Overexpression and purification of Escherichia coli holo-acyl carrier protein and synthesis of acyl carrier protein

Abstract:

[Objective] To investigate the mechanism of fatty acids, lipid A and N-acylhomoserine lactones biosynthesis of bacteria by using high quality Escherichia coli holo-ACP and varied length chain acyl-ACPs as substrates. [Methods and Results] Using PCR technique we amplified the acpP and acpS gene fragments from genomic DNA of E. coli strain MG1655. Ligating these gene fragments with plasmids pBAD24 or pET28b respectively, we obtained 3 expression plasmids of acyl carrier protein: pBAD-ACP, pET-ACP and pET-ACP-ACPS, and one expression plasmid of holo-acyl carrier protein synthase: pBAD-ACPS. Then we constructed 3 acyl carrier protein producer strains: DH5a/pBAD-ACP、BL21 (DE3)/pET-ACP and BL21(DE3)/pET-ACP-ACPS by transforming E. coli strains DH5a or BL21(DE3)with pBAD-ACP, pET-ACP or pET-ACP-ACPS, respectively. Although these 3 strains could produce more acyl carrier protein under induc-tion than strain DK574, which was used to purify holo-acyl carrier protein in general, the yield of holo-acyl carrier protein of these strains was still lower. In order to increase the yield of holo-acyl carrier protein in these strains, we introduced pBAD-ACPS into these strains. The assay of expressions of new strains was shown the that strain DH5a harbored pBAD-ACP and pBAD-ACPS double plasmids produced more holo-acyl carrier protein than strain DK574, and the purity of holo-acyl carrier protein was also increased (up to 99%). Then we purified high quality holo-acyl carrier protein from the culture of the strain DH5α harbored pBAD-ACP and pBAD-ACPS by using UNOsphere Q anion-exchange chroma-tography. Utilizing holo-acyl carrier protein and long chain fatty acids as substrates and under Vibrio harveyi acyl-acyl carrier protein synthetase catalyzing, we synthesized several different acyl-acyl carrier proteins. [Conclusion] From this study we obtained a high holo-ACP producer strain and demonstrated that co-expressing acpP with acpS, E.coli strains could produce more holo-ACP.