The production of recombinant proteins in Escherichia coli is an important application of biotechnology. 2-Oxoglutarate-dependent l-pipecolic acid hydroxylase derived from Xenorhabdus doucetiae (XdPH) is an excellent biocatalyst that catalyzes the hydroxylation of l-pipecolic acid to produce cis-5-hydroxy-l-pipecolic acid. However, the enzyme tends to form aggregates in the E. coli expression system. Our group established two rules, namely, the "α-helix rule" and the "hydropathy contradiction rule," to select residues to be altered for improving the heterologous recombinant production of proteins, by analyzing their primary structure. We rationally designed XdPH variants that are expressed in highly soluble and active forms in the E. coli expression system using these hotspot prediction methods, and the L142R variant showed a remarkably high soluble expression level compared to the wild-type XdPH. Further mutations were introduced into the L142R gene by site-directed mutagenesis. Moreover, the I28P/L142R and C76Y/L142R double variants displayed improved soluble expression levels compared to the single variants. These variants were also more thermostable than the wild-type XdPH. To analyze the effect of the alteration on one of the hotspots, L142 was replaced with various hydrophilic and positively charged residues. The remarkable increase in soluble protein expression caused by the alterations suggests that the decrease in the hydrophobicity of the protein surface and the enhancement of the interaction between nearby residues are important factors determining the solubility of the protein. Overall, this study demonstrated the effectiveness of our protocol in identifying aggregation hotspots for recombinant protein production and in basic biochemical research.
© 2022 The Authors. Published by American Chemical Society.