Team:CityU HK/project/module tesA

Construction and overexpression of ‘TesA in Escherichia coli

In Escherichia coli, tesA codes for the thioesterase I enzyme which is expressed in the periplasm. Although the actual physiological role of thioesterase I is not known, it has been demonstrated that thioesterase I has the ability to cleave the thioester bond in fatty acyl-CoA (Cho & Cronan, 1993). It has been previously shown that deleting the leader sequence of the TesA protein results in the enzyme being expressed as a cytosolic enzyme (Cho & Cronan, 1995). This altered form of the enzyme without the leader sequence was named ‘TesA and overexpression of the cytosolic ‘TesA has been used to increase the yield of free fatty acids (FFAs) in biotechnology as a feedstock for biofuel production (Janßen & Steinbüchel, 2014).

During the transport of long chain fatty acids (LFCAs), the FadD protein in the inner membrane activates LFCA to form fatty acyl-CoA, which may be broken down to acetyl-CoA through β-oxidation. To retard β-oxidation of fatty acyl-CoA, the ‘tesA gene is cloned and overexpressed in E. coli. It is predicted that overexpression of ‘TesA in E. coli would favor the conversion of fatty acyl-CoA to free fatty acids (FFAs) in the cytosol (Figure 1). By co-transforming the ‘tesA plasmid with the Δ9- Δ12-Δ15-desaturase gene cluster plasmid into E. coli cells, increased conversion of FFAs to α-linolenic acid (ALA) will be expected.

Figure 1. Conversion of fatty acyl-CoA to free fatty acid.

Gene Construct

To create the ‘tesA gene construct, the coding sequence (without the leader sequence) of tesA was amplified by PCR and the PCR amplicon was ligated to the RBS (BBa_B0034) and PBAD promoter (BBa_I13453) fragments.

References:

Cho, H. & Cronan, J. E. (1993). Escherichia coli thioesterase I, molecular cloning and sequencing of the structural gene and identification as a periplasmic enzyme. Journal of Biological Chemistry 268(13), 9238-9245.

Cho, H. & Cronan, J.E. (1995). Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis. Journal of Biological Chemistry 270(9), 4216-4219

Janßen, H. J. & Steinbüchel, A. (2014). Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnology for biofuels 7(1), 7.

Lee, L., Lee, Y., Leu, R. & Shaw, J. (2006). Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1. Biochem. J 397, 69-76.