通过改换大肠杆菌腺苷酸代谢途径来增加胞内能荷水平

Engergy charge changing by pathway manipulation of adenosine triphosphate metabolism in Escherichia coli

摘要:

原核生物细胞能量和物质代谢的途径是一个很复杂的网络, 改变代谢途径中的基因会对能量和物质流产生怎样的影响, 仍然不是很清楚。以往的文献和研究已经将大肠杆菌的腺嘌呤核苷酸补救合成途径研究的很透彻。使用HPLC对删除了add基因的大肠杆菌细胞内腺苷类核苷酸分析表明, 在腺嘌呤核苷酸补救途径中单一基因的途径操作不能有效改变腺嘌呤类核苷酸的代谢流向。实验中通过删除大肠杆菌JM83株中的add基因(编码腺苷脱氨酶[EC:3.5.4.4][1, 2]), deoD基因(编码嘌呤核苷磷酸酶[EC:2.4.2.1][3, 4]), amn基因(编码AMP核苷酶 [EC:3.2.2.4][5])并引入外源ado1基因(来自酵母编码腺苷激酶[EC:2.7.1.20][6,7,8]), 构建了菌株J991+ (add-, deoD-, amn-, ado1+, JM83), 将其在含腺苷的LB培养基培养, 使用HPLC分析其胞内腺苷类能量物质发现, ATP, ADP, AMP胞内含量都有所增加, 分别都比对照JM83菌株提高一倍左右, 大大加强了腺苷转化AMP的代谢流量, 实现了改变物质代谢流向并使ATP积累的目的。该菌种实现了高产ATP代谢通路的构建, 为下游生物工程发酵提供了较野生菌更高效的菌种, 有望通过发酵工程优化培养, 大幅提高ATP产量。同时, “尝试改变AMP的浓度而非直接针对ATP调节代谢途径, 达到ATP积累的目”这一思路为同类研究提供参考。最后也表明在腺嘌呤核苷酸补救代谢途径中, 为达到物质代谢流改变的目的, 多基因联合操作较之单基因敲除更为有效。

Abstract:

Endocystic metabolism of energy and material is a complex matrix. We do not know the consequence of replacing the genes controlling the metabolic fluxes. Single gene(add) deletion cannot change the direction of metabolic fluxes of adenosine. Through deleting 3 genes, namely add (encoding adenosine deaminase [EC:3.5.4.4]), deoD (encoding purine-nucleoside phosphorylase [EC:2.4.2.1]) and amn (encoding AMP nucleosidase [EC:3.2.2.4]), and introducing ado1 gene(encoding adenosine kinase in S. cerevisiae[EC:2.7.1.20]), we modified the salvage pathway of adenine metabolism, and constructed a strain named J991. Extract of J991 was analyzed by HPLC. The endocystic concentration of ATP, ADP and AMP raised 2-fold as the control, and the metabolic fluxes of adenosine are also changed. It is a new way for ATP stimulation. Multi-gene manipulation is more effective than single-gene manipulation in salvage pathway of adenine.