Regulation Mechanism Of Trans-encoded SRNA Nc117 Related To Short-chain Alcohols Tolerance In Synechocystis Sp.PCC 6803

Cyanobacteria Synechocystis sp.PCC 6803,a prokaryotic microorganism,can grow only with solar energy,CO₂ and water.A variety of biofuels have been synthesized in the engineered Synechocystis sp.PCC 6803,demonstrating its feasibility as a photosynthetic cell factory.However,the low tolerance of cyanobacterial chassis to the toxicity of biofuels limits their potential for biofuels production and industrial application.In order to explore the regulatory elements related to biofuel stress response in cyanobacteria,a trans-encoded small RNA Nc117was found involved in the tolerance of Synechocystis sp.PCC 6803 to ethanol and1-butanol in our previous small RNA analysis.However,its detailed mechanism remains to be determined.In this study,the function of small RNA Nc117 was further characterized.In short,3'RACE was employed to determine the exact length of small RNA Nc117 as102 nucleotides,and the positive regulatory effect of Nc117 on short-chain alcohols tolerance was further confirmed.Subsequently,combined with computational target prediction and transcriptome analysis,the potential target of Nc117 was discussed.By comparative transcriptomic analysis,119 up-regulated genes and 116 down-regulated genes were identified in the Nc117 overexpression strain compared with the wild type,of which the upstream regions of 5 genes were overlapped with those predicted by the computational target prediction approach.Through phenotypic analysis of gene deletion and overexpression strains under short-chain alcohols stress,a gene slr0007encoding D-glycero-?-D-manno-heptose-1-phosphate guanylyltransferase was identified as a potential target for Nc117,suggesting that the synthesis of lipopolysaccharide or S-layer glycoprotein may be the one reason for the increased alcohol tolerance.Further small RNA regulation experiments in vivo showed that small RNA Nc117 promoted the stability of m RNA transcribed from slr0006-slr0007operon at post-transcriptional level.This study reports for the first time the positive regulation of trans-coding small RNA on biofuel tolerance in cyanobacteria,which provides evidence for the new regulation mechanism of trans-encoded small RNA in cyanobacteria and valuable information for the rational construction of photosynthetic autotrophic chassis with high tolerance.