大肠杆菌L-色氨酸合成的代谢流分析  

Metabolic Flux Analysis of L-Tryptophan Synthesis in Escherichia coli

作　　者：申彤[1,2] 徐庆阳[1] 张成林[1] 谢希贤[1] 

机构地区：[1]天津科技大学生物工程学院,工业发酵微生物教育部重点实验室,天津300457 [2]新疆大学生命科学与技术学院,新疆乌鲁木齐830046

出　　处：《生物技术通讯》2014年第1期49-52,共4页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2012AA02A703)

摘　　要：目的：从代谢流的层面研究育种过程中基因操作对色氨酸积累的影响，为色氨酸菌种选育的设计思路提供理论指导和验证。方法：根据实验菌株的代谢特点构建￡一色氨酸代谢网络图，对出发菌株TRTH0709，及其重组菌株TRTH1013、TRTH1105和TRTH1107在30L发酵罐中进行分批流加发酵试验，在发酵进入稳定期后的26．28h，分别检测主要胞外代谢物的浓度并计算变化速率。结果和结论：得到了各菌株在拟稳态下的代谢流分布图。转酮酶基因（tktA）和磷酸烯醇式丙酮酸合成酶基因（ppsA）过表达能显著影响中心代谢途径，使代谢流向有利于色氨酸合成的方向改变，贮碳因子基因（csrA）敲除的影响较小，但在tktA和ppsA过表达质粒存在的情况下对色氨酸合成的代谢流有明显的促进作用。进一步的菌种改造仍有待进行，葡萄糖转运系统的替代和三羧酸循环的减弱是主要方向。Objective： To analysis the effect of gene manipulation on accumulation of L-tryptophan at the level of metabolic flow in the process of strain breeding. To provide theoretical guidance and validation for strains breed ing design. Methods： According to the metabolic characteristics of test strain, metabolism network of L-tryptophan was constructed. The fed-batch fermentation of L-tryptophan by original strain TRTH0709 and recombination strains （TRTHI013, TRTHll05 and TRTHll07） were carrried out in 30 L fermentor. The concentrations of extra-cellu lar metabolites were determined under pseudo-steady state of the batch culture（26-28 h）. Results ＆ Conclusion： The metabolic flux distribution maps of the four strains were obtained, compared and analyzed. Overexpression of transketolase（tktA） and PEP synthase（ppsA） can significantly influence the central metabolic pathways, make the metabolic flow redirect to tryptophan synthesis way. The influence of carbon store regulator A （csrA） disruption is relatively small on the metabolic pathways, but has significant role in promoting the metabolic flow of L-trypto- phan synthesis at the presence of the tktA and ppsA expression pIasmid. Metabolic flow analysis also shows that further breeding efforts remains to be performed, replace of the glucose transport system（PTS） and the reduction of the Krebs cycle are the main directions.

关 键 词：L-色氨酸' 代谢流' 大肠杆菌' 转酮酶' 磷酸烯醇式丙酮酸合成酶' 贮碳因子 

分 类 号：TQ922[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]