Functional Characterization Of Site-2-protease In The Stress Response Of Cyanobacterium Synechocystis Sp.PCC6803

In the face of constant changes in the external environment,organisms will adjust through the corresponding response regulation mechanism to maintain homeostasis and adapt to various environmental stresses.The intracellular proteolysis mechanism mediated by S2 P protease is a conservative mechanism for cells to respond to environmental stresses.As the earliest photosynthetic autotroph on the earth,Synechocystis sp.PCC6803 has a simple structure and is a model organism optimized for studying organisms to adapt to the environmental stresses.It contains four S2 P proteases,which are: Sll0862,Slr0643,Sll0528 and Slr1821,all of which are involved in the response of Synechocystis sp.PCC6803 to various environmental stresses.In this paper,the functions of two S2 P proteases,Slr0643 and Sll0528,in adapting to environmental stresses were investigated,and the regulation mechanism of Slr0643 in participating in glucose mixture was analyzed by transcriptomics and metabolomics techniques.At the same time,a CRISPRi system was constructed in Synechocystis sp.PCC6803 to simultaneously inhibit 4 S2 P protease genes,so as to further explore the various functions of S2 P protease in the future.The following conclusions were obtained.(1)The genome-wide transcriptome analysis revealed that when glucose was added for four hours,the pathways of iron uptake and metabolism,nitrogen utilization,and the synthesis of ATP and protein were up-regulated,as well as inorganic carbon uptake pathway was downregulated.While for ?slr0643 mutant,the obvious upregulation of the genes for iron uptake and metabolism was impeded or even inverted,indicating that Slr0643 might played a positive role in the regulation of iron uptake and metabolism.By studying the phenotypes of ?slr0643 mutant in iron-limiting condition,it is found in the medium without iron,the growth of ?slr0643 mutant is not affected in the short term(5 days),and after a long period of training(20 days),the color of culture fluid was yellow,and the cells were unhealthy.Combined with the results of transcriptome analysis,slr0643 might be involved in the regulation of genes related to the absorption and utilization of iron ions,thus affecting the absorption of iron ions by cells.(2)The metabolic mechanism of Slr0643 protein in the adaptation of glucose mixotrophic acclimation was studied by gas chromatography-mass spectrometry(GC-MS).By identifying the intracellular metabolites in wild type and ?slr0643 in the process of adding 2.5 m M glucose,it was found that there were 11 differential metabolites in the wild type and mutant,and the expression level of the 9 differential metabolites in the wild type increased significantly.Through the enrichment analysis of the metabolic pathways involved in the different metabolites of mutants relative to the wild type at each culture time,it was found that the differential metabolites were mainly concentrated in 8 metabolic pathways,including Tricarboxylic acid cycle,pyruvate metabolism,butanoate metabolism,glutamic acid metabolism,valine,leucine and isoleucine biosynthesis,starch and sucrose metabolism,niacinamide metabolism and inositol phosphate metabolism,suggesting that Slr0643 protein was involved in regulating these metabolic pathways to make Synechocystis sp.PCC6803 adapt to glucose mixture.These results indicated that the deletion of slr0643 damaged various metabolic pathways in the cell,thus making the cell unable to use glucose normally,revealing that Slr0643 plays an important role in the adaptation of glucose mixotrophic acclimation for Synechocystis sp.PCC6803.(3)By comparing the growth curves of sll0528 knockout strain ?sll0528,over-expressed strain Osll0528 and wild type in the ethanol stress,it is found in the condition with different ethanol concentration(1.5%,2.0%,2.5%,3.0%),the growth of Osll0528 were better than the other two,and ?sll0528 was strongly inhibited and could hardly grow.The results showed that sll0528 gene had a positive effect on ethanol tolerance of algae collecting cells,and the Osll0528 could improve ethanol tolerance of Synechocystis sp.PCC6803.(4)The recombinant plasmids P0168 sgRNA and P3031 d Cas9 were constructed,which were necessary for the CRISPRi system to inhibit 4 S2 P proteases at the same time,and P0168 sgRNA was successfully transformed into Synechocystis sp.PCC6803,and the sgRNA transformation strain was obtained.