Author:
Rao Yi,Yang Jingyao,Wang Jiaqi,Yang Xinyuan,Zhang Mengxi,Zhan Yangyang,Ma Xin,Cai Dongbo,Wang Zhangqian,Chen Shouwen
Abstract
AbstractTerminators serve as the regulatory role in gene transcription termination; however, few researches about terminator optimization have been conducted, which leads to the lack of available and universal terminator for gene expression regulation in Bacillus. To solve this problem and expand synthetic biology toolbox of Bacillus licheniformis, the terminator T1 of endogenous α-amylase gene (amyL) was characterized in this research, with a termination efficiency of 87.81%. Then, we explored and optimized the termination strength of terminator T1 from four aspects: the distance between stop codon and terminator, GC content at the bottom of stem structure, loop size, and U-tract length, and the best terminator T24 was attained by combination optimization strategy, which termination efficiency was increased to 97.97%, better than the commonly used terminator T7 (T7P) from Escherichia coli. Finally, terminator T24 was applied to protein expression, which, respectively, led to 33.00%, 25.93%, and 11.78% increases of green fluorescence intensity, red fluorescence intensity, and keratinase activity, indicating its universality in protein expression. Taken together, this research not only expands a plug-and-play synthetic biology toolbox in B. licheniformis but also provides a reference for the artificial design of versatile intrinsic terminator.
Graphical Abstract
Funder
National Key Research and Development Program of China
Science and Technology Project of Hubei Tobacco Company
Open Project Funding of the State Key Laboratory of Biocatalysis and Enzyme Engineering
Publisher
Springer Science and Business Media LLC
Subject
Renewable Energy, Sustainability and the Environment,Biomedical Engineering,Food Science,Biotechnology
Cited by
1 articles.
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