Abstract: As an acyl donor, the acyl-acyl carrier protein (acyl-ACP) plays pivotal roles in biosynthesis of various natural products, including fatty acids, polyketones and so on. At present the acyl-CoA has been used as a substitute for uncommercialized acyl-ACP to perform in vitro study of the relevant enzyme activity. By using acyl-CoA, the catalytic specificity of the enzymes can't be revealed correctly because the substrate protein acyl-ACP could interact with the relevant enzymes through the ACP. Based on the plasmid pET-28a(+) -ACP derived from Escherichia coli, 12 single-site ACP mutants were designed, and the corresponding holo-ACPs were expressed and purified. These holo-ACPs were subsequently used as substrates to synthesize C16:0-ACP and C18:1-ACP. The HPLC results show that, ACP mutant with change in a single amino acid can effect on the acyl-ACP feature. In particular, the ACP mutant with change in T40 residue has notable impacts on hydrophobicity, ultraviolet absorption response and stability of acyl-ACP as well.