基于RNA-Seq的裂殖壶菌响应氧因子调控的分子机制研究

Docosahexaenoic acid (DHA) is one of the important ω-3 long-chain polyunsaturated fatty acids, which plays a major role in promoting the development of neurons cells and preventing angiocardiopathy. Recently, microbial oil production has attracted much attention. The problem of the unclear regulation mechanism of double bonds formation and carbon chain enlongation in oleaginous microorganism is concerned in this study. Taking the phenomenon that oxygen could regulate the synthetic efficiency of saturated fatty acids and unsaturated fatty acids as a breakthrough, transcriptomics will be applied to get differential intracellular gene expression profiles of Schizochytrium sp. under different oxygen supply conditions and different fermentation stages. Based on bioinformatics database and statistical software, the key regulatory genes related to double bonds formation and carbon chain enlongation will be achieved. At the same time, genetic transformation system will be developed to knockout or overexpress the specific key genes. The intrinsic mechanism of the key regulatory factor for DHA synthesis by Schizochytrium sp. will also be elaborated. In addition, the regulation mechanism will be revealed to explain how oxygen factor induced the metabolic shift of saturated fatty acid and unsaturated fatty acid pathways in Schizochytrium sp. A new research line will be proposed to get key regulatory factors based on transcriptomics for microorganism who have no genomic information. What we discussed will also be significant to reveal the regulation mechanism of unsaturated fatty acid biosynthesis in oleaginous microorganism.

二十二碳六烯酸（DHA）是一种重要的ω-3长链多不饱和脂肪酸，具有促进神经细胞发育、预防心血管疾病等功效。近年来，利用微生物产油已成为研究热点。本研究针对产油微生物中不饱和双键形成及碳链延长调控机制不明晰这一问题，以氧刺激可改变裂殖壶菌胞内饱和脂肪酸与不饱和脂肪酸合成效率这一现象为切入点，采用转录组学的研究手段，利用RNA-Seq技术获取在不同氧供应条件及生长产油期裂殖壶菌胞内基因差异表达图谱，结合生物信息学和统计学软件挖掘与不饱和双键形成、碳链延长相关的关键调控基因；构建裂殖壶菌遗传转化体系对关键基因进行过量表达或定点敲除，阐述关键调控因子对裂殖壶菌合成DHA的内在作用机制，揭示氧因子诱导裂殖壶菌胞内饱和与不饱和脂肪酸合成途径偏转的调控机理，发展一条适用于非模式微生物基于转录组样本获取关键调控因子的新路线，对揭示产油微生物不饱和脂肪酸的合成及调控机制具有重要意义。

二十二碳六烯酸（DHA）是一种重要的ω-3长链多不饱和脂肪酸，具有促进神经细胞发育、预防心血管疾病等功效。近年来，利用微生物产油已成为研究热点。本项目主要围绕产油微生物中不饱和双键形成及碳链延长调控机制不明晰这一问题展开，主要开展三方面的研究：（1）前体研究平台搭建，完成了裂殖壶菌全基因组测序，并构建了裂殖壶菌遗传操作平台和细胞组分分析平台；（2）对不同氧刺激条件和不同发酵时期下的裂殖壶菌进行转录组测序和分析，发现结合和单细胞过程很可能与细胞从生长转入产油阶段相关，氧化还原过程在油脂由积累到返耗过程转变起到关键作用；同时进一步分析了中心代谢中的14个基因，脂肪酸合成过程的8个基因以及甘油酯代谢中的12个基因；此外10个转录因子可能与氧因子控制脂肪酸合成密切相关；（3）基于转录组信息的基因功能验证及调控，基于转录组分析结果，通过敲除并替换PKS酶中的AT模块，改变了胞内EPA的含量，EPA含量增加至5%；通过过表达omega-3去饱和酶基因将胞内3%的DPA转化为DHA，为从基因层面调控多不饱和脂肪酸的双键形成和比例奠定了基础。