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

•A fusion tag strategy was applied to tune the expression of activator.•A silent mutation strategy was applied to improve the expression of β-subunit.•The expression levels of three ORFs were balanced and the final activity improved. 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 α-subunit, β-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 β-subunit was lower than that of α-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 β-subunit, a silent mutation strategy was applied in the overlapping sequence with α-subunit around its translation initial region. Finally, the expression of β-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.