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Int. J. Mol. Sci. 2016, 17(11), 1813; doi:10.3390/ijms17111813

Directed Evolution of Dunaliella salina Ds-26-16 and Salt-Tolerant Response in Escherichia coli

1
Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, No. 94 Weijin Rd., Tianjin 300071, China
2
Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, No. 94 Weijin Rd., Tianjin 300071, China
*
Authors to whom correspondence should be addressed.
Academic Editor: Patrick C. Y. Woo
Received: 19 September 2016 / Revised: 17 October 2016 / Accepted: 25 October 2016 / Published: 29 October 2016
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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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 from Dunaliella salina, encodes a transcription factor that can confer salt tolerance to a number of organisms including Escherichia coli (E. coli), Haematococcus pluvialis and 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 an E. coli expression 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 the E. coli transformant enduring salt concentrations up to 1.54 M, in comparison with 1.03 M for the wild type gene. Besides, Ds-26-16 and EP-5 also conferred E. coli transformant 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 of Ds-26-16 and EP-5 maintained normal cellular morphology, increased the intracellular antioxidant enzymatic activity, reduced malondialdehyde content, and stimulated Nitric Oxide synthesis, thus enhancing salt tolerance to E. coli transformants. View Full-Text
Keywords: Ds-26-16; directed evolution; salt-tolerant response; abiotic stress; Escherichia coli Ds-26-16; directed evolution; salt-tolerant response; abiotic stress; Escherichia coli
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Guo, Y.; Dong, Y.; Hong, X.; Pang, X.; Chen, D.; Chen, X. Directed Evolution of Dunaliella salina Ds-26-16 and Salt-Tolerant Response in Escherichia coli. Int. J. Mol. Sci. 2016, 17, 1813.

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