乙酰乳酸合成酶基因的克隆与高效表达

Cloning and effective expression of acetolactate synthase

摘要:

【目的】乙酰乳酸合成酶(ALS)是异丁醇生物合成中的关键酶, 实现ALS的高效表达对调控异丁醇代谢途径有重要意义。【方法】根据GenBank中ALS的基因序列(alsS)设计引物, 以枯草芽孢杆菌168基因组DNA为模板通过PCR扩增技术得到目标酶基因, 目的片段全长为1 713 bp。将alsS连接到pET-30a(+)上, 得到重组质粒pET-30a(+)-alsS, 并在Escherichia coli BL2l(DE3)中实现表达。【结果】对表达条件进行了优化, 获得最佳表达条件为: 诱导温度30 °C, 诱导起始菌体OD600为0.6?0.8, 诱导剂IPTG浓度为1?mmol/L, 诱导时间为6 h。表达的乙酰乳酸合成酶大部分以可溶性形式存在于菌体内, 优化后酶活可达到24.4 U/mL, 比优化前提高了7.13倍。经HisTrapTMFF亲和层析后获得电泳纯的ALS, 比活为95.2 U/mg。【结论】ALS的有效表达为在大肠杆菌体内构建异丁醇代谢途径打下了基础。

Abstract:

[Objective] Acetolactate synthase (ALS) is the key enzyme in isobutanol biosynthetic pathway. Efficient expression of ALS is of great significance for the regulation of isobutanol metabolic pathway. [Methods] The acetolactate synthase gene (alsS) from Bacillus subtilis was amplified by PCR with primers designed according to the sequence of alsS in GeneBank, which is 1 713 bp. Then the alsS was cloned into the expression vector of pET-30a(+). The resulted recombinant plasmid was transformed into Escherichia coli BL2l(DE3) for the overexpression of alsS. [Results] The heterologous expression condition was optimized to be inducted at an OD600 of 0.6?0.8, 30 °C with 1 mmol/L IPTG for 6 h. ALS was mostly expressed in the supernatant with the activity of 24.4 U/mL, which was improved for 7.13 times. Electrophoretically pure ALS was obtained after HisTrapTMFF affinity chromatography with the specific activity of 95.2 U/mg. [Conclusion] These results contributed to the construction of isobutanol biosynthetic pathway in E. coli.