大肠杆菌ppsA和tktA基因的串联表达  

Co-expressions of Phosphoenolpyruvate Synthetase A(ppsA) and Transketolase A (tktA) Genes of Escherichia coli

作　　者：李永辉[1] 刘云[1] 王世春[1] 童朝阳[1] 徐琪寿[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850

出　　处：《生物工程学报》2003年第3期301-306,共6页Chinese Journal of Biotechnology

基　　金：军事医学科学院创新基金资助项目 (No .990 2 5 0 2 )~~

摘　　要：ppsA和tktA是芳香族氨基酸生物合成中心途径的两个关键酶基因 ,在大肠杆菌中 ,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。采用PCR方法从大肠杆菌K 12株中扩增到ppsA和tktA ,并实现了两基因的高效表达 ,其中ppsA活性提高了 10 .8倍 ,tktA活性提高了 3.9倍 ,当这两个基因串联在一个质粒上导入大肠杆菌进行表达时 ,PpsA的活性变化较大 (2 .1～ 9.1倍 ) ,TktA的活性相对稳定 (3.9～ 4 .5倍 ) ,且这两个基因单独表达和串联表达都能使芳香族氨基酸生物合成共同途径中关键中间产物DAHP的产量提高 ,且串联表达比单独表达较高。Metabolic engineering 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,and tyrosine are essential amino acids for human and animals.In addition, phenylalanine is used in aspartame production. Escherichia coli and many other microoganism synthesize aromatic amino acids through the condensation reaction between phospho enolpyruvate(PEP) and erythrose 4 phosphate(E4P) to form 3 deoxy D arabinoheptulosonate 7 phosphate(DAHP). But 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. To channel more carbon flux into the aromatic pathway, one has to overcome pathways competing for PEP. pps A and tkt A are the key genes in central metabolism of aromatic amino acids biosynthesis. pps A encoding phosphoenolpyrucate synthetase A(PpsA) which catalyzes pyruvate into PEP; tkt A encoding transketolase A which plays a major role in erythrose 4 phosphate (E4P) production of pentose pathway. We amplified pps A and tkt A from E.coli K 12 by PCR and constructed recombinant plasmids of them in pBV220 vector containing P RP L promoter. Because of each gene carrying P L promoter, four productions of ligation were obtained. The monoclonal host containing recombinant plasmids was routinely grown in Luria Bertani(LB) medium added Ampicillin at 37℃ overnight, and then inoculated in LB(Ap r) medium by 3%～5% in flasks on a rotary shaker at 30℃, induced at 42℃ for 4.5 hours when OD 600 ≈0.4, cells were obtained by centrifugation at 10000 r/min at 4℃.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

关 键 词：大肠杆菌 tktA基因 串联表达 ppsA基因 芳香族氨基酸 

分 类 号：Q786[生物学—分子生物学]