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| Characterization of Promoters in the Glycolytic Pathway and Tricarboxylic Acid Cycle of E. coli and Its Application |
XUAN Mei-juan,ZHANG Xiao-yun,GAO Ying,Li-GAO Ying,WU Jia-jing,MA Mei,WANG Yan-mei,KOU Hang,LU Fu-ping,LI Ming( ) |
| Key Laboratory of Industrial Fermentation Microbiology(Tianjin University of Science and Technology), Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, Biotechnology College, Tianjin University of Science and Technology, Tianjin 300457, China |
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Abstract The promoters are important components that regulate gene transcription, and key parts in synthetic biology research and cell factory design. Glycolytic pathway and tricarboxylic acid (TCA) cycle are the central metabolisms of carbohydrate catabolism and are strictly regulated by promoter strength. In order to screen some endogenous constitutive promoters with various strength necessary for synthetic biology studies and cell factory design of Escherichia coli, the strength and core structural elements of 27 promoters in glycolytic pathway and TCA cycle of E. coli were systematically studied using the red fluorescent protein (RFP) mCherry as the reporter gene and online analysis software. The results showed that the strength range of these promoters varied greatly, and the strength of the strongest promoter PgapA was 43.6 times that of the weakest promoter PacnA. Moreover, the -10 and -35 sequences of promoters are not exactly same as their consistent sequences, and the spacer between them is 17±3 bp. However, the strength of the promoters was basically consistent with the structural characteristics of the promoters. Using the strongest promoter PgapA, Phosphoenolpyruvate carboxylase and pyruvate kinase were expressed in recombinant E. coli DH5αΔpck, respectively. Their enzyme activity was increased by 0.32 and 1.57 times, respectively, and the production of citric acid was also increased by 124.7% and 75.5%. These promoters with different strength have laid a foundation for the study of synthetic biology and the design of cell factory of E. coli.
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Received: 20 December 2019
Published: 23 June 2020
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Corresponding Authors:
Ming LI
E-mail: liming09@tust.edu.cn
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Cite this article:
XUAN Mei-juan,ZHANG Xiao-yun,GAO Ying,Li-GAO Ying,WU Jia-jing,MA Mei,WANG Yan-mei,KOU Hang,LU Fu-ping,LI Ming. Characterization of Promoters in the Glycolytic Pathway and Tricarboxylic Acid Cycle of E. coli and Its Application. China Biotechnology, 2020, 40(6): 20-30.
URL:
https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1912034 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I6/20
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|
|
| [1] |
Dellomonaco C, Clomburg J M, Miller E N , et al. Engineered reversal of the beta-oxidation cycle for the synthesis of fuels and chemicals. Nature, 2011,476:355-359.
|
|
|
| [2] |
Machado H B, Dekishima Y, Luo H , et al. A selection platform for carbon chain elongation using the CoA dependent pathway to produce linear higher alcohols. Metab Eng, 2012,14:504-511.
|
|
|
| [3] |
Choi Y J, Lee S Y . Microbial production of short-chain alkanes. Nature, 2013,502:571-574.
|
|
|
| [4] |
Kallio P, Pasztor A, Thiel K , et al. An engineered pathway for the biosynthesis of renewable propane. Nat Commun, 2014,5:4731. doi: 10.1038/ncomms5731.
doi: 10.1038/ncomms5731
|
|
|
| [5] |
Doroshenko V G, Livshits V A, Airich L G , et al. Metabolic engineering of Escherichia coli for the production of phenylalanine and related compounds. Appl Biochem Microbiol, 2015,51(7):733-750.
|
|
|
| [6] |
Yim H, Haselbeck R, Niu W , et al. Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nat Chem Biol, 2011,7:445-452.
pmid: 21602812
|
|
|
| [7] |
Barton N R, Burgard A P, Burk M J , et al. An integrated biotechnology platform for developing sustainable chemical processes. J Ind Microbiol Biotechnol, 2015,42:349-360.
|
|
|
| [8] |
Xia X X, Qian Z G, Ki C S , et al. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proc Natl Acad Sci USA, 2010,107:14059-14063.
|
|
|
| [9] |
Yang J E, Choi Y J, Lee S J , et al. Metabolic engineering of Escherichia coli for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose. Appl Microbiol Biotechnol, 2014,98:95-104.
|
|
|
| [10] |
Choi K R, Shin J H, Cho J S , et al. Systems metabolic engineering of Escherichia coli. EcoSal Plus, 2016, doi: 10.1128/ecosalplus.ESP-0010-2015.
doi: 10.1128/ecosalplus.ESP-0010-2015
pmid: 30681066
|
|
|
| [11] |
Nandagopal N, Elowitz M B . Synthetic biology: integrated gene circuits. Science, 2011,333:1244-1248.
|
|
|
| [12] |
Engstrom M D, Pfleger B F . Transcription control engineering and applications in synthetic biology. Synth Syst Biotechnol, 2017,2:176-191.
|
|
|
| [13] |
Chen X L, Gao C, Guo L , et al. DCEO biotechnology: tools to design, construct, evaluate, and optimize the metabolic pathway for biosynthesis of chemicals. Chem Rev, 2018,118:4-72.
pmid: 28443658
|
|
|
| [14] |
Alper H, Fischer C, Nevoigt E , et al. Tuning genetic control through promoter engineering. Proc Natl Acad Sci USA, 2005,102:12678-12683.
doi: 10.1073/pnas.0504604102
pmid: 16123130
|
|
|
| [15] |
Blount BA, Weenink T, Vasylechko S , et al. Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology. PLoS One, 2012,7(3):e33279-e332809.
|
|
|
| [16] |
Xu N, Wei L, Liu J . Recent advances in the applications of promoter engineering for the optimization of metabolite biosynthesis. World J Microb Biot, 2019,35:33.
|
|
|
| [17] |
Sendy B . Studies on transcription in Escherichia coli. Birmingham: University of Birmingham, 2017.
|
|
|
| [18] |
Chae T U, Choi S Y, Kim J W , et al. Recent advances in systems metabolic engineering tools and strategies. Curr Opin Biotechnol, 2017,47:67-82.
pmid: 28675826
|
|
|
| [19] |
Zaslaver A, Bren A, Ronen M , et al. A comprehensive library of fluorescent transcriptional reporters for Escherichia coli. Nature Methods, 2006,3(8):623-628.
doi: 10.1038/nmeth895
pmid: 16862137
|
|
|
| [20] |
Fernie A R, Carrari F, Sweetlove L J . Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport. Curr Opin Plant Biol, 2004,7:254-261.
|
|
|
| [21] |
Shimada T, Yamazaki Y, Tanaka K , et al. The whole set of constitutive promoters recognized by RNA polymerase RpoD holoenzyme of Escherichia coli. PLoS One, 2014,9(3):e90447. doi: 10.1371/journal.pone.0090447.
doi: 10.1371/journal.pone.0090447
pmid: 24603758
|
|
|
| [22] |
Mohedano M L, García-Cayuela T, Pérez-Ramos A , et al. Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus. J Ind Microbiol Biotechnol, 2015,42(2):247-253.
|
|
|
| [23] |
陈英, 王培娟, 张文君 , 等. mCherry红色荧光标记乳酸菌的融合表达系统构建及应用. 生物工程学报, 2019,35(3):492-504.
|
|
|
| [23] |
Chen Y, Wang P J, Zhang W J , et al. Construction and application of mCherry red fluorescent protein fusion expression system in lactic acid bacteria. Chin J Biotech, 2019,35(3):492-504.
|
|
|
| [24] |
Pietrocola F, Galluzzi L, Bravo-San Pedro J M , et al. Acetyl coenzyme A: a central metabolite and mecond messenger. Cell Metab, 2015,21:805-821.
pmid: 26039447
|
|
|
| [25] |
Sidoli S, Trefely S, Garcia B A , et al. Integrated analysis of acetyl-CoA and histone modification via mass spectrometry to investigate metabolically driven acetylation. Methods Mol Biol, 2019,1928:125-147.
|
|
|
| [26] |
Shi L, Tu B P . Acetyl-CoA and the regulation of metabolism: mechanisms and consequences. Curr Opin Cell Biol, 2015,33:125-131.
pmid: 25703630
|
|
|
| [27] |
Akram M . Citric acid cycle and role of its intermediates in metabolism. Cell Biochem Biophys, 2014,68(3):475-478.
|
|
|
| [28] |
Hopp A K, Grüter P, Hottiger M O . Regulation of glucose metabolism by NAD + and ADP-ribosylation. Cells, 2019,8, 890. doi: 10.3390/cells8080890.
doi: 10.3390/cells8080890
|
|
|
| [29] |
Tayara H, Tahir M, Chong KT . Identification of prokaryotic promoters and their strength by integrating heterogeneous features. Genomics, 2019, https://doi.org/10.1016/j.ygeno.2019.08.009. In press.
|
|
|
| [30] |
Solovyev V, Salamov A . Automatic annotation of microbial genomes and metagenomic sequences. In metagenomics and its applications in agriculture, biomedicine and environmental studies. Nova Science Publishers, 2011: 61-78.
|
|
|
|
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