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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (4): 41-50    DOI: 10.13523/j.cb.2210051
研究报告     
低聚磷酸盐促进cAMP发酵合成的全局转录组分析*
卢南巡1,王荔巍1,刘玫秀1,张中华1,常景玲1,2,李志刚1,2,**()
1.河南科技学院生命科技学院 新乡 453003
2.现代生物育种河南省协同创新中心 新乡 453003
Global Gene Transcriptome Analysis for Enhanced Cyclic Adenosine Monophosphate Fermentation Performance by Polyphosphates
LU Nan-xun1,Wang Li-wei1,LIU Mei-xiu1,ZHANG Zhong-hua1,CHANG Jing-ling1,2,LI Zhi-gang1,2,**()
1. School of Life Science and Technology, Henan Institute of Science and Technology, Xinxiang 453003, China
2. Collaborative Innovation Center of Modem Biological Breeding of Henan Province, Xinxiang 453003, China
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摘要：

目的:以节杆菌Arthrobacter sp. CCTCC 2013431为研究对象,探究低聚磷酸盐作为能量供体促进环磷酸腺苷(cyclic adenosine monophosphate,cAMP)发酵合成的作用机理。方法:在7 L发酵罐中添加低聚磷酸盐,对发酵性能、转录组学、关键酶活性以及重要代谢物水平进行分析,揭示低聚磷酸盐促进cAMP发酵合成的机理。结果:发酵24 h添加2 g/L六偏磷酸钠,cAMP产量显著提高,达到3.64 g/L,与对照组相比提高了33.82%。转录组数据分析表明,由于六偏磷酸钠的添加,227个基因表达量显著上调,265个基因表达量显著下调;磷酸戊糖途径和cAMP合成途径中多数酶编码基因的转录水平,电子传递链中复合体III、复合体IV和F0F1-ATP酶以及聚磷酸盐激酶编码基因的转录水平,硫氧还蛋白、过氧化氢酶及CLP蛋白酶等编码基因的转录水平均显著提高。此外,对丙酮酸激酶、6-磷酸葡萄糖脱氢酶、腺苷琥珀酸合成酶、腺苷酸环化酶、过氧化氢酶、聚磷酸盐激酶活性以及胞内活性氧、ATP和NADPH等进行测定,进一步证明了转录组分析结果。结论:六偏磷酸钠能够强化磷酸戊糖途径与cAMP合成途径,提高ATP供应,同时维持胞内氧化还原平衡,促进产物合成与积累。
关键词： 低聚磷酸盐;  转录组分析;  氧化还原平衡;  能量代谢;  环磷酸腺苷    
Abstract:

Objective: To explore the mechanism for enhanced cAMP fermentation production by polyphosphates, Arthrobacter sp. CCTCC 2013431 culture was carried out under low-polyphosphates addition condition as the starting strain. Methods: Fermentations with/without hexametaphosphate addition were conducted in a 7 L bioreactor and the fermentation performance, global gene transcriptome, key enzymes activities together with important metabolites levels were analyzed systematically. Results: With 2 g/L-broth sodium hexametaphosphate added at 24 h, cAMP concentration reached 3.64 g/L with an increment of 33.82% higher than that of control group and the fermentation performance was also promoted obviously. Transcriptome analysis showed that 227 genes were up-regulated significantly and 265 genes were down-regulated significantly due to the addition of hexametaphosphate. For glycometabolism, the transcription levels of key enzyme genes in pentose phosphate pathway and cAMP synthesis pathway were enhanced significantly and for energy metabolism the transcription levels of complex Ⅲ, complex Ⅳ as well as F0F1-ATPase in electron transport chain and polyphosphate kinase gene were also increased significantly by which sufficient carbon skeleton and ATP were provided for cAMP biosynthesis. In addition, transcription levels of reductase genes, such as thioredoxin, catalase and CLP protease, were also increased significantly whereby intracellular redox balance was maintained conducive to cell metabolism and product synthesis. Finally, the activities of pyruvate kinase, 6-phosphoglucose dehydrogenase, adenylosuccinate synthetase, adenylate cyclase, catalase, polyphosphate kinase and intracellular ROS, ATP and NADPH levels under different fermentation conditions were measured to further support the transcriptome analysis results. Conclusion: Sodium hexametaphosphate addition enhanced the carbon flux distribution in pentose phosphate pathway and cAMP synthesis pathway and energy metabolism for ATP synthesis. At the same time, intracellular redox balance was also maintained. Furthermore, cAMP fermentation synthesis and accumulation was promoted significantly.
Key words: Polyphosphates    Transcriptome analysis    Redox balance    Energy metabolism    Cyclic adenosine monophosphate (cAMP)
收稿日期: 2022-10-29 出版日期: 2023-05-04
ZTFLH:  Q819  
基金资助: 河南省高等学校重点科研(23A416006)
通讯作者: **电子信箱:lizhigang25@126.com   
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卢南巡
王荔巍
刘玫秀
张中华
常景玲
李志刚

引用本文:

卢南巡, 王荔巍, 刘玫秀, 张中华, 常景玲, 李志刚. 低聚磷酸盐促进cAMP发酵合成的全局转录组分析*[J]. 中国生物工程杂志, 2023, 43(4): 41-50.

LU Nan-xun, Wang Li-wei, LIU Mei-xiu, ZHANG Zhong-hua, CHANG Jing-ling, LI Zhi-gang. Global Gene Transcriptome Analysis for Enhanced Cyclic Adenosine Monophosphate Fermentation Performance by Polyphosphates. China Biotechnology, 2023, 43(4): 41-50.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2210051    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I4/41

低聚磷酸盐 添加量/(g·L-1-broth) 添加时间/h
0 12 24 36 48
Na5P3O10 1 1.74±0.02 1.86±0.03 2.01±0.07 1.95±0.03 1.78±0.02
2 1.88±0.04 2.17±0.02 2.24±0.05 2.13±0.04 2.03±0.05
3 1.81±0.03 2.09±0.04 2.12±0.06 2.07±0.07 1.97±0.03
4 1.72±0.05 1.94±0.07 2.07±0.03 1.91±0.09 1.83±0.06
(NaPO3)6 1 1.84±0.08 2.13±0.06 2.47±0.02 2.24±0.06 2.12±0.02
2 2.01±0.04 2.54±0.03 2.82±0.04 2.67±0.04 2.36±0.04
3 1.93±0.02 2.37±0.05 2.53±0.07 2.38±0.03 2.14±0.03
4 1.87±0.03 2.15±0.04 2.21±0.06 2.16±0.08 2.04±0.07
对照 0 1.66±0.04 - - - -
表1  低聚磷酸盐添加量和添加时间对cAMP发酵生产的影响
图1  添加六偏磷酸钠对cAMP发酵性能的影响
图2  显著差异表达基因数目统计
图3  显著差异表达基因的GO功能分类
图4  添加六偏磷酸钠对糖代谢及cAMP合成途径中关键酶编码基因转录水平的影响
Name Gene ID Fold change Description
qcrB Gene 2735 1.24 Cytochrome bc complex cytochrome b subunit
cydB Gene 1177 1.38 Cytochrome d ubiquinol oxidase subunit II
coxC Gene 2738 1.33 Cytochrome C oxidase subunit III
coxA Gene 2731 1.26 Cytochrome C oxidase subunit I
atpA Gene 0526 1.21 F0F1 ATP synthase subunit alpha
atpB Gene 0530 1.27 F0F1 ATP synthase subunit A
atpC Gene 0523 1.29 ATP synthase epsilon chain
ppx Gene 0866 1.26 Exopolyphosphatase
ccdA Gene 1569 1.54 Cytochrome C biogenesis protein
表2  添加/不添加六偏磷酸钠条件下与能量代谢相关的差异表达基因分析
Name Gene ID Fold change Description
katE Gene 0919 1.22 Catalase
katG Gene 3837 1.21 Catalase/peroxidase HPI
trxA Gene 0119 1.50 Thioredoxin family protein
cydCD Gene 1178 1.40 Cysteine transport
ybbN Gene 0513 1.34 Thioredoxin
clpS Gene 0502 1.25 Protein catabolic process
clpX Gene 2585 1.23 ATP-dependent Clp protease ATP-binding subunit
nei Gene 2591 1.50 Fpg/Nei family DNA glycosylase
mutM Gene 0424 1.49 Formamidopyrimidine DNA glycosylase
表3  添加/不添加六偏磷酸钠条件下与维持胞内氧化还原平衡相关差异表达基因分析
图5  六偏磷酸钠对cAMP合成相关途径中关键酶活性及重要代谢产物水平的影响
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