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

【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,thlA2和thlA3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(K_m),但ThlA1对四碳底物的催化效率(k_(cat)/K_m)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。

[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 kluyveriproduced 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.