产油酵母脂滴区室化调控代谢途径合成脂肪酸衍生物

In eukaryotic cells, the vast majority of proteins and their metabolic pathways are localized into the cell compartments. Compartmentalization of enzymes and pathways in membrane-bound organelles can increase yields of synthetic proteins, improve the underlying kinetics of bottle-neck enzymes, sequester toxic compounds, isolate synthetic pathways from competing pathways, and provide a compact and suitable environment for biosynthesis. Lipid droplets (LD) are specific organelles where fatty acids are stored, degraded and transported in oleaginous yeast; adequate supply of lipid substrates makes them a potential workhouse for production of fatty acid derived biofuels. Ldp1 is an LD structural protein with amphipathic helices domain, when C-terminal GFP-tagged Ldp1 (Ldp1-GFP) overexpressed in Saccharomyces cerevisiae, Ldp1-GFP localized on the LD surface and facilitated giant LD formation. Hereby, we propose to use the Rhodosporidium toruloides as the model system, via techniques such as bioinformatics analysis, gene deletion & complementing, RNA interference, site-directed mutagenesis, lipid droplet proteomics analysis, lipid droplet lipidomics analysis, metabolic engineering and synthetic biology to (1) elucidate the molecular basis of of lipid droplet protein targeting, (2) create the library of lipid droplet target peptide/domains, (3) compartmentalize pathway into lipid droplet of R. toruloides to biosynthesis of fatty acid ethyl ester and long chain alkenes, (4) find out the rule of towards repurposing the yeast lipid droplet for compartmentalizing heterologous metabolic pathways. The anticipated results will advance our understanding on the rule of the lipid droplet for compartmentalizing metabolic pathways, and the results will also launch fundamentals for oleaginous yeast based cell factory for value-added fatty acid derivatives and be of great significance for improving the economics of microbial oils.

真核生物代谢途径及其关键酶多限定在细胞内特定区室，这种区室化将有效促进酶和底物接触，避免竞争途径对代谢前体和中间体的争夺，为生物合成提供更适的局部微环境。前期研究发现：1）产油酵母脂滴具备成为脂肪酸衍生燃料区室化生产车间的潜力；2）产油酵母脂滴结构蛋白Ldp1促进“大”脂滴的形成，并可借助融合表达使GFP定位于脂滴。基于此，本项目拟将脂肪酸衍生燃料生产途径区室化至圆红冬孢酵母脂滴，内容包括：1）产油酵母脂滴靶定肽发现和优化，建立蛋白质脂滴靶定新方法；2）实现目标蛋白对脂滴的特异性靶定；3）脂滴区室化脂肪酸乙酯和脂肪烃合成途径重构；4）结合脂滴比较蛋白质组学和比较脂质组学分析，揭示脂滴区室化调控脂肪酸乙酯和脂肪烃合成的规律。本项目将丰富对脂滴功能的科学认识，发展出新型亚细胞代谢区室化调控策略；研究成果将为构建高值脂肪酸衍生物细胞工厂奠定科学基础，对改善微生物油脂的经济性具有重要参考价值。

前期研究发现，产油酵母脂滴结构蛋白Ldp1促进“大”脂滴的形成，并可借助融合表达使GFP定位于脂滴，产油红酵母脂滴具备成为脂肪酸衍生物区室化生产车间的潜力。基于此，本项目以圆红酵母为研究对象，对其脂滴蛋白及基功能进行了系统研究：1)利用CRISPR-Cas9基因编辑技术确定了候选脂滴结构蛋白LDP1和CALs的功能。2）利用生物信息学分析脂滴蛋白LDP1中潜在的脂滴靶定基序。3）以GFP为报告基因，分析了含有脂滴靶定肽核心基序的LDP1截短体对脂滴的靶定效率。4）利用LPD1脂滴靶定功能，完成了脂肪酸衍生物圆红冬孢酵母菌株的构建，并进行了脂质成份分析和功能评价。5)建立了红酵母CRISPR-Cas9基因编辑技术。总之，本项目丰富了对脂滴功能的科学认识，发展出了新型亚细胞代谢区室化调控策略，获得了一批红酵母脂滴区室化元件和工程菌株，研究成果将为构建高值脂肪酸衍生物细胞工厂奠定科学基础，对改善微生物油脂的经济性具有重要参考价值。

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