Abstract
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.
Highlights
Biofuels have become a hot research area in recent decades due to their potential of replacing fossil (Atsumi et al, 2008)
The trans-encoded small RNA (sRNA) Nc117 was demonstrated to be involved in the tolerance to biofuels ethanol and 1-butanol in Synechocystis in our previous study (Pei et al, 2017)
For the other four genes, the similar phenotype in alcohols resistance was not observed as slr0007, which might be due to several possible reasons: (i) the significance of involvement in alcohols tolerance: these genes may be involved in alcohols tolerance, but we failed to identify them under the tested conditions; (ii) the changes of these genes may be secondary responses of genes regulated by Nc117, or be involved in other functions instead of short chain alcohols resistance that are still yet to be investigated; (iii) the false positive from the deficiency of computational target prediction and RNA-seq transcriptomic analysis
Summary
Biofuels have become a hot research area in recent decades due to their potential of replacing fossil (Atsumi et al, 2008). For the resistance mechanism to biofuels, based on several previous studies, cyanobacteria employed a combination of multiple resistance systems to adapt to biofuels stress or nutrient limited environments, including cell membrane tolerance mechanism, transport vector system, intracellular transformation and degradation, cell surface structure and morphology changes, as well as common pressure response tolerance mechanisms (Jie et al, 2012; Qiao et al, 2012, 2013; Huang et al, 2013; Tian et al, 2013; Xu et al, 2014) This brings great challenges to further improve tolerance by conventional sequential multi-gene modification approaches (Gao X. et al, 2016). “transcriptional engineering” for tolerance improvement (Anders and Huber, 2010), especially the sRNAs engineering that has the advantages such as rapid response, low metabolic burden and flexible and precise control, could be an applicable approach (Gaida et al, 2013)
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