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iterative saturated mutagenesis

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
Ge Qu, Jing Zhao, Ping Zheng, Jibin Sun, Zhoutong Sun
Screening is the bottleneck of directed evolution. In order to address this problem, a series of novel semi-rational designed strategies have been developed based on combinatorial active-site saturation test and iterative saturation mutagenesis, including single code saturation mutagenesis, double code saturation mutagenesis and triple code saturation mutagenesis. By creation of "small and smart" high qualified mutant libraries and combinatorial mutagenesis of specific sites, these new strategies have been successfully applied in multiparameter optimization, e...
January 25, 2018: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Shu-Ping Zou, Yu-Guo Zheng, Qun Wu, Zhi-Cai Wang, Ya-Ping Xue, Zhi-Qiang Liu
Enantioselective hydrolysis of epoxides by epoxide hydrolase (EH) is one of the most attractive approaches for the synthesis of chiral epoxides. So far, attempts to develop an efficient epoxide hydrolase -mediated biotransformation have been limited by either the low activity or insufficient enantioselectivity of epoxide hydrolase. In this study, iterative saturation mutagenesis (ISM) of epoxide hydrolase from Agrobacterium radiobacter AD1 (ArEH) was performed for efficient production of (R)-epichlorohydrin...
January 2018: Applied Microbiology and Biotechnology
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 29, 2017: Organic & Biomolecular Chemistry
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...
December 2017: Applied Microbiology and Biotechnology
Grey S Chen, Siang Wun Siao, Claire R Shen
Iterative ketoacid elongation has been an essential tool in engineering artificial metabolism, in particular the synthetic alcohols. However, precise control of product specificity is still greatly challenged by the substrate promiscuity of the ketoacid decarboxylase, which unselectively hijacks ketoacid intermediates from the elongation cycle along with the target ketoacid. In this work, preferential tuning of the Lactococcus lactis ketoisovalerate decarboxylase (Kivd) specificity toward 1-pentanol synthesis was achieved via saturated mutagenesis of the key residue V461 followed by screening of the resulting alcohol spectrum...
September 12, 2017: Scientific Reports
Guangyue Li, Jian-Bo Wang, Manfred T Reetz
Enzymes have been used for a long time as catalysts in the asymmetric synthesis of chiral intermediates needed in the production of therapeutic drugs. However, this alternative to man-made catalysts has suffered traditionally from distinct limitations, namely the often observed wrong or insufficient enantio- and/or regioselectivity, low activity, narrow substrate range, and insufficient thermostability. With the advent of directed evolution, these problems can be generally solved. The challenge is to develop and apply the most efficient mutagenesis methods which lead to highest-quality mutant libraries requiring minimal screening...
May 11, 2017: Bioorganic & Medicinal Chemistry
Kaiyuan Tian, Kee Tai, Bryan Jian Wei Chua, Zhi Li
Engineering a methanol tolerant lipase is of great importance in biodiesel production. Here, the first semi-rational method for directed enzyme evolution to enhance methanol tolerance by targeting high B-factor residues for iterative saturation mutagenesis (ISM) is reported. The best double mutant, TLL-S105C/D27R, retained 71% of its original activity after incubation in methanol, showing 30% greater methanol tolerance than TLL. TLL-S105C/D27R also displayed 27% higher activity over TLL. Structure modelling suggested that the increased stability of TLL-S105C/D27R was caused by the formation of a new hydrogen bond which stabilized the protein structure...
May 19, 2017: Bioresource Technology
Xue-Qing Li, Qin Wu, Die Hu, Rui Wang, Yan Liu, Min-Chen Wu, Jian-Fang Li
To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly(21) with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11A(G21I), was selected from the mutagenesis library, E...
December 2017: AMB Express
Yu Jiang, Fenghui Qian, Junjie Yang, Yingmiao Liu, Feng Dong, Chongmao Xu, Bingbing Sun, Biao Chen, Xiaoshu Xu, Yan Li, Renxiao Wang, Sheng Yang
Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86-100%...
May 4, 2017: Nature Communications
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...
April 2017: Applied Microbiology and Biotechnology
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
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
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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