基于代谢网络模型系统解析浓香型白酒窖泥梭菌生理代谢特征

The representative aroma compounds of strong-flavor baijiu (SFB) are predominantly ethyl hexanoate. Hexanoate, as the precursor of ethyl hexanoate, was mainly biosynthesized by Clostridium strains from pit mud of SFB. A comprehensive analysis of physiological characteristics of Clostridium strains from pit mud of SFB is the key to the understanding of the formation of flavor compounds in strong-flavor baijiu. In this study, 4 Clostridium strains isolated from the pit mud of SFB will be studied by genome sequencing and functional annotation firstly, then genome scale metabolic model (GSMM) of the 4 Clostridium strains will be constructed on the basis of genome annotations, bioinformatics database and experimental data. These 4 GSMMs together with the constraint-based methods, fermentation experiments, and metabolites analysis, will be used as the platform for comprehensive understanding of the physiological and metabolic characteristics. Furthermore, a systematic comparison of GSMMs of Clostridium strains selected from pit mud and other environment will be performed to investigate the evolutionary metabolic characteristics of Clostridium stains after the domestication of pit mud. Results presented in this study will be helpful for the understanding of the flavor compounds producing metabolism of Clostridium strains in pit mud, and providing theoretical support for the development and application of Clostridium strains in SFB production.

浓香型白酒主体呈香物质为己酸乙酯，其前体物质己酸主要由窖泥中梭菌生产。全局解析窖泥梭菌生理代谢特性是理解白酒风味物质形成机制的关键。本项目以筛选自浓香型白酒窖泥的4株梭菌为研究对象，通过基因组测序与功能注释，结合生物信息数据库和实验数据，构建窖泥梭菌基因组规模代谢网络模型（genome scale metabolic model, GSMM），并以此为平台，结合基于约束的算法分析、生理实验和代谢产物分析，全局解析窖泥梭菌生理功能和碳代谢机制。另外，通过系统比较窖泥梭菌和非窖泥梭菌GSMM，揭示窖泥梭菌驯化后的进化特性。研究结果将有助于深入理解窖泥中梭菌的代谢产香机制，并为窖泥梭菌在浓香型白酒应用中提供一定的理论支持。

梭菌是浓香型白酒微生态环境中重要的微生物，其能够利用多种碳源物质生产浓香型白酒风味物质前体如乙酸、丁酸、己酸等。为了全局解析白酒梭菌的生理代谢特性，本项目以前期筛选的四种梭菌C. tyrobutyricum、C. beijerinckii、C. butyricum、C. guangxiense 为研究对象，通过构建全基因组规模代谢网络模型（genome scale metabolic model, GSMM）与泛基因组分析研究了这四种梭菌的生理代谢与进化特征。主要研究内容包括：四株梭菌的全基因组测序、功能基因组注释以及泛基因组分析。通过泛基因组分析发现白酒梭菌C. beijerinckii和C. butyricum相对于其他生态位的同种梭菌具有298和181个特异基因家族，可能属于窖泥梭菌的特有进化特征，但大多数功能未知。同时C. tyrobutyricum、C. beijerinckii、C. butyricum均具有大量的可移动遗传元件。基于基因组功能注释数据、碳源利用实验与代谢组学数据构建了4种梭菌的高质量GSMMs，并解析了其碳源利用和代谢产物合成的途径。分析四个梭菌GSMMs发现其适宜白酒生态环境的代谢特征包括：多种碳源（多糖、二糖、单糖、甘油等）的转运和同化能力；能够合成多种小分子短链脂肪酸如乙酸、丁酸、己酸等；均能够合成氢气，用于维持窖泥系统的微生物群落稳定；能够转运和代谢窖泥中的主要氮源铵离子；C. beijerinckii和C. butyricum能够代谢腐胺，可能会降低白酒中生物胺的含量。白酒梭菌GSMM的构建能够为白酒梭菌生理调控、组学数据解析、代谢工程改造以及种间相互作用机制解析提供理论基础和计算平台。同时，也能用于指导开发白酒梭菌作为资源微生物生产短链脂肪酸、氢气以及人工窖泥制备等方面。