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

The Escherichia coli 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 E. coli enzyme. In such cases, when this foreign protein is overexpressed in E. coli, 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 E. coli. 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 E. coli 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 E. coli ACP (AcpP) residues that can prevent this in vivo 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 E. coli, presenting D35N mutagenesis as an attractive approach to prevent undesired modification of AcpP in vivo. The strategy was tested on two other non-native type II ACPs that express predominantly as phosphopantetheinylated proteins in E. coli, Mus musculus mitochondrial FAS ACP (mACP), and Salmonella Typhimurium invasion acyl carrier protein (IacP). A single D35N mutation in the "DSL" motif of these ACPs prevented their in vivo phosphopantetheinylation by AcpS, demonstrating D35N mutagenesis as a viable strategy to express apo-ACP in E. coli.