Cloning and effective expression of acetolactate synthase
[Objective] Acetolactate synthase (ALS) is the key enzyme in isobutanol biosyn-thetic 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 BL21(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 ℃ 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 chro-matography with the specific activity of 95.2 U/mg.[Conclusion] These results contributed to the construction of isobutanol biosynthetic pathway in E.coli.
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万方数据
维普
