利用多功能裂合酶CpcE/F合成非天然重组藻胆蛋白

藻胆蛋白裂合酶是催化藻胆色素与脱辅基藻胆蛋白亚基共价连接的裂合酶,在藻胆蛋白的生物合成途径中发挥着重要作用。本实验以藻蓝蛋白裂合酶Cpc E/F为研究对象,采用基因工程组合表达技术,构建了pCDFDuet-apcA-cpcE/F-hox1-pebS、pCDFDuet-pecA-cpcE/F-hox1-pebS等重组质粒,并导入大肠杆菌中,在IPTG的诱导下,成功表达得到两种非天然藻胆蛋白ApcA-PEB和PecA-PEB,这表明Cpc E/F不仅可以催化藻蓝胆素和脱辅基藻蓝蛋白的结合,还可以催化藻红胆素(PEB)与别藻蓝蛋白α-亚基(ApcA)和藻红蓝蛋白α-亚基(PecA)的结合,因此是一种多功能的裂合酶。根据重组产物的光谱特征峰分析发现,ApcA-PEB和PecA-PEB作为两种非天然存在的重组藻胆蛋白,具有与对应天然藻胆蛋白有着相近的特征吸收光谱和荧光发射峰,但同时具有明显的波长偏移并伴有色素异化现象,其光谱学性质主要由其所携带的色素基团决定。另外,荧光寿命衰减分析发现,不同脱辅基蛋白对于重组藻胆蛋白的光谱稳定性具有重要的影响。

Biosynthesis of unnatural phycobiliproteins using universal bilin lyase CpcE/F

Phycobilin lyase enzymes can attach phcobilins to their cognate apoproteins, which play an important role in the biosynthesis of phycobiliproteins. To verify the universal function of lyase cpcE/F, two recombinant plasmids: pCDFDuet-apcA-cpcE/F-hox1-pebS and pCDFDuet-pecA-cpcE/F-hox1-pebS were constructed using genetic engineering methods, and then two unnatural phycobiliproteins, ApcA-PEB and PecA-PEB, were successfully expressed in Escherichia coli, which indicated that cpcE/F could catalyze not only the ligation of PCB with different apoproteins, but also that of PEB and different apoproteins, and thus are universal bilin lyases. The absorption and fluorescence spectrum analysis showed that both unnatural proteins exhibit similar characteristic peaks to those of natural phycobiliproteins with some peaks of isomers and wavelength shifts, and their spectroscopic characteristics were mainly determined by the bilin type carried on them. Furthermore, different apoproteins could also regulate the absorption spectrum and show important influences on the photostability of phycobilins. Our work will provide important information to better understand the biosynthetic mechanism of the phycocybiliproteins and production of unnatural phycobiliproteins with a better fluorescence quality.