基于转录组学的嗜热厌氧杆菌共利用戊糖己糖产氢分子机制研究

Overcome the microbial metabolic barrier for o-fermentation of pentose and hexose is essential to achieve efficient utilization of lignocellulosic biomass. Understanding the molecular mechanism of pentose and hexose co-metabolism is upmost important to reveal the nature of hydrogen production by pentose and hexose co-fermentation, and achieving large-scale hydrogen production. In this study, autonomously screened mutant Thermoanaerobacterium thermosaccharolyticum GZ-25 that can utilize pentose and hexose simultaneously to produce hydrogen will be taken as the object. From the anti-metabolite engineering point of view, whole genome sequencing of the mutant strain will be carried out to locate differentially expressed genes related with the pentose and hexose co-fermentation and hydrogen production. Meanwhile, transcriptomic sequencing will be used to analyze the difference in expression levels of related genes with different carbon sources and different growth stages, the molecular mechanism behind hydrogen production by co-fermentation of pentose and hexose of the strain will be unveiled. On this basis, engineered potential strains with high utilization rate of pentose and hexose and high hydrogen production rate will be constructed through the gene over-expression and knockout. Based on the innovative ideas and advanced methods that will be performed in this project, it is quite possible to reveal a new theory on pentose and hexose co-fermentation for hydrogen production and to provide important theoretical and technical basis to realize maximum biological hydrogen production capacity.

戊糖己糖共利用产氢是突破微生物代谢障碍实现木质纤维素生物质高效利用的关键环节。认识和发现该过程中微生物共代谢戊糖己糖规律与分子机制，是从源头上揭示戊糖己糖共利用产氢本质、实现规模化产氢的突破口。本课题以前期自主筛选得到的嗜热厌氧杆菌突变株GZ-25为研究对象，解析其共利用戊糖己糖产氢代谢规律。从反向代谢工程角度，对突变株的基因组重测序探明与戊糖己糖共代谢产氢相关的差异基因，然后通过不同碳源及不同生长阶段的样品转录组分析，获得相关基因表达水平及调控机制基础数据，解析嗜热厌氧杆菌突变株具有戊糖己糖共利用产氢特性的分子机制。在此基础上，通过基因超量表达及敲除技术，分析和验证关键功能基因对戊糖己糖共利用及发酵产氢的影响，为构建戊糖己糖利用能力强，产氢量高的工程潜力菌株奠定理论基础。本课题研究思路和方法具有创新性，为实现木质纤维素中戊糖己糖高效利用，趋近最大生物产氢能力提供重要的理论基础。

木质纤维素生物制氢的关键技术挑战之一就是提高微生物对戊糖生物转化能力的同时实现戊糖和己糖的同步高效转化，这也是目前国内外利用木质纤维素类生物质制备生物能源研究的焦点。前期研究发现嗜热厌氧杆菌可以利用戊糖产生氢气，同时该类细菌同步利用戊糖己糖产氢的独特优势可有效克服现有木质纤维素水解液中戊糖难以被有效利用等缺点。据此，本项目开展系列研究，并取得以下成果：（1）完善嗜热厌氧杆菌共利用戊糖己糖产氢底物利用、菌体生长、产物生成的相关参数分析，明确不同条件下戊糖己糖共利用产氢效能及底物代谢规律；（2）通过基因组及转录组测序分析，解析嗜热厌氧杆菌戊糖己糖共利用的代谢调控机制，确定增强混合糖中戊糖代谢产氢的关键靶控因子；（3）提出了基于废弃生物质循环利用的生物产氢效能强化策略，构建以“生物质发酵产氢-产氢残余物制备生物炭-生物炭强化产氢”的闭合循环强化废弃生物质发酵产氢体系，通过生物产氢过程定向代谢调控来大幅提高生物产氢能力。三年来，在基金的支持下，申请国家专利4项（已授权3项），已发表论文12篇（其中SCI收录论文11篇，CSCD核心源刊论文1篇）。本项目研究成果已在国内外同行中产生重要影响，研究成果可为戊糖己糖共利用产氢机制提供理论基础，对推动木质纤维素生物制氢的工程应用具有指导价值。