Uncovering and engineering complex phenotypes in bacteria using regulatory noncoding RNAs

Microorganisms exhibit amazing capabilities to sustainably produce biofuels, pharmaceuticals, and other chemicals. However, industrial stresses hinder efficient production. Metabolic engineering aims to overcome this challenge by genetically enhancing the metabolism of the organism, but existing approaches fail to fully control the complex cellular responses to stress, which are dynamic and involve large sets of genes. One untapped resource is the pool of native regulators known as noncoding RNAs that instinctively coordinate stress responses. Despite widespread discovery noncoding RNAs, few strategies harness their power to meet industrial goals. Most noncoding RNA functions remain unknown and lack foreseeable roles in producing phenotypes of interest. Moreover, existing methods to uncover noncoding RNA functions are slow and aimless, requiring laborious biochemical experiments and resulting in hit-or-miss relevance to any engineering goal. To address this, we developed computational methods to efficiently identify noncoding RNAs relevant to a target phenotype. In the context of local, single-gene RNA regulators, we developed a strategy to identify 5’ untranslated regions from transcriptome data and to rapidly screen their responses to stress in vivo. With this discovery pipeline, we exposed the first 5’ untranslated regions in ethanol-producing bacterium Zymomonas mobilis and illuminated their roles in stress responses. We also uncovered a network of multi-gene noncoding RNA regulators involved in the natural ethanol tolerance of Z. mobilis. To enable rapid identification of multi-gene noncoding RNA regulators like these in other organisms, we developed a computational approach to mine transcriptome data for noncoding RNAs with greatest potential impact on a phenotype of interest. Taken together, this work accelerates complex phenotype engineering in a variety of organisms by expanding the set of regulators available for engineering to include noncoding RNAsChemical Engineerin