全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用：99美元
投递稿件

查看量下载量

相关文章

更多...

基于二维电泳高表达蛋白斑点的谷氨酸棒杆菌强启动子P1937的鉴定
Identification of the Strong Promoter P1937 in Corynebacterium glutamicum Based on the High Expression Protein Bolt of Two-Dimensional Electrophoresis

DOI: 10.12677/AMB.2021.101004, PP. 21-29

Keywords: 谷氨酸棒杆菌，二维电泳，内源强启动子，启动子活性检测
Corynebacterium glutamicum, Two-Dimensional Electrophoresis, Endogenous Strong Promoter, Promoter Activity Test

Full-Text   Cite this paper   Add to My Lib

Abstract:

为了构建高效谷氨酸棒杆菌(Corynebacterium glutamicum)细胞工厂，需要开发出更多的高效基因表达元件。强启动子的使用是使基因高效表达的关键环节，然而关于谷氨酸棒杆菌强启动子鉴定的报道十分少见。本试验基于谷氨酸棒杆菌细胞全蛋白二维电泳，对其中分子量大小约为30 KDa、等电点约为4.0的斑点进行研究，分析其对应蛋白编码基因的启动子。根据试验结果表明，Cgl1937基因的启动子P1937具有更高的转录活性。将强启动子Ptac-M和启动子P1937分别连接到启动子探测载体pDXW-11上，通过电转化进谷氨酸棒杆菌C. glutamicum ATCC13032感受态细胞，以获得工程菌株C. glutamicum/pDXW-11-Ptac-M和C. glutamicum/pDXW-11-P1937。测试工程菌株对氯霉素的耐受性，实验结果表明，菌株C. glutamicum/ pDXW-11-Ptac-M和C. glutamicum/pDXW-11-P1937的耐受性分别为30 μg/mL和40 μg/mL；对报告蛋白氯霉素酰基转移酶CAT酶活进行检测，结果表明，C. glutamicum/pDXW-11-Ptac-M和C. glutamicum/ pDXW-11-P1937的酶比活力分别为0.85 U/mg蛋白质和9.53 U/mg蛋白质；通过荧光定量PCR检测两种工程菌株氯霉素乙酰基转移酶cat基因转录水平，实验结果显示，cat基因在启动子P1937的控制下，其转录水平是Ptac-M的2.07倍。以上结果表明，谷氨酸棒杆菌Cgl1937基因的启动子P1937为强启动子。
It is very necessary to develop more gene expression elements for constructing efficient Corynebacterium glutamicum cell factories. The usage of strong promoter is a key to gene efficient expression. However, there are few reports on identification of strong promoters in C. glutamicum. In this study, based on the two-dimensional electrophoresis of the whole cell protein of C. glutamicum, we selected the protein blot (MD, approx. 30 KDa; pI, approx. 4.0) to analyze the promoters of the genes. The results of promoter prediction showed that the promoter of the Cgl1937 gene, P1937, has a strong activity. The strong promoter Ptac-M and the promoter P1937 were inserted into the promoter-probe vector pDXW-11, and transformed into C. glutamicum ATCC13032, generating the engineered strains C. glutamicum/pDXW-11-Ptac-M and C. glutamicum/pDXW-11-P1937. The results of the chloramphenicol gradient test showed that the tolerance of the strains C. glutamicum/pDXW-11-Ptac-M and C. glutamicum/pDXW-11-P1937 were 30 μg/mL and 40 μg/mL, respectively. The activity test of the reporter protein chloramphenicol acyltransferase CAT showed that the activities of the two strain were 0.85 U/mg protein and 9.53 U/mg protein. The qPCR test showed that the transcription level of the cat gene under the control of P1937 was 2.07 times higher than that under the control of the strong promoter Ptac-M. These results demonstrate that the promoter P1937 of gene Cgl1937 is a strong promoter in C. glutamicum.

References

[1]  Kinoshita, S., Udaka, S., Shimono, M., et al. (1957) Applied Microbiology, 3, 193.
https://doi.org/10.2323/jgam.3.193
[2]  Hartbrich, A., Schmitz, G., Weuster-Botz, D., et al. (1996) Development and Application of a Membrane Cyclone Reactor for in Vivo NMR Spectroscopy with High Microbial Cell Densities. Biotechnology and Bioengineering, 51, 624-635.
https://doi.org/10.1002/(SICI)1097-0290(19960920)51:6<624::AID-BIT2>3.0.CO;2-J
[3]  Nakamura, Y., Nishio, Y., Ikeo, K., et al. (2003) The Genome Stability in Corynebacterium Species Due to Lack of the Recombination Repair System. Gene, 317, 149-155.
https://doi.org/10.1016/S0378-1119(03)00653-X
[4]  龙梦飞, 徐美娟, 张显, 等. 合成生物学与代谢工程在谷氨酸棒杆菌产氨基酸中的应用[J]. 中国科学, 2019, 49(5): 541-552.
[5]  李慧萍, 姚兴伟, 郭楠, 等. 影响启动子活性测定的关键影响因素[J]. 中国实验诊断学, 2010, 14(7): 1017-1020.
[6]  Xu, N., Wei, L. and Liu, J. (2019) Recent Advances in the Applications of Promoter Engineering for the Optimization of Metabolite Biosynthesis. World Journal of Microbiology and Biotechnology, 35, 33.
https://doi.org/10.1007/s11274-019-2606-0
[7]  Albersmeier, A., Pfeifer-Sancar, K., Ruckert, C., et al. (2017) Genome-Wide Determination of Transcription Start Sites Reveals New Insights into Promoter Structures in the Actinomycete Corynebacterium glutamicum. Journal of Biotechnology, 257, 99-109.
https://doi.org/10.1016/j.jbiotec.2017.04.008
[8]  Jensen, R.R. and Hammer, K. (1998) Artificial Promoters for Metabolic Optimization. Biotechnology and Bioengineering, 58, 191-195.
https://doi.org/10.1002/(SICI)1097-0290(19980420)58:2/3<191::AID-BIT11>3.0.CO;2-G
[9]  Zhang, W., Zhao, Z.H., Yang, Y.K., et al. (2003) Construction of an Expression Vector That Uses the aph Promoter for Protein Expression in Corynebacterium glutamicum. Plasmid, 94, 1-6.
https://doi.org/10.1016/j.plasmid.2017.09.001
[10]  Gruber, T.M. and Gross, C.A. (2003) Multiple Sigma Subunits and the Partitioning of Bacterial Transcription Space. Annual Review of Microbiology, 57, 441-66.
https://doi.org/10.1146/annurev.micro.57.030502.090913
[11]  Liu, X.X., Yang, Y.K., Zhang, W., et al. (2015) Expression of Recombinant Protein Using Corynebacterium glutamicum: Progress, Challenges and Applications. Critical Reviews in Biotechnology, 20, 1-13.
[12]  Xu, D., Tan, Y., Shi, F., et al. (2011) Construction of a Novel Promoter Vector and Its Application for Screening Strong Promoter for Brevibacterium flavum Metabolic Engineering. World Journal of Microbiology and Biotechnology, 27, 961-968.
https://doi.org/10.1007/s11274-010-0539-8
[13]  张献, 张玮祎, 李川敏, 等. 谷氨酸棒杆菌Cgl0864基因强启动子的鉴定[J]. 河北农业大学学报, 2020, 43(2): 76-82.
[14]  Sambrook, J., Fritsh, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Habor Press, Cold Spring Habor.
[15]  Xu, D., Tan, Y., Huan, X., et al. (2010) Construction of a Novel Shuttle Vector for Use in Brevibacterium flavum, an Industrial Amino Acid Producer. Journal of Microbiological Methods, 80, 86-92.
https://doi.org/10.1016/j.mimet.2009.11.003
[16]  Shaw, W.V. (1975) Chloramphenicol Acetyltransferase from Chloramphenicol-Resistant Bacteria. Methods in Enzymology, 43, 737-755.
https://doi.org/10.1016/0076-6879(75)43141-X
[17]  Zhao, Z.H., Liu, X.X., Zhang, W., et al. (2016) Construction of Genetic Parts from the Corynebacterium glutamicum Genome with High Expression Activities. Biotechnology Letters, 38, 2119-2126.
https://doi.org/10.1007/s10529-016-2196-y
[18]  Livak, K.J. and Schmittgen, T.D. (2001) Analysis of Relative Gene Expression Data Using Real-Timing Quantitative PCR and the 2？ΔΔCT. Methods, 24, 70-79.
https://doi.org/10.1006/meth.2001.1262
[19]  徐友强, 马翠卿, 陶飞. 细菌启动子识别及应用研究进展[J]. 生物工程学报, 2010, 26(10): 1393-1403.
[20]  Jeschek, M., Gerngross, D. and Panke, S. (2016) Rationally Reduced Libraries for Combinatorial Pathway Optimization Minimizing Experimental Efforts. Nature Communications, 7, Article No. 11163.
https://doi.org/10.1038/ncomms11163

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133