克氏梭菌硫解酶的鉴定及其功能研究
Identification and functional analysis of the three thiolases from Clostridium kluyveri
投稿时间:2018-02-09  修订日期:2018-05-04
DOI:10.13343/j.cnki.wsxb.20180074
中文关键词:克氏梭菌  己酸  硫解酶  转录组
英文关键词:Clostridium kluyveri  hexanoate  thiolase  transcriptome
基金项目:国家自然科学基金(21706097,31530055)
作者单位E-mail
杨娇 江南大学生物工程学院, 酿酒科学与酶技术研究中心, 江苏 无锡 214122  
任聪 江南大学生物工程学院, 酿酒科学与酶技术研究中心, 江苏 无锡 214122
江南大学教育部工业生物技术重点实验室, 江苏 无锡 214122 
 
徐岩 江南大学生物工程学院, 酿酒科学与酶技术研究中心, 江苏 无锡 214122
江南大学教育部工业生物技术重点实验室, 江苏 无锡 214122 
yxu@jiangnan.edu.cn 
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中文摘要:
      [目的]硫解酶是梭菌属微生物合成短中链脂肪酸的关键酶。克氏梭菌(Clostridium kluyveri)具有3个高度同源的硫解酶编码基因,对这3个基因的功能鉴定是解析克氏梭菌高己酸合成能力的关键。[方法]通过发酵动力学分析确定克氏梭菌的己酸和丁酸生成动力学特征;转录组测序结合反转录-荧光定量RCR分析克氏梭菌3个硫解酶编码基因的表达水平和时序表达特征;在大肠杆菌中异源表达这3个硫解酶,并对其硫解酶动力学参数进行测定。[结果]克氏梭菌生成丁酸、己酸、辛酸,其中己酸为主要代谢产物;转录组数据显示,在乙酸消耗完全之前,thlA1基因维持恒定表达,thlA2基因表达时序上调,thlA3基因表达时序下调,转录组测序表明3个硫解酶编码基因均具有较高水平的转录活性,thlA2thlA3的最高表达量分别约为thlA1的29%和43%;硫解酶动力学参数测定结果表明,克氏梭菌3个硫解酶对于四碳底物均显示出相似的底物亲和力(Km),但ThlA1对四碳底物的催化效率(kcat/Km)略低于ThlA2和ThlA3。[结论]克氏梭菌的3个硫解酶均具有催化活性,在克氏梭菌体内均呈活跃表达,表明克氏梭菌拥有3个具有催化活性的硫解酶,这为后续深入研究克氏梭菌己酸合成机理奠定了基础。
英文摘要:
      [Objective] Clostridium kluyveri genome encodes for three highly homologous thiolases. To identify the function of these three thiolases will help us to understand how Clostridium kluyveri can efficiently produce hexanoate. [Methods] The characteristics of hexanoate and butyrate production were examined via fermentation kinetics analysis. The transcriptome and reverse transcription-quantitative RCR were used to analyze the expression profiles of thiolase-encoding genes during fermentation. Thiolases from Clostridium kluyveri were heterologously expressed in Escherichia coli and their enzyme kinetic parameters were examined. [Results] Clostridium kluyveri produced butyrate, hexanoate and octanoate, of which hexanoate was the major product. Transcription analysis showed that thlA1 gene was constitutively expressed, thlA2 gene was up-regulated and thlA3 gene was down-regulated before the depletion of acetate. The three thiolase-encoding genes all had higher transcription levels, and the highest expression levels of thlA2 and thlA3 were approximately 29% and 43% that of thlA1, respectively. The enzyme kinetic parameters with four-carbon substrate demonstrated that the three thiolases from Clostridium kluyveri had similar affinity. However, the catalytic efficiency (kcat/Km) of ThlA1 for four-carbon substrates was lower than that of ThlA2 and ThlA3. [Conclusion] All of the three thiolases from Clostridium kluyveri had catalytic activities, and also actively expressed in vivo.
杨娇,任聪,徐岩.克氏梭菌硫解酶的鉴定及其功能研究.微生物学报,2019,59(1):79-92
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