N-terminal engineering of overlapping genes in the nitrile hydratase gene cluster improved its activity

Nitrile hydratase which catalyzes the hydration of nitriles to the corresponding amides is operon-encoded. However, when heterologously expressed, genes in the same operon are usually not equally expressed, and the ratio needs to be fine-tuned. A gene cluster of three genes (corresponding to alpha-subunit, beta-subunit and activator) encoding the nitrile hydratase was cloned from Aurantimonas manganoxydans ATCC BAA-1229 and expressed in Escherichia coli. However, difficulty was encountered in heterologous expression of the activator and the expression level of beta-subunit was lower than that of alpha-subunit, which together resulted in low catalytic efficiency. To improve the expression of activator, a set of SKIK tags were fused to the N-terminus of the activator. To elevate the expression level of beta-subunit, a silent mutation strategy was applied in the overlapping sequence with alpha-subunit around its translation initial region. Finally, the expression of beta-subunit and activator were improved and the maximum activity of NHase1229 was doubled, reaching 160 U/mL towards 3-cyanopyridine. These results indicate that N-terminal engineering is an efficient strategy for optimizing the expression of multiple genes in operons.