从2,3-丁二醇代谢角度构建工程微生物群体及其生态学机制研究

2,3-Butanediol (2,3-BD) is a platform chemical of great significant which can be applied in many fields of national economy. In order to get over the pathogenic defects of the strains and enhance the lower productivity of safety 2,3-BD production in monoculture fermentation, ecological principle will be utilized to solve the problem found in metabolic engineering. In this study, Saccharomyces cerevisiae genetic engineering strain will be obtained by knocking out branch metabolic pathways and over-expressing relevant genes of 2,3-BD metabolic pathway. On the basis of the engineering microbial communities (EMC) constructed by co-culturing the engineering strain and acetogens, the effects on 2,3-BD production by acetic acid and the regulation mechanism of 2,3-BD metabolism will be investigated by taking advantage of proteomics, metabonomics and transcriptomics; the relationship between 2,3-BD metabolism and Quorum Sensing(QS) within the S. cerevisiae population will be determined. Furthermore, stable factors of the EMC and QS within the populations will be illuminated and communication mechanism of signaling molecules between populations will be decoded from synthetic ecology point of view. All of which not only provide microbial resources for industrial production of 2,3-BD, but also provide breakthrough for QS within populations and between populations of eucaryotic microbes, making the QS theory more perfect.

2,3-丁二醇（2,3-BD）是一种重要的平台化合物，广泛应用在国民经济的多个领域。为解决目前单一微生物发酵及菌株致病性给2,3-BD生产带来的影响并进一步提高2,3-BD产量，本研究将从生态学角度出发，利用生态学原理解决代谢工程问题。通过敲除支路代谢和过表达2,3-BD代谢路径相关基因，获得酿酒酵母基因工程菌株。继而在构建以该工程菌株和产乙酸细菌组合的工程微生物群体（EMC）基础上，利用蛋白质组学、代谢组学和转录组学技术，探讨乙酸对2,3-BD产生的影响，阐明2,3-BD代谢的调控机理；明确酿酒酵母种群内部2,3-BD代谢与群体效应的关系；从合成生态学角度，揭示EMC保持稳定的因素以及群体内部群体效应现象，解码种群间信号分子交流的方式及机制。这不仅为2,3-BD的工业化生产提供了菌种资源，同时也为真核微生物种群内及种群间群体效应现象提供新的发现，充实并完善群体效应理论。

2,3-丁二醇及其衍生物是具有极高利用价值的化工原料。本研究分别以酿酒酵母与肺炎克雷伯氏菌作为出发菌株，通过敲除代谢支路相关基因和过表达2,3-丁二醇代谢路径相关基因的方式提高其产量；同时探讨乙酸对2,3-BD产生的影响。结果表明：重组菌株W141-01（异源表达alsD、alsS和bdh1）及pdc1、pdc5基因双敲除菌株H5-02均可以提高2,3-BD的产量，说明改变代谢路径可以有效的获得目标菌株；在乙酸作用条件下，其可上调2,3-BD代谢途径关键酶基因ilv2和bdh1的表达，最终可以提高2,3-BD产量，并且在S. cerevisiae与A. pasteurianus共培养时，A. pasteurianus的代谢产物乙酸同外源添加乙酸起到了相同的信号分子作用，这说明乙酸可以作为信号分子促进2,3-BD的产生。同时在K. pneumoniae体内也发现和酿酒酵母类似的情况，也就是说敲除HD79菌株的ldh、 ack和aldA基因以及过表达budA、budB和budC基因，均不同程度提高2,3-BD产量（最高可达33.357 g/L）。HD79和HD79-02转录组学分析发现4628差异表达基因中404个显著上调，162个显著下调。过表达budR基因可以使BudA和BudB受到调控从而使其活性上升，使得2,3-BD产量提高，说明乙酸作为信号分子增强budR基因的转录。本研究不仅为2,3-BD的工业化生产提供了菌种资源，为深度理解2,3-BD代谢方式和加强其代谢调控提供理论和实践模型，同时也为真核微生物种群内及种群间群体效应现象提供新的发现。