Functions Analysis Of Regulatory Molecules In The Model Cyanobacterium Synechocystis Sp. PCC 6803

Unicellular model cyanobacterium Synechocystis sp.PCC 6803 has been considered as a promising microbial chassis for biofuel production due to utilize solar energy and CO2 as the sole energy and carbon sources.However,complicated regulatory mechanisms in Synechocystis and its low tolerance to biofuels toxicity have restricted their potential application.In addition to ongoing efforts to optimize the existing pathways and to discover and construct novel pathways,one option to achieve high productivity is to explore regulator molecules in Synechocystis,and improve cellular tolerance to toxic biofuel products using transcriptional engineering approach.Two-component signal transduction systems(TCSTSs)are well known to be involved in response to stress responses and regulation of central carbohydrate and energy metabolism in Synechocystis.In this study,by constructing and growth analysis of knockout mutants of 44 response regulators(RRs)of the TCSTSs,several RR mutants with differential growth patterns under autotrophic or photomixotrophic condition were identified.Metabolomic profiling analysis revealed significant functional overlapping of the response regulators in regulating central metabolism.By a co-response correlation analysis,a wide spread crosstalk of TCSTSs in regulating central metabolism were identified.Furthermore,an integrative analysis of evolutionary relationships and metabolomic profiling revealed several pairs of RRs with highly functional convergence,suggesting possible conserved functions that these TCSTSs were involved.To further investigate the mechanism of Synechocystis in response to biofuel toxicity,a protein co-expression network was constructed using large proteomic datasets of Synechocystis under various biofuel stresses and environmental perturbation,and then was combined with the protein-protein interaction(PPI)network.Proteins with statistically higher topological overlap in the network were identified as common responsive proteins to both biofuels stress and environmental perturbations,mostly enriched in ROS response pathway,transporters and cell membrane permeability.In addition,a WGCNA network was constructed to distinguish unique responses to biofuels from those to environmental perturbations,and to uncover metabolic modules and proteins uniquely associated with biofuels stress.The results showed most proteins enriched in photosynthesis,chlorophyll biosynthesis and amino acid metabolism,indicating the cells tended to employ a combination of multiple resistance mechanisms in dealing with biofuel stresses.For rational construction of high-tolerant chassis,transcriptional systems for tolerance improvement instead of conventional sequential multi-gene modification,especially the sRNA engineering approach,could be an applicable approach,due to its rapid response,flexible and precise control,ready restoration,and low metabolic burden.Based on genome-wide sRNA sequencing combined with systematic analysis of previous transcriptomic and proteomic data under the same perturbations,we reported the identification of 133 trans-encoded sRNA transcripts in Synechocystis,including 23 novel sRNAs identified for the first time.Notably,experimental validation revealed the overexpression of s RNA nc117 could improve the tolerance of Synechocystis response to ethanol and butanol stresses.Further targets prediction and qRT-PCR validation revealed most involved in transporter proteins and cell wall/membrane modifications.These findings provide important technical possibilities by utilizing small RNA engineering for cyanobacteria chassis modification.In this study,two most important regulatory molecules in Synechocystis,including TCSTS and sRNA were systematically analyzed.Results led to identified multiple TCSTSs involved in cell growth regulation during autotrophic and photomixtrophic cultivation.In addition,the systematic analysis of sRNA combined with biofuels tolerance mechanism in Synechocystis led to identify the first biofuels associated sRNA.These results are important to further investigate the function of regulatory molecules in Synechocystis.