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RESEARCH PRODUCT

Applying a New REFINE Approach in Zymomonas mobilis Identifies Novel sRNAs That Confer Improved Stress Tolerance Phenotypes

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As global controllers of gene expression, small RNAs represent powerful tools for engineering complex phenotypes. However, a general challenge prevents the more widespread use of sRNA engineering strategies: mechanistic analysis of these regulators in bacteria lags far behind their high-throughput search and discovery. This makes it difficult to understand how to efficiently identify useful sRNAs to engineer a phenotype of interest. To help address this, we developed a forward systems approach to identify naturally occurring sRNAs relevant to a desired phenotype: RNA-seq Examiner for Phenotype-Informed Network Engineering (REFINE). This pipeline uses existing RNA-seq datasets under different growth conditions. It filters the total transcriptome to locate and rank regulatory-RNA-containing regions that can influence a metabolic phenotype of interest, without the need for previous mechanistic characterization. Application of this approach led to the uncovering of six novel sRNAs related to ethanol tolerance in non-model ethanol-producing bacterium Zymomonas mobilis. Furthermore, upon overexpressing multiple sRNA candidates predicted by REFINE, we demonstrate improved ethanol tolerance reflected by up to an approximately twofold increase in relative growth rate compared to controls not expressing these sRNAs in 7% ethanol (v/v) RMG-supplemented media. In this way, the REFINE approach informs strain-engineering strategies that we expect are applicable for general strain engineering.