谷氨酸棒状杆菌果糖代谢阻断工程菌的构建

Construction of Engineered Strain of Corynebacterium glutamicum Capable of Blocking Fructose Metabolism

谷氦酸棒状杆菌不仅可以利用葡萄糖和果糖作为碳源进行糖类代谢,也可以利用这些碳源作为底物生产葡萄糖酸、甘露醇及山梨醇等产品。为了提高底物利用率和目的产物的积累量,利用代谢工程阻断糖类代谢的磷酸烯醇式丙酮酸一糖磷酸转移酶系统(phosphoenolpyruvate carbohydrate phosphotransferase system,PTS)和失活相应磷酸激酶。是实现此目标的有效手段。本实验利用同源重组和反向筛选等技术手段,分别获得ptsF单基因缺失工程菌CG△ptsF和ptsF、ptsH、ptsI三基因缺失工程菌CG△ptsF△ptsH△ptsI。工程菌的生长情况研究表明:在以葡萄糖为唯一碳源的培养基上,工程菌CG△ptsF和工程菌CG△ptsF△ptsH△ptsI与野生型生长情况基本一致,说明葡萄糖代谢不受3个基因影响;在以蔗糖为唯一碳源的培养基上,工程菌CG△ptsF和工程菌CG△ptsF△ptsH△ptsI的生长速率分别是野生型的48.4%和29.7%,菌体浓度分别是野生型的61.6%和34.1%;在以果糖为唯一碳源的培养基上,工程菌CG△ptsF菌体浓度是野生型43.2%,工程菌CG△ptsF△ptsH△ptsI生长为0,证明其完全阻断了果糖代谢,同时说明果糖的PTS系统受ptsF、ptsH和ptsI基因编码的PTS相关蛋白的联合控制。果糖代谢阻断工程菌的获得,为进一步构建以果糖原型为底物的甘露醇或山梨醇生产工程菌株提供了遗传资源,也为谷氦酸棒状杆菌的糖类代谢研究提供了理论依据。

Corynebacterium glutamicum is a food-grade microorganism widely used to produce amino acids,proteins and other chemical products in the fields of biological transformation,food additives,animal feed,cosmetics,medicine and health care.It can not only use glucose and fructose as the carbon sources,but also can use them as the substrates to produce gluconic acid,mannitol,sorbitol and other products.Blocking sugar metabolism in which the phosphoenolpyruvate carbohydrate phosphotransferase system（PTS） and phosphokinase are involved through metabolic engineering is an effective avenue to improve substrate utilization and the accumulation of desired products.In this research,the mutant strains CG △ptsF lacking the ptsF gene and CG △ptsF △ptsH △ptsI lacking the ptsF,ptsH and ptsI genes were constructed by homologous recombination and reverse screening.It was shown that both CG △ptsF and CG △ptsF △ptsH △ptsI were grown in medium with glucose as the only carbon source,while glucose metabolism was not affected by the lack of these three genes.Compared to the wild-type strain,the growth rates of CG △ptsF and CG △ptsF △ptsH △ptsI were 48.4% and 29.7% and cell concentrations of CG △ptsF and CG △ptsF △ptsH △ptsl were 61.6% and 34.1%,respectively,when the mutant strains were grown in medium using sucrose as the sole carbon source.However,when they were grown using fructose as the sole carbon source,the growth rates of CG △ptsF and CG △ptsF △ptsH △ptsI were 43.2% and 0 compared to the wild-type strain.It turned out that the fructose PTS system was controlled by combination of the ptsF,ptsH and ptsl genes encoding proteins associated with PTS.These engineered bacteria capable of blocking fructose metabolism can provide a genetic resource to construct mannitol or sorbitol-producing strains with fructose as the substrate and also lay the theoretical basis for the study of carbohydrate metabolism in Corynebacterium glutamicum.