微生物乳酸代谢调控蛋白LldR的结构及机制研究

Lactate metabolism of pathogenic bacteria plays an important role in their resistance to the body's immune system and infection with the human. The lactate metabolism mechanism in different Pseudomonas strains was studied in our previous works. The lactate metabolism regulator LldR in the opportunistic pathogen Pseudomonas aeruginosa XMG was cloned, exogenous expressed and its biological function was successfully identified. LldR belongs to the transcriptional regulatory protein FadR family. The proteins in this family regulate a series of important physiological and metabolic processes including pyruvic acid, lactic acid, glycolic acid and fatty acid metabolism of bacteria. Preliminary studies show that there might have significant differences in the structure and regulatory mechanism between the LldR and the well studied FadR. In this study, the crystal structure of LldR in P. aeruginosa XMG would be analyzed by protein crystallization and X-ray diffraction techniques. Using site-directed mutagenesis, electrophoretic mobility shift assay and DNA hydrolase I footprinting analysis, the key amino acids sites in the binding of LldR to DNA would be determined. Using isothermal titration calorimetry, surface plasmon resonance, and circular dichroism, the regulation mechanisms of LldR in P. aeruginosa XMG would be clarified. This study would deepen the researchers' knowledge of the regulation mechanisms in FadR family proteins and reveal the mechanisms of the FadR family regulated important microbial metabolic processes.

致病菌乳酸代谢在其抵抗人体免疫系统攻击、感染人体过程中起重要作用。课题组前期从事假单胞菌乳酸代谢机制研究，成功实现了条件致病菌铜绿假单胞菌XMG中乳酸代谢调控蛋白LldR的基因克隆，外源表达与生物学功能鉴定。LldR属于转录调控蛋白中的FadR家族，该家族蛋白调控微生物丙酮酸、乳酸、乙醇酸以及脂肪酸代谢等一系列重要生理代谢过程。前期研究表明，LldR与已报道的FadR在结构与调控机制方面可能存在明显区别。本申请拟以XMG中的LldR为研究对象，基于蛋白质结晶与X-射线衍射技术，解析LldR的晶体结构；采取定点突变、凝胶阻滞、DNA水解酶I足迹分析等技术确定LldR与DNA结合的关键氨基酸位点；利用等温滴定量热、表面等离子共振、圆二色谱等技术，阐明XMG中LldR调控该菌株乳酸代谢的具体机制；为深入揭示该家族蛋白调控的重要微生物代谢过程奠定理论基础。

乳酸分解代谢在致病菌抵抗人体免疫系统攻击、感染人体过程中发挥重要作用。项目组前期确定Pseudomonas aeruginosa XMG中调控蛋白LldR调控该菌株中乳酸分解代谢。LldR属于FadR家族转录调控蛋白，该家族蛋白调控微生物中丙酮酸、乳酸、乙醇酸以及脂肪酸代谢等一系列重要生理过程。本项目外源表达、分离纯化了P. aeruginosa XMG中LldR，通过对LldR的结晶条件筛选、优化、X射线衍射分析，获得了分辨率为2.55 Å的LldR衍射数据，后续通过对LldR的序列相似性网络分析及结构模拟，获得了P. aeruginosa XMG中LldR的结构模型。结合凝胶阻滞、DNA水解酶I足迹分析、等温滴定量热等技术，推测LldR调控乳酸分解代谢机制如下：LldR包含两个结构域，无乳酸存在时，N端结构域通过关键氨基酸位点R38、R48、R52及G69，与操纵基因中反向重复序列5’-AATTGGTCTTACCAATT-3’ 按照2：1比例结合，阻止下游乳酸分解代谢关键酶编码基因的转录；D-乳酸或L-乳酸存在时，其与LldR的C端结构域发生相互作用，阻止N端结构域与操纵基因的结合，从而促进乳酸分解代谢关键酶编码基因的转录。上述研究深化了LldR所属FadR家族蛋白调控机制的认识，为揭示FadR家族蛋白调控的其他重要微生物代谢过程奠定了基础。乳酸分解代谢关键酶NAD非依赖型乳酸脱氢酶在生物催化2-酮基羧酸方面具有较大潜力，考虑到假单胞菌中乳酸分解代谢关键酶均受到LldR的负控诱导调控，本项目对Pseudomonas putida KT2440中LldR进行分子改造，获得的突变菌株在利用廉价底物甘油为碳源生长时组成型表达NAD非依赖型乳酸脱氢酶，后续利用该突变菌株全细胞为催化剂，成功实现了两种2-酮基羧酸：丙酮酸和2-酮基丁酸的高效生产。