology and Bioengineering
Leyuan Ma, Jeffrey I Boucher, Janet Paulsen, Sebastian Matuszewski, Christopher A Eide, Jianhong Ou, Garrett Eickelberg, Richard D Press, Lihua Julie Zhu, Brian J Druker, Susan Branford, Scot A Wolfe, Jeffrey D Jensen, Celia A Schiffer, Michael R Green, Daniel N Bolon
Developing tools to accurately predict the clinical prevalence of drug-resistant mutations is a key step toward generating more effective therapeutics. Here we describe a high-throughput CRISPR-Cas9-based saturated mutagenesis approach to generate comprehensive libraries of point mutations at a defined genomic location and systematically study their effect on cell growth. As proof of concept, we mutagenized a selected region within the leukemic oncogene BCR-ABL1 Using bulk competitions with a deep-sequencing readout, we analyzed hundreds of mutations under multiple drug conditions and found that the effects of mutations on growth in the presence or absence of drug were critical for predicting clinically relevant resistant mutations, many of which were cancer adaptive in the absence of drug pressure...
October 31, 2017: Proceedings of the National Academy of Sciences of the United States of America
Feng-Jiao Zhao, Yun Jin, Zhongchuan Liu, Chao Guo, Tong-Biao Li, Zi-Yi Li, Ganggang Wang, Zhong-Liu Wu
ChKRED20 is an efficient and robust anti-Prelog ketoreductase that can catalyze the reduction of ketones to chiral alcohols as pharmaceutical intermediates with great industrial potential. To overcome its limitation on the bioreduction of ortho-substituted acetophenone derivatives, the X-ray crystal structure of the apo-enzyme of ChKRED20 was determined at a resolution of 1.85 Å and applied to the molecular modeling and reshaping of the catalytic cavity via three rounds of iterative saturation mutagenesis together with alanine scanning and recombination...
October 24, 2017: Applied Microbiology and Biotechnology
Alexander Dennig, Alexandra Maria Weingartner, Tsvetan Kardashliev, Christina Andrea Müller, Erika Tassano, Martin Schürmann, Anna Joëlle Ruff, Ulrich Schwaneberg
The aromatic hydroxylation of pseudocumene (1a) and mesitylene (1b) with P450 BM3 yields key phenolic building blocks for α-tocopherol synthesis. The P450 BM3 wild-type (WT) catalyzed selective aromatic hydroxylation of 1b (94%), whereas 1a was hydroxylated to a large extent on benzylic positions (46-64%). Site-saturation mutagenesis generated a new P450 BM3 mutant, herein named "variant M3" (R47S, Y51W, A330F, I401M), with significantly increased coupling efficiency (3 to 8-fold) and activity (75 to 230-fold) for 1a and 1b conversion...
October 9, 2017: Chemistry: a European Journal
Emil Hamnevik, Thilak Reddy Enugala, Dirk Maurer, Siphosethu Ntuku, Ana Oliveira, Doreen Dobritzsch, Mikael Widersten
Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A...
September 30, 2017: FEBS Journal
Ulrich Elling, Reiner A Wimmer, Andreas Leibbrandt, Thomas Burkard, Georg Michlits, Alexandra Leopoldi, Thomas Micheler, Dana Abdeen, Sergei Zhuk, Irene M Aspalter, Cornelia Handl, Julia Liebergesell, Maria Hubmann, Anna-Maria Husa, Manuela Kinzer, Nicole Schuller, Ellen Wetzel, Nina van de Loo, Jorge Arturo Zepeda Martinez, David Estoppey, Ralph Riedl, Fengtang Yang, Beiyuan Fu, Thomas Dechat, Zoltán Ivics, Chukwuma A Agu, Oliver Bell, Dieter Blaas, Holger Gerhardt, Dominic Hoepfner, Alexander Stark, Josef M Penninger
The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers...
October 5, 2017: Nature
Pia Skoczinski, Kristina Volkenborn, Alexander Fulton, Anuseema Bhadauriya, Christina Nutschel, Holger Gohlke, Andreas Knapp, Karl-Erich Jaeger
BACKGROUND: Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production...
September 25, 2017: Microbial Cell Factories
Benjamin J Cole, Meghan E Feltcher, Robert J Waters, Kelly M Wetmore, Tatiana S Mucyn, Elizabeth M Ryan, Gaoyan Wang, Sabah Ul-Hasan, Meredith McDonald, Yasuo Yoshikuni, Rex R Malmstrom, Adam M Deutschbauer, Jeffery L Dangl, Axel Visel
Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system...
September 2017: PLoS Biology
Adam N Yadon, Kashmeel Maharaj, John H Adamson, Yi-Pin Lai, James C Sacchettini, Thomas R Ioerger, Eric J Rubin, Alexander S Pym
Tuberculosis chemotherapy is dependent on the use of the antibiotic pyrazinamide, which is being threatened by emerging drug resistance. Resistance is mediated through mutations in the bacterial gene pncA. Methods for testing pyrazinamide susceptibility are difficult and rarely performed, and this means that the full spectrum of pncA alleles that confer clinical resistance to pyrazinamide is unknown. Here, we performed in vitro saturating mutagenesis of pncA to generate a comprehensive library of PncA polymorphisms resultant from a single-nucleotide polymorphism...
September 19, 2017: Nature Communications
Shaima Kammoonah, Brinda Prasad, Priyadarshini Balaraman, Hemanshu Mundhada, Ulrich Schwaneberg, Erika Plettner
Cytochrome P450cam (a camphor hydroxylase) from the soil bacterium Pseudomonas putida shows potential importance in environmental applications such as the degradation of chlorinated organic pollutants. Seven P450cam mutants generated from Sequence Saturation Mutagenesis (SeSaM) and isolated by selection on minimal media with either 3-chloroindole or the insecticide endosulfan were studied for their ability to oxidize of 3-chloroindole to isatin. The wild-type enzyme did not accept 3-chloroindole as a substrate...
September 18, 2017: Biochimica et Biophysica Acta
Abbas Ismail, Rosli Md Illias
The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type...
December 2017: Journal of Industrial Microbiology & Biotechnology
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