大肠杆菌糖酵解途径和三羧酸循环启动子的表征及其应用 <sup>*</sup>

doi:10.13523/j.cb.1912034

中国生物工程杂志

2020, Vol. 40

Issue (6): 20-30 DOI: 10.13523/j.cb.1912034

研究报告

大肠杆菌糖酵解途径和三羧酸循环启动子的表征及其应用 ^*

玄美娟,张晓云,高莹,高丽影,吴佳婧,马梅,王艳梅,寇航,路福平,黎明(

)

工业发酵微生物教育部重点实验室天津市工业微生物重点实验室天津科技大学生物工程学院天津 300457

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

全文: PDF(1051 KB) HTML

摘要：

启动子是控制基因转录的重要元件,也是合成生物学研究和细胞工厂设计的关键环节。糖酵解途径和三羧酸循环是糖类分解代谢的中心代谢,受到包括启动子强度在内的严格调控。为了筛选一系列能满足合成生物学研究和细胞工厂设计需要的不同强度的内源性组成型启动子,利用报告基因——红色荧光蛋白mCherry和在线分析软件,系统研究了大肠杆菌糖酵解和三羧酸循环中27个启动子的强度和核心结构元件。结果表明:这些启动子的强度范围变化很大,最强启动子PgapA的强度是最弱启动子PacnA强度的43.6倍;启动子的-10序列和-35序列与它们的一致序列也不完全相同,两者之间的距离为17±3bp;但是,启动子的强度和启动子的结构特征基本一致。应用最强启动子PgapA在重组大肠杆菌DH5αΔpck中分别表达磷酸烯醇式丙酮酸羧化酶基因和丙酮酸激酶基因,它们的酶活性分别提高了0.32和1.57倍,柠檬酸产量也提高了124.7%和75.5%。这些不同强度的启动子为大肠杆菌的合成生物学研究和细胞工厂设计奠定了一定的基础。

关键词： 启动子; 糖酵解途径; 三羧酸循环; 磷酸烯醇式丙酮酸羧化酶; 丙酮酸激酶

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.

Key words: Promoter Glycolytic pathway Tricarboxylic acid cycle Pyruvate kinase Phosphoenolpyruvate carboxylase

收稿日期: 2019-12-20 出版日期: 2020-06-23

ZTFLH:

Q814

基金资助: * 国家自然科学基金(21176190)

通讯作者: 黎明 E-mail: liming09@tust.edu.cn

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	路福平
	黎明

引用本文:

玄美娟,张晓云,高莹,高丽影,吴佳婧,马梅,王艳梅,寇航,路福平,黎明. 大肠杆菌糖酵解途径和三羧酸循环启动子的表征及其应用 ^*[J]. 中国生物工程杂志, 2020, 40(6): 20-30.

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.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1912034 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I6/20

表1 本研究所用的菌株和质粒

图1 大肠杆菌糖酵解途径及三羧酸循环

Number		Gene		Primer name and primer sequence(5'→3')	Restriction site
1		glk		glkF: CAGGATCCGGCATCGCTGCAATTGGTGC	BamH Ⅰ
				glkR: GGGGTACCACTGCTCCGCTAAAGTCAAAATAATTCTTT	KpnⅠ
2		pgm		pgmF: CGGGATCCCGCGTTGTCGGGTTAGGGTATTAT	BamH Ⅰ
				pgmR: CCGGTACCATCCAGTGCAGTGTCTTAGCTGAATAC	Kpn Ⅰ
3		pgi		pgiF: CGGGATCCACTGGCTCCTCCAACACCGTTACTT	BamH Ⅰ
				pgiR: CCGGTACCTACTCTTCTGATTTTGAGAATTGTGAC	Kpn Ⅰ
4		pfkA		pfkAF: GACGGATCCCAGGGAGGGTAAACGGTCTATGCTT	BamH Ⅰ
				pfkAR: CCGGTACCTACCTCTGAACTTTGGAATGCAA	Kpn Ⅰ
5		pfkB		pfkBF: CCGGATCCAAGGATCAAAGATTAGCGTCCCTGG	BamH Ⅰ
				pfkBR: CGGGTACCTCCTCCTATAGGCTGATTTCAGTCTG	Kpn Ⅰ
6		fbaB		fbaBF: CCGGATCCAGGTAATGTAAGGTACGCGATGACAA	BamH Ⅰ
				fbaBR: CCGGTACCTGCTCCCGTAAATTCCGATTGG	Kpn Ⅰ
7		fbaA		fbaAF: CCGGATCCTCATACTCTAAATAATTCGAGTTGCAGGAA	BamH Ⅰ
				fbaAR: CCGGTACCTGTCCTGTATCGTCGGGCCTTATA	Kpn Ⅰ
8		gapA		gapAF: CAGGATCCGGCTGCACCTAAATCGTGATGAA	BamH Ⅰ
				gapAR: CCGGTACCTCCACCAGCTATTTGTTAGTGAATAAAAG	Kpn Ⅰ
Number	Gene		Primer name and primer sequence(5'→3')			Restriction site
9	pgk		pgkF: GCGGATCCGTCAGATAAATGTCTTCTTCGGCTGGA			BamH Ⅰ
			pgkR: CCGGTACCCTCCTGCAAGGTTTTCCCTGAGCAA			Kpn Ⅰ
10	gpmA		gpmAF: CCGGATCCGTTCAAATCACCAGCAAACACCGACAT			BamH Ⅰ
			gpmAR: CCGGTACCTACTCCTCAAATCATCTTTTAATGATAATAATT			Kpn Ⅰ
11	gpmM		gpmMF: CTGGATCCCGCGTTAACTGGAATGCAATTT			BamH Ⅰ
			gpmMR: CCGGTACCAACCTCATACTCAAGAGTCAAAATTTGCG			Kpn Ⅰ
12	eno		enoF: CGGGATCCTTGCCAGTTCCATCCGGAGTTT			BamH Ⅰ
			enoR: CCGGTACCTTCCTCAAGTCACTAGTTAAACTGAAACTCC			Kpn Ⅰ
13	pykF		pykFF: GGGGATCCTATAATGCGCGCCAATTGACT			BamH Ⅰ
			pykFR: CCGGTACCGTCTTAGTCTTTAAGTTGAGAAGGATGGG			Kpn Ⅰ
14	pykA		pykAF: GTGGATCCGGTCAAAGAAGCGCTGAAGGAA			BamH Ⅰ
			pykAR: GGGGTACCTACTCCGTTGACTGAAACAACCAGG			Kpn Ⅰ
15	aceE		aceEF: CCGGATCCTCTCTGCGTCGTCTGGAGCAAC			BamH Ⅰ
			aceEhR: CCGGTACCTATTCCTTATCTATCTAATAACGTTGAGTTTTCT			Kpn Ⅰ
16	gltA		gltAF: CCGGATCCAGTGTGGAAGTATTGACCAATTCATTC			BamH Ⅰ
			gltAR: CTGGTACCTCTCCTTAGCGCCTTATTGCG			Kpn Ⅰ
17	acnA		acnAF: CGGGATCCATGCCAGCATAGTGACAATGAAACAG			BamH Ⅰ
			acnAR: CCGGTACCCCTCCTTAATGACAGGGTTGCG			Kpn Ⅰ
18	acnB		acnBF: CCGGATCCGATTATCACCATGCGAATTAACG			BamH Ⅰ
			acnBR: CCGGTACCTCTCCTCGCTCTCATTGTCATAGT			Kpn Ⅰ
19	icd		icdF: CGGGATCCTTATAGCCTAATAACGCGCATCTTTCAT			BamH Ⅰ
			icdR: CCGGTACCCTCTCCTTCGAGCGCTACTGGTTT			Kpn Ⅰ
20	sucA/B		sucA,sucBF: TGGGATCCTTATCCGGCCTACAAGTCATTACCC			BamH Ⅰ
			sucA,sucBR: ACGGTACCATCCCTTAAGCATCTTTTTTATGC			Kpn Ⅰ
21	sucC/D		sucC,sucDF: TCGGATCCACCAATGTAGGTCGGATAAGGCG			BamH Ⅰ
			sucC,sucDR: CCGGTACCTGTCCATCCTTCAGTAATCGTTATC			Kpn Ⅰ
22	sdhA/B/C/D		sdhF: CCGGATCCCAGCCTATACTGCCGCCAGGT			BamH Ⅰ
			sdhR: CCGGTACCCGCCCACATGCTGTTCTTATTATTC			Kpn Ⅰ
23	frdA/B/C/D		frdF: CGGGATCCAGGAATCAAACAGCGGTGGGC			BamH Ⅰ
			frdR: CCGGTACCTTCCTCCAGATTGTTTTTATCCCACAG			Kpn Ⅰ
24	fumA/C		fumF: CCGGATCCCATGATCAATCCCTGTTTTAATGTGG			BamH Ⅰ
			fumR: CCGGTACCCTCTCACTTACTGCCTGGTTTGGTTATG			Kpn Ⅰ
25	mqo		mqoF: TCGGATCCCGGACGGCATAATATTACGACGC			BamH Ⅰ
			mqoR: CCGGTACCAATGCCTTACTTTTAGTCGCTTTATTGC			Kpn Ⅰ
26	mdh		mdhF: CGGGATCCTGAGAAACATGCCTGCGTCACG			BamH Ⅰ
			mdhR:CCGGTACCCTCCTTATTATATTGATAAACTAAGATATGTTGCTCC			Kpn Ⅰ
27	ldhA		ldhAF: CGGGATCCTCAGTAATAACAGCGCGAGAACG			BamH Ⅰ
			ldhAR: CCGGTACCTTCTCCAGTGATGTTGAATCACA			Kpn Ⅰ
28	PgapA		ETgapAF1: GGCCGCGCGCTGCACCTAAATCGTGATGAAAATC			BssH Ⅱ
			ETgapAR1: GCTCTAGACAGCTATTTGTTAGTGAATAAAAGGTTGCCTG			Xba Ⅰ
29	ppc		ETppcF: GGTTAACCATATGAACGAACAATATTCCGCATTGCGT			Nde Ⅰ
			ETppcR: GGACTAGTTTAGCCGGTATTACGCATACCTGCC			Spe Ⅰ
30	pykF		ETpykFF: GGTTAACCATATGAAAAAGACCAAAATTGTTTGCACCATC			Nde Ⅰ
			ETpykFR: GGACTAGTTTACAGGACGTGAACAGATGCGG			Spe Ⅰ

表2 扩增启动子和目的基因引物序列

图2 重组质粒pET-promoter-RFP的构建过程

图3 重组质粒pET-promoter-RFP 酶切验证

图4 不同基因启动子相对于启动子PacnA的相对强度

表3 启动子功能元件分析

图5 重组质粒pET-gapA-PPC与pET-gapA-PykF构建

图6 重组质粒pET-gapA-ppc(a)与pET-gapA-PykF(b)酶切验证

图7 PPC(a)与PykF(b)酶活力测定

图8 重组菌株产柠檬酸的含量

[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.
	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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