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A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences

文献类型： 外文期刊

作者： Xiong, Ai-Sheng 1 ; Peng, Ri-He 1 ; Zhuang, Jing 1 ; Liu, Jin-Ge 1 ; Gao, Feng 1 ; Xu, Fang 1 ; Cai, Bin 1 ; Yao, Quan-Ho 1 ;

作者机构： 1.Shanghai Acad Agr Sci, Biotechnol Res Inst, Shanghai 201106, Peoples R China

关键词： beta-galactosidase;chemical synthesis;degenerate oligonucleotide;directed evolution;semi-rational design;BETA-GALACTOSIDASE;ESCHERICHIA-COLI;IN-VITRO;PYROCOCCUS-WOESEI;PROTEIN EVOLUTION;CODON USAGE;GENE;MUTAGENESIS;FAMILY;GLUCURONIDASE

期刊名称：BIOLOGICAL CHEMISTRY （ 影响因子：3.915； 五年影响因子：3.922 ）

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收录情况： SCI

摘要： Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable P-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified P-galactosidase from YH8757 exhibited a lower specific activity at 25 degrees C than those purified from mutated YG6755 and YG8252.

  • 相关文献

[1]Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity. Xiong, Ai-Sheng,Peng, Ri-He,Zhuang, Jing,Li, Xian,Xue, Yong,Liu, Jin-Ge,Gao, Feng,Cai, Bin,Chen, Jian-Min,Yao, Quan-Hong.

[2]Directed evolution of beta-galactosidase from Escherichia coli into beta-glucuronidase. Xiong, Ai-Sheng,Peng, Ri-He,Zhuang, Jing,Liu, Jin-Ge,Xu, Fang,Cai, Bin,Guo, Zhao-Kui,Qiao, Yu-Shan,Chen, Jian-Min,Zhang, Zhen,Yao, Quan-Hong. 2007

[3]Temperature Sensitivity of alpha-enolase from Native and Introduced Ducks: Differences in Gene Expression and Sequence Correlation with Geographic Distribution. Zeng, Tao,Liu, Yali,Wang, Deqian,Lu, Lizhi,Jiang, Xueyuan,Liu, Hui.

[4]PCR-aided synthesis and stable expression in E.coli of the cryIA(c)Bt gene. Peng, RH,Xiong, AS,Li, X,Fan, HQ,Huang, XM,Yao, QH. 2001

[5]Concurrent mutations in six amino acids in beta-glucuronidase improve its thermostability. Xiong, Ai-Sheng,Peng, Ri-He,Cheng, Zong-Ming,Li, Yi,Liu, Jin-Ge,Zhuang, Jing,Gao, Feng,Xu, Fang,Qiao, Yu-Shan,Zhang, Zhen,Chen, Jian-Min,Yao, Quan-Hong. 2007

[6]Expression and Function of a Modified AP2/ERF Transcription Factor from Brassica napus Enhances Cold Tolerance in Transgenic Arabidopsis. Xiong, Ai-Sheng,Wang, Feng,Jiang, Hai-Hua,Peng, Ri-He,Jin, Xiao-Fen,Zhu, Bo,Yao, Quan-Hong,Zhuang, Jing,Zhang, Jian.

[7]Kinetic characters and resistance to inhibition of crude and purified brain acetylcholinesterase of three freshwater fishes by organophosphates. Li, SN,Xie, XC,Zhu, GN,Tan, YJ.

[8]Cloning, Mutagenesis and Expression of the Housefly Acetylcholinesterase in Pichia pastoris, and its Potential Application in the Detection of Pesticide Residues. Tan, Furong,Wang, Ligang,Wang, Jinbin,Wu, Xiao,Zhu, Hong,Zhao, Kai,Jiang, Lingxi,Tao, Shiru,Tang, Xueming,Tan, Furong,Wang, Ligang,Wang, Jinbin,Wu, Xiao,Zhu, Hong,Zhao, Kai,Jiang, Lingxi,Tao, Shiru,Tang, Xueming,Yang, Yan. 2010

[9]Enhanced pesticide sensitivity of novel housefly actylcholinesterases: a new tool for the detection of residual pesticide contamination. Tan, Furong,Wang, Ligang,Wang, Jinbin,Wu, Xiao,Zhu, Hong,Jiang, Lingxi,Tao, Shiru,Zhao, Kai,Tang, Xueming,Tan, Furong,Wang, Jinbin,Wu, Xiao,Zhu, Hong,Jiang, Lingxi,Tao, Shiru,Zhao, Kai,Tang, Xueming,Wang, Ligang,Yang, Yan.

[10]Co-ordinate expression of glycine betaine synthesis genes linked by the FMDV 2A region in a single open reading frame in Pichia pastoris. Wang, Sanhong,Yao, Quanhong,Tao, Jianmin,Qiao, Yushan,Zhang, Zhen.

[11]Near-infrared reflectance Spectroscopy-based methods for phytase registration in feed industry. Yang, Haifeng,Lv, Xiaowen,Wang, Jing,Li, Junguo,Li, Hui,Qin, Yuchang.

[12]High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris. Xiong, Ai-Sheng,Yao, Quan-Hong,Peng, Ri-He,Zhang, Zhen,Xu, Fang,Liu, Jin-Ge,Han, Pei-Lai,Chen, Jian-Min.

[13]A delta-endotoxin encoded in Pseudomonas fluorescens displays a high degree of insecticidal activity. Peng, R,Xiong, A,Li, X,Fuan, H,Yao, Q.

[14]AtCopeg1, the unique gene originated from AtCopia95 retrotransposon family, is sensitive to external hormones and abiotic stresses. Duan, Ke,Zhu, Hong,Pan, Aihu,Huang, Jianhua,Ding, Xiangzhen,Zhang, Qiong. 2008

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