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

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，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。

Abstract:

[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 (k_cat/K_m) 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.