Burkholderia sp.IDO3对粪臭素的响应及下游代谢基因  

Response of Burkholderia sp.IDO3 to skatole and downstream metabolic gene analysis

作　　者：李玉洁 张佳鑫 王彩红 马桥 LI Yujie;ZHANG Jiaxin;WANG Caihong;MA Qiao(College of Environmental Science and Engineering,Dalian Maritime University,Dalian 116026,China)

机构地区：[1]大连海事大学环境科学与工程学院,大连116026

出　　处：《应用与环境生物学报》2024年第3期579-585,共7页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(32170121)资助。

摘　　要：粪臭素是一种具有强烈粪臭味的氮杂环芳烃污染物,广泛存在于动物肠道、粪便、垃圾填埋场和海洋等环境介质中,较为稳定且难以降解.以Burkholderia sp.IDO3为研究对象,系统研究菌株在粪臭素压力下的生理生化响应,并利用RT-qPCR和异源表达技术探究下游降解功能基因.结果显示,粪臭素对Burkholderia sp.IDO3最小抑制浓度为1.5mmol/L;粪臭素促进菌膜形成和提高菌株表面疏水性,且呈剂量依赖性.氧化应激实验表明粪臭素提高Burkholderia sp.IDO3的超氧化物歧化酶和过氧化氢酶活性,抑制菌株脂质过氧化(丙二醛)以及还原型谷胱甘肽含量.结合前期组学分析推测邻苯二酚1,2-双加氧酶基因(DCN14_04105)可能为下游代谢关键基因,RT-qPCR实验表明该基因在粪臭素压力下上调3.70倍.将该基因在大肠杆菌中表达并获得纯酶,结果表明该酶最适pH为8.5,最适温度为30℃,Zn^(2+)、Co^(2+)、Cd^(2+)、Se4+、Sn^(2+)、Ca^(2+)对其具有抑制作用,Km值为25.98μmol/L,kcat值为10.70 s-1.本研究表明粪臭素影响了Burkholderia sp.IDO3菌株的生理生化性能,菌株代谢粪臭素中心代谢途径为邻苯二酚1,2-双加氧酶途径.该结果可为粪臭素的高效降解提供新的微生物资源,也为粪臭素的功能研究提供参考.(图7表1参30)Skatole is a nitrogen containing heterocyclic aromatic pollutant with a strong fecal odor.It is widely distributed in intestines,feces,landfills,and marine environments.It is relatively stable and difficult to degrade.Burkholderia sp.IDO3 was assessed for physiological and biochemical indices under skatole exposure.Downstream metabolic genes were analyzed using RT-qPCR and heterologous gene expression studies.The minimum inhibitory concentration(MIC)of skatole for Burkholderia sp.IDO3 was 1.5 mmol/L.Skatole promotes biofilm formation and hydrophobicity in strain IDO3 in a dose-dependent manner.Oxidative stress experiments revealed that skatole increases superoxide dismutase and catalase activities and inhibits lipid peroxidation(malondialdehyde)and glutathione production.Based on preliminary omics analyses,we speculated that catechol 1,2-dioxygenase(DCN14_04105)may be the key gene involved in downstream metabolism.RTqPCR showed that this gene was upregulated 3.70-fold under skatole exposure.The gene was expressed in Escherichia coli to obtain pure protein.Pure enzyme experiments revealed an optimal pH of 8.5 and an optimal temperature of 30℃.Zn^(2+),Co^(2+),Cd^(2+),Se4+,Sn^(2+),and Ca^(2+)exhibited inhibitory effects.The Km value was 25.98μmol/L,and kcat was 10.70 s-1.This study demonstrated the physiological and biochemical properties of Burkholderia sp.IDO3 was used to assess the effect of skatole exposure.The central metabolic pathway of skatole degradation is likely catalyzed by catechol 1,2-dioxygenase.These results provide a new microbial resource for efficient skatole degradation,and facilitate functional studies.

关 键 词：粪臭素 伯克霍尔德菌 功能响应 生物降解 邻苯二酚1 2-双加氧酶 

分 类 号：X172[环境科学与工程—环境科学]