Journal
ENZYME AND MICROBIAL TECHNOLOGY
Volume 117, Issue -, Pages 9-14Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2018.05.015
Keywords
Nitrile hydratase; Activator; Gene cluster; Fusion tag; Silent mutation
Categories
Funding
- National Natural Science Foundation of China [21476199, 21676240]
- National Basic Research Program of China (973 Program) [2011CB710800]
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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.
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