Pseudomonas putida KT2440中2,3-丁二醇分解代谢机制研究

Microbial 2,3-butanediol (BD) metabolism have a significant impact on physiological processes of its symbiotic animals, plants, microorganisms. However, different from the BD synthesis mechanism, there are still several unclear key steps in microbial catabolism of BD, which limits the investigation of BD metabolism-related physiological processes. Pseudomonas putida KT2440 is a model strain of environmental microorganism. It can simultaneously use the three isomers of BD as the sole carbon source. In this project, P. putida KT2440 would be used as the research object for the study of BD catabolism. Roles of the three kinds of 2,3-butanediol dehydrogenase in production of acetoin from the three isomers of BD would be identified, respectively. Mechanisms of the dehydrogenation and splitting of acetoin by the acetoin dehydrogenase would be clarified. Based on the protein purification and transcriptomics techniques, the acetoin racemase encoding gene and its roles in interconversion of the two kinds of acetoin and BD utilization would be determined. Transcriptome would also be used in the project to determine the downstream metabolic pathways of acetoin lysates and to analyze the gene transcription of other physiological processes related to BD utilization. Based on the result of the project, the theoretical basis for the deeply revealing of the microbial BD metabolism mechanism and related physiological process would be established.

微生物的2,3-丁二醇（BD）代谢对与其共生的动物、植物、微生物的生理代谢过程有着重要的影响。相对于BD的合成机制，微生物的BD分解代谢机制仍有多个关键步骤未能阐明，限制了BD代谢相关生理过程研究的开展。环境微生物模式菌株Pseudomonas putida KT2440可同时利用BD的3种异构体为唯一碳源生长。本项目拟以P. putida KT2440为研究对象，分析该菌株中3种2,3-丁二醇脱氢酶分别在3种BD异构体脱氢生成乙偶姻过程中的作用；揭示乙偶姻脱氢酶催化乙偶姻脱氢裂解的具体机制；蛋白分离纯化与转录组学技术确定乙偶姻消旋酶编码基因，阐明乙偶姻消旋酶参与2种乙偶姻互变及BD代谢的内在机理；转录组学与关键基因敲除及回补相结合，解析乙偶姻裂解产物下游代谢途径，分析BD利用对其它生理过程相关基因转录的影响。该项目可为深入揭示微生物BD分解代谢机制及相关生理过程奠定理论基础。

2,3-丁二醇是一种微生物初级代谢产物，广泛存在于不同生境。相对于2,3-丁二醇的合成机制，微生物的2,3-丁二醇的分解代谢机制仍有多个关键步骤未能阐明，限制了2,3-丁二醇代谢相关生理过程研究的开展。本项目发现环境微生物模式菌株Pseudomonas putida KT2440和条件致病菌模式菌株Pseudomonas aeruginosa PAO1均能以2,3-丁二醇的三种手性异构体(2R,3R)-2,3-丁二醇、(2S,3S)-2,3-丁二醇、meso-2,3-丁二醇为唯一碳源生长，后续通过生物化学、分子生物学手段揭示了上述菌株中2,3-丁二醇的三种手性异构体经不同2,3-丁二醇脱氢酶催化生成(3S)-乙偶姻或(3R)-乙偶姻、(3S)-乙偶姻或(3R)-乙偶姻经乙偶姻脱氢酶复合体脱氢裂解进入中心代谢的具体机制。研究过程中发现P. putida KT2440和P. aeruginosa PAO1中2,3-丁二醇代谢关键基因均位于同一操纵子中，受转录调控蛋白AcoR的激活调控，P. aeruginosa PAO1中AcoR的效应物为乙醛，而P. putida KT2440中AcoR的效应物为乙偶姻。已有研究报道2,3-丁二醇可以增强P. aeruginosa的致病性并影响肺部微生物组结构，本项目另外对2,3-丁二醇影响P. aeruginosa毒力的机制开展了部分研究，确定2,3-丁二醇不能作为信号分子诱导P. aeruginosa致病相关基因的表达，但2,3-丁二醇代谢可以增加P. aeruginosa生物量并提高群体感应信号分子浓度，最终促进毒力因子的产生。本项目揭示了2种模式微生物中2,3-丁二醇经脱氢、裂解最终进入中心代谢的完整途径与表达调控机制，为深入认识2,3-丁二醇代谢相关生理过程、指导2,3-丁二醇代谢导致感染的防治奠定了理论基础。