大肠杆菌DH5α及其耐乙酸突变株DA19在氮源限制下的代谢和关键酶特性研究

Study on the Metabolism of Escherichia coli DH5α and Its Acetate-tolerant Mutant DA19 Based on Key Enzyme Activity Analysis

摘要:

在氮源限制的基本培养基中对大肠杆菌DH5α及其耐乙酸突变株DA19进行连续培养，通过测定中心代谢途径关键酶的活性分析二者代谢差异。结果表明，DA19的6-磷酸葡萄糖脱氢酶(G6PDH)和异柠檬酸脱氢酶(ICDH)活性高于DH5α，而磷酸果糖激酶(PFK)和乙酸激酶(ACK)活性低于DH5α，反映了DA19进入磷酸戊糖途径(PPP)的碳流增加，而进入酵解和乙酸产生(Ack-Pta)途径的碳流减少。因此，关键酶活差异与DA19菌体关于葡萄糖得率提高、副产物乙酸和丙酮酸的生成减少相一致。添加腺嘌呤后，DH5α的G6PDH和ICDH活性增加，PFK和ACK活性降低，而DA19各酶活变化不明显。乙酸钠的添加导致除PFK外的其他酶活性均降低，尤其是DH5α的ICDH明显降低，这些结果反映的中心代谢途径流量变化也与二者生长和代谢副产物积累的变化一致。

Abstract:

The differences in metabolism in Escherichia coli DH5α and its acetate-tolerant mutant DA19 were analyzed based on the activity of key enzymes involved in central metabolism when both strains were continuously cultured in nitrogen source-limited defined media. The activity of glucose-6-phosphate dehydrogenase (G6PDH) and isocitrate dehydrogenase (ICDH) in DA19 increased as compared with those in DH5α, while acetate kinase (ACK) and phosphofructosekinase (PFK) decreased. These indicated that more carbon flux of DA19 entered the phosphopentose pathway (PPP) and less entered the glycolytic (EMP) pathway and acetic acid production (Ack-Pta) pathway. Therefore，the differences in activity of key enzyme coincided with increased cell yield based on consumed glucose (Y_X/G) and decreased production of acetic acid and pyruvate of DA19. G6PDH and ICDH in DH5α were up-regulated by addition of adenine, while ACK and PFK were down-regulated. On the other hand, adenine had little effect on those in DA19. The enzymes except PFK in both strains were down-regulated by sodium acetate, especially the activity of ICDH in DH5α. These results suggested changed flux of central metabolic pathways were also consistent with the changes of growth properties and byproducts formation.