基于杨木发酵产异丁醇大肠杆菌工程菌构建

doi:10.11707/j.1001-7488.20150718

摘要/Abstract

摘要：

[目的] 克隆异丁醇生物合成途径中的2个关键酶基因——乙醇脱氢酶基因(adh2)和2-酮酸脱羧酶基因(kivd),构建产异丁醇的工程大肠杆菌;进一步以杨木水解液为底物进行发酵,以期能利用木质纤维原料发酵生产异丁醇,为异丁醇的可再生生产提供研究基础。[方法] 分别以酿酒酵母总DNA和乳酸乳球菌总DNA为模板,通过PCR扩增异丁醇生物合成途径关键酶基因adh2和kivd。同时构建重组质粒pSTV-29-adh2和pSTV-29-kivd,分别转化大肠杆菌DH5α,构建重组菌株E.coli DH5α-pSTV-29-adh2和E.coli DH5α-pSTV-29-kivd。进一步构建串联表达质粒pSTV-29-adh2-kivd,导入大肠杆菌DH5α中,构建重组工程菌E.coli DH5α-pSTV-29-adh2 -kivd。各重组菌株经IPTG诱导表达后,采用BCA 蛋白定量试剂盒测定其粗酶提取液中目的蛋白含量,并通过SDS-PAGE 电泳检测重组菌株E.coli DH5α-pSTV-29-adh2,E.coli DH5α-pSTV-29-kivd和E.coli DH5α-pSTV-29-adh2-kivd中目的基因adh2和kivd的蛋白表达量。利用各重组菌E.coli DH5α分别以葡萄糖和杨木水解液为原料发酵生产异丁醇,采用气相色谱检测发酵液中异丁醇含量。[结果] 重组质粒pSTV-29- adh2 ,pSTV-29-kivd和pSTV-29-adh2-kivd经菌液PCR、酶切及测序验证,得到的片段大小与目的基因adh2(1 067 bp)和kivd(1 667 bp)大小和质粒载体pSTV-29(2 999 bp)大小相符,adh2和kivd基因测序结果与NCBI中对应基因序列完全一致,表明表达载体pSTV-29-adh2,pSTV-29-kivd和pSTV-29-adh2-kivd构建成功。蛋白含量测定结果和SDS-PAGE电泳检测结果显示, 目的基因adh2,kivd的蛋白在各重组菌中均得到表达,在电泳图谱中的对应位置40 kD(Adh2蛋白分子质量40 kD)和70 kD(Kivd蛋白分子质量70 kD)处均有明显的蛋白特征条带,且adh2基因编码的乙醇脱氢酶蛋白比kivd基因编码的2-酮酸脱羧酶蛋白表达量高,而空白对照菌株未出现任何蛋白特征条带,表明各重组菌E.coli DH5α-pSTV-29-adh2,E.coli DH5α-pSTV-29-kivd和E.coli DH5α-pSTV-29-adh2-kivd构建成功。在发酵试验中,只有重组菌E.coli DH5α-pSTV-29-adh2-kivd能产异丁醇,以葡萄糖为底物的发酵液中异丁醇产量为4.1 g·L^-1,以杨木水解液为底物的发酵液中异丁醇产量为0.14 g·L^-1,而重组菌E.coli DH5α-pSTV-29-adh2和E.coli DH5α-pSTV-29-kivd的发酵液中均未检测到异丁醇。结果表明,只有adh2和kivd在重组菌E.coli DH5α体内同时表达时才能利用缬氨酸途径生物合成异丁醇。[结论] 成功构建了产异丁醇大肠杆菌工程菌E.coliDH5α-pSTV-29-adh2-kivd,实现了异丁醇关键酶基因在大肠杆菌中的表达及木质纤维发酵产异丁醇,为发酵法生产异丁醇提供了一条新的途径。

关键词: 异丁醇, 重组大肠杆菌, 乙醇脱氢酶基因, 2-酮基异戊酸脱羧酶, 木质纤维水解液

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

中图分类号:

Q291

王启业, 王义强, 田宇, 彭牡丹, 陈章靖, 陆利. 基于杨木发酵产异丁醇大肠杆菌工程菌构建[J]. 林业科学, 2015, 51(7): 157-164.

Wang Qiye, Wang Yiqiang, Tian Yu, Peng Mudan, Chen Zhangjing, Lu Li. Construction of Engineering Escherichia coli Strains Used for Isobutanol Production from Poplar Hydrolysate Fermentation[J]. Scientia Silvae Sinicae, 2015, 51(7): 157-164.

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