Study On Modification And Reconstitution Of PBS Structure By Expressing Pebs In Synechocystis Sp.PCC 6803

Phycobilisome(PBS)is the major light-harvesting complex of cyanobacteria and red algae,that plays an irreplaceable role in the primary production of whole biosphere.PBS absorbs green and orange light in the range of about 500-650 nm,and transfers light energy to photosynthetic systems(PSs).Its light-harvesting function comes from a variety of phycobilins with different absorption spectra connected with phycobiliproteins.The diverse chromophores and their broad absorption spectra range make PBS an excellent material for engineering light-harvesting components for the construction of artificial photosynthesis systems.But the super structure and complex assembly of PBS have always been an obstacle.In order to explore the comprehensive engineering of wild type PBS,this study simplified the PBS structure of Synechocystis sp.PCC 6803(Synechocystis).The mutant strains with deletion of core and rod structure were constructed respectively.The difference of PBS structure and their spectral characteristics,as well as the effects on phenotypes and photosynthetic physiology were studied.Based on these mutants as chassis strains,the phycoerythrobilin(PEB)synthesis pathway was constructed into Synechocystis with two methods for changing the spectral characteristics of PBS.The two methods are as follows: replacing phycocyanobilin(PCB)synthetase PcyA with the special PEB synthetase PebS from cyanophage P-SSM2;and coexpression of PebS and PcyA.The expression of PebS induced new phycobiliprotein structures with special spectral characteristics and affected the photosynthetic physiological functions of Synechocystis.In the work of PebS replacing PcyA,it was found that PEB replacing PCB could inhibit phycobiliproteins aggregation,and the role of phycobilins in PBS assembly was explored through structure modeling.A special phytochromobilin(PΦB)was isolated and identified from the new phycobiliprotein structure,reflecting the new catalytic path of phycobilins.This work enriches the understanding of PBS structure assembly,synthesis of phycobilins,and connecting of phycobilins and phycobiliproteins.These were the basics for research and engineering of PBS.Moreover,in this work,a novel dichromatic allophycocyanin(Dic-APC)trimer was obtained and characterized.In a small(110 k Da)unit,the two chromophores cover a wide spectral range(550 to 660nm)and conduct efficient energy transfer.It provides an effective light harvesting element for artificial photosynthesis,and also shows the potential of engineering PBS.At the same time,this work synthesized Dic-APC in vitro using core-linker ptotein Apc C,and provided a method for the targeting.In the work of coexpression with PebS and PcyA,the effects of photosynthetic physiology and PBS structure caused by PEB expression were studied.When PEB and PCB were coexpressed,the state transition effect of PBS was remained,but caused the blue-shift of terminal energy emitter of PBS,which resulted in the reduction of photosynthetic efficiency and the increase of light tolerance of Synechocystis cells.In addition,the coexpression of these two phycobilins also formed a variety of new phycobiliprotein structures with different aggregation states and unique spectral characteristics.The above research shows that the engineering and reconsitution of PBS could be realized through structural simplification and construction of new phycobilin synthesis pathway.The new light-harvesting elements with different structure and light-harvesting range would be applied to the construction of artificial photosynthesis system.