顶头孢霉中己糖激酶编码基因Achka的功能研究  

The function of a hexokinase encoding gene Achka in Acremonium chrysogenum

作　　者：苏豫[1,2] 陈依军[1] 车永胜[2] 刘钢[3] 

机构地区：[1]中国药科大学化学生物学研究室,江苏南京210009 [2]军事医学科学院毒物药物研究所抗毒药物与毒理学国家重点实验室,北京100850 [3]中国科学院微生物研究所真菌学国家重点实验室,北京100101

出　　处：《菌物学报》2017年第3期323-331,共9页Mycosystema

基　　金：国家自然科学基金(31470177)~~

摘　　要：己糖激酶在真菌中广泛存在,参与葡萄糖磷酸化等生理过程。本文从头孢菌素产生菌顶头孢霉中克隆并鉴定了一个己糖激酶编码基因,命名为Achka。通过RT‐PCR证明Achka含有4个内含子,其推测的编码蛋白含有484个氨基酸,分子量为53.7k Da。在顶头孢霉中敲除Achka后发现,突变株在以果糖、蔗糖或甘露糖为唯一碳源的培养基上生长受到严重限制。我们在大肠杆菌中异源表达了Achka,并对表达的重组蛋白AcHKA进行了分离纯化。酶学动力学分析表明,AcHKA对果糖的最大反应速率要大于葡萄糖,但是却对葡萄糖有更高的亲和力。上述结果进一步证明AcHKA为己糖激酶,在顶头孢霉体内主要负责果糖、蔗糖和甘露糖等的代谢。Hexokinases are generally believed to play pleiotropic roles in biological processes such as sugar sensing and phosphorylation. A hexokinase-like protein encoding gene Achka was identified in the cephalosporin producing strain Acremonium chrysogenum. RT-PCR analysis revealed that Achka contained four introns. The deduced protein of Achko contains 484 amino acids with a calculated molecular mass of 53.7kDa. Achka was expressed in E, coli and the recombinant AcHKA was purified. Kinetic characterization showed that AcHKA had higher affinity, but lower phosphorylation efficiency for glucose compared to fructose. Disruption of Achko significantly delayed the fungal growth in MMCB media when fructose, sucrose or mannose was used as the sole carbon source. These results suggest that AcHKA is a hexokinase and plays the key roles in the utilization of fructose, sucrose and mannose in Acremoniurn chrysogenurn.

关 键 词：顶头孢霉 己糖激酶 Achka 己糖利用 

分 类 号：Q78[生物学—分子生物学] Q936