Table_3_Regulation Mechanism Mediated by Trans-Encoded sRNA Nc117 in Short Chain Alcohols Tolerance in Synechocystis sp. PCC 6803.XLSX

Microbial small RNAs (sRNAs) play essential roles against many stress conditions in cyanobacteria. However, little is known on their regulatory mechanisms on biofuels tolerance. In our previous sRNA analysis, a trans-encoded sRNA Nc117 was found involved in the tolerance to ethanol and 1-butanol in Synechocystis sp. PCC 6803. However, its functional mechanism is yet to be determined. In this study, functional characterization of sRNA Nc117 was performed. Briefly, the exact length of the trans-encoded sRNA Nc117 was determined to be 102 nucleotides using 3′ RACE, and the positive regulation of Nc117 on short chain alcohols tolerance was further confirmed. Then, computational target prediction and transcriptomic analysis were integrated to explore the potential targets of Nc117. A total of 119 up-regulated and 116 down-regulated genes were identified in nc117 overexpression strain compared with the wild type by comparative transcriptomic analysis, among which the upstream regions of five genes were overlapped with those predicted by computational target approach. Based on the phenotype analysis of gene deletion and overexpression strains under short chain alcohols stress, one gene slr0007 encoding D-glycero-alpha-D-manno-heptose 1-phosphate guanylyltransferase was determined as a potential target of Nc117, suggesting that the synthesis of LPS or S-layer glycoprotein may be responsible for the tolerance enhancement. As the first reported trans-encoded sRNA positively regulating biofuels tolerance in cyanobacteria, this study not only provided evidence for a new regulatory mechanism of trans-encoded sRNA in cyanobacteria, but also valuable information for rational construction of high-tolerant cyanobacterial chassis.