基于油质蛋白oleosin鉴定短序列脂滴定位信号

Identification of Short Peptides from Oleosin for Lipid Droplet Localization in Xanthophyllomyces dendrorhous

目的:人工区室化是代谢工程及合成生物学的重要技术手段,针对脂滴区室化技术未得到有效评价的现状,以产油微生物红法夫酵母为检验体系,评价筛选脂滴定位信号。方法:基于芝麻油质蛋白oleosin的三维结构及其跨膜结构域分析,设计短序列脂滴定位信号,构建定位信号与eGFP荧光蛋白基因融合的表达载体,转化红法夫酵母,采用荧光显微观察评价定位信号的定位功能。结果:完整的oleosin蛋白Ols1(_((1-145)))和肽段Ols2(_((1-63)))、Ols4((32-63))、Ols5((44-63))均能引导eGFP定位于红法夫酵母的脂滴内部,肽段Ols3((64-145))不能有效引导eGFP定位于脂滴,仅在细胞质中游离表达。结论:筛选得到了一个短序列脂滴定位信号Ols5((44-63)),其仅含19个氨基酸,能够有效引导eGFP定位于脂滴内部。

Objective:Artificial compartmentalization is among the important approaches in metabolic engineering and synthetic biology.Due to the insufficient evaluation of compartmentalization in lipid droplets(LDs),the aim of this work is to identify efficient LD localization signals in the oleaginous yeast Xanthophyllomyces dendrorhous.Methods:Based on the three-dimensional structure and the predicted transmembrane domain(TMD)of sesame oleosin,LD localization signals were predicted and fused with eGFP to generate a series of expression vectors,which were used to transform X.dendrorhous.Green and orange fluorescence was observed and used to assess the efficiency of the predicted LD localization signals.Results:The intact oleosin protein Ols1_((1-145))and truncated peptides Ols2_((1-63)),Ols4(32-63),and Ols5(44-63)successfully guided eGFP is localization in LDs,while truncated peptide Ols3(64-145)was ineffective and most eGFP proteins were expressed in cytoplasm.Conclusion:A short LD localization signal Ols5(44-63)consisting of 19 amino acids was identified,which can precisely lead the eGFP to localize inside the LDs.