Enhanced Acetate Tolerance and Recombinant Protein Accumulation in Escherichia coli by Transgenic Expression of a Heat Shock Protein from Carrot (Daucus carota L.)

Abstract

Background: In Escherichia coli (E. coli) culture, acetate accumulates as an undesirable by-product of aerobic fermentation on glucose and inhibits cell growth and recombinant protein production.

Objectives: We examined whether the heterologous expression of a eukaryotic heat shock protein (Hsp) can confer tolerance to acetate in E. coli.

Materials and methods: Transgenic cell lines (TCLs) heterologously expressing a small heat shock protein (sHsp) from carrot (Daucus carota L.), DcHsp17.7, were exposed to heat, sodium acetate, and alkaline conditions. The cell growth and cell viability were examined by measuring O.D.₆₀₀ and colony-forming units (CFU), respectively. The His-tagged recombinant alcohol dehydrogenase (ADH) gene cloned in a pET11a expression vector was introduced into TCL1 and expressed by isopropyl β-D-1-thiogalactopyranoside treatment. After purifying using Ni-NTA affinity chromatography, its accumulation levels were examined using SDS-PAGE in the presence of acetate.

Results: TCLs constitutively expressing DcHsp17.7 showed improved growth, cell density, and cell viability under the stress conditions of heat, acetate, and alkaline compared to an empty vector control line. In acetate stress conditions, TCL1 accumulated more cellular proteins (approximately 130%) than the control. The recombinant ADH accumulated to a higher level in TCL1 (2.2-fold at 16 °C) than the control. The addition of acetate reduced the recombinant ADH level by 70% in the control when compared with the absence of acetate. In contrast, recombinant ADH accumulation was not affected by acetate in TCL1. In the presence of acetate, TCL1 accumulated 6.4-fold more recombinant ADH than did the control. Furthermore, recombinant ADH produced in TCL1 showed 1.5-fold higher enzyme activity than that produced in the control in the presence or absence of acetate.

Conclusion: Our study showed that heterologously expressed DcHsp17.7 from carrot can alleviate the negative effects of acetate on E. coli.

Keywords: Alcohol dehydrogenase; Alkaline tolerance; Enzyme activity; Heat tolerance; Molecular chaperone.