Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformis
Abstract Terminators 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 s...
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| Format: | Article |
| Language: | English |
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SpringerOpen
2022-10-01
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| Series: | Bioresources and Bioprocessing |
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| Online Access: | https://doi.org/10.1186/s40643-022-00597-1 |
| _version_ | 1811182257777410048 |
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| author | Yi Rao Jingyao Yang Jiaqi Wang Xinyuan Yang Mengxi Zhang Yangyang Zhan Xin Ma Dongbo Cai Zhangqian Wang Shouwen Chen |
| author_facet | Yi Rao Jingyao Yang Jiaqi Wang Xinyuan Yang Mengxi Zhang Yangyang Zhan Xin Ma Dongbo Cai Zhangqian Wang Shouwen Chen |
| author_sort | Yi Rao |
| collection | DOAJ |
| description | Abstract Terminators 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 |
| first_indexed | 2024-04-11T09:29:41Z |
| format | Article |
| id | doaj.art-02c0cee9e4814bb88101035bfef22146 |
| institution | Directory Open Access Journal |
| issn | 2197-4365 |
| language | English |
| last_indexed | 2024-04-11T09:29:41Z |
| publishDate | 2022-10-01 |
| publisher | SpringerOpen |
| record_format | Article |
| series | Bioresources and Bioprocessing |
| spelling | doaj.art-02c0cee9e4814bb88101035bfef221462022-12-22T04:31:55ZengSpringerOpenBioresources and Bioprocessing2197-43652022-10-019111010.1186/s40643-022-00597-1Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformisYi Rao0Jingyao Yang1Jiaqi Wang2Xinyuan Yang3Mengxi Zhang4Yangyang Zhan5Xin Ma6Dongbo Cai7Zhangqian Wang8Shouwen Chen9State Key Laboratory of Biocatalysis and Enzyme EngineeringEnvironmental Microbial Technology Center of Hubei ProvinceCollege of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme EngineeringEnvironmental Microbial Technology Center of Hubei ProvinceCollege of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme EngineeringEnvironmental Microbial Technology Center of Hubei ProvinceCollege of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme EngineeringEnvironmental Microbial Technology Center of Hubei ProvinceCollege of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme EngineeringEnvironmental Microbial Technology Center of Hubei ProvinceCollege of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme EngineeringEnvironmental Microbial Technology Center of Hubei ProvinceCollege of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme EngineeringEnvironmental Microbial Technology Center of Hubei ProvinceCollege of Life Sciences, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme EngineeringEnvironmental Microbial Technology Center of Hubei ProvinceCollege of Life Sciences, Hubei UniversityHubei Engineering Research Center for Deep Processing of Green Se-Rich Agricultural Products, School of Modern Industry for Selenium Science and Engineering, National R&D Center for Se-Rich Agricultural Products Processing, Wuhan Polytechnic UniversityState Key Laboratory of Biocatalysis and Enzyme EngineeringEnvironmental Microbial Technology Center of Hubei ProvinceCollege of Life Sciences, Hubei UniversityAbstract Terminators 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 Abstracthttps://doi.org/10.1186/s40643-022-00597-1Intrinsic terminatorSynthetic biologyProtein expressionBacillus licheniformis |
| spellingShingle | Yi Rao Jingyao Yang Jiaqi Wang Xinyuan Yang Mengxi Zhang Yangyang Zhan Xin Ma Dongbo Cai Zhangqian Wang Shouwen Chen Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformis Bioresources and Bioprocessing Intrinsic terminator Synthetic biology Protein expression Bacillus licheniformis |
| title | Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformis |
| title_full | Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformis |
| title_fullStr | Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformis |
| title_full_unstemmed | Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformis |
| title_short | Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformis |
| title_sort | minimization and optimization of α amylase terminator for heterologous protein production in bacillus licheniformis |
| topic | Intrinsic terminator Synthetic biology Protein expression Bacillus licheniformis |
| url | https://doi.org/10.1186/s40643-022-00597-1 |
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