【作者】 李永辉；

【导师】 徐琪寿；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2003， 博士

【摘要】 本研究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。芳香族氨基酸包括酪氨酸、苯丙氨酸和色氨酸，是人体和动物体内的必需氨基酸。苯丙氨酸既是合成新型甜味剂Aspartame的原料，又是合成一些抗癌药物的中间体和良好载体，市场需求日益增加。 大肠杆菌和许多微生物一样，其合成芳香族氨基酸的起始物是由磷酸烯醇式丙酮酸(PEP)和4-磷酸赤癣糖(E4P)二者缩合形成的3-脱氧-α-阿拉伯庚酮糖酸-7-磷酸(DAHP)。PEP和E4P是DAHP合成的限制性底物，在大肠杆菌中，PEP又是许多酶的竞争性底物，特别是负责葡萄糖转运的糖磷酸基转运系统。ppsA基因编码磷酸烯醇式丙酮酸合成酶A(PpsA)，该酶催化丙酮酸合成磷酸烯醇式丙酮酸；tktA基因编码转酮酶A，该酶在磷酸戊糖途径中生成4-磷酸赤藓糖起主要作用。芳香族氨基酸的合成步骤有七步是共同的，亦即从DAHP到分支酸的合成步骤，其中脱氢奎宁酸合成酶(AroB)、5-烯醇式丙酮酰莽草酸合成酶(AroA)和分支酸合成酶(AroC)是此代谢途径的关键酶。分支酸是芳香族氨基酸合成途径的分支点，与苯丙氨酸合成有关，双功能酶分支酸变位酶-预苯酸脱水酶(pheA基因编码)是关键酶。CsrA是整体调控网络的调控基因，可负调控指数生长后期诱导的一些代谢途径，包括糖原的生物合成、糖原的分解代谢、糖异生，而对糖酵解的一些重要基因的表达则执行正调控功能，CsrA也调控直接参与PEP代谢的三个酶的活性水平。另外，一些间接影响PEP的酶也被CsrA所调控，因此csrA的敲除不但可以使细胞内的PEP的量大大增加，而且打破了细胞固有的生理代谢协调，使碳代谢流尽可能多的流向苯丙氨酸的生物合成方向。基于上述分析，本文主要从以下三个方面对苯丙氨酸基因工程菌进行了改造： 1．从大肠杆菌ATCC31884的基因组中克隆并表达了PEP合成酶基因ppsA和合成E4P的关键基因tkt4，用原核高效表达载体pBV220分别实现了两种蛋白的高效表达，其酶活性分别比对照菌提高了10.8和3.9倍；考乏季芝羌凄了毫履{全渺分成;送窟屏务乡论乎乡之亨孑灵苏亥穿2.构建了这两个基因的不同形式的串联表达重组子:PPT-I、PPT-ll、PTP一I、PTP一n，其中PPsA的活性升高了2一9倍，TktA的活性升高了3 .9一4.5倍;通过DAHP含量的测定，结果表明串联重组子中DAHP的含量比对照菌高约一倍，筛选并确定PPT-H、PTP一工这两种串联方式效率最高，提示我们:多基因串联表达时，其各个酶活性比较协调一致的重组子可能为最佳组合。3.从大肠杆菌中克隆了莽草酸途径关键酶基因aro刁、aroC、aroB及整体代谢调控基因CsrA及其侧翼序列;从质粒pET28a中克隆了Kan抗性基因;4.构建了基因盒子aroAar口CaroBKan，在其前面加入了强启动子Ptac，初步证明了pta。和Kan是有活性的;5.成功的构建了线性化片断CsrA尸tacaro月aroCaroBKdnCsrA，为基因敲除和基因替换做好了准备;利用Red重组系统成功地进行了基因敲除和基因替换，PCR和Southem blot鉴定其为正确的，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31884△c甲B;6.观察到菌落形态和生长状况的改变，实验证实大肠杆菌31884△C甲B比对照菌大肠杆菌31884△T在产酸率上高4.53倍，而糖消耗上两者差异并不大，这充分说明前者能在细胞内积累更多的糖原，并得到有效的利用。莽草酸途径的最优化和整体调控基因CsrA的敲除正是上述改变的分子基础，同时也为三种芳香族氨基酸的基因工程菌的构建打下了基础;7.在国内外首次实现了共同途径限制性底物关键酶PPsA刁无‘」及aroG与分支途径关键酶基因pheA的串联高效表达，所构建的重组质粒PTGA，其PPsA、TktA、AroG、CM和PD的酶活分别比对照提高了3、2、2，5、4、2.3倍，且其酶活比较协调一致;8.将PTGA导入到筛选的基因敲除和基因替换菌株大肠杆菌3 1 884△C甲B中，摇瓶发酵证实比以往所构建的基因工程菌株具有较高的phe产量和糖转化率率，分别为0.448%和22.4%。在BIOFLO3000 Bateh/Continuous Bioreaetor(SL)进行发酵试验结果证实，发酵液中副生杂酸较少，产品经初步纯化后纯度较高，可达93%。熔点、旋光度测定和红外光谱分析证实产物为苯丙氨酸。 总之，从工业化生产上所要求的产量、产率和产品纯度这三方面考虑，本试验所构建的基因工程菌株都达到或基本达到要求，从而为工业化生产打下了基础，为今后氨基酸生物合成特别是芳香族氨基酸的生物合成提供了一个新思路。更多还原

【Abstract】 This work is the analysis of metabolic pathway and designing rational genetic modification to optimize cellular properties by using principle of molecular biology. Aromatic metabolites such as tryptophan, phenylalanine, tyrosine which can only be synthesized by plants and microbes are essential amino acids for human and animals. In addition, phenylalanine is used in aspartame production and acts as intermediate and well vector in synthesis of some anti-cancer drugs. So it has increasing commercial demand.Escherichia coli and many other microoganism synthesize aromatic amino acids through the condensation reaction between phosphoenolpyru vate(PEP) and erythrose-4-phosphate(E4P) to form 3-deoxy-D- arabinohep tulosonate 7-phosphate(DAHP). PEP and E4P are limiting substrates for formation of DAHP. In bacterial, many enzymes compete for intracellular PEP, especially the phosphotransferase system which is responsible for glucose transport in E. coli. This system uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. ppsA and tktA are the key genes in central metabolism of aromatic amino acids biosynthesis. ppsA encoding phosphoenolpyrucate synthetase A(PpsA) which catalyzes pyruvate into PEP; tktA encoding transketolase A which plays a major role in erythrose-4-phosphate (E4P) production of pentose pathway. The common biosynthesis pathway of aromatic amino acids includes seven steps from DAHP to chorismate acid. For the common pathway, 3-dehydroquinate(DHQ) synthase(encoded by aroB), 5-enolpyruv- oylshikimate S-phosphate(EPSP) synthase(encoded by aroA), and chorisma- te synthase(encoded by aroC] are rate-limiting enzymes. Chorismate acid is branch point in aromatic amino acids biosynthesis, related to phenylalanine, bifunctional enzymes chorismate mutase/prephenate dehydratase(encoded by pheA) is rate-limiting enzyme. The global regulator CsrA of E. coli is a specific mRNA-binding protein. CsrA negatively regulates several metabolic pathways that are induced post-exponentially, including glycogen biosynthesis, gluconeogenesis, and glycogen catabolism; positively controls gene expression within the glycolytic pathway; and also CsrA modulates the levels ofenzymes that participate directly in PEP metabolism. Several enzymes that indirectly affect PEP are also regulated by CsrA. Thus, a CsrA mutation not only causes a significant elevation in intracellular PEP, but also breaks cellular intrinsic metabolic coordination and channel more carbon flux to phenylalanine biosynthesis. Based on above analysis, this thesis improved genetic engineering bacteria of phenylalanine biosynthesis on the following three respects:1. We amplified ppsA and tktA from E.coli K-12 by PCR and constructed recombinant plasmids of them in pBV220 vector containing PRPL promoter. Because of each gene carrying PL promoter, four productions of ligation were obtained, that are PPT-Ⅰ ,PPT- Ⅱ ,PTP-Ⅰ , and PTP-Ⅱ. The results of SDS-PAGE demonstrated that the bands at 84kD and 73kD were more intensive than the same ones of the controls. The specific activity of PpsA in crude extracts was increased by 10.8-fold, and TktA, by 3.9-fold.2. When both genes were co-expressed in E.coli, the activity of PpsA varied from 2.1-9.1 fold comparing to control, but the activity of TktA was relatively stable(3.9-4.5 fold) . Whatever the two genes were expressed respectively or cooperatively, both could promote the production of DAHP, the first intermediate of the common aromatic pathway, but co-expression was more effective on forming DAHP and screened PPT-Ⅱ and PTP-Ⅰ as more effective. The results demonstrate that co-expression of ppsA and tktA can improve the production of DAHP, and what’s more, when multigenes co-expressed, the recombinant which has coordinated enzymes activity is optimum.3. We amplified aroA, aroC, aroB of common pathway, CsrA and its flanking sequence of global regulation network from E. colt, and Kan resistant gene from plasmid pET28a.4. Centerpi更多还原