Studies On Biosynthesis And Application Of Biliproteins Of Cyanobacteria

Cyanobacteria is the photosynthesis microorganism. Phycobilisomes are main light-harvesting complexes. Phycobiliproteins are the function composition of the complexes in the algae photosynthesis. Biosynthesis research of phycobiliprotein is of great significance, contributing to the understanding of the structural basis and the biochemical mechanism of cyanobacteria photosynthesis, which can complete the assembly of the phycobilisomes, and also providing the necessary conditions for biosynthesis in vitro of these fluorescent biliproteins. By heterologous recombination in vivo in E. coli, the thesis confirmed that CpeY/Z or CpeY can catalyze covalent attachment of PEB to CpeA of PEIs from Synechococcus sp. Strain WH8102, and PEB-CpeA has a higher yield under catalysis of CpeY/Z, but neither CpeY/Z nor CpeY catalyze covalent attachment of PEB or PUB to MpeA of PEIIs. Further experiments found that the lyases MpeU, MpeY and so on didn’t catalyze the attachment of either PEB or PUB to MpeA.Although biosynthesis in vitro of phycoerythrin-bound PUB has not yet been clarified, biosynthesis of attaching PUB to phycocyanin in Synechococcus sp. Strain WH8102made a breakthrough. The thesis found that PecE/F can catalyzed the binding PEB to PecA and then isomerize it into PUB and generated biliprotein PUB-PecA. This is the first time in vitro synthesis of PUB that is abundant in marine algae. Moreover, the thesis found that RpcG from Synechococcus sp. strain WH8102catalyzed the attachment and isomerization of PEB or PCB to RpcA and produced biliprotein PUB-RpcA or PVB-RpcA. PUB-RpcA had the same spectral characteristics with native a-R-PC from Synechococcus sp. strain WH8102. Similarly to a-PEC, PVB-RpcA is of photochromic effect. The same as PecE/F, RpcG also can catalyze the attachment and isomerization of PCB or PEB to PecA. Taking part in the synthesis process of PEB-CpcB, PEB-PecB and PEB-CpeB, the lyase-isomerase RpcG cannot exhibit isomerase function, which indicated that isomerization needs coordination between lyases and the corresponding apoprotein.Biliproteins like PVB-RpcA that do not exist in cyanobacteria are able to be synthesized in vitro by the crisscross of different bilin substrates, lyases and apoproteins. They are of great value in the application. Biliproteins generated in this way generally have a higher fluorescence quantum yield. They are excellent fluorescent labeled materials. However, biliproteins synthesis in vivo requires at least four coding genes for co-expression. In order to simplify biosynthetic pathway of biliproteins, encoding gene of phycobilin synthases (HO1, and PcyA or PebS) were fused with encoding gene of lyase (RpcG, CpcS or CpcT), and then coexpressed together with encoding gene of apoprotein (PecA or CpcB) in E. coli. Spectral characteristics of produced biliprotein PVB-PecA, PUB-PecA, PCB-CpcB(C-84), PCB-CpcB(C-155), PEB-CpcB(C-84) and PEB-CpcB(C-155) are comparable to those of before fusing. Observed by fluorescence microscopy, a variety of colored and bright fluorescent cells were seen.The most advantage of phycobiliproteins is of high fluorescence quantum yield, but requiring both phycobilin synthases and lyase is a very obvious flaw. Recently, the research of cyanobacteriochromes has been made some progress. GAF domains from cyanobacteriochromes autocatalytically bind kinds of bilins, such as PΦB, BV, PCB and PVB. They have a good advantage over phycobiliproteins. The thesis showed that coding genes of GAF3domain obtained from slr1393of Synechocystis sp. PCC6803and phycobilin synthase encoding genes, which had been constructed into expression vectors, transferred into E. coli for co-expression, and then produced biliprotein PCB-GAF3, PΦB-GAF3. Spectral analysis indicated that they are red, and even far red fluorescent protein of maximum fluorescence emission at670nm and690nm, and have a unique photochromism.As we know, GFP as a classic fluorescence probe does not require other auxiliary proteins for co-expression, but synthesis of biliproteins based on GAF domain still involve bilin synthases. The problem can be solved by the end-to-end gradual fusion gene method. The two genes(ho1and pcyA) were fused at first and introduced the ho1.pcyA construct, and then the gene gaf3coding for the apoprotein was fused to hol:pcyA at the5’-end. Experiments indicated that the expression plasmid containing the fusion gene gaf3:hol:pcyA can produce biliprotein PCB-GAF3:HO1:PcyA, which has the same spectral characteristics with PCB-GAF3. It also exhibited reversible photochromism. The results were demonstrated by the way of spectroscopy, fluorescence microscopy and PVA film. Instead of gaf3, when the gene apcA coding for phycobiliprotein was fused to hol.pcyA at the5’-end, the construct apcA.hol:pcyA also can expressed fluorescent biliprotein PCB-ApcA:HO1:PcyA, which indicated that the fusion gene method is of universal significance. Fluorescent biliprotein with excellent spectral properties is a good complement of GFP family. Especially, their red fluorescence and photoswitchable effect will facilitate depth imaging, super-resolution microscopy, and even three-dimensional data-storage.