热稳定性丙酮酸:铁氧还蛋白氧化还原酶异源表达及其在乙酰辅酶A合成中的应用 <sup>*</sup>

doi:10.13523/j.cb.1908013

中国生物工程杂志

2020, Vol. 40

Issue (3): 72-78 DOI: 10.13523/j.cb.1908013

技术与方法

热稳定性丙酮酸:铁氧还蛋白氧化还原酶异源表达及其在乙酰辅酶A合成中的应用 ^*

乐易林,傅毓,倪黎,孙建中(

)

江苏大学环境与安全工程学院生物质能源研究所镇江 212013

Expression and Characterization of a Thermostable Pyruvate Ferredoxin Oxidoreductase from the Hyperthermophile Thermotoga neapolitana and Its Application in Acetyl-CoA Production

LE Yi-lin,FU Yu,NI Li,SUN Jian-zhong(

)

Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University, Jiangsu 212013, China

全文: PDF(749 KB) HTML

摘要：

乙酰辅酶A被广泛应用到生物医学研究中,使用TPP代替昂贵的ATP为辅因子合成乙酰辅酶A受到广泛关注.新阿波罗栖热袍菌(Thermotoga neapolitana)来源的丙酮酸:铁氧还蛋白氧化还原酶(TnPFOR)在大肠杆菌中进行了重组表达,分析了其酶学特性,并探讨了利用嗜热酶(TnPFOR)酶法合成乙酰辅酶A.采用pET-20b(+)载体,将新阿波罗栖热袍菌来源的四亚基组成的嗜热酶(TnPFOR)在大肠杆菌中进行异源表达;通过热处理和阴离子交换层析法纯化嗜热酶(TnPFOR);重组表达的嗜热酶(TnPFOR)的最适反应温度和pH分别为90℃和6.5,TnPFOR在90℃下孵育1h时保留了50%活性.利用嗜热酶(TnPFOR),以TPP为辅酶合成了乙酰辅酶A,并探讨了不同温度,丙酮酸钠底物浓度和反应时间对乙酰辅酶A合成的影响.得到的优化条件为:最适反应温度为90℃,丙酮酸钠浓度为1.5mmol/L,反应时间为2min.

关键词： 大肠杆菌; 重组表达; 嗜热酶; 嗜热菌; 合成乙酰辅酶A

Abstract:

Pyruvate ferredoxin oxidoreductase (PFOR) catalyzes the synthesis of acetyl-CoA from pyruvate and coenzyme A (CoA) using thiamine pyrophosphate (TPP) as coenzyme. The four subunit-type TnPFOR from T. neapolitana was expressed in Escherichia coli and characterized. The gene of TnPFOR from T. neapolitana was cloned into pET-20b(+). TnPFOR was purified by a heat treatment followed by an ion exchange chromatography. The TnPFOR had an optimal condition for its maximum activity at 90℃ and pH 6.5 and it was indeed thermostable with a half-life of more than 1h at 90℃. The application of TnPFOR to catalyze the conversion of pyruvate into acetyl-CoA was also evaluated. The influence of different reaction conditions (reaction temperature, pyruvate concentrations and reaction time) on the synthesis of acetyl-CoA was discussed. The optimal reaction temperature is 90℃, pyruvate concentrations is 1.5mmol/L and the reaction time is 2min.

Key words: Escherichia coli Recombinant expression Thermophilic enzyme Thermophiles Synthesis of acetyl-CoA

收稿日期: 2019-08-06 出版日期: 2020-04-18

ZTFLH:

Q946.5

基金资助: * 国家自然科学基金(31772529);国家重点科技项目(2018YF0107100)

通讯作者: 孙建中 E-mail: jzsun1002@ujs.edu.cn

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引用本文:

乐易林,傅毓,倪黎,孙建中. 热稳定性丙酮酸:铁氧还蛋白氧化还原酶异源表达及其在乙酰辅酶A合成中的应用 ^*[J]. 中国生物工程杂志, 2020, 40(3): 72-78.

LE Yi-lin,FU Yu,NI Li,SUN Jian-zhong. Expression and Characterization of a Thermostable Pyruvate Ferredoxin Oxidoreductase from the Hyperthermophile Thermotoga neapolitana and Its Application in Acetyl-CoA Production. China Biotechnology, 2020, 40(3): 72-78.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1908013 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I3/72

图1 TnPFOR酶四个亚基共表达示意图

图2 丙酮酸:铁氧还蛋白氧化还原酶基因PCR扩增结果(a)和重组质粒pET-PFOR双酶切验证胶图(b)

图3 重组丙酮酸:铁氧还蛋白氧化还原酶表达和纯化SDS-PAGE(a)和Native-PAGE(b)电泳图谱

图4 温度,pH和氧气对TnPFOR酶活性和酶稳定性的影响

图5 HPLC检测热稳定性丙酮酸:铁氧还蛋白氧化还原酶合成乙酰辅酶A

图6 反应温度(a),丙酮酸钠底物浓度(b)和反应时间(c)对乙酰辅酶A合成的影响

[1]	Nielsen J . Synthetic biology for engineering acetyl coenzyme A metabolism in yeast. Mbio, 2014,5(6):e02153.
[2]	Koskinen A M, Karisalmi K . Polyketide stereotetrads in natural products. Chem Soc Rev, 2005,34(8):677-690.
[3]	Go M K, Chow J Y, Cheung V W , et al. Establishing a toolkit for precursor-directed polyketide biosynthesis: exploring substrate promiscuities of acid-CoA ligases. Biochemistry, 2012,51(22):4568-4579.
[4]	Nes W D . Biosynthesis of cholesterol and other sterols. Chem Rev, 2011,111(10):6423-6451.
[5]	Ouyang T, Walt D R . A new chemical method for synthesizing and recycling acyl coenzyme A thioesters. J Org Chem, 1991,56(11):3752-3755.
[6]	Lu X, Liu Y, Yang Y , et al. Constructing a synthetic pathway for acetyl-coenzyme A from one-carbon through enzyme design. Nature Communications, 2019,10(1):1378.
[7]	Dubey N C, Tripathi B P, Müller M , et al. Enhanced activity of acetyl CoA synthetase adsorbed on smart microgel: an implication for precursor biosynthesis. ACS Appl Mater Interfaces, 2015,7(3):1500-1507.
[8]	Dubey N C, Tripathi B P, Stamm M , et al. Smart core-shell microgel support for acetyl coenzyme A synthetase: a step toward efficient synthesis of polyketide-based drugs. Biomacromolecules, 2014,15(7):2776-2783.
[9]	Takenaka M, Yoon K S, Matsumoto T , et al. Acetyl-CoA production by encapsulated pyruvate ferredoxin oxidoreductase in alginate hydrogels. Bioresour Technol, 2017,227:279-285.
[10]	任增亮, 堵国成, 陈坚 , 等. 大肠杆菌高效表达重组蛋白策略. 中国生物工程杂志, 2007,27(9):103-109.
	Zeng R L, Du G C, Chen J , et al. Strategies for high-level expression of recombinant protein in Escherichia coli. China Biotechnology, 2007,27(9):103-109.
[11]	罗漫杰, 谢渊, 钱志刚 , 等. 超嗜热酯酶在不同宿主中的异源高效表达研究. 中国生物工程杂志, 2014,34(12):36-44.
	Luo M J, Xie Y, Qian Z G , et al. High-level heterogenous expression of a hyperthermophilic esterase in different hosts. China Biotechnology, 2014,34(12):36-44.
[12]	马蓉, 徐昊, 丁锐 , 等. 大肠杆菌多基因共表达策略. 中国生物工程杂志, 2012,32(4):117-122.
	Ma R, Xu H, Ding R , et al. The strategy of gene coexpression in Escherichia coli. China Biotechnology, 2012,32(4):117-122.
[13]	Dipasquale L ,D' Ippolito G, Fontana A. Capnophilic lactic fermentation and hydrogen synthesis by Thermotoga neapolitana: an unexpected deviation from the dark fermentation model. International Journal of Hydrogen Energy, 2014,39(10):4857-4862.
[14]	Kletzin A, Adams M W . Molecular and phylogenetic characterization of pyruvate and 2-ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosus and pyruvate ferredoxin oxidoreductase from Thermotoga maritima. J Bacteriol, 1996,178(1):248-257.
[15]	Yoon K S, Ishii M, Kodama T , et al. Purification and characterization of pyruvate: ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6. Archives of Microbiology, 1997,167(5):275-279.
[16]	杜明伦, 黄君君, 马香 , 等. 大肠杆菌基因组中重叠基因注释的机器学习优化方法. 中国生物化学与分子生物学报, 2018,34(8):861-867.
	Du M L , H J J, Ma X , et al. Machine learning optimization method for overlapping genes annotation in Escherichia coli genomes. Chinese Journal of Biochemistry and Molecular Biol, 2018,34(8):861-867.

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