Abstract:

[Objective] Alcohol dehydrogenase and 2- keto acid decarboxylase are two key enzymes for isobutanol production. To construct a recombinant Escherichia coli strain for isobutanol production, the cDNA sequence of the two enzymes genes (adh2 and kivd) neede to be cloned from Saccharomyces cerevisiae and Lactobacillus lactis, respectively. The poplar hydrolysate was further used as a fermentation substrate to produce isobutanol with the recombinant E. coli to investigate whether the recombinant E. coli strain can utilize lignocellulosic materials to produce isobutanol. This study also intends to provide a research basis for the renewable production of isobutanol. [Method] We amplified the gene of adh2 by PCR with S. cerevisiae genome as a template and amplified the kivd with L.lactis genome as a template. We constructed the recombinant plasmid pSTV-29-adh2 and pSTV-29-kivd, and then transformed the plasmids into E.coli DH5α respectively, obtained the recombinant strains E. coli DH5α-pSTV-29-adh2 and E. coli DH5α-pSTV-29-kivd. Further, the tandem expression plasmid pSTV-29-adh2-kivd was constructed and then integrated into the E. coli DH5α for constructing the recombinant strain E. coli DH5α- pSTV-29-adh2-kivd. Isopropyl beta-D-thiogalactopyranoside(IPTG) was used to induce the recombinant strains E. coli DH5α for recombinant protein expression. Alcohol dehydrogenase and 2-Ketoisovalerate decarboxylase activities in E. coli DH5α-pSTV-29-adh2, E. coli DH5α-pSTV-29-kivd and E. coli DH5α-pSTV-29-adh2-kivd were measured. The amount of protein expression was analyzed with gray gel scan through SDS-PAGE. The fermentation of recombinant E. coli DH5α strains showed that the proteins encoded by adh2 and kivd were expressed simultaneously. The fermentation of recombinant E. coli DH5α strains was conducted in flask with glucose and poplar wood hydrolysate as fermentation substrates to produce isobutanol separately. The isobutanol concentration was detected by chromatography. [Results] The recombinant plasmid pSTV-29-adh2, pSTV-29-kivd and pSTV-29-adh2-kivd were verified by bacteria liquid PCR, enzyme digestion and DNA sequencing. The obtained fragment size was consistent with the size of target genes (adh2 and kivd) and plasmid vector pSTV-29, the results of adh2 and kivd gene sequencing were in complete accord with corresponding gene sequences in NCBI. The results indicated that the expression vector pSTV-29-adh2, pSTV-29-kivd and pSTV-29-adh2-kivd were successfully constructed. Protein content measurement and SDS-PAGE electrophoresis analysis showed that the proteins encoded by adh2 and kivd were respectively expressed in E. coli DH5α-pSTV-29-adh2 and E. coli DH5α-pSTV-29-kivd, and simultaneous expressed in E. coli DH5α-pSTV-29-adh2-kivd. In gray gel scan, the corresponding position of 40 kD (Adh2 protein molecular weight) and 70 kD (Kivd protein molecular weight) had obvious characteristics of protein bands, and the expression quantity of adh2 encoding protein exceeded that of kivd encoding protein, while the blank control strain didn't appear any protein characteristic bands. All of the results showed that the recombinant strains E. coli DH5α-pSTV-29-adh2, E. coli DH5α-pSTV-29-kivd and E. coli DH5α-pSTV-29-adh2-kivd were successfully constructed. In the fermentation experiments, only the E. coli DH5α-pSTV-29-adh2 could produce isobutanol with glucose and poplar wood hydrolysate as fermentation substrates, and the yields were 4.1 g·L^-1 and 0.14 g·L^-1, respectively. The strains E. coli DH5α-pSTV-29-adh2 and E.coli DH5α-pSTV-29-kivd didn ’t produce isobutanol. Thus, isobutanol could produce only with the adh2 and kivd expressed simultaneously in E. coli DH5α-pSTV-29-adh2-kivd. [Conclusion] The key genes of isobutanol synthetic pathway were expressed in E. coli and the recombinant strain E. coli DH5α-pSTV-29-adh2-kivd would utilize lignocellulose to produce isobutanol. This study offered an alternative strategy for isobutanol biosynthesis.

Key words: isobutanol, recombinant Escherichia coli, alcoholdehy drogenase, 2-ketoisovalerate decarboxylase, lignocellulose hydrolysate