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"Saturation mutagenesis"

Gur Pines, Ryan T Gill
Saturation mutagenesis is conveniently located between the two extremes of protein engineering, namely random mutagenesis, and rational design. It involves mutating a confined number of target residues to other amino acids, and hence requires knowledge regarding the sites for mutagenesis, but not their final identity. There are many different strategies for performing and designing such experiments, ranging from simple single degenerate codons to codon collections that code for distinct sets of amino acids...
2018: Methods in Molecular Biology
Zhi-Qiang Liu, Ming-Ming Lu, Xin-Hong Zhang, Feng Cheng, Jian-Miao Xu, Ya-Ping Xue, Li-Qun Jin, Yuan-Shan Wang, Yu-Guo Zheng
Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacturing of chelating agents, glyphosate herbicides and surfactants. To improve activity and tolerance to the substrate for IDA production, Acidovorax facilis nitrilase was selected for further modification by the gene site saturation mutagenesis method. After screened by a two-step screening method, the best mutant (Mut-F168V/T201N/S192F/M191T/F192S) was selected. Compared to the wild-type nitrilase, Mut-F168V/T201N/S192F/M191T/F192S showed 136% improvement in specific activity...
May 9, 2018: International Journal of Biological Macromolecules
Min Zhang, Chengqi Wang, Thomas D Otto, Jenna Oberstaller, Xiangyun Liao, Swamy R Adapa, Kenneth Udenze, Iraad F Bronner, Deborah Casandra, Matthew Mayho, Jacqueline Brown, Suzanne Li, Justin Swanson, Julian C Rayner, Rays H Y Jiang, John H Adams
Severe malaria is caused by the apicomplexan parasite Plasmodium falciparum. Despite decades of research, the distinct biology of these parasites has made it challenging to establish high-throughput genetic approaches to identify and prioritize therapeutic targets. Using transposon mutagenesis of P. falciparum in an approach that exploited its AT-rich genome, we generated more than 38,000 mutants, saturating the genome and defining mutability and fitness costs for over 87% of genes. Of 5399 genes, our study defined 2680 genes as essential for optimal growth of asexual blood stages in vitro...
May 4, 2018: Science
Hailin Chen, Meijie Li, Changqing Liu, Haibo Zhang, Mo Xian, Huizhou Liu
BACKGROUND: Lycopene is a terpenoid pigment that has diverse applications in the food and medicine industries. A prospective approach for lycopene production is by metabolic engineering in microbial hosts, such as Escherichia coli. Isopentenyl diphosphate isomerase (IDI, E.C. is one of the rate-limiting enzymes in the lycopene biosynthetic pathway and one major target during metabolic engineering. The properties of IDIs differ depending on the sources, but under physiological conditions, IDIs are limited by low enzyme activity, short half-life and weak substrate affinity...
April 30, 2018: Microbial Cell Factories
Yunus Ensari, Gaurao V Dhoke, Mehdi D Davari, Anna Joelle Ruff, Ulrich Schwaneberg
Positions identified in directed evolution campaigns or by (semi-) rational design can be recombined iteratively or simultaneously. Iterative recombination (CAST method) yielded many success stories and is beneficially used if screening capabilities are limited (four iterative SSMs generated 20x4= 80 different enzyme variants). Simultaneous site saturation mutagenesis offers a significantly higher diversity (204=160000 variants) and enables to find higher improvements especially if the selected positions are in close proximity to each other (cooperative effects)...
April 30, 2018: Chembiochem: a European Journal of Chemical Biology
Taylor L Mighell, Sara Evans-Dutson, Brian J O'Roak
Phosphatase and tensin homolog (PTEN) is a tumor suppressor frequently mutated in diverse cancers. Germline PTEN mutations are also associated with a range of clinical outcomes, including PTEN hamartoma tumor syndrome (PHTS) and autism spectrum disorder (ASD). To empower new insights into PTEN function and clinically relevant genotype-phenotype relationships, we systematically evaluated the effect of PTEN mutations on lipid phosphatase activity in vivo. Using a massively parallel approach that leverages an artificial humanized yeast model, we derived high-confidence estimates of functional impact for 7,244 single amino acid PTEN variants (86% of possible)...
April 23, 2018: American Journal of Human Genetics
Rongming Liu, Liya Liang, Alaksh Choudhury, Marcelo C Bassalo, Andrew D Garst, Katia Tarasava, Ryan T Gill
Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in Escherichia coli for improved production of the platform chemical 3-hydroxypropionic acid (3HP)...
April 14, 2018: Metabolic Engineering
Li-Qiang Fan, Ming-Wei Li, Yong-Jun Qiu, Qi-Ming Chen, Si-Jing Jiang, Yu-Jie Shang, Li-Ming Zhao
Gamma-amino butyric acid (GABA) is an important bio-product used in pharmaceuticals, functional foods, and a precursor of the biodegradable plastic polyamide 4 (Nylon 4). Glutamate decarboxylase B (GadB) from Escherichia. coli is a highly active biocatalyst that can convert L-glutamate to GABA. However, its practical application is limited by the poor thermostability and only active under acidic conditions of GadB. In this study, we performed site-directed saturation mutagenesis of the N-terminal residues of GadB from Escherichia coli to improve its thermostability...
April 13, 2018: Journal of Biotechnology
Johan A Kers, Robert E Sharp, Anthony W Defusco, Jae H Park, Jin Xu, Mark E Pulse, William J Weiss, Martin Handfield
Lantibiotics offer an untapped pipeline for the development of novel antibiotics to treat serious Gram-positive (+) infections including Clostridium difficile . Mutacin 1140 (MU1140) is a lantibiotic produced by Streptococcus mutans and acts via a novel mechanism of action, which may limit the development of resistance. This study sought to identify a lead compound for the treatment of C. difficile associated diarrhea (CDAD). Compounds were selected from a saturation mutagenesis library of 418 single amino acid variants of MU1140...
2018: Frontiers in Microbiology
Lin Ge, Dongdong Li, Tao Wu, Linguo Zhao, Gang Ding, Zhenzhong Wang, Wei Xiao
The α-L-Rhamnosidase is an important enzyme with applications in the food and pharmaceutical industries because it can release terminal L-rhamnose residues from various natural products. In this study, the B-factor-saturation mutagenesis strategy was used to increase the thermostability of α-L-rhamnosidase from Aspergillus terreus. The D594Q and G827K/D594Q mutant enzymes were obtained by screening a series of mutants; they prolonged the half-life of the enzyme at 70 °C by 2.1-fold and 2.3-fold, respectively...
March 29, 2018: Journal of Biotechnology
Fei Zheng, Tao Tu, Xiaoyu Wang, Yuan Wang, Rui Ma, Xiaoyun Su, Xiangming Xie, Bin Yao, Huiying Luo
Background: Cellulases of glycosyl hydrolase (GH) family 5 share a (β/α)8 TIM-barrel fold structure with eight βα loops surrounding the catalytic pocket. These loops exposed on the surface play a vital role in protein functions, primarily due to the interactions of some key amino acids with solvent and ligand molecules. It has been reported that motions of these loops facilitate substrate access and product release, and loops 6 and 7 located at the substrate entrance of the binding pocket promote proton transfer reaction at the catalytic site motions...
2018: Biotechnology for Biofuels
Chiaki Hori, Kenta Oishi, Ken'ichiro Matsumoto, Seiichi Taguchi, Toshihiko Ooi
In our previous study, artificial polyhydroxyalkanoate (PHA) poly[(R)-2-hydroxybutyrate] [P(2HB)] was successfully biosynthesized from racemic 2HB in recombinant Escherichia coli using an engineered PHA synthase, PhaC1Ps (S325T/Q481K). Although P(2HB) has promising material properties, the low level of polymer production was a drawback. In this study, we performed directed evolution of PhaC1Ps towards enhanced P(2HB) accumulation in E. coli by site-directed dual saturation mutagenesis at the positions 477 and 481, which was known for their potential in enhancing natural PHA accumulation...
March 21, 2018: Journal of Bioscience and Bioengineering
Feng Cheng, Jianhua Yang, Marco Bocola, Ulrich Schwaneberg, Leilei Zhu
Protein engineering of enzyme loop regions is an effective strategy to improve enzymatic properties. Previous studies that aimed to boost the activity of PpADI (an arginine deiminase from Pseudomonas plecoglossicida) under physiological conditions yielded several significantly improved variants that harbor substitutions predominantly located in active-site-decorating loops. A multi-site saturation mutagenesis at four positions in loop 1 (37, 38, 42, and 43) and three positions in loop 4 (402, 403, and 404) was performed to elucidate the importance of these loops in modulating the substrate affinity of PpADI...
March 19, 2018: Biochemical and Biophysical Research Communications
Jung-Hoon Bae, Bong Hyun Sung, Jung-Hoon Sohn
The 56th residue of ribosomal protein L42 (Rpl42) determines the sensitivity of yeast cells to the antibiotic cycloheximide (CYH). In this study, we identified the relationship between the 56th residue of Rpl42 and the function of the ribosome by site saturation mutagenesis. The resulting 20 RPL42 mutants harbouring one of 20 amino acids at the 56th residue were classified into five groups: sensitive to CYH (RPL42aP); weak resistance (RPL42aA, RPL42aM, RPL42aC, RPL42aN, RPL42aD, RPL42aS, and RPL42aT), moderate resistance (RPL42aL, RPL42aI, RPL42aV, RPL42aG, and RPL42aH), and strong resistance (RPL42aQ, RPL42aE, RPL42aR, and RPL42aK) to CYH; and non-functional (RPL42aF, RPL42aY, and RPL42aW)...
March 16, 2018: FEMS Microbiology Letters
John B McArthur, Hai Yu, Nova Tasnima, Christie M Lee, Andrew J Fisher, Xi Chen
The lack of α2-6-linkage specific sialidases limits the structural and functional studies of sialic acid-containing molecules. Photobacterium damselae α2-6-sialyltransferase (Pd2,6ST) was shown previously to have α2-6-specific, but weak, sialidase activity. Here we develop a high-throughput blue-white colony screening method to identify Pd2,6ST mutants with improved α2-6-sialidase activity from mutant libraries generated by sequential saturation mutagenesis. A triple mutant (Pd2,6ST S232L/T356S/W361F) has been identified with 101-fold improved activity, high α2-6-sialyl linkage selectivity, good activity in cleaving two common sialic acid forms N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc)...
March 15, 2018: ACS Chemical Biology
Alessandra Piccirilli, Paola Sandra Mercuri, Moreno Galleni, Massimiliano Aschi, André Matagne, Gianfranco Amicosante, Mariagrazia Perilli
GES-type β-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the Ω-loop of GES-1 and GES-5 was investigated. GES-1P174E and GES-5P174E mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1P174E and GES-5P174E mutants exhibited lower k cat and k cat / Km values for cephalosporins and penicillins. Concerning carbapenems, GES-1P174E shared higher k cat values but lower Km values than those calculated for GES-1...
May 2018: Antimicrobial Agents and Chemotherapy
Kritika Gupta, Raghavan Varadarajan
Where convenient phenotypic readouts are available, saturation mutagenesis coupled to deep sequencing provides a rapid and facile method to infer sequence determinants of protein structure, stability and function. We provide brief descriptions and currently available options for the various steps involved, and mention limitations of current implementations. We also highlight recent applications such as estimating relative stabilities and affinities of protein variants, mapping epitopes, protein model discrimination and prediction of mutant phenotypes...
March 2, 2018: Current Opinion in Structural Biology
Fucheng Zhu, Tianyue Jiang, Bin Wu, Bingfang He
Metalloprotease PT121Y114S , an effective catalyst for Z-aspartame synthesis under the substrate (Z-Asp:l-Phe-OMe) molar ratio of 1:5, was obtained previously. Herein, a computational strategy combining molecular dynamics simulation of the enzyme-substrate complex with binding free energy (ΔG) calculations was established to guide the further engineering of PT121Y114S . One His224 residue proximal to the PT121Y114S active site was selected on the basis of the difference in ΔG decomposition of PT121Y114S toward l-Phe-NH2 and l-Phe-OMe...
July 1, 2018: Food Chemistry
Shubhangi Kaushik, Sérgio M Marques, Prashant Khirsariya, Kamil Paruch, Lenka Libichova, Jan Brezovsky, Zbynek Prokop, Radka Chaloupkova, Jiri Damborsky
The traditional way of rationally engineering enzymes to change their biocatalytic properties utilizes the modifications of their active sites. Another emerging approach is the engineering of structural features involved in the exchange of ligands between buried active sites and the surrounding solvent. However, surprisingly little is known about the effects of mutations that alter the access tunnels on the enzymes' catalytic properties, and how these tunnels should be redesigned to allow fast passage of cognate substrates and products...
April 2018: FEBS Journal
Chao Wang, Shuang Chen, Hong-Bin Zhang, Yao Li, Xue-Qin Hu
Dextran produced by dextransucrase hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown. Previous studies have investigated the relationships between structure and linkage specificity of the dextransucrase DSR from Leuconostoc mesenteroides by the site-directed mutagenesis of the catalytic pocket. The glycosidic linkage of dextran produced by mutant enzymes changed slightly by 3% to 20%. The mutagenesis dextransucrases were constructed by inserting an amino acid into a catalytic pocket to investigate the product specificities of dextransucrase thoroughly...
June 2018: International Journal of Biological Macromolecules
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