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iterative mutagenesis

Rafael G Saer, Valentyn Stadnytskyi, Nikki C Magdaong, Carrie Goodson, Sergei Savikhin, Robert E Blankenship
In this paper we report the steady-state optical properties of a series of site-directed mutants in the Fenna-Matthews-Olson (FMO) complex of Chlorobaculum tepidum, a photosynthetic green sulfur bacterium. The FMO antenna complex has historically been used as a model system for energy transfer due to the water-soluble nature of the protein, its stability at room temperature, as well as the availability of high-resolution structural data. Eight FMO mutants were constructed with changes in the environment of each of the bacteriochlorophyll a pigments found within each monomer of the homotrimeric FMO complex...
April 2017: Biochimica et Biophysica Acta
Ivan Campeotto, Adi Goldenzweig, Jack Davey, Lea Barfod, Jennifer M Marshall, Sarah E Silk, Katherine E Wright, Simon J Draper, Matthew K Higgins, Sarel J Fleishman
Many promising vaccine candidates from pathogenic viruses, bacteria, and parasites are unstable and cannot be produced cheaply for clinical use. For instance, Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is essential for erythrocyte invasion, is highly conserved among field isolates, and elicits antibodies that neutralize in vitro and protect in an animal model, making it a leading malaria vaccine candidate. However, functional RH5 is only expressible in eukaryotic systems and exhibits moderate temperature tolerance, limiting its usefulness in hot and low-income countries where malaria prevails...
January 31, 2017: Proceedings of the National Academy of Sciences of the United States of America
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
Lee J Sweetlove, Jens Nielsen, Alisdair R Fernie
The goal of increasing crop productivity and nutrient-use efficiency is being addressed by a number of ambitious research projects seeking to re-engineer photosynthetic biochemistry. Many of these projects will require the engineering of substantial changes in fluxes of central metabolism. However, as has been amply demonstrated in simpler systems such as microbes, central metabolism is extremely difficult to rationally engineer. This is because of multiple layers of regulation that operate to maintain metabolic steady state and because of the highly connected nature of central metabolism...
December 22, 2016: Plant Journal: for Cell and Molecular Biology
Karin Bjerre, Mette D Cantor, Jan V Nørgaard, Hanne D Poulsen, Karoline Blaabjerg, Nuria Canibe, Bent B Jensen, Birgitte Stuer-Lauridsen, Bea Nielsen, Patrick M F Derkx
OBJECTIVES: To generate tryptophan-overproducing Bacillus subtilis strains for in situ use in pigs, to reduce the feed cost for farmers and nitrogen pollution. RESULTS: A novel concept has been investigated-to generate B. subtilis strains able to produce tryptophan (Trp) in situ in pigs. Mutagenesis by UV was combined with selection on Trp and purine analogues in an iterative process. Two mutants from different wild types were obtained, mutant 1 (M1) produced 1 mg Trp/l and mutant 2 (M2) 14 mg Trp/l...
February 2017: Biotechnology Letters
Yemin Wang, Zhengsheng Tao, Hualiang Zheng, Fei Zhang, Qingshan Long, Zixin Deng, Meifeng Tao
Many high-value secondary metabolites are assembled by very large multifunctional polyketide synthases or non-ribosomal peptide synthetases encoded by giant genes, for instance, natamycin production in an industrial strain of Streptomyces gilvosporeus. In this study, a large operon reporter-based selection system has been developed using the selectable marker gene neo to report the expression both of the large polyketide synthase genes and of the entire gene cluster, thereby facilitating the selection of natamycin-overproducing mutants by iterative random mutagenesis breeding...
October 13, 2016: Metabolic Engineering
Chengtuo Niu, Linjiang Zhu, Xin Xu, Qi Li
Higher thermostability is required for 1,3-1,4-β-glucanase to maintain high activity under harsh conditions in the brewing and animal feed industries. In this study, a comprehensive and comparative analysis of thermostability in bacterial β-glucanases was conducted through a method named spatial compartmentalization of mutational hotspots (SCMH), which combined alignment of homologous protein sequences, spatial compartmentalization, and molecular dynamic (MD) simulation. The overall/local flexibility of six homologous β-glucanases was calculated by MD simulation and linearly fitted with enzyme optimal enzymatic temperatures...
February 2017: Applied Microbiology and Biotechnology
Jun Huang, Zhen Yu, Mei-Hong Li, Ji-Dong Wang, Hua Bai, Jun Zhou, Yu-Guo Zheng
UNLABELLED: Spinosad, a highly effective insecticide, has an excellent environmental and mammalian toxicological profile. Global market demand for spinosad is huge and growing. However, after much effort, there has been almost no improvement in the spinosad yield from the original producer, Saccharopolyspora spinosa Here, we report the heterologous expression of spinosad using Saccharopolyspora erythraea as a host. The native erythromycin polyketide synthase (PKS) genes in S. erythraea were replaced by the assembled spinosad gene cluster through iterative recombination...
September 15, 2016: Applied and Environmental Microbiology
Kenan Jijakli, Basel Khraiwesh, Weiqi Fu, Liming Luo, Amnah Alzahmi, Joseph Koussa, Amphun Chaiboonchoe, Serdal Kirmizialtin, Laising Yen, Kourosh Salehi-Ashtiani
Through iterative cycles of selection, amplification, and mutagenesis, in vitro selection provides the ability to isolate molecules of desired properties and function from large pools (libraries) of random molecules with as many as 10(16) distinct species. This review, in recognition of a quarter of century of scientific discoveries made through in vitro selection, starts with a brief overview of the method and its history. It further covers recent developments in in vitro selection with a focus on tools that enhance the capabilities of in vitro selection and its expansion from being purely a nucleic acids selection to that of polypeptides and proteins...
August 15, 2016: Methods: a Companion to Methods in Enzymology
Yan Duan, Lina Ba, Jianwei Gao, Xianxing Gao, Dunming Zhu, René M de Jong, Daniel Mink, Iwona Kaluzna, Zhanglin Lin
ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket...
October 2016: Applied Microbiology and Biotechnology
Kai Wu, Hualei Wang, Lifeng Chen, Haiyang Fan, Zhiqiang Zhao, Dongzhi Wei
Enantiopure styrene oxide (SO) and its derivatives are important building blocks for chiral synthesis. In this study, we developed an attractive "1-pot, 2-step" chemoenzymatic approach for producing enantiopure SO with 100 % theoretical yield. This approach involved asymmetric reduction of α-chloroacetophenone by an alcohol dehydrogenase (ADH; step 1), followed by base-induced ring closure (epoxidation) of enantiopure 2-chloro-1-phenylethanol produced by the ADH (step 2). By-product formation during epoxidation was suppressed to <1 % by adding methyl tert-butyl ether (MTBE) as the second phase...
October 2016: Applied Microbiology and Biotechnology
Xiao-Jing Luo, Jian Zhao, Chun-Xiu Li, Yun-Peng Bai, Manfred T Reetz, Hui-Lei Yu, Jian-He Xu
Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling...
November 2016: Biotechnology and Bioengineering
Meng Wang, Huimin Zhao
The advantages of computational design and directed evolution are complementary, and only through combined and iterative use of both approaches, a daunting task such as protein-ligand interaction design, can be achieved efficiently. Here, we describe a systematic strategy to combine structure-guided computational design, iterative site saturation mutagenesis, and yeast two-hybrid system (Y2H)-based phenotypic screening to engineer novel and orthogonal interactions between synthetic ligands and human estrogen receptor α (hERα) for the development of novel gene switches...
2016: Methods in Molecular Biology
Zhoutong Sun, Ylva Wikmark, Jan-E Bäckvall, Manfred T Reetz
Directed evolution of stereo- and regioselective enzymes constitutes a prolific source of catalysts for asymmetric transformations in organic chemistry. In this endeavor (iterative) saturation mutagenesis at sites lining the binding pocket of enzymes has emerged as the method of choice, but uncertainties regarding the question of how to group many residues into randomization sites and how to choose optimal upward pathways persist. Two new approaches promise to beat the numbers problem effectively. One utilizes a single amino acid as building block for the randomization of a 10-residue site, the other also employs only one but possibly different amino acid at each position of a 9-residue site...
April 4, 2016: Chemistry: a European Journal
Yun Hee Choi, Jong Hoon Kim, Bum Seok Park, Byung-Gee Kim
α1,3-Fucosyltransferase (α1,3-FucT) is essential for the biosynthesis of biologically active α1,3-fucosyloligosacchairdes (3-FOs) from human milk oligosaccharides (HMO), particularly 3-fucosyllactose (3-FL) trisaccharide. α1,3-FucT from Helicobacter pylori 26695 (FutA) accepts lactose and LacNAc as glycan acceptors and has a very low level of expression in Escherichia coli, and it shows a low catalytic activity for lactose in the large-scale synthesis of 3-FL. To overcome the poor solubility of FutA, codon optimization, and systematic truncation of the protein at the C-terminus with only one heptad repeat remaining (Δ52 FutA) were conducted to yield 150-200 mg/L of soluble protein of FutA and resulting in more than an 18-fold increase in the 3-FL yield...
August 2016: Biotechnology and Bioengineering
Yanru Liu, Liqing Qiu, Jianzhong Huang, Bingchun Zhao, Zuozhen Wang, Xiaolan Zhu, Yuanyuan Gao, Zhengyu Shu
OBJECTIVE: We improved the thermostability of LipA from Burkholderia cecapia ZYB002 by protein engineering technology to expand the application of lipase LipA. METHOD: On the basis of B-factor value of lipase LipA, series of potential mutation hotspots were selected for iterative saturation mutagenesis and the corresponding small mutation gene libraries were then constructed to screen the hyperthermal variants. RESULTS: From the above mutation libraries, we obtained a series of mutants whose enzyme half-life at 55 degrees C increased by 1...
June 4, 2015: Wei Sheng Wu Xue Bao, Acta Microbiologica Sinica
Christoph G W Gertzen, Lina Spomer, Sander H J Smits, Dieter Häussinger, Verena Keitel, Holger Gohlke
TGR5 (Gpbar-1, M-Bar) is a class A G-protein coupled bile acid-sensing receptor predominately expressed in brain, liver and gastrointestinal tract, and a promising drug target for the treatment of metabolic disorders. Due to the lack of a crystal structure of TGR5, the development of TGR5 agonists has been guided by ligand-based approaches so far. Three binding mode models of bile acid derivatives have been presented recently. However, they differ from one another in terms of overall orientation or with respect to the location and interactions of the cholane scaffold, or cannot explain all results from mutagenesis experiments...
November 2, 2015: European Journal of Medicinal Chemistry
Jinxiang Yuan, Ming Li, Yasaira Rodriguez Torres, Courtney S Galle, Jeffery L Meier
UNLABELLED: Triggers and regulatory pathways that effectively link human cytomegalovirus (HCMV) major immediate early (MIE) latent-lytic switch activation with progeny production are incompletely understood. In the quiescently infected human NTera2 cell model of primitive neural stem cells, we found that costimulation with vasoactive intestinal peptide (V) and phorbol ester (P) synergistically activated viral infection, but this effect waned over time. Coupling retinoic acid (R), an inducer of neuronal differentiation, to VP pulse stimulation attenuated the decline in viral activity and promoted the spread of the active infection through concentric layers of neighboring cells as cellular differentiation progressed...
December 2015: Journal of Virology
Quy Le, Nancy Maizels
AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance...
September 2015: PLoS Genetics
Xin Yin, Jian-Fang Li, Chun-Juan Wang, Die Hu, Qin Wu, Ying Gu, Min-Chen Wu
Feruloyl or ferulic acid esterase (Fae, EC catalyzes the hydrolysis of ester bonds between polysaccharides and phenolic acid compounds in xylan side chain. In this study, the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii was increased by iterative saturation mutagenesis (ISM). Two amino acids, Ser33 and Asn92, were selected for saturation mutagenesis according to the B-factors analyzed by B-FITTER software and ΔΔG values predicted by PoPMuSiC algorithm. After screening the saturation mutagenesis libraries constructed in Pichia pastoris, 15 promising variants were obtained...
December 2015: Applied Microbiology and Biotechnology
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