基于合成生物学研究解脂耶氏酵母高效转化油酸和亚油酸合成γ-癸内酯的新途径

γ-Decalactone (GDL, C10H18O2) is a well-known flavor compound produced by fruits and widely used in food, beverages, chemicals, pharmaceuticals and detergents. In addition, there are no commercially optical GDL and its analogues. Yarrowia lipolytica is one of the most successful microbe in biosynthesis of GDL. However, transformation efficiency is much decreased due to the inhibition of β-oxidization auxiliary enzymes which prevent opening double carbon covalent bond in the ricinoleic acid. Moreover, Y. lipolytica can’t use fatty acid without hydroxyl group, such as oleic acid and linoleic acid, although they are much cheaper raw materials. Therefore, it is necessary to transform hydroxylase genes to the Y. lipolytica to introduce the hydroxyl group, and then to construct a new approach of biotransformation of oleic acid and linoleic acid to GDL. Because hydroxyl group is probably inserted in different sites, several GDL analogues might be obtained too. In order to reduce the cost for the GDL, the proposal aims at reconstructing the Y. lipolytica genome to endow it with the ability to enroll oleic acid and linoleic acid into its metabolic pathway. In this proposal, 8 typical enzymes will be selected and constructed into E. coli to add hydroxyl group to the fatty acid. Then, the constructed E. coli will be used together with Y. lipolytica to catalyze the biosynthesis of GDL and its analogues from oleic acid and linoleic acid. In this step, HPLC, Mass Spectrometry, NMR will be used in the analysis of the biosynthetic products. The genes of these enzymes studied in the previous step will be selected, optimized and constructed into Y. lipolytica genome to directly catalyze the formation of GDL and its analogues from oleic acid and linoleic acid. The fermentation condition will be also optimized to increase the productivity of the target products. In summary，this project is aimed to ⅰ) decrease the cost of GDL produced by Y. lipolytica through genetic engineering to broaden substrates and improve transformation efficiency, and to ⅱ) biosynthesize some analogues of GDL and provide a solid foundation to innovate new flavor compounds.

γ-癸内酯具有诱人的桃香味，天然存在于水果中，国际上还没有天然旋光性及结构类似物产品供应。解脂耶氏酵母生物转化蓖麻油酸合成γ-癸内酯时，因受β-氧化辅助酶限制，较难打开蓖麻油酸双键，极大地影响了转化效率。在酵母中导入羟化酶基因，有望在打开更为廉价的油酸和亚油酸双键的同时引入羟基，构建生物转化合成γ-癸内酯的新途径，解除限制，降低成本，且因羟基接入位点的不同，可能会创制出多种γ-癸内酯结构类似物。本项目拟采用大肠杆菌重组表达8种不饱和脂肪酸羟化酶；重组酶协同解脂耶氏酵母催化油酸和亚油酸，优化脂肪酸羟化酶基因，实现多个基因在酵母中组成性协同表达，直接催化油酸和亚油酸为γ-癸内酯及类似物；通过高效液相色谱、质谱和核磁共振定性定量分析转化合成的γ-癸内酯及类似物；通过代谢工程和发酵工程等技术提高产物转化效率和生产强度。研究成果有望拓展解脂耶氏酵母底物利用范围，为新香料物质的创制奠定基础。

项目背景：随着香料产业的发展，人们对于天然香料的需求日益增大，γ-癸内酯（γ-decalacton，GDL）是一种芳香化合物，天然存在于一些水果和发酵品中，GDL在香精制作以及制药等方面都有不可替代的作用。化学合成的GDL是没有光学活性的消旋体，且安全性低，而通过生物法获得的GDL，与从水果、植物中提取获得的结构相似，具有光学活性，具有天然等同效应，因此用生物法生产GDL具有更广阔的发展前景。解脂耶氏酵母生物转化蓖麻油酸合成γ-癸内酯时，因受β-氧化辅助酶限制，较难打开蓖麻油酸双键，极大地影响了转化效率。.研究内容、重要结果及关键数据：本研究在酵母中导入羟化酶基因，打开油酸和亚油酸双键的同时引入羟基，构建生物转化合成γ-癸内酯的新途径。本研究选择了源自Bifidobacterium breve、C. purpurea和Pseudomonas sp菌株的3个油酸水合酶基因，源自Lactobacillus acidophilus和Lactobacillus acidophilus的2个亚油酸水合酶基因，源自F. oxysporum的1个亚油酸氧化酶基因，源自Nostoc punctiforme和Bacillus megaterium的2个二元醇合酶基因，克隆至大肠杆菌表达载体pQE60，然后转化至大肠杆菌，诱导表达，获得相应的重组酶，分离纯化各酶。以油酸为底物，测定8个重组酶的纯化物催化合成相对应羟基脂肪酸的催化能力，研究了8个重组酶催化反应的温度、pH稳定性、最适反应温度、pH和动力学性质等酶学性质。研究了8个重组酶发酵特性，并获得粗酶液，与解脂耶氏酵母协同转化油酸和亚油酸合成γ-癸内酯，其中源自C. purpurea的油酸水合酶基因，经大肠杆菌表达的重组酶与解脂耶氏酵母协同转化时，获得了γ-癸内酯的结构类似物5-羟基-4-癸内酯，产量为0.02g/L。将这些酶基因构建重组表达质粒，转化至解脂耶氏酵母，仅源自Pseudomonas sp中的油酸水合酶基因和源自Nostoc punctiforme二元醇合酶基因表达成功，优化重组菌发酵工艺，以油酸为底物，γ-癸内酯的产率达到5.47 g/L，油酸转化为GDL转化率为27.35%。.科学意义：本研究成果有望拓展解脂耶氏酵母底物利用范围，为新香料物质的创制奠定基础。该重组菌能够转好地转化饲料原料中的油脂。