基于嗜盐古菌phaP/phaR重组表达的PHA颗粒粒径控制研究

The “granule”nature of polyhydroxyalkanoates (PHAs) opens up a broad range of applications in various biomedical applications, from protein purification to vaccine vehicles. However，the particle size of native PHA granules cannot be controlled as easily as the artificial PHA particles, which would greatly hinder its development in biomedical application. In our preliminary study in the haloarchaeon Haloferax mediterranei, the well cooperation between the PhaR and the PhaP was proved to play a central role in the regulation of PHA granule morphology. This project will mainly focus on the modification of the PhaR/PhaP regulatory system. Through an integrative approach of promoter replacement, promoter mutation, and gene site-mutation, the phaP and phaR genes will be engineered to express controllably and differently. Then the particle size of PHA granules would be measured by transmission electron microscopy and laser diffraction. Combine with the quantitative study of the proteins, the impact of the ratio of the expression level of phaP and phaR on the morphology of PHA granules will be assessed. Also, the key regions or key sites of PhaR that account for the size control of PHA granules will be characterized. In light of the evaluation, the regulatory model for the fine control of the particle size will be suggested to produce size-controllable native PHA granules by means of genetic engineering. Taken together, this project will provide a better understanding of the coordination of these modules involved in the PHA granule morphology control, which would not only greatly promote the application of native PHA granules with lower cost, but also suggest constructive new strategies for the produce native PHA granules with designated size in PHA accumulation microbes.

天然聚羟基脂肪酸酯（polyhydroxyalkanoate：PHA）的颗粒特性使其可用于疫苗制备、蛋白纯化等生物医学领域，但其粒径难如人工合成颗粒般可控，极大限制了其应用。基于前期研究，申请人发现嗜盐古菌——地中海富盐菌中PHA颗粒结构蛋白PhaP和调节蛋白PhaR的协同关系是最重要的颗粒形态决定因子。本项目拟以PhaR和PhaP为研究核心，通过启动子置换和突变、功能基因的点突变等手段实现基因可控表达，以透射电镜等技术分析颗粒粒径，结合相关蛋白的定量研究，系统揭示phaP/phaR不同表达强度和基因结构与嗜盐古菌PHA颗粒粒径的关系，最终实现基于基因工程的天然PHA颗粒粒径的精确控制。目前，天然PHA颗粒粒径控制研究鲜有报道，本项目探索性剖析PHA颗粒粒径调控各模块之间的全局协调关系，其结果不但极大促进颗粒应用效果，降低工业生产成本，也为其他PHA产生菌的粒径控制提供建设性的启示。

聚羟基脂肪酸酯（polyhydroxyalkanoate：PHA）颗粒作为一类生物可降解聚酯纳米/微米颗粒，天然PHA颗粒表面含有大量的结合蛋白，可直接通过基因工程设计与颗粒结合蛋白融合表达获得高丰度的目的蛋白，用于制备亚单位疫苗或蛋白纯化等领域。然而由于在微生物产生PHA过程中，天然颗粒粒径不易控制，极大限制了其在生物医学领域的研发与应用。. 本项目立足于天然PHA颗粒应用需求导向，从天然PHA颗粒粒径控制这个核心问题出发，重点开展了基于基因工程的天然PHA颗粒粒径控制研究，并拓展分析了颗粒粒径对天然PHA颗粒的蛋白表达纯化应用的影响，取得了一系列原创性的成果。本项目通过控制嗜盐古菌PHA颗粒主要结合蛋白PhaP和PhaR的不同表达水平和更换背景菌株，实现了嗜盐古菌PHA颗粒粒径大小的梯度控制，收获了一批可以生产平均粒径从约60nm到550nm的六个梯度的PHA颗粒的菌株，并对PhaR/ PhaP在颗粒粒径控制中的作用机制进行了补充；同时发挥天然PHA颗粒的应用优势，建立了嗜盐古菌中基于PHA颗粒的蛋白表达纯化系统；最后，融入了上述颗粒粒径控制策略，先后采用了不同蛋白标签和背景菌株，对该蛋白表达纯化系统进行优化，使蛋白产量提高了4倍，获得了高效便捷的嗜盐古菌蛋白表达和纯化系统。. 这些科学发现为天然PHA颗粒应用领域提供新的认识，为优化疫苗制备等其他天然PHA颗粒相关研究提供了实验方法学基础和实验材料，有利于拓展嗜盐古菌天然PHA颗粒的应用，并为其他PHA积累微生物的天然PHA颗粒的改造及应用提供理论指导。