Cloning And Expression Of The Key Enzymes In The Isobutanol Biosynthetic Pathway

Isobutanol becomes new biofuel with high octane、high energy, low vapor pressure、low hygroscopic, and biosynthesis of isobutanol becomes research focus with the development of synthetic biology.In this paper, firstly, alsS gene was got by PCR using B.subtilis168genome as template and the expression plasmid pET30a(+)-alsS was constructed, ilvC、ilvD、 yqhD genes were got by PCR using E.coli MG1655genome as template, and the expression plasmids pET22b(+)-ilvC、pET22b(+)-ilvD、pET30a(+)-yqhD were constructed. These recombinant plasmids were all successfully transferred into Escherichia coli BL21(DE3). The acetolactate synthase(AHAS)、acetohydroxyacid iosmeroreductase (AHAIR)、dihydroxyacid dehydratase (DHAD)、dehydrogenase (ADH) with molecular weight of62.8、54.1、67.9、42.7kDa respectivly were all effectively expressed, the concentration of total protein were0.41、0.34、0.83、2.12mg/mL, the enzyme activity analysis method of AHAS、AHAIR was established, the measured activity of two enzymes were3.42U/mL and4.02U/mL respectively.Secondly, the expression condition of AHAS was optimized for it played an important role in the isobutanol metabolic pathway, and the best expression condition was obtained. When the cell was cultivated to OD600between0.6～0.8, IPTG was added to the final concentration of1mmol/L. The bacteria was induced for6h at30℃and200r/min, and the enzyme was mostly expressed in soluble form in vivo with the maximum enzyme activity of24.4U/mL, which improved7.13times. The electrophoretically pure AHAS was got after nickel affinity chromatography with the specific activity of95.2U/mg, which improved1.8times.Finally, the coexpression of AHAS and AHAIR in the single plasmid with bio-cistron model and in the two plasmids with two resistance model were compared. The total protein and the enzyme activity of two enzymes in two plasmids with two resistance were higher than that in single-vector model. Then the influence of SD sequence on the expression of two genes in bicistronic plasmid system was studied. The results showed that two genes were unexpressed without SD sequence among them and SD sequence inserted differently among two genes will not only increase the expression level of the second gene, but also lower the expression level of the first gene. So GAAGGAGATATACAT was added before ilvD gene as the SD sequence to ligate ilvD after the obtained gene alsS-ilvC to construct the recombinant plasmid of three genes. AHAS、AHAIR、DHAD were also expressed under two models. The metabolic intermediate of2-ketoisovalerate was all detected indicating that three genes were all expressed successfully under two expression models.