嗜热厌氧乙醇杆菌调控因子PadhE-1的分离、重组表达及其与adhE基因启动子的结合

Expression and Identification of the PadhE-1 from Thermoanaerobacter ethanolicus JW200 and Its Binding with adhE Promoter in vitro

摘要:

嗜热厌氧乙醇杆菌发酵是生产纤维素乙醇的有效途径, 而嗜热厌氧乙醇杆菌醛/醇脱氢酶(AdhE)是其乙醇代谢途径中的关键酶。以adhE启动子区域的DNA序列(TRRadhE)作为诱饵, 通过制备核酸亲和层析柱, 利用调控因子与DNA特异性结合的特点, 得到调控因子PadhE-1。经过蛋白测序和NCBI数据库的比对, 发现其N端序列与Thermoanaerobacter pseudethanolicus 39E的LacI家族转录调控因子 (GenBank accession No. YP_001665770)有90%的同源性。根据同源性设计引物, 从嗜热厌氧乙醇杆菌JW200基因组中通过PCR扩增得到调控因子PadhE-1的基因, 并克隆到表达载体pET-28a得到重组质粒pET-PadhE。转化大肠杆菌JM109 (DE3), 经IPTG诱导表达, Ni2+亲和柱纯化, 得到重组表达的调控因子PadhE-1。体外凝胶阻滞实验证实PadhE-1能与TRRadhE特异性结合。这将有助于我们了解嗜热厌氧乙醇杆菌的乙醇代谢调控机理。

Abstract:

Thermoanaerobacter ethanolicus produces ethanol from biomass derived substrates at temperatures above 70°C. AdhE, which exhibited bifunctional acetaldehyde/ethanol dehydrogenase activities, play a key role in producing ethanol. Proteins interacting with the transcriptional regulatory region (TRR) of adhE were for the first time isolated and identified by affinity chromatography. One of the proteins, PadhE-1, a homolog of LacI family, was cloned and expressed in Escherichia coli. Through electromobility shift assays, it was determined that the interaction between PadhE-1 and TRRadhE was specific. The research would be helpful to construct the bacteria with the extremely thermophilic and high ethanol-producing characteristics.