基于生物膜培养的微藻碳代谢和分配机制研究

Shortage of cheap algal biomass feedstock is the bottleneck limiting the commercialization of microalgae resource utilization. Biofilm cultivation system may achieve breakthrough for its potential in enhancing microalgae biomass productivity compared with conventional aqua-suspended microalgae cultivation method. With this biofilm cultivation system, cells are separated from most of the aqua and transmission medium between carbon source and algal cells is reduced significantly so that light and CO2 reach the algal cells easier. Consequently, it results in great increase of biomass and target compounds such as lipid accumulation based on previous studies. Carbon metabolism and carbon distribution may play important roles in the change of biomass and lipid synthesis under different cultivation mode, however, the physiological mechanism remains unclear. In this research, oleaginous microalgae Scenedesmus dimorphus is selected as the target microalgae. The biofilm cultivation system is employed as the experimental platform, with the suspended cultivation as the control. The physiological and biochemical character variations of S. dimorphus will be firstly investigated to explore the external carbon flux change responding to different cultivation systems. Subsequently, the internal regulation discipline of carbon metabolic network will be analyzed using multi-omics techniques. Meanwhile, the key genes of S. dimorphus that may involve in the regulation pathways of carbon metabolism in response to water environment change will be cloned. According to their functions, the molecular regulation mechanism will be clarified. And then, by comprehensive analysis of these results, the carbon metabolism and carbon distribution mechanism in biofilm cultivation is expected to be revealed. The accomplishment of this research would generate new knowledge for better understanding the mechanism and regulation of carbon fixation, metabolism and target compound storage in S. dimorphus cells, providing research foundation for the regulation strategy based on the biofilm cultivation for target compound enrichment in microalgae.

生物量产率低是微藻资源规模化利用的瓶颈问题，微藻生物膜培养技术与传统液体悬浮培养方法相比具有显著增加生物量产率的优势。藻体与大水体分离使得藻细胞所处光环境和CO2传递环境发生变化是生物膜培养的主要特点，前期研究发现这些变化极大地提高了微藻生物量和油脂等目标产物积累，我们推测碳代谢与分配行为在其中发挥着重要作用，但目前其机理尚不明确。本项目以栅藻为研究对象，以液体悬浮培养作为对照，首先通过研究栅藻生理生化指标变化，掌握栅藻响应水环境改变的碳代谢流向宏观变化；在此基础上，利用多组学联合分析等技术，解析栅藻为应对水环境改变的碳代谢调控规律；最后，挖掘和筛选可能参与栅藻碳代谢和调控的关键基因并进行功能验证，进而全面揭示生物膜培养时栅藻碳代谢和分配机制。本研究将有助于获得有关微藻固碳、目标产物积累及调控等新知识，为基于生物膜培养的微藻高产高附加值产品调控策略的建立提供依据和研究基础。

微藻生物膜培养模式下由于大水体减少对藻细胞生理及碳代谢造成的影响和作用机制尚不清楚。本项目以栅藻为实验藻种，以液体悬浮培养为对照，研究不同培养模式对栅藻生长、光合活性和产物积累的影响，栅藻碳代谢网络中基因转录水平、关键酶活性和代谢物物含量变化，碳代谢途径关键酶基因异源表达的生物学功能，以期解析栅藻为应对水环境改变的碳代谢和分配机制。研究发现与液体悬浮培养相比，生物膜培养的栅藻生物量、油脂积累和群体光合效率提高。不同培养模式下栅藻的转录组和关键酶酶活差异分析显示，生物膜培养显著影响栅藻光合作用、碳代谢途径以及脂肪酸合成等途径。生物膜培养时，碳代谢关键酶基因PEPC、ME、PFK和脂肪酸合成中的FASN、ACCase等基因显著上调表达，且碳代谢关键酶Rubisco、PK和G6PDH活性也有所提高，说明在生物膜培养时，藻细胞碳同化和碳代谢效率增强并且促进脂肪酸的合成。不同培养模式下代谢组分析显示，生物膜培养显著影响栅藻的代谢物积累。生物膜培养时，上调较显著的差异代谢物多为脂类，而下调较显著的差异代谢物多为有机酸类物质。且谷胱甘肽代谢和鞘脂类等代谢途径上调趋势较显著，可能参与了微藻生长、油脂积累和抵御胁迫的生理过程。在莱茵衣藻中异源表达栅藻碳固定关键酶PFK基因，转基因衣藻的生物量有显著提升。将栅藻PFK和ME基因分别在烟草叶片中瞬时表达，转基因烟草叶片的PFK和ME酶活性比野生型以及转空载有显著提高。以上研究说明与悬浮培养相比，生物膜培养时微藻表现出整体较高的光合效率与生物量和油脂产率，其原因是微藻碳捕获和碳固定能力提高，除了为藻细胞生长提供碳骨架，还会通过碳代谢引导碳流更多流向脂类等高能代谢产物合成的方向进行碳分配。研究结果将有助于深入了解微藻应对水环境改变的生理机制，为微藻产物代谢调控策略和提高藻类与高等植物固碳能力的定向育种应用提供借鉴和理论依据。

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