异戊二烯共培养清洁发酵新体系的建立和蓝细菌-大肠杆菌(或枯草芽孢杆菌）相互作用关系的研究

With the depletion of the nonrenewable petrochemical resources, bio-isoprene will be a promising alternative towards the petroleum-isoprene, while the environmental pollution problem of the fermentation industry has imposed restrictions on the industrialization of the bio-isoprene production. Although co-culture system can play important roles in developing the technology of clean production, the efficient theoretical guidance for the establishment of clean co-culture system is not available yet. For in depth understanding the establishment strategy of clean co-culture system, and the microbial interactions in the co-culture system, we put forward to establish binary co-culture system for clean isoprene production in the present project and then adopt the methods of microbiome to analyze microbial interactions and the synergy mechanism in the co-culture system. Firstly, the cyanobacterium– Escherichia coli coculture system for clean isoprene production will be established. Bacillus subtilis will be substituted for E. coli in the alternative plan. Secondly, the cyanobacterium– E. coli (or B. subtilis) interactions in the co-culture systems will be investigated to find the rules of material exchange and energy transfer, to obtain the key signal molecules and to analyze the mechanism of gene expression regulation through transcriptome analysis, proteome analysis and metabolome analysis. The mechanisms of cyanobacterium– E. coli (or B. subtilis) interactions and clean isoprene production in the co-culture system will be put forward and verified, which could provide the scientific basis for the establishment of clean co-culture systems and rational design of microbial community. Besides, this study is also going to be an important case in point of microbiome.

随着化石资源日益枯竭，生物法制备异戊二烯将成为替代石化来源异戊二烯的重要方法。然而发酵行业的污染问题却成为了制约异戊二烯生物技术产业化的重要因素。虽然共培养体系对于实现清洁发酵能够起到关键作用，但是共培养清洁发酵研究还处在起步阶段，缺乏有效的理论指导。为了深入研究共培养清洁发酵体系，本项目提出建立异戊二烯二元共培养体系，并进行微生物组学研究分析。首先，拟采用模式生物蓝细菌和大肠杆菌，构建异戊二烯共培养清洁发酵体系。以枯草芽孢杆菌替代大肠杆菌为备选方案。然后，通过转录组学、蛋白质组学、代谢物组学分析，对蓝细菌和大肠杆菌（或枯草芽孢杆菌）的相互作用关系进行研究，获得物质交换和能量传递的规律，寻找关键的信号分子，解析基因表达调控机制，提出并验证微生物共生和异戊二烯大量合成的机制。本研究将为清洁无污染共培养方法的建立提供翔实的科学依据，为微生物群落的理性设计提供例证，并将推动微生物组学的发展。

目前大多数微生物发酵采用分批或补料分批的方式，存在工艺复杂和耗水量大等问题。微生物的连续生产有助于发酵工业的绿色可持续发展。光自养型和异养型共培养体系对建立生物基化学品的连续发酵模式具有重要意义，然而关于藻菌关系的基础研究还处在起步阶段，模式藻菌共培养体系的研究不足。本研究建立了聚球藻-大肠杆菌共培养体系，并实现长时间稳定的异戊二烯合成。与纯培养相比，共培养的发酵时间从100h延长到400h，异戊二烯产量提高了8倍。为了深入解析这个新系统，我们采用了多组学的方法进行了分析研究。S. elongatus PCC 7942光合作用产生的氧化压力触发了大肠杆菌BL21(DE3)一系列的转录、蛋白和代谢层面的反应，这些变化在不同的时空尺度上相互关联。应对氧化压力的代谢通路可能有助于实现长期稳定发酵。虽然该共培养体系的整体效率仍然较低，但是根据组学分析发现的基本规律，未来可以进一步提高和改善藻菌共培养体系的性能。本研究对揭示非自然共生的光自养和异养微生物之间共同的相互作用具有重要意义，可为微生物群落的理性设计提供科学依据。