窖泥来源厌氧产孢菌群利用葡萄糖高效合成己酸的机理研究

Medium-chain fatty acids are types of fatty acids containing 6-12 carbons. They include caproic, caprylic, capric and lauric acid, of which caproic acid (caproiate) has a large demand. Caproiate can be produced from chemical as well as fermentation. However, chemical synthesis is not an environmental friendly approach, and no mature fermentation technology is available. High efficient caproiate production through low cost feed stocks is essential to enable feasible caproiate fermentation technology. Our previous study identified the core microbiota involved in short- and medium-fatty acid production in the pit mud. Based on this knowledge, we modified the culture conditions for sporulation bacteria. Interesting, high levels of caproiate can be produced from sporulation microbiota enriched from pit mud by using glucose as carbon source. As we know, high levels of caproiate can only be produced by Clostridium kluyveri and Megasphaera elsdenii, and the former one is a sporulation bacteria utilizing ethanol as carbon source and the latter one is a non-sporulation bacteria utilizing glucose as carbon source. Hence, we speculate that the newly enriched microbiota may contain unknown caproiate-producing bacteria and synthetic pathway. This study will focus on the microbial mechanisms of caproiate production from microbiota enriched from pit mud . Microbiome study approaches will be used to elucidate the microbiota structure, community functions and interaction between butyrate-producing and caproiate-producing communities, to reveal the microbial and biochemical mechanisms of the caproiate synthesis via the pit mud sporulation microbiota. Therefore, this study may shed a light on the efficient biosynthesis of caproiate from renewable resource such as glucose-based biomass.

己酸广泛应用于食品、医药等行业，主要通过化学法合成。生物发酵法制备己酸具有更优的环境友好性和食品安全性，但受菌种和成本限制，尚未进行规模化的应用，亟需开发以廉价原料高效合成己酸的绿色使能技术。从我国传统食品酿造环境（窖泥）中富集得到的厌氧产孢菌群可以具有高效利用葡萄糖合成己酸的能力。目前报道的高效己酸合成菌种主要为乙醇营养型的克氏梭菌（产孢型）和葡萄糖营养型的埃氏巨球型菌（非产孢型）。本研究中新发现的己酸合成菌群属于葡萄糖营养产孢型，推测其具有未被鉴定的己酸合成菌及合成酶系。本项目拟以不同来源的窖泥厌氧产孢菌群为研究对象，在明确其己酸发酵动力学基础上，利用微生物组学技术对己酸合成菌群的物种组成结构、短中链脂肪酸合成途径和丁酸菌-己酸菌相互作用关系等进行研究，揭示新型厌氧产孢菌群高效合成己酸的生物学基础及其合成酶系编码基因，为后续新型己酸绿色生物合成技术研发奠定理论基础。

从我国传统食品酿造环境（窖泥）中富集得到的厌氧产孢菌群具有高效利用葡萄糖合成己酸的能力，但何种微生物产生了己酸，如何产生己酸的机制不清。本项目建立起产酸菌群富培养技术和降酸菌群寡培养技术，可实现窖泥厌氧菌群中高丰度物种的可培养化。运用产酸菌群富培养技术，鉴定出白酒窖泥中至少具有四种产己酸菌，其中三种为瘤胃菌科新种，一种为传统认知的己酸菌（克氏梭菌），瘤胃菌科己酸菌能以葡萄糖为碳源进行定向富集。利用微生物组学技术揭示出乙酸和高浓度氮源是影响产己酸菌群结构和表型稳定性的关键因素。从葡萄糖富集菌群中分离得到瘤胃菌科微生物Ruminococcaceae bacterium LBM19010及LBM19011，这些菌株代表的物种为厌氧产孢菌群中的主体产己酸菌，也是白酒酿造窖泥体系中的主体产己酸菌。此外，还分离得到了非主体己酸菌新种，命名为解淀粉己杆菌（Caproicibacterium amylolyticus LBM18003）。分离得到的两种己酸菌为亲缘关系接近的新种，属于瘤胃菌科下的一个未分类新属，被定义为己杆菌属（Caproicibacterium）。己酸菌 LBM19010/LBM19011具有广泛的底物利用谱，除可以利用葡萄糖外，还可以利用淀粉、麦芽糖、乳酸等多种碳源，该菌与其他己酸菌相比，在富集菌群和窖泥中均具有极强的鲁棒性，利用葡萄糖时展现出良好的生长优势，而利用乳酸时呈现出降酸发酵特征。分别基于葡萄糖碳源和乳酸碳源建立起两项产己酸菌群定向富集技术，在白酒酿造中展现出良好的应用价值。综上，本项目解析出了窖泥己酸菌的种类，并建立起快速从窖泥中建立稳定产己酸菌群的富集培养技术，通过纯培养菌株分离，明确了主体己酸菌的物种类型，并揭示了其基本代谢特征。