The Construction Of Recombinant E.Coli Producing Isobutanol

Along with the depletion of fossil fuel resources and the increasing environment problem, biofuel is expected to become new energy instead of fossil fuel in future. Compared with traditional biofuel (fuel ethanol), isobutanol has the features of difficult water absorption, low volatility, high octane value and high energy density, which make it a potential new generation of renewable energy. At present, using microorganism with glucose substrate to product isobutanol has not yet reached the level of industrialization. To change the host bacteria by genetic engineering for synthesizing and producing isobutanol highly becomes an important research direction. Alcohol dehydrogenase and 2-Keto acid decarboxylase are two key enzymes in the whole process of isobutanol biosynthesis. Due to lack of 2-Keto acid decarboxylase in E.coli,2-Keto acid (precursor substance) cannot be converted into the relevant aldehyde, thus not producing isobutanol. The article aimed to introduce exogenous pathway of isobutanol metabolism in E.coli by genetic modification and make isobutanol synthesized in E. coli.The alcohol dehydrogenase gene (adh2) was amplified from total DNA of S.cerevisiae by PCR amplification reaction, then connected it to the vector of pMD18-T after adding A. After verifying the vector by sequencing, the recombinant plasmid pSTV-29-adh.2 was constructed by inserting adh2 into expression vector pSTV-29, and then transformed E.coli DH5a. The results showed that alcohol dehydrogenase gene was expressed in E.coli by electrophoresis analysis, and that isobutyl aldehyde metabolism pathway is constructed in E.coli DH5oc.The 2-Ketoisovalerate decarboxylase gene (kivd) was amplified from total DNA of L.lactis by PCR amplification reaction, then connected it to the vector of pMD18-T after adding A. The vector was verified by sequencing, the recombinant plasmid, pSTV-29-kivd was constructed by inserting kivd into expression vector pSTV-29, and then transformed E.coli DH5a. The results showed that 2-Ketoisovalerate decarboxylase gene was expressed in E.coli by electrophoresis analysis, and that 2-Ketoisovalerate metabolism pathway is constructed in E.coli DH5α.Tandem expression plasmid pSTV-29-adh2-kivd was constructed by inserting kivd into pSTV-29-adh2, and then transformed E.coli DH5a. The recombinant strains induced by IPTG to express alcohol dehydrogenase gene and 2-Ketoisovalerate decarboxylase at the same time. The electrophoresis analysis of SDS-PAGE showed that the protein encoded by adh2 and kivd was expressed simultaneously. Recombinant strains produced 0.14g/L isobutanol from 10% glucose under the condition of 200rpm,37℃ and 24 hours through gas chromatography detection.The research successfully introduce the novel pathway for fermenting glucose to produce isobutanol in E.coli by expressing adh2 and kivd.