Research On Combinational Biosynthesis Of Fluorescent Phycocyanin And Evolution Of Cyanobacterial Phycobilisomes Linker Polypeptides

Cyanobacteria are among the oldest life on earth with the capacity of oxygenic photosynthesis. As a light-harvesting complex, phycobilisomes (PBSs) play a crucial role in the energy absorbing and transferring of photosynthesis in cyanobacteria. PBSs are highly organized complexes, and are composed of various biliproteins and linker polypeptides. A series of researches on PBSs have been done since they have comprehensive application in the fields of food, medicine and biotechnology, which becomes a hotspot in the research of algae. More stable and single-effect PBSs subunits are urgent for wide applications in the above fields.Five genes cpcA(S6),cpcE,cpcF,ho1 and pcyA were cloned from Synechocystis sp. PCC6803 (S6) genomic DNA. A vector pCDF-cpcA (S6)-cpcE-cpcF, ho1-pcyA (V1) was successfully constructed resulting in combinational biosynthesis of a fluorescent holo-α-phycocyanin (S6) in Escherichia coli BL21. In addition, a strategy in co-expression of multigenes by using one expression vector was presented, which is a promising method to produce lager-scale fluorescent phycobiliproteins (PBPs) in biotechnological applications.Based on former research about optically active holo-α-PC (S6), the gene cpcA (Sp) cloned from Spirulina platensis (Sp) genomic DNA, and a new vector pCDF-cpcA (Sp)-cpcE-cpcF, ho1-pcyA (V2) was constructed. The vector could play a role to synthesize PCB, apo-PC (Sp), and lyase CpcE/F (S6) catalyzing the connection of them in E. coli BL21, which finally produced fluorescentα-subunit C-PC of Sp (rHHPC). The recombinant E. coli BL21 was cultured to OD₆₀₀ = 27 in a 5L fermentor. The fermentation technique was improved to reduce the production of inclusion bodies. Inhibition effect on hydroxyl and peroxyl radicals makes rHHPC as a potent antioxidant.In order to biosynthesize fluorescent holo-β-PC (Sp) in E. coli BL21, special chromophore lyases genes CpeS and CpcT were carried out BLASTP in S6 genome, and two high homologous genes Slr2049 and Slr1649 were obtained. Sites 82 and 153 inβ-PC (Sp) were site-directed mutated. Two groups of co-expression vectors were constructed and transformed into E. coli BL21, which were pCDF-cpcB(C153A)- slr2049, ho1-pcyA and pCDF-cpcB(C82I)-slr2049, ho1-pcyA, pCDF-cpcB(C82I)- slr1649, ho1-pcyA and pCDF-cpcB(C153I)-slr1649, ho1-pcyA. The result indicates that Slr2049 and Slr1649 catalyzed the 82β- and 153β-PC (Sp) linking to the PCB, respectively. Fluorescent cpcB(C153A)-PCB and cpcB(C82I)-PCB were achieved. In addition, the spectroscopy characteristic between several recombinant and natural PBPs were analyzed.All recombinant proteins were purified by metal chelating affinity chromatography, and were analyzed by SDS-PAGE, chromoprotein Zn2+ electrophoresis, spectrum detect and so on.Recent availability of the whole genome database of cyanobacteria provides us a global and further view on the complex structural PBSs. A total of 192 putative linker genes including 167 putative PBSs-associated linker genes and 25 Ferredoxin-NADP+ oxidoreductase (FNR) genes were detected through whole genome analysis of all 25 cyanobacterial genomes (20 finished and 5 in draft). A phylogenetic analysis based on protein data demonstrates a possibility of six classes of the linker family in cyanobacteria. Emergence, divergence, and disappearance of cyanobacterial linkers among cyanobacterial species were due to speciation, gene duplication, gene transfer, or gene loss, and acclimation to various environmental selective pressures especially light.This study provides a method for the production of the fluorescent phycocyanin in the research of PBSs construction and biotechnological applications, which would helps to find the mechanism of fluorescent and biological activities in PBPs and approach to construct artificial PBSs. Research on cyanobacterial linker family plays an important role in divergence and evolution of cyanobacterial light-harvesting systems.