自养-异养转化小球藻葡萄糖跨膜转运与油脂合成的C/N调控

Enhancement of lipid synthesis resulted from heterotrophic growth with glucose as nutrient source in Chlorella protothecoides 0710 depends not only on glucose concentration but also on C/N of growth medium. Until now most studies on lipid synthesis for biodiesel production in microalgae has been focused on its technological application; For transmembrane transport of hexose with relative proteins/genes in microalgae, N regulation and control has not been paid attention to and nor connected with the metabolic flux change of lipid synthesis. Studies in this proposal based on photosynthesis-fermentation model (PFM) of C. protothecoides 0710 include (1) Control by C/N over glucose absorption, heterotrophic transformation and lipid accumulation in C. protothecoides 0710; (2) The differential expression of glucose transport gene; (3) Metabolomics and construction of central metabolic network; (4) Dynamic changes of sub-cell structure and characteristics of glucose transporter; (5) Glucose transmembrane transport process controlled by C/N. The aim of this study is to understand regulation and control by C/N over glucose transmembrane transport which resulted in transformation of this algal strain from autotrophic to heterotrophic metabolism in PFM and great increase lipid yield in cells. It is very significant in basic research for heterotrophic metabolism of microalgae and its energy exchange. Above studies will be also helpful to reduce the cost in microalgae biodiesel refinery by means of using both CO2 and organic carbon to reduce the sugar consumption in PFM, therefore represents a potential value in renewable bioenergy application.

Chlorella protothecoides 0710细胞吸收葡萄糖进入异养生长并导致油脂合成增加，不仅受限于葡萄糖供给浓度，还受控于培养液中C/N值。迄今有关微藻油脂合成及生物柴油研究侧重于应用技术方面，微藻己糖跨膜转运及相关蛋白/基因的基础研究未考虑氮调控因素，也不联系藻细胞油脂合成代谢通路变化。本申请基于小球藻的光合发酵模型（PFM），研究内容包括： C/N对小球藻葡萄糖吸收、异养转化和油脂积累的控制作用；葡萄糖跨膜转运基因差异表达分析；代谢物组分析和中心C代谢网络构建；亚细胞结构动态变化和葡萄糖转运蛋白特征分析；C/N调控葡萄糖跨膜转运过程分析等。认识实验藻株PFM中C/N调控葡萄糖跨膜转运导致由自养转化为异养并大幅提高细胞油脂产量原因，对于深入认识微藻异养代谢及能量转换规律具有重要的理论意义，还有助于高效利用CO2和有机碳两种原料增加PFM中油脂合成。

在小球藻由自养向异养转化过程中，首次观察到细胞自嗜现象，为揭示细胞油脂及功能脂类的积累机理和碳代谢规律提供了重要的细胞生物学证据。筛选出一株光合自养缺陷型小球藻突变体，为我们以该突变体为对照，揭示野生型小球藻在自养－异养转化过程中受C/N调控的规律提供了可行性。转录组、蛋白组和代谢组学分析揭示了小球藻细胞自养－异养转化及表型变化过程最关键基因的表达差异及部分分子生物学调控机制。完成了自养-异养小球藻中心C代谢网络构建和13C标记代谢流量分析，揭示了Chlorella protothecoides高效利用葡萄糖及油脂合成代谢的性质。首次认识了异养小球藻中糖转运蛋白的分子生物学特征 。具体揭示了C. protothecoides不同C/N比下的葡萄糖吸收存在叶绿体-淀粉体-油体的演变过程。还发现光照对异养小球藻葡萄糖摄取与代谢具有显著影响。上述主要进展反映，我们通过控制实验藻株的自养向异养转化过程和不同C/N输入，分析实验藻株葡萄糖转运基因的差异表达与代谢物组变化，同时分析细胞与生物化学特征变化，揭示了PFM过程C/N协同调控作用下自养-异养转化小球藻葡萄糖跨膜转运的规律及其油脂合成的规律。达到了项目任务书的目标。