氧化苦参碱促进cAMP补救合成的生理机制研究  

Physiological Mechanism for Enhanced Cyclic Adenosine Monophosphate Salvage Synthesis by Oxymatrine

作　　者：刘玫秀 陈晨 朱云龙 刘伟 张中华 常景玲[1] 李志刚 LIU Meixiu;CHEN Chen;ZHU Yunlong;LIU Wei;ZHANG Zhonghua;CHANG Jingling;LI Zhigang(School of Life Sciences,Henan Institute of Science and Technology,Xinxiang 453003,China)

机构地区：[1]河南科技学院生命科学学院,新乡453003

出　　处：《中国生物工程杂志》2024年第12期13-20,共8页China Biotechnology

基　　金：河南省高等学校重点科研项目(23A416006);河南省科技攻关项目(222102110195、232102110127);河南省研究生教育改革与质量提升工程项目(YJS2024JD19)资助项目。

摘　　要：目的:补救途径合成cAMP时,伴随鸟苷等副产物的大量生成,通过添加肌苷酸脱氢酶抑制剂减少副产物合成促进cAMP发酵合成。方法:通过摇瓶实验确定最适肌苷酸脱氢酶抑制剂种类及添加条件。在7 L发酵罐中进行发酵试验,从发酵整体性能、关键酶活性、副产物水平及转录组分析等方面研究氧化苦参碱促进cAMP发酵合成的机制。结果:发酵24 h时添加20 mg/L氧化苦参碱,cAMP浓度达到4.30 g/L,与对照相比,cAMP浓度显著提高了58.67%。对关键酶活性和副产物分析表明,氧化苦参碱显著提高了产物合成相关酶活性,抑制了肌苷酸脱氢酶和5′-核苷酸酶活性,副产物鸟苷合成明显下降。转录组测序分析结果表明,糖酵解(EMP)、从头合成及副产物合成等途径中相关酶基因转录水平明显降低,同时,TCA循环、PPP途径、补救途径中相关酶基因转录水平显著上升。结论:氧化苦参碱能明显抑制副产物合成,提高cAMP合成途径的碳流和ATP供应。Objective:When cAMP was synthesized via the salvage pathway,guanosine was also produced in large amounts as a major by-product.In order to enhance cAMP salvage synthesis,an inosine monophosphate dehydrogenase(IMPDH)inhibitor was screened and utilized to reduce by-product production.Methods:The optimum addition conditions for IMPDH were determined by shake flask experiments and the fermentations with oxymatrine addition were carried out in a 7 L fermentor.Then,the fermentation performance,key enzyme activities,by-product levels and transcriptome analysis were systematically investigated.Results:With the addition of 20 mg/L oxymatrine for 24 h,the cAMP concentration reached 4.30 g/L with an increase of 58.67%higher than that of the control,and the fermentation performance was also obviously promoted.The results of enzyme activities and by-product levels showed that IMPDH together with 5'-nucleotidase were strongly inhibited and the enzyme activities for cAMP synthesis were enhanced,obviously leading to higher cAMP levels and lower guanosine production.The transcriptome analysis results showed that due to the addition of oxymatrine,the transcription levels of key enzyme genes in EMP,de novo synthesis and by-product synthesis pathway were significantly decreased,and at the same time,those in TCA cycle,PPP pathway and salvage pathway were significantly increased.Conclusion:Oxymatrine could effectively decrease by-product synthesis and promote cAMP synthesis by regulating metabolism of enzyme activity and transcription level,resulting in more carbon flow and ATP supply directed into cAMP savage pathway for product synthesis.

关 键 词：氧化苦参碱 转录组分析 肌苷酸脱氢酶 鸟苷 环磷酸腺苷 

分 类 号：Q819[生物学—生物工程]