奎尼酸生物合成的代谢工程研究
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摘要
奎尼酸是一种具有极高价值的精细化工产品和医药中间体，在医药工业、食品工业、化工等行业均有较大的用途。本研究是将代谢工程技术应用于产奎尼酸基因工程菌的构建。根据代谢工程原理系统分析了细胞代谢网络，并利用DNA重组技术合理设计细胞代谢途径及其遗传修饰，进而完成细胞特性改造。目的是在大肠杆菌中构建奎尼酸生物合成途径，将碳代谢流最大程度的引向奎尼酸生成的方向。
利用大肠杆菌莽草酸途径合成新的代谢物奎尼酸，需要提高上游几种代谢中间产物PEP、E4P、DAHP、DHQ的含量，与之对应的编码其合成酶的几种基因分别是ppsA、tktA、aroG、aroB。另外须在宿主细胞引入编码与奎尼酸合成有关的异源酶基因扩展代谢途径，然后串联表达酶基因，同时适量增加不同种属的多个关键酶的有效含量，改善限速反应。利用同源重组进行基因整合和基因破坏，改造染色体结构定向改变微生物代谢途径。本文主要从以下几个方面对奎尼酸基因工程菌进行了改造：
1．编码奎尼酸脱氢酶的qutB基因是来自于构巢曲霉。在现有的基础上构建了一系列包含qutB基因的表达重组质粒。SD序列与起始密码子不同距离的pBVqutB1，pBVqutB2；具有qutB双拷贝三基因串联的pBVqutBqutBaroG；具有ppsA、tktA、qutB三基因串联的pBVPTB，以及新构建的单基因重组质粒pETqutB，pBVtacqutB，pTrcqutB。并对含有qutB基因的一系列重组质粒在大肠杆菌中进行了表达与否的验证。结果显示pTrcqutB，pBVtacqutB没有特异的蛋白表达带，说明pTrc99a和pBVtac载体不适合用于qutB基因的表达。pETqutB经过IPTG诱导后有特异的蛋白表达，并且在2—5小时内随着时间的延长而增加，但是时间更长(7h)则因为蛋白降解而出现表达蛋白减少的迹象。pBVqutB1和pBVqutB2同本实验室保存的pBVqutB没有出现明显的蛋白表达量上的差异，这说明利用pBV220表达载体表达qutB基因，起始密码子与SD序列之间距离的远近并不是影响其表达量的关键。含有两个qutB基因和一个aroG基因的多基因串连质粒pBVqutBqutBaroG，经过热诱导，也出现了非常明显的表达条带。
2．对含有qutB基因在大肠杆菌中可以表达几种重组质粒进行酶活测定，与含有pBVqutB的对照菌相比，含有pBVqutB1，pBVqutB2的菌株的奎尼酸脱氢酶的酶活没有明显变化，三基因串联的pBVqutBqutBaroG甚至出现了酶活降低的现象。
3．成功地从粗糙脉孢菌的基因组中克隆了奎尼酸脱氢酶的基因qa-3，并与几种表达载体连接得到了pBVqa3、pETqa3、pBVtacqa3、pTrcqa3，但是这些质粒重组子在大肠杆菌中均没有观察到该基因的特异表达条带。
4．结合qa-3基因的密码子的特点和计算机模拟的mRNA二级结构，对qa-3基因的密码子和mRNA二级结构进行改造，优化该基因的密码子结构，降低形成mRNA二级结构的自由能，得到新的基因qa3RG~m。构建的pBVqa3RG~m重组质粒在大肠杆菌中成功的表达了奎尼酸脱氢酶，酶活也比对照明显提高。同时还构建了ppsA、tktA、qa3RG~m三基因串联的重组质粒pBVPTA。
5．成功的构建了pUC-DK、pUC-DAK、pUC-DBK并制备了线性化片段DK、DAK、DBK，为基因敲除和基因替换做好了准备。利用Red重组系统成功的地进行了基因敲除和基因替换，并稳定了敲除和替换菌株的基因型，把这株菌株定名为大肠杆菌31K。
6．成功的构建奎尼酸发酵的工程菌5、AP、B、A、BP、5K、APK、BK、AK、BPK、220K。通过实验室摇瓶初步发酵，在硅胶板上可以初步确定，菌株AP(31BK/pBVPTA)产生的奎尼酸量最大，也最稳定。
7．将菌株AP扩大摇瓶发酵的规模，然后经过树脂富集，HPLC制备，得到了微量的奎尼酸样品。经过红外光谱、核磁共振、电子喷雾质谱以及紫外、液质联用等的鉴定可以确定与奎尼酸标准品无差异，由此可以确定我们利用生物合成并且分离鉴定了奎尼酸。
总之，为了合成奎尼酸并提高其产量，本研究从分子进化、基因重组、网络构建等不同的方面对奎尼酸生物合成进行了许多有益尝试，既为研究奎尼酸的生物合成奠定了基础，也为基因工程应用于代谢工程提供一定的借鉴意义。
Quinic acid is an important fine chemical product and intermediates of drag synthesis, which can be numerously used in drag synthesis, food and chemistry industry. In this study metabolic engineering are applied for genetic engineering bacteria of quinic acid. This work about the analysis of metabolic pathway and designing rational genetic modification is to optimize cellular properties by using principle of molecular biology. The objection is to establish an approach to quinic acid biosynthesis in E.coli and change the carbon flow to redirect into the quinic acid biosynthesis branch.
Production of a new metabolite quinic acid, used shikimate pathway in E.coli, it is necessary to enhance upriver metabolic intermediate , such as PEP、E4P、DAHP and DHQ which corresponding gene is ppsA、tktA、aroG、aroB, respectively. It is necessary to extend metabolic pathway by introduction of a heterogenous gene qutB or qa-3 into the host cell. Double specific enzyme genes or three ones co-expressed in a single plasmid vector to improve the enzymes' rate-limiting reactions. Both disruption of the aroD gene and directed-site insertion of the aroB or qa-3 or qutB gene make use of homologous recombine to change chromosome structure and directionally shift metabolic pathway of microorganism. This thesis improved genetic engineering bacteria of quinic acid biosynthesis on the following respects:
1. A series of recombinant plasmid including qutB gene encoded quinate dehydrogenase from Aspergillus nidulans were constructed, such as pBVqutB1 and pBVqutB2 involved different distance between SD sequence and start codon; pBVqutBqutBaroG of three genes in series; pBVPTB contained ppsA、tktA、qutB, as well as pETqutB, pBVtacqutB, pTrcqutB contained single gene qutB. These recombinant plasmids were expressed in E.coli and the results showed that pTrcqutB and pBVtacqutB lacked specific protein band, explaining the vectors pTrc99a and pBVtac are not suitable for the expression that used for the gene of qutB. The pETqutB expresses the specific protein band after IPTG induced, and in 2-5 hours the expression increases along with horary extension but time is long (7 h) then to appear the evidence that expression protein reduce for protein degradation. pBVqutB1 and pBVqutB2 compared with the pBVqutB kept in this laboratory don't appear the obvious difference about protein expression. This elucidation shows that the distance between start codon and SD sequence was not the key to affect the expression of qutB gene when vector pBV220 was used. pBVqutBqutBaroG implied two qutB and one aroG also appears very obviously specific protein band.
2. Enzyme activity of quinate dehydrogenase expressed by pBVqutB1, pBVqutB2 is not obvious distinguish compared with reference by pBVqutB, but by pBVqutBqutBaroG decreases.
3. qa-3 gene was cloned successfully from genome of Neurospora crassa which also encodes quinate dehydrogenase and constructed into vectors to obtain pBVqa3、pETqa3、pBVtacqa3、pTrcqa3 . But they don't express in E.coli.
4. According to codon usage of qa-3 and computer simultating its mRNA secondary structure, some codons were changed and lowered free energy a lot from -374.3 kJ/mol to -80.5 kJ/mol. New gene qa3RG~m was expressed in E. coli and also the enzyme activity of quinate 5-dehydrogenase could be accurately surveyed and increased obviously. pBVPTA including ppsA、tktA、qa3RG~m also was constructed.
5. pUC-DK、pUC-DAK、pUC-DBK were constructed successfully and linear fragment DK、DAK、DBK were prepared for gene knockout and gene replacement. In order to increasing the in vivo catalytic activity of specific enzymes, the chromosomal genome of E. coli was modifizated by using Red recombination system. Stabilizing the genotype and new strain was named as 31BK.
6. After transforming the recombinant plasmid into 31BK, the flasks fermentation demonstrate that strain AP (31BK/pBVPTA) has higher and stabler quinic acid production in engineering bacterium 5、AP、B、A、BP、5K、APK、BK、AK、BPK、220K through silica gel chromatography.
7. The flasks fermentation of strain AP has been enlarged and gained a dram of quinic acid sample passed through enrichment by resin, HPLC preparation et al. NMR, FT-IR Microscope, Ion Trap (HCT) Spectrum, LC-MS and UV identified the sample was not difference with standard. This conforms that we utilize the method of biosynthesis to synthesize, purify and identify the production quinic acid.
In summary, this study tries to actively experiment from different aspects e.g. molecular evolution, gene recombination and network construction to produce QA and enhances its yield. And these settle the groundwork for biosynthesis of quinic acid and provide a reference for genetic engineering applied to metabolic engineering.
利用大肠杆菌莽草酸途径合成新的代谢物奎尼酸，需要提高上游几种代谢中间产物PEP、E4P、DAHP、DHQ的含量，与之对应的编码其合成酶的几种基因分别是ppsA、tktA、aroG、aroB。另外须在宿主细胞引入编码与奎尼酸合成有关的异源酶基因扩展代谢途径，然后串联表达酶基因，同时适量增加不同种属的多个关键酶的有效含量，改善限速反应。利用同源重组进行基因整合和基因破坏，改造染色体结构定向改变微生物代谢途径。本文主要从以下几个方面对奎尼酸基因工程菌进行了改造：
1．编码奎尼酸脱氢酶的qutB基因是来自于构巢曲霉。在现有的基础上构建了一系列包含qutB基因的表达重组质粒。SD序列与起始密码子不同距离的pBVqutB1，pBVqutB2；具有qutB双拷贝三基因串联的pBVqutBqutBaroG；具有ppsA、tktA、qutB三基因串联的pBVPTB，以及新构建的单基因重组质粒pETqutB，pBVtacqutB，pTrcqutB。并对含有qutB基因的一系列重组质粒在大肠杆菌中进行了表达与否的验证。结果显示pTrcqutB，pBVtacqutB没有特异的蛋白表达带，说明pTrc99a和pBVtac载体不适合用于qutB基因的表达。pETqutB经过IPTG诱导后有特异的蛋白表达，并且在2—5小时内随着时间的延长而增加，但是时间更长(7h)则因为蛋白降解而出现表达蛋白减少的迹象。pBVqutB1和pBVqutB2同本实验室保存的pBVqutB没有出现明显的蛋白表达量上的差异，这说明利用pBV220表达载体表达qutB基因，起始密码子与SD序列之间距离的远近并不是影响其表达量的关键。含有两个qutB基因和一个aroG基因的多基因串连质粒pBVqutBqutBaroG，经过热诱导，也出现了非常明显的表达条带。
2．对含有qutB基因在大肠杆菌中可以表达几种重组质粒进行酶活测定，与含有pBVqutB的对照菌相比，含有pBVqutB1，pBVqutB2的菌株的奎尼酸脱氢酶的酶活没有明显变化，三基因串联的pBVqutBqutBaroG甚至出现了酶活降低的现象。
3．成功地从粗糙脉孢菌的基因组中克隆了奎尼酸脱氢酶的基因qa-3，并与几种表达载体连接得到了pBVqa3、pETqa3、pBVtacqa3、pTrcqa3，但是这些质粒重组子在大肠杆菌中均没有观察到该基因的特异表达条带。
4．结合qa-3基因的密码子的特点和计算机模拟的mRNA二级结构，对qa-3基因的密码子和mRNA二级结构进行改造，优化该基因的密码子结构，降低形成mRNA二级结构的自由能，得到新的基因qa3RG~m。构建的pBVqa3RG~m重组质粒在大肠杆菌中成功的表达了奎尼酸脱氢酶，酶活也比对照明显提高。同时还构建了ppsA、tktA、qa3RG~m三基因串联的重组质粒pBVPTA。
5．成功的构建了pUC-DK、pUC-DAK、pUC-DBK并制备了线性化片段DK、DAK、DBK，为基因敲除和基因替换做好了准备。利用Red重组系统成功的地进行了基因敲除和基因替换，并稳定了敲除和替换菌株的基因型，把这株菌株定名为大肠杆菌31K。
6．成功的构建奎尼酸发酵的工程菌5、AP、B、A、BP、5K、APK、BK、AK、BPK、220K。通过实验室摇瓶初步发酵，在硅胶板上可以初步确定，菌株AP(31BK/pBVPTA)产生的奎尼酸量最大，也最稳定。
7．将菌株AP扩大摇瓶发酵的规模，然后经过树脂富集，HPLC制备，得到了微量的奎尼酸样品。经过红外光谱、核磁共振、电子喷雾质谱以及紫外、液质联用等的鉴定可以确定与奎尼酸标准品无差异，由此可以确定我们利用生物合成并且分离鉴定了奎尼酸。
总之，为了合成奎尼酸并提高其产量，本研究从分子进化、基因重组、网络构建等不同的方面对奎尼酸生物合成进行了许多有益尝试，既为研究奎尼酸的生物合成奠定了基础，也为基因工程应用于代谢工程提供一定的借鉴意义。
Quinic acid is an important fine chemical product and intermediates of drag synthesis, which can be numerously used in drag synthesis, food and chemistry industry. In this study metabolic engineering are applied for genetic engineering bacteria of quinic acid. This work about the analysis of metabolic pathway and designing rational genetic modification is to optimize cellular properties by using principle of molecular biology. The objection is to establish an approach to quinic acid biosynthesis in E.coli and change the carbon flow to redirect into the quinic acid biosynthesis branch.
Production of a new metabolite quinic acid, used shikimate pathway in E.coli, it is necessary to enhance upriver metabolic intermediate , such as PEP、E4P、DAHP and DHQ which corresponding gene is ppsA、tktA、aroG、aroB, respectively. It is necessary to extend metabolic pathway by introduction of a heterogenous gene qutB or qa-3 into the host cell. Double specific enzyme genes or three ones co-expressed in a single plasmid vector to improve the enzymes' rate-limiting reactions. Both disruption of the aroD gene and directed-site insertion of the aroB or qa-3 or qutB gene make use of homologous recombine to change chromosome structure and directionally shift metabolic pathway of microorganism. This thesis improved genetic engineering bacteria of quinic acid biosynthesis on the following respects:
1. A series of recombinant plasmid including qutB gene encoded quinate dehydrogenase from Aspergillus nidulans were constructed, such as pBVqutB1 and pBVqutB2 involved different distance between SD sequence and start codon; pBVqutBqutBaroG of three genes in series; pBVPTB contained ppsA、tktA、qutB, as well as pETqutB, pBVtacqutB, pTrcqutB contained single gene qutB. These recombinant plasmids were expressed in E.coli and the results showed that pTrcqutB and pBVtacqutB lacked specific protein band, explaining the vectors pTrc99a and pBVtac are not suitable for the expression that used for the gene of qutB. The pETqutB expresses the specific protein band after IPTG induced, and in 2-5 hours the expression increases along with horary extension but time is long (7 h) then to appear the evidence that expression protein reduce for protein degradation. pBVqutB1 and pBVqutB2 compared with the pBVqutB kept in this laboratory don't appear the obvious difference about protein expression. This elucidation shows that the distance between start codon and SD sequence was not the key to affect the expression of qutB gene when vector pBV220 was used. pBVqutBqutBaroG implied two qutB and one aroG also appears very obviously specific protein band.
2. Enzyme activity of quinate dehydrogenase expressed by pBVqutB1, pBVqutB2 is not obvious distinguish compared with reference by pBVqutB, but by pBVqutBqutBaroG decreases.
3. qa-3 gene was cloned successfully from genome of Neurospora crassa which also encodes quinate dehydrogenase and constructed into vectors to obtain pBVqa3、pETqa3、pBVtacqa3、pTrcqa3 . But they don't express in E.coli.
4. According to codon usage of qa-3 and computer simultating its mRNA secondary structure, some codons were changed and lowered free energy a lot from -374.3 kJ/mol to -80.5 kJ/mol. New gene qa3RG~m was expressed in E. coli and also the enzyme activity of quinate 5-dehydrogenase could be accurately surveyed and increased obviously. pBVPTA including ppsA、tktA、qa3RG~m also was constructed.
5. pUC-DK、pUC-DAK、pUC-DBK were constructed successfully and linear fragment DK、DAK、DBK were prepared for gene knockout and gene replacement. In order to increasing the in vivo catalytic activity of specific enzymes, the chromosomal genome of E. coli was modifizated by using Red recombination system. Stabilizing the genotype and new strain was named as 31BK.
6. After transforming the recombinant plasmid into 31BK, the flasks fermentation demonstrate that strain AP (31BK/pBVPTA) has higher and stabler quinic acid production in engineering bacterium 5、AP、B、A、BP、5K、APK、BK、AK、BPK、220K through silica gel chromatography.
7. The flasks fermentation of strain AP has been enlarged and gained a dram of quinic acid sample passed through enrichment by resin, HPLC preparation et al. NMR, FT-IR Microscope, Ion Trap (HCT) Spectrum, LC-MS and UV identified the sample was not difference with standard. This conforms that we utilize the method of biosynthesis to synthesize, purify and identify the production quinic acid.
In summary, this study tries to actively experiment from different aspects e.g. molecular evolution, gene recombination and network construction to produce QA and enhances its yield. And these settle the groundwork for biosynthesis of quinic acid and provide a reference for genetic engineering applied to metabolic engineering.
引文
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