微藻油脂合成积累路径关键酶基因的挖掘甄选及调控机制研究

The improvement of microalgal lipid accumulation will be one of key technologies for achieving low-cost commercialization of microalgal biodiesel. However, due to the cognitive deficiency and relative scarcity of precise research for some key enzyme genes associated with microalgal lipid accumulation, the corresponding lipid metabolic regulation mechanisms are still unknown. This will eventually result in the lack of theoretical guidances for enhancing cell lipid accumulation through genetic engineering strategies. In this proposal, fresh-water Chlorella vulgaris, seawater Nannochloropsis gaditana, and polar-region Coccomyxa subellipsoidea three relative alga species will be adopted as the experimental subjects to carry out study. The tandem omics means based on cytomics, transcriptomics, proteomics, and bioinformatics will be employed to firstly analysize the quantitative relationship between lipid accumulation efficiency in three nitrogen-starvation stressed microalgal cells and time course; The related correspondence analysis among the differentially expressed gene profiles will be further performed. The aims of this proposal is to screen and select congeneric key regulatory enzymes or new regulatory enzymes associated with lipid accumulation among three algal species; The gene interferece and gene over-expression strategies will be used to confirm the positive or negative correlations between gene expression regulation mode and lipid accumulation. The main effect degree of the regulatory enzyme genes associated with lipid accumulation will be cleared. Finally, the molecular mechanism of lipid accumulation regulated by the key enzyme genes response to nitrogen stress will be revealed. Expected outcomes of this proposal will provide theoretical foundations for accurate alteration of gene engineering microalgal species and promote the large-scale development of microalga bioenergy.

提高微藻油脂积累是实现微藻生物柴油低成本产业化的关键技术之一。然而由于目前对微藻油脂合成积累路径关键调控酶基因的认知不足及精准研究相对匮乏，导致脂代谢调控机制尚不明确，增强藻细胞脂质积累的相关策略也缺乏一定理论指导。本项目拟以潜力淡水小球藻、海水微拟球藻和极地胶球藻三近缘极差藻株为受试对象，基于细胞组–转录组–蛋白组–生物信息组串联递进分析平台技术，先解析氮饥饿藻细胞脂质积累与时间效应的定量关系；在此基础上，依托其全基因组信息，关联对应三藻株细胞转录组与蛋白组差异表达基因，挖掘甄选三藻株中油脂合成积累同种关键调控酶基因及可能发现的新调控因子；再以基因干扰、超量表达等方法确认这些酶基因表达调控模式与油脂积累正负相关性，同时廓清其调控油脂合成积累主效程度，进而揭示关键酶基因对油脂合成积累的调控机制。该研究为准确进行富油微藻基因工程改造奠定理论基础，并推进微藻能源规模化的进程。

提高微藻脂质积累是实现微藻能源低成本产业化之关键。然而由于目前对微藻脂质积累路径关键调控酶基因的认知不足及精准研究相对匮乏，导致脂代谢调控机制尚不清晰，增强微藻脂质积累的相关策略缺乏理论指导。本项目以淡水小球藻、海水微拟球藻和极地胶球藻为受试对象，基于细胞组-转录组-代谢组-生物信息串联递进分析技术，解析了氮限诱导藻细胞脂质积累与时间的量效关系，小球藻、胶球藻和微拟球藻分别在0.25 g/L、0.5 g/L 和0 g/L NaNO3诱导24 h、12 h、12 h达到最大油脂含量57%、63.84%和59.95%；在三藻株油脂高效富集关键时间点基础上，依托其全基因组信息，筛选调控脂质积累的差异基因，结果显示小球藻、胶球藻和微拟球藻中分别有9115、541和668个基因显著差异表达；同理，以多维和单维相结合方法筛选三藻株中显著性差异表达代谢分别为139、145和233个；将三藻株显著性差异表达基因和差异代谢物进行PCA比较、KEGG Pathway Venn分析、KEGG Pathway富集关联和O2PLS分析，共有10个显著性差异基因及调控路径中的16个差异代谢物关联对应，其糖酵解路径ppc和pdha、磷脂路径pgd、油脂路径accA、fata、acsl、fabD和fabF以及激素信号路径necd基因与其调控路径代谢产物如油酸、PS、PI、PE、PA、PC、ABA等显示一致性上调，而类胡萝卜路径lcby基因与其调控代谢物叶黄素一致性下调，表明这些基因通过增强糖酵解和脂肪酸路径以及下调类胡萝卜素路径和启动激素信号通路，为油脂合成积累提供前驱物，促进油脂高效富集。进一步以超量表达法验证了代表性pdha基因表达模式与油脂积累的正相关性，并以胞外抑制实验确认了pgd调控的代谢物PC和PE是脂质积累前驱物。研究成果为利用基因工程策略增强脂质积累及推进微藻能源规模化进程提供理论依据。