A Single Mutation in the "DSL" Motif of the Acyl Carrier Protein Can Prevent Its Phosphopantetheinylation by Holo-Acyl Carrier Protein Synthase (AcpS).

The expression system is the method of choice to obtain high yields of a pure protein. Since most biological pathways are evolutionarily conserved from bacteria to mammals, there is always a chance that a non-native protein shares sequence or structural homology with the natural substrate of an enzyme. In such cases, when this foreign protein is overexpressed in , it may be processed as a substrate by that enzyme, resulting in its modification. A notable example is the heterologous expression of Type II acyl carrier proteins (ACPs) in . Due to the conservation of a type II fatty acid synthesis pathway (FAS) across bacteria to mammals, the non-native type II ACPs are often recognized as a substrate by the 4'-phosphopantetheinyl transferase, also known as the Holo-acyl carrier protein synthase (AcpS). This undesirable modification is a concern when the objective is to obtain milligram amounts of apo-ACP. Here, using an approach combining mutagenesis, enzyme activity, and NMR, we have probed for the ACP (AcpP) residues that can prevent this modification. Taking cues from the AcpP-AcpS crystal structure (PDB entry 1F80), five charge-neutralization mutations were designed on the AcpP surface, i.e., D35N, E41A, E47A, E48A, and E47A/E48A, to disrupt the AcpP-AcpS interaction. All the AcpP mutants except D35N expressed as partially phosphopantetheinylated proteins in , presenting D35N mutagenesis as an attractive approach to prevent undesired modification of AcpP The strategy was tested on two other non-native type II ACPs that express predominantly as phosphopantetheinylated proteins in , mitochondrial FAS ACP (mACP), and Typhimurium invasion acyl carrier protein (IacP). A single D35N mutation in the "DSL" motif of these ACPs prevented their phosphopantetheinylation by AcpS, demonstrating D35N mutagenesis as a viable strategy to express apo-ACP in .