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

Publication date: October 2018Source: Enzyme and Microbial Technology, Volume 117Author(s): Zhengfei Yang, Xiaolin Pei, Gang Xu, Jianping Wu, Lirong YangAbstractNitrile 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.
Source: Enzyme and Microbial Technology - Category: Biotechnology Source Type: research