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 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.

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

“…They have reduced levels of lysine and cysteine residues, and a higher content of aspartate and small hydrophobic residues. Structurally, they contain higher amounts of random coil structure at the expense of α-helix [145][146][147][148][149][150]. A recent report from Warden et al demonstrated that the extended binding of cations by acidic residues with complementary chelating partners and interactions through the highly ordered hydration shells of the cations are primary mechanisms of halotolerance [145].…”