title: Directed Evolution of Dunaliella salina Ds-26-16 and Salt-Tolerant Response in Escherichia coli.
abstract: Identification and evolution of salt tolerant genes are crucial steps in developing salt tolerant crops or microorganisms using biotechnology.Ds-26-16, a salt tolerant gene that was isolated fromDunaliella salina, encodes a transcription factor that can confer salt tolerance to a number of organisms includingEscherichia coli(E.coli),Haematococcus pluvialisand tobacco. To further improve its salt tolerance, a random mutagenesis library was constructed using deoxyinosine triphosphate-mediated error-prone PCR technology, and then screened using anE.coliexpression system that is based on its broad-spectrum salt tolerance. Seven variants with enhanced salt tolerance were obtained. Variant EP-5 that contained mutation S32P showed the most improvement with theE.colitransformant enduring salt concentrations up to 1.54 M, in comparison with 1.03 M for the wild type gene. Besides,Ds-26-16andEP-5also conferredE.colitransformant tolerance to freezing, cold, heat, Cu2+and alkaline. Homology modeling revealed that mutation S32P in EP-5 caused the conformational change of N- and C-terminal α-helixes. Expression ofDs-26-16andEP-5maintained normal cellular morphology, increased the intracellular antioxidant enzymatic activity, reduced malondialdehyde content, and stimulated Nitric Oxide synthesis, thus enhancing salt tolerance toE.colitransformants.
authors: Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, No. 94 Weijin Rd., Tianjin 300071, China. guoyuan09143121@126.com.
authors: Yuan
authors: Y
authors: Guo
authors: Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, No. 94 Weijin Rd., Tianjin 300071, China. ypdong@163.com.
authors: Yanping
authors: Y
authors: Dong
authors: Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, No. 94 Weijin Rd., Tianjin 300071, China. jenelolen@outlook.com.
authors: Xiao
authors: X
authors: Hong
authors: Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, No. 94 Weijin Rd., Tianjin 300071, China. fightingnk@163.com.
authors: Xiaonan
authors: X
authors: Pang
authors: Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, No. 94 Weijin Rd., Tianjin 300071, China. chendefu@nankai.edu.cn.
authors: Defu
authors: D
authors: Chen
authors: Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, No. 94 Weijin Rd., Tianjin 300071, China. xiwenchen@nankai.edu.cn.
authors: Xiwen
authors: X
authors: Chen
mesh_terms: D020418:Algal Proteins / Q000235:genetics*; D000975:Antioxidants / Q000378:metabolism; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005786:Gene Expression Regulation; D016296:Mutagenesis; D009154:Mutation; D009569:Nitric Oxide / Q000096:biosynthesis; D055049:Salt Tolerance / Q000235:genetics*; D012965:Sodium Chloride / Q000633:toxicity; D016823:Volvocida / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*
chemical_list: D020418:Algal Proteins; D000975:Antioxidants; D009569:Nitric Oxide; D012965:Sodium Chloride
keywords: Ds-26-16; Escherichia coli; abiotic stress; directed evolution; salt-tolerant response