腺嘌呤对大肠杆菌DH5α和其耐乙酸突变株DA19 代谢流分布的影响

【目的】本文通过分析在基本培养基中添加腺嘌呤对大肠杆菌DH5α和其耐乙酸突变株DA19代谢流分布的影响,从而进一步了解二者在代谢调控方面的差异。【方法】对2个菌株分别在氮源限制基本培养基及添加腺嘌呤的氮源限制基本培养基中进行连续培养,分析两者代谢流变化差异,并与酶活测定结果进行比较。【结果】添加腺嘌呤降低了DH5α的葡萄糖比消耗速率和乙酸的比生成速率,提高了菌体关于葡萄糖的得率,而丙酮酸比生成速率变化不明显。与MN培养基相比,添加腺嘌呤后DH5α降低了乙酸分流比,提高了分泌丙酮酸和三羧酸循环分流比,同时明显改变了磷酸果糖激酶、6-磷酸葡萄糖脱氢酶和乙酸激酶酶活。与DH5α不同,添加腺嘌呤使得DA19的丙酮酸比生成速率增加了近57%,而其它参数无明显改变。与MN培养基相比,DA19在添加腺嘌呤后降低了三羧酸循环分流比,大大提高了分泌丙酮酸分流比,而关键酶活未发生明显改变。酶活变化与代谢流结果基本一致。【结论】由于大肠杆菌DH5α和DA19嘌呤核苷酸从头合成途径能力存在差异,因此添加腺嘌呤对两个菌株的代谢流分布产生了完全不同的影响。

Effect of adenine on metabolic fluxes in Escherichia coli DH5alpha and its acetate-tolerant mutant DA19

[Objective] In order to further understand the difference in metabolic regulation between Escherichia coli DH5α and its acetate-tolerant mutant DA19,we analyzed the effect of supplementing adenine in defined media on metabolic fluxes in the two strains.[Methods] E.coli DH5α and DA19 were continuously cultured in nitrogen-limited defined media without or with supplemented adenine.Based on mass balance and metabolic reaction stoichiometry,the metabolic fluxes in DH5α and DA19 at different culture condition were calculated,and the results were compared with the activities of key enzymes.[Results] The supplementation of adenine decreased specific glucose consumption rate and specific acetate production rate,and improved growth yield coefficient on glucose of DH5α.Nevertheless,specific pyruvate production rate did not significantly change.Furthermore acetate split ratio decreased whereas pyruvate and TCA split ratios increased.Obvious changes were observed in the activities of phosphofructokinase,6-phosphoglucose dehydrogenase and acetokinase.Compared with DH5α,the supplementation of adenine increased rate specific pyruvate production rate of DA19 nearly 57%,and other parameters did not change.In addition,it showed decreased TCA split ratio and greatly increased pyruvate split ratio,whereas no changes in the key enzyme activities were observed.The differences of enzyme activities in the two strains were reasonably consistent with those in metabolic fluxes.[Conclusion] Because there were differences in the de novo biosynthesis capacity of purine nucleotides between DH5α and DA19,supplementation of adenine had completely different effect on metabolic fluxes in two strains.