Cloning and effective expression of acetolactate synthase

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.