A single mutation in the DSL motif of the acyl carrier protein can prevent its in vivo modification by E. coli Holo-acyl carrier protein synthase (AcpS)
E. coli expression system is the method of choice to obtain high yields of a pure protein. However, there is always a chance that an overexpressed protein shares structural or sequence homology with the substrate of an E. coli enzyme. In such cases, the expressed protein may be partially or fully converted into the product. A notable example is the expression of acyl carrier proteins (ACP) in E. coli. Since most type II ACPs of the fatty acid synthesis pathway (FAS) have a conserved helix II, the carrier proteins are recognized as a substrate by Holo-acyl carrier protein synthase (AcpS). Thus, most ACPs express as partially or fully loaded proteins in E. coli. 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 probed for residues in ACP that can prevent this in vivo modification, without affecting Sfp (Surfactin synthetase activating enzyme) function. Taking cues from the E. coli ACP-AcpS structure (PDB 5VCB), charge neutralization mutations were designed at five different positions in EcACP that participate in ion-pair interaction with AcpS. Three of the mutants expressed solely as apo-ACP in E. coli viz. D35N, E41A and E47A/E48A. However, only the D35N mutant could be converted into holo-/acyl-ACP using Sfp in vitro, establishing mutagenesis as a viable strategy to prevent undesired modifications in vivo. As proof of principle, the mutation was applied to two unrelated ACPs that express primarily as modified proteins in E. coli -Mus musculus mitochondrial FAS ACP (MmACP) and Salmonella Typhimurium invasion acyl carrier protein (IacP). Single D35N mutation of the ACPs prevented their in vivo modification by AcpS, and the mutants were efficiently converted into holo-ACP by Sfp in vitro. These results demonstrate that D35N mutagenesis is a useful strategy to express apo-ACP in E. coli and is applicable across all type II ACPs. Furthermore, we show that holo-IacP and holo-MmACP are not recognized as substrates by AcpH (E. coli Acyl carrier protein hydrolase), and therefore they express predominantly as modified proteins in E. coli.