大腸桿菌內CRP對醣類代謝基因調控功能研究

Abstract

碳水化合物的代謝是一種常見於生物體內的機制，功能為提供能量以及生合成的必要物質。在諸多碳源中，碳水化合物比起脂質、肽類及酒精等要易被生物體利用。以大腸桿菌而言，碳水化合物的代謝主要是受環磷酸腺苷cAMP及其受體蛋白CRP之複合物cAMP-CRP所調控。cAMP-CRP可以透過「代謝抑制」使大腸桿菌選擇利用的碳源。此外，CRP也能和代謝基因，以及該基因之轉錄因子形成「前饋迴路」。前饋迴路是已經被證實存在於多種碳水化合物代謝基因調控的模式裡，且具有重大意義。在先前的研究中顯示，大腸桿菌碳水化合物代謝基因有約70%受到CRP調控，這點與其運輸基因相近似。而這些基因裡約有50%能與CRP形成前饋迴路，因此我們便以調查碳水化合物代謝基因及運輸基因之前饋迴路關聯為研究目的，並發現前饋迴路的數量與醣類代謝之間的關係。 在前饋迴路調控碳水化合物代謝中，我們進一步研究前饋迴路對麥芽糖代謝之生理意義。結果發現麥芽糖代謝基因受CRP調控。在此調控機制中，CRP可以刺激麥芽糖代謝基因轉錄因子(malT)的表現，但又同時抑制麥芽糊精磷酸化酶(malP)、麥芽糖轉葡糖基酶(malQ)和麥芽糊精葡萄糖甘脢(malZ)的表現。在雙碳源實驗結果中，亦發現malT失去CRP調控後會減緩葡萄糖耗盡時轉換利用麥芽糖的速度。我們認為，前饋迴路的存在能夠使大腸桿菌透過特定基因的調控來快速適應環境的變化。

Carbohydrate metabolism is an important biochemical reaction that provides energy and builds functional materials in various life forms. Carbohydrate is the preferred carbon source over lipid, peptide, organic acids, and alcohol. Carbohydrate metabolism in Escherichia coli, a well-established model in research of microorganisms, is mainly controlled by cyclic adenosine monophosphate (cAMP) and its receptor protein (CRP) complex cAMP–CRP. The cAMP–CRP can form a mechanism known as “catabolite repression,” which allows E. coli to rapidly select different carbohydrate sources. CRP can form a close loop called “feed-forward loop (FFL)” with CRP-mediated genes and CRP-mediated transcription factors (TFs). An FFL demonstrates significant regulatory patterns and can be detected in many types of gene mediation and in carbohydrate metabolism. Our previous study demonstrated that in E. coli carbohydrate degradation, more than 70% genes were mediated by CRP; this percentage is similar to that in carbohydrate transportation. In addition, 50% of these genes can form FFLs with CRPs and their TFs. Therefore, we aim to determine the FFL relationship between degradation and transportation, and have discovered the relation between carbonhydrate metabolism and the numbers of FFLs. To precisely determine the FFL mechanism, we selected the maltose degradation system, which the CRP-mediated FFL remained unknown. In this case, CRP can activate malT to facilitate the utilization of maltose by E. coli but repress the degradation genes malZ and malPQ. In maltose diauxic growth test, malT without CRP mediated had delayed growth rate. After studying the FFL, we hypothesize that E. coli can adapt to environmental shifts with specific gene regulation.