基于杨木发酵产异丁醇大肠杆菌工程菌构建
기우양목발효산이정순대장간균공정균구건
Construction of Engineering Escherichia coli Strains Used for Isobutanol Production from Poplar Hydrolysate Fermentation
  • 万方数据
    林业科学 임업과학
    Scientia Silvae Sinicae
    2015年 7期 157-164 ,共8页

    王启业%王义强%田宇%彭牡丹%陈章靖%陆利

    왕계업%왕의강%전우%팽모단%진장정%륙리

    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 異丁醇%重組大腸桿菌%乙醇脫氫酶基因%2 -酮基異戊痠脫羧酶%木質纖維水解液
    이정순%중조대장간균%을순탈경매기인%2 -동기이무산탈최매%목질섬유수해액
    isobutanol%recombinant Escherichia coli%alcoholdehy drogenase%2-ketoisovalerate decarboxylase%lignocellulose hydrolysate
    【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(1067 bp)和 kivd(1667 bp)大小和质粒载体 pSTV-29(2999 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． coli DH5α-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。
    【목적】극륭이정순생물합성도경중적2개관건매기인———을순탈경매기인( adh2)화2－동산탈최매기인( kivd)，구건산이정순적공정대장간균；진일보이양목수해액위저물진행발효，이기능이용목질섬유원료발효생산이정순，위이정순적가재생생산제공연구기출。【방법】분별이양주효모총 DNA 화유산유구균총 DNA 위모판，통과 PCR확증이정순생물합성도경관건매기인 adh2화 kivd。동시구건중조질립 pSTV-29-adh2화 pSTV-29-kivd，분별전화대장간균 DH5α，구건중조균주 E． coli DH5α-pSTV-29-adh2화 E． coli DH5α-pSTV-29-kivd。진일보구건천련표체질립 pSTV-29-adh2-kivd，도입대장간균 DH5α중，구건중조공정균 E． coli DH5α-pSTV-29-adh2-kivd。각중조균주경IPTG유도표체후，채용BCA 단백정량시제합측정기조매제취액중목적단백함량，병통과SDS-PAGE 전영검측중조균주 E． coli DH5α-pSTV-29-adh2，E． coli DH5α-pSTV-29-kivd 화 E． coli DH5α-pSTV-29-adh2-kivd 중목적기인 adh2화 kivd적단백표체량。이용각중조균 E． coli DH5α분별이포도당화양목수해액위원료발효생산이정순，채용기상색보검측발효액중이정순함량。【결과】중조질립pSTV-29-adh2，pSTV-29-kivd화pSTV-29-adh2-kivd경균액 PCR、매절급측서험증，득도적편단대소여목적기인 adh2(1067 bp)화 kivd(1667 bp)대소화질립재체 pSTV-29(2999 bp)대소상부，adh2화 kivd기인측서결과여 NCBI중대응기인서렬완전일치，표명표체재체 pSTV-29-adh2， pSTV-29-kivd화 pSTV-29-adh2-kivd구건성공。단백함량측정결과화 SDS-PAGE 전영검측결과현시，목적기인adh2，kivd적단백재각중조균중균득도표체，재전영도보중적대응위치40 kD( Adh2단백분자질량40 kD)화70 kD ( Kivd단백분자질량70 kD)처균유명현적단백특정조대，차 adh2기인편마적을순탈경매단백비 kivd 기인편마적2－동산탈최매단백표체량고，이공백대조균주미출현임하단백특정조대，표명각중조균 E． coli DH5α-pSTV-29-adh2，E． coli DH5α-pSTV-29-kivd 화 E． coli DH5α-pSTV-29-adh2-kivd 구건성공。재발효시험중，지유중조균 E． coli DH5α-pSTV-29-adh2-kivd능산이정순，이포도당위저물적발효액중이정순산량위4.1 g·L －1，이양목수해액위저물적발효액중이정순산량위0.14 g·L －1，이중조균 E． coli DH5α-pSTV-29-adh2화 E． coli DH5α-pSTV-29-kivd적발효액중균미검측도이정순。결과표명，지유 adh2화 kivd재중조균 E． coli DH5α체내동시표체시재능이용힐안산도경생물합성이정순。【결론】성공구건료산이정순대장간균공정균 E． coli DH5α-pSTV-29-adh2-kivd，실현료이정순관건매기인재대장간균중적표체급목질섬유발효산이정순，위발효법생산이정순제공료일조신적도경。
    [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.
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