高山被孢霉多不饱和脂肪酸合成途径中关键酶的底物选择特性和催化位点专一性研究

高山被孢霉是目前产多不饱和脂肪酸(PUFAs)真菌中唯一具有正式安全性评估的菌种(GRAS)，其PUFAs含量极为丰富，总的脂肪含量达到菌体生物量的50%，其中ω-3达到总脂肪酸含量的3%，而ω-6达到总脂肪酸含量的近30%，是名副其实的油脂细胞工厂。在课题组前期已完成的M. alpina ATCC32222基因组测序结果基础上，本项目选择其PUFAs合成途径上的关键脱饱和酶(ω3 Des、Δ12 Des和Δ9-I Des)进行研究，建立膜结合脱饱和酶的高效表达、纯化策略，在考察其底物选择特性和催化位点专一性基础上，完善M. alpina ATCC32222中PUFAs的合成途径，进而通过获得膜结合脱饱和酶的结构信息，解析其结构与功能的关系，阐明脂肪酸脱饱和酶具有如此底物选择特性和催化位点专一性的原因，这也将为利用微生物高产二十碳五烯酸(EPA)等ω3脂肪酸的潜在应用提供理论依据。

Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50% of its dry weight in the form of triacylglycerols, mostly composed of high quantities of arachidonic acid (ARA, 20:4n-6), and a small amount of eicosapentaenoic acid (EPA, 20:5n-3). It has been used for the industrial production of ARA as the ingredient of infant formulas with a record of complete safety. Here we choose three key desaturases (ω3 Des、Δ12 Des and Δ9-I Des) from our sequenced M. alpina ATCC32222, to establish an efficient expression and purification strategy for membrane-bound desaturases and to obtain their structural information, which will help to elucidate substrate selectivity and catalytic regioselectivity of membrane-bound fatty acid desaturases and lay the basis for future high production of EPA and other ω3 fatty acids in M. alpina.