大肠杆菌ATCase抗反馈抑制突变体的构建及其对胞苷积累的影响

为解决胞苷生物合成途径中天冬氨酸氨甲酰转移酶受胞苷三磷酸反馈抑制调节的问题,通过对其碱基序列和蛋白质结构分析,利用基因定点突变的方法构建了大肠杆菌的ATCase突变酶,得到三个突变体:M1(H20L)、M2(K60E)、M3(K94E),并在E.coli DH5α中对融合蛋白进行了表达.酶活测定表明,M1、M2、M3的ATCase酶相对活性都比野生型M0的高,分别为野生型M0的1.10、1.22和1.37倍,且比活力都有不同程度提高.与含野生型pyrBI基因的M0相比,含突变型基因的M1、M2和M3均对15,mmol/L的CTP具有强的抗反馈抑制作用,且M1、M2和M3的抗CTP反馈抑制作用分别是M0的5.4、6.0和8.5倍.最后将各突变质粒转入到E.coli Cyt10(Δcdd)中进行发酵培养,结果表明,与未含突变基因菌株相比,各含突变基因菌株的胞苷积累量均有不同程度的提高,说明ATCase定点突变使胞苷的合成积累途径得到了不同程度的强化.

Construction of ATCase Mutants with Feedback Inhibition Resistance and their Effect on the Cytidine Production in E. coli

In order to obtain E.coli ATCase mutants with high catalytic activities and feedback inhibition resistance,sitedirected mutagenesis,homologial analysis of the related amino acid sequences of ATCase and PCR were used to construct three mutants:M1(H20L),M2(K60E),M3(K94E).First,pyrBI gene of M0(wild type)was ligated to plasmid pSTV28 vector,and site-directed mutagenesis was carried out by PCR.The fusion proteins were expressed in E.coli DH5α,and the erzyme activity analysis showed that the original ATCase relative activity of M0 not only remained but also increased by 1.10-fold,1.22-fold and 1.37-fold respectively.Their specific activities were also increased.Measuring of the feedback inhibition resistance of the enzyme showed that E.coli DH5α strains containing the mutation gene had a strong feedback inhibition resistance against 15 mmol/L of CTP concentration.The enzymic activities found in M1,M2 and M3 strains were 5.4-,6.0-and 8.5-fold of M0 in CTP feedback inhibition resistance of ATCase regulation.Finally,all mutant plasmids were transformed into E.coli Cyt10(Δcdd).After fermentation,ATCase synthesis of site-directed mutagenesis indicated that the accumulation of cytidine pathway got strengthened.