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摘要:为探究新型产油酵母菌解脂耶罗威亚酵母(Yarrowialipolytica)的油脂代谢工程机理，本研究运用生物信息学方法对该酵母脂肪酸合成途径和甘油酯合成途径中的关键酶基因进行了序列比对、聚类分析及其酶活位点的预测、改造与稳定性评价等等研究旨在深入认识该酵母产油关键酶基因的序列、表达、进化特征及其蛋白三维结构特点。研究发现1)脂肪酸合成关键酶包括FAS1、fabF和ACSL等5种关键酶，三酰甘油合成关键酶包括GLYCTK、GLYK、gldA、LPIN、GPAT3_4、MBOAT1_2和GLYK等22种关键酶都与该酵母高产油的特性相关；2)对耶氏酵母、酿酒酵母、裂殖酵母和玉米黑粉病等7种真菌油脂合成关键酶的同源基因进行聚类分析的结果显示，关键酶蛋白被分为FAS2、gldA、FAS1、fabG等23个类群(如图1)；3)关键酶活性位点的预测与改造发现比如当gldA酶活性位点的筛选条件设定为㈠序列上下游遇gap即结束㈡保守区段长度为3个氨基酸且≥1／2，筛选发现有3个活性位点，将第43号氨基酸序列的P转换为Q，第68号氨基酸序列的G转换为L，第75号氨基酸序列的T转换为L其酶活更强，蛋白功能表达更加充分。这些研究为后续重构产油基因的表达调控网络即定向改变油脂代谢流的方向并使其通量最大化提供了理论基础。43756
毕业论文关键词：产油酵母菌 解脂耶罗威亚酵母 甘油三酯 柠檬酸 序列比对
Study on metabolic engineering of new oil producing yeast
Abstract: In order to explore the new oil-producing yeast Yarrowialipolytica yeast’s mechanism of lipid metabolism, we use the method of bioinformatics to research the study of key enzymes’s Sequence alignment, cluster analysis and forecasting activity sites, and so on , of the yeast fatty acid synthesis pathway and glycerides synthesis pathway. So that we can understand the sequence of the key oil-producing yeast gene expression and protein three-dimensional characteristics deeply. The study found1)Fatty acid biosynthetic enzymes include FAS1, fabP and ACSL and other five key enzyme triacylglycerol synthesis key enzymes include GLYCTK, GLYK, gldA, LPIN, GPAT3_4, MBOAT1_2 and GLYK and other 22 kinds of key enzymes,which related to the characteristics of the yeast yielding oil；2)the results for Yarrowia yeast, Saccharomyces cerevisiae, Schizosaccharomycespombe and corn smut fungi seven fat synthesis key enzymes homologous gene cluster analysis revealed that a key enzyme protein is pided FAS2, gldA, FAS1, fabG etc. 23 groups (Figure 1); 3)the discovery of prediction and reconstruction of key enzyme active site, forexample When the filter condition gldA the enzyme active site sequence (i) is set as the upstream and downstream ends (ii) in case of gap that is conserved segment length of 3 amino acids and ≥1 / 2, screening found three active sites, the 43 amino acid sequence the P is converted to Q, G converts the amino acid sequence No. 68 as No. 75 to convert the amino acid sequence of L T, therefore Its activity is stronger, fuller expression of protein function. These studies for the subsequent reconstruction of oil-producing gene expression regulatory networks that is oriented to change the direction of the oil flow and metabolic flux maximize it provides a theoretical basis.
Key words: Oleaginous yeast；Yarrowialipolytica；Triglycerides；Citric acid；Sequence Alignment.
目录
摘要 1
引言 2
1材料与方法 4
1.1 酵母及模式物种的选取 5
1.2 油脂合成代谢关键酶选择与序列下载 5
1.3 油脂合成关键酶的序列比对与聚类分析