基于“动态”蛋白质组技术对藻蓝色素蛋白生物合成及组装机制的研究

Phycocyanin, existed in cyanobacteria cells, is a kind of widely used food colorants. It is mainly produced through microbial synthesis, but the biosynthesis and protein assembly mechanism of phycocyanin is still unknown, the production of phycocyanin still have low transformation rate and poor stability problems. In this study, we intends to adopt a novel dynamic proteomics method, which could eliminate the interference of protein accumulation, accurately capture the newly synthesized proteins and establish the protein "synthesis rate" expression map through 35S pulse labeling of samples. The key enzymes involved in phycocyanin biosynthesis and assembly process will be screened and identified by comparing cyanobacteria PCC6803 and its induced strain (phycocyanin high-yield and low-yield mutant strains) based on differential synthesized proteins. The genes encoding key enzymes will be cloned and over-expressed, its function in phycocyanin biosynthesis and assembly will be detected through Blue-Native, spectrometry and pigment stability experiments. Then the dynamic synthesis rules of key enzymes will be analyzed and the protein regulation network will be established using Metacore software, which will reveal the molecular mechanism of phycocyanin biosynthesis and assembly. This program will provide fundamental theoretical basis and technical support for efficient synthesis of high stable phycocyanin by microbial method, which has important research value and application prospect.

藻蓝来源于蓝藻等细胞，是国标GB2760-2014规定允许使用的天然食品着色剂，目前主要通过微生物合成法获得，但其生物转化途径及色素蛋白组装机理尚不完全明确，藻蓝合成存在转化率较低、稳定性差等问题。本项目拟采用一种新型动态蛋白质组技术，该技术利用35S对样本进行短至30min的脉冲标记，能够排除蛋白累积的干扰，精准捕获标记时间内新合成的蛋白质，建立蛋白“合成速率”表达谱；通过比对蓝藻PCC6803与其高产、低产藻蓝突变株的蛋白合成差异，筛查藻蓝生物合成组装相关的关键酶；克隆其编码基因并构建超表达工程菌，通过Blue-Native、光谱法以及稳定性检测等技术明确关键酶在藻蓝合成组装中的功能；采用Metacore软件分析关键酶的动态合成规律，构建蛋白调控网络，进而揭示藻蓝生物合成组装的分子机制。研究成果将为微生物法高效合成高稳定性的藻蓝色素提供理论基础和技术支持，具有重要的研究价值和应用前景。

藻蓝色素是一类天然功能性食用色素，具有多种生物活性。本项目主要从藻蓝色素的生物合成和下游功能调控机制两部分进行了探究。对于生物合成，尝试采用诱变育种对螺旋藻进行诱变，但由于难度较大，后期将工作的重心放在了藻蓝色素下游的功能研究中，主要在四种NSCLC细胞中研究了藻蓝色素的抗肿瘤活性，对细胞进行了增殖、凋亡、迁移等方面的检测；同时采用高通量转录组技术发现TIRAP和RIPK1两个关键的蛋白在其中可能参与重要的调控作用；进一步采用miRNA-seq技术对藻蓝色素调控过程中的miRNA进行了筛选，并结合前期的两个关键靶点，最终挖掘了藻蓝色素-miRNA-关键靶点-细胞功能的调控机制，为藻蓝色素功能开发和利用奠定了理论基础。