辅助能量物质强化环磷酸腺苷发酵合成机制 <sup>*</sup>

doi:10.13523/j.cb.1905019

中国生物工程杂志

2020, Vol. 40

Issue (1-2): 102-108 DOI: 10.13523/j.cb.1905019

研究报告

辅助能量物质强化环磷酸腺苷发酵合成机制 ^*

李志刚^1,²,陈宝峰²,张中华²,常景玲^1,^2,^**(

)

1 现代生物育种河南省协同创新中心新乡 453003
2 河南科技学院生命科技学院新乡 453003

The Physiological Mechanism for Enhanced Cyclic Adenosine Monophosphate Biosynthesis by Auxiliary Energy Substance

LI Zhi-gang^1,²,CHEN Bao-feng²,ZHANG Zhong-hua²,CHANG Jing-ling^1,^2,^**(

)

1 Collaborative Innovation Center of Modem Biological Breeding of Henan Province, Xinxiang 453003,China
2 School of Life Science and Technology, Henan Institute of Science and Technology, Xinxiang 453003,China

全文: PDF(801 KB) HTML

摘要：

微生物代谢过程中,环磷酸腺苷(cAMP)由ATP直接环化形成,强化ATP合成有利于产物的积累。在分批发酵24h添加3g/L-broth丙酮酸钠(辅助能量物质),cAMP浓度达到4.13g/L,比对照批次提高了24.4%,发酵性能得到明显改善。对关键酶活性及能量代谢水平的测定结果表明,由于丙酮酸钠的添加,丙酮酸激酶的活性显著下降,而6-磷酸葡萄糖脱氢酶、琥珀腺苷酸合成酶和腺苷酸环化酶等产物合成途径中酶的活性均明显提高;异柠檬酸脱氢酶、琥珀酸脱氢酶和呼吸链脱氢酶等酶活性,以及辅因子NADH/NAD ⁺、ATP/AMP均明显提高。表明添加丙酮酸钠改变了糖酵解和磷酸戊糖途径间的碳流分配,使更多碳流向产物合成途径,同时提高了整体能量代谢水平,更利于ATP的生成,为产物的合成提供了物质和能量基础,进而促进了cAMP的合成与积累。

关键词： 丙酮酸钠; 环磷酸腺苷; 呼吸链脱氢酶; NADH; 关键酶

Abstract:

High ATP level is beneficial to cyclic adenosine phosphate (cAMP) accumulation. Due to 3g/L-broth sodium pyruvate addition into the broth at 24h, cAMP final content achieved 4.13g/L which was enhanced 24.4% when compared with control, with obvious improved fermentation performance. NADH, ATP concentrations and activities of key enzymes contained in related metabolic pathways were detected to explain the mechanism. The results declare that pyruvate kinase was inhibited obviously by sodium pyruvate and the activities of key enzymes for cAMP synthesis (glucose-6-phosphate dehydrogenase, adenylosuccinate synthetase and adenylate cyclase) were enhanced greatly, which indicated that more carbon flux was directed into pentose phosphate pathway and purine synthesis pathway from glycolytic pathway for AMP production. In addition, activities of key enzymes in TCA (isocitrate dehydrogenase and succinate dehydrogenase), NADH/NAD ⁺, ATP/AMP and respiratory chain activity were enhanced greatly which indicated that pyruvate activated energy metabolism to supply sufficient energy for cAMP synthesis. Sufficient energy supplement and enhanced carbon flux accounts for the physiological mechanism of higher cAMP accelerated by sodium pyruvate.

Key words: Pyruvate Cyclic adenosine monophosphate Respiratory chain activity NADH Key enzymes

收稿日期: 2019-05-12 出版日期: 2020-03-27

ZTFLH:

Q819

基金资助: * 河南省科技攻关项目(社会发展)(162102310419)

通讯作者: 常景玲 E-mail: changjl001@126.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	李志刚
	陈宝峰
	张中华
	常景玲

引用本文:

李志刚,陈宝峰,张中华,常景玲. 辅助能量物质强化环磷酸腺苷发酵合成机制 ^*[J]. 中国生物工程杂志, 2020, 40(1-2): 102-108.

LI Zhi-gang,CHEN Bao-feng,ZHANG Zhong-hua,CHANG Jing-ling. The Physiological Mechanism for Enhanced Cyclic Adenosine Monophosphate Biosynthesis by Auxiliary Energy Substance. China Biotechnology, 2020, 40(1-2): 102-108.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1905019 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I1-2/102

表1 丙酮酸钠不同添加时间和添加量对cAMP发酵产苷的影响

图1 添加丙酮酸钠对cAMP发酵性能的影响

图2 丙酮酸钠对琥珀腺苷酸合成酶、腺苷酸环化酶、丙酮酸激酶和6-磷酸葡萄糖脱氢酶活性的影响

图3 丙酮酸钠对异柠檬酸脱氢酶和琥珀酸脱氢酶活性的影响

图4 丙酮酸对NADH/NAD+值及ATP/AMP值的影响

图5 丙酮酸对细胞呼吸链脱氢酶活性的影响

图6 添加丙酮酸钠条件下cAMP代谢合成示意图

[1]	Mcphee I, Gibson L, Kewney J , et al. Cyclic nucleotide signalling: a molecular approach to drug discovery for Alzheimer’s disease. Biochemical Society Transactions, 2005,33(6):1330-1332.
[2]	Hong D, Peng X R . Role of the cAMP in immunological liver injury in mice: comparing LPS-induced model with LPS. Chinese Pharmacology Bull, 2003,19(8):940-943.
[3]	Li L, Chen X C, Cheng J , et al. Bi-stage control of dissolved oxygen to enhance cyclic adenosine monophosphate production by Arthrobacter A302. Bioprocess and Biosystems Engineering, 2012,35(9):1281-1286.
[4]	Chen X C, Song H, Fang T , et al. Enhanced cyclic adenosine monophosphate production by Arthrobacter A302 through rational redistribution of metabolic flux. Bioresource Technology, 2010,101(9):3159-3163.
[5]	陈晓春 . 环磷酸腺苷高产菌的选育及其代谢调控. 南京: 南京工业大学, 2010.
	Chen X C . Breeding high-yield strain for cyclic adenosine monophosphate production and metabolic regulation. Nanjing: Nanjing University of Technology, 2010.
[6]	Liu D, Chen Y, Li A , et al. Enhanced uridine 5'-monophosphate production by whole cell of Saccharomyces cerevisiae through rational redistribution of metabolic flux. Bioprocess and Biosystems Engineering, 2012,35(5):729-737.
[7]	Niu H Q, Chen Y, Yao S W , et al. Metabolic flux analysis of Arthrobacter sp. CGMCC 3584 for cAMP production based on 13C tracer experiments and gas chromatography-mass spectrometry . Journal of Biotechnology, 2013,168(4):355-361.
[8]	徐若烊, 王大慧, 许宏庆 , 等. 丙酮酸钠促进S-腺苷蛋氨酸和谷胱甘肽联合高产及其生理机制. 食品与工业科技, 2018,39(2):113-118.
	Xu R Y, Wang D H, Xu H Q , et al. Sodium pyruvate improves the co-production of S-adenosylmethionine and glutathione and its physiological mechanism. Science and Technology of Food Industry, 2018,39(2):113-118.
[9]	Hara K, Kondo A . ATP regulation in bioproduction. Microbial Cell Factories, 2015,14(1):198-207.
[10]	李志刚, 陈宝峰, 方智博 , 等. 基于柠檬酸盐与次黄嘌呤偶合添加的环磷酸腺苷发酵工艺. 食品与发酵工业, 2018,44(11):154-158.
	Li Z G, Chen B F, Fang Z B , et al. A novel fermentation process for cyclic adenosine monophosphate production based on citrate coupling hypoxanthine addition in pulses. Food and Fermentation Industries, 2018,44(11):154-158.
[11]	Cao Y, Enock M, Shi Z P , et al. A novel metabolic model incorporating directed signal flow diagram with enzymatic activities data for evaluating glutamate yield in glutamate fermentation. Biochemical Engineering Journal, 2013,77(5):136-146.
[12]	Hatzinger P, Palmer P, Smith R , et al. Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria. Journal of Microbiological Methods, 2003,52(1):47-58.
[13]	郑志永, 姚善泾 . 比色法测定大肠杆菌呼吸链脱氢酶活性. 浙江大学学报(工学版), 2005,39(3):119-122.
	Zheng Z Y, Yao S J . Colorimetric assay for respiratory chain dehydrogenase activity in Escherichia coli. Journal of Zhejiang University (Engineering Science), 2005,39(3):119-122.
[14]	Goel A, Lee J, Michael M , et al. Suppressed acid formation by cofeeding of glucose and citrate in Bacillus cultures: emergence of pyruvate kinase as a potential metabolic engineering site. Biotechnology Progress, 1995,11(1):380-385.
[15]	Zhang X M, Lai L H, Xu G Q , et al. Effects of pyruvate kinase on the growth of Corynebacterium glutamicum and L-serine accumulation. Process Biochemistry, 2017,55(11):32-40.

[1]	苗轶男,李敬知,王帅,李春,王颖. 萜烯生物合成中关键酶的研究进展^*[J]. 中国生物工程杂志, 2021, 41(6): 60-70.
[2]	王光路, 王梦园, 周忆菲, 马科, 张帆, 杨雪鹏. 吡咯喹啉醌生物合成研究进展 ^*[J]. 中国生物工程杂志, 2021, 41(1): 103-113.
[3]	陈宝峰,李志刚,张中华,常景玲. 低聚磷酸盐与次黄嘌呤偶合添加提高环磷酸腺苷发酵性能 ^*[J]. 中国生物工程杂志, 2019, 39(8): 25-31.
[4]	程丽娜,陆海燕,曲淑玲,张轶群,丁娟娟,邹少兰. 微生物发酵法生产环磷酸腺苷研究进展 ^*[J]. 中国生物工程杂志, 2018, 38(2): 102-108.
[5]	刘珊, 李元, 祝俊. 偶联基于酮还原酶的NADH再生系统一锅法酶催化合成L-2-氨基丁酸[J]. 中国生物工程杂志, 2017, 37(1): 64-70.
[6]	胡桂元, 杨套伟, 饶志明, 刘梅, 徐美娟, 张显. 增强胞内NDAH水平和乙偶姻还原酶活力提高2,3-丁二醇产量[J]. 中国生物工程杂志, 2016, 36(6): 57-64.
[7]	马力, 吴昊, 王斌斌, 乔建军, 朱宏吉. 转录调控因子Rex的功能及调节机制研究进展[J]. 中国生物工程杂志, 2016, 36(10): 94-100.
[8]	李小鑫, 高明昊, 张苗苗, 刘晓晨, 乔长晟. 溶解氧对γ-聚谷氨酸合成的影响[J]. 中国生物工程杂志, 2015, 35(3): 42-48.
[9]	孙焕民, 过敏, 伊日布斯. 胞内氧化还原水平对嗜热厌氧乙醇菌发酵代谢的影响[J]. 中国生物工程杂志, 2012, 32(07): 73-78.
[10]	肖丽霞, 师明磊, 王洋, 张彦, 沈文龙, 李德彬, 赵玉军, 赵志虎. NADH依赖型酶活性检测方法的建立[J]. 中国生物工程杂志, 2012, 32(04): 72-75.
[11]	吴琼周应群孙超陈士林. 人参皂苷生物合成和次生代谢工程[J]. 中国生物工程杂志, 2009, 29(10): 102-108.
[12]	黄志华,张延平,黄兴,王宝光,曹竹安. 在Klebsiella pneumoniae醛脱氢酶失活菌中构建NADH再生系统[J]. 中国生物工程杂志, 2006, 26(12): 75-80.
[13]	甘烦远, 沈月毛, 郝小江. 紫杉醇生物合成的研究进展[J]. 中国生物工程杂志, 2000, 20(1): 52-56.

Viewed

Full text

Abstract

Cited

Shared

Discussed