甲基营养菌解除PpsR负调控叶绿素合成的机制研究

Plant-colonizing methylotrophic bacteria in the phyllosphere under low methanol condition activate the biosynthetic genes of bacteriochlorophyll to utilize sunlight for accessory energy conservation. However, the potential mechanism is still unclear so far. In our previous works, the transcriptional level of photopigment suppressor protein R (PpsR) responsible for bacteriochlorophyll biosynthesis was found to be regulated by a global stress regulator (PhyR) through knocking out ppsR gene and bioinformatic analysis. Moreover ppsR gene was identified not to be controlled by PhyR directly. These results suggested that this regulation process in methylotrophic bacteria was inconsistent with well-known mechanism of anaerobic photosynthetic bacteria. To this end, we propose to develop a high-throughput screen through CRISPRi for identifying the transcriptional regulators of ppsR by interfering the expression of effector genes of PhyR. Furthermore, molecular interaction mechanism between the identified regulator and ppsR promoter will be demonstrated by using a combined methodology of three dimensional protein structure and promoter structure analysis. Next, the effect of either knocking out or overexpressing the regulator of ppsR on methylotrophic bacteria will be evaluated via analyzing the growth change and bacteriochlorophyll synthesis. Overall, this proposal will provide an insight into understanding how methylotrophic bacteria activate the biosynthesis of bacteriochlorophyll to adapt to stress from poor carbon source, and also will help to explain the potential mechanism of photosynthesis regulation in aerobic bacteria. Ultimately, the regulation process of PpsR demonstrated here will provide a new strategy to produce extra ATP by manipulating photosynthesis in the engineered methylotrophic bacteria.

定植于植物叶片的甲基营养菌为应对贫瘠碳源的生存条件，能够激活细菌叶绿素合成，捕获光能补偿生长所需能量，但是目前叶绿素合成的激活机制尚不清楚。前期申请者通过基因敲除与生物信息学分析发现甲基营养菌叶绿素合成关键负调控因子PpsR的转录水平受胁迫抗性全局调控因子PhyR的影响，但是PhyR不能直接作用于ppsR，这一调控过程不同于厌氧光合作用细菌。基于此，本项目拟利用CRISPRi打靶干扰技术首先从PhyR效应基因中高通量筛选出ppsR的转录调控因子；继而从蛋白质三维结构和启动子结构两个层面解析转录调控因子与ppsR的分子互作机制，并进一步研究敲除与过表达ppsR的转录调控因子对甲基营养菌生长和光合色素合成代谢的影响。本项研究将为阐明甲基营养菌适应逆境启动光合作用的过程提供理论依据，并扩充对好氧型细菌光合作用调控机制的认知，未来也将为代谢工程改造甲基营养菌利用光合代谢产能提供新的思路。

甲基杆菌是定殖于植物叶际的主要植物益生菌群之一，可促进植物生长，是重要的植物益生菌剂，而其在植物叶际较低的定殖能力是限制应用的重要因素。为适应叶际贫瘠环境，甲基杆菌在植物叶际环境下合成细菌叶绿素，捕获光能一补偿其生长繁殖所需能量，而在实验室以充足甲醇为碳源进行生长时检测不到细菌叶绿素的合成，其细菌叶绿素合成调控机制的研究对于提高甲基杆菌在植物叶际的适应性及通过代谢工程改造甲基杆菌利用光合代谢产能具有重要意义。本项目在甲基杆菌模式菌—扭脱甲基杆菌AM1中确定了实验室下细菌叶绿素合成的条件，探究了其细菌叶绿素合成调控机制，并通过解除扭脱甲基杆菌细菌叶绿素合成的负调控提高了其在植物叶际的定殖能力以及高耗能产品3-羟基丙酸的产量。通过碳源种类、光照强度、光照模式、摇床转速等条件改变，确定其在实验室条件下产生细菌叶绿素的最佳条件，发现细菌叶绿素在光暗循环培养条件下可促进扭脱甲基杆菌AM1的生长，提高细胞内ATP水平。针对扭脱甲基杆菌胞内细菌也速率水平较低的问题，通过比较转录组分析预测限制细菌叶绿素合成的调控基因及结构基因，并对其进行敲除或过表达，构建扭脱甲基杆菌AM1工程菌，使其组成型合成细菌叶绿素并提高胞内细菌叶绿素含量水平。该工程菌在植物叶际定殖能力提高50%；以该工程菌作为底盘，在给予光照条件下可提高高耗能产品3-羟基丙酸的产量25%。该项目首次在甲基杆菌利用光合产能提高其植物叶际定殖能力以及利用光合产能提高高耗能产品产量。本项目实施期间发表SCI论文一篇，目前在投高水平SCI论文一篇，申请国家专利一项，培养硕士研究生一名。本项目实施为在甲基杆菌中利用光合产能提供了新的思路，并为提高甲基杆菌在植物叶际定殖能力、开发利用甲基杆菌益生菌剂提供了有力支持。