This study summarizes the effort to identify genes of Escherichia coli that confer tolerance to 3-hydroxypropanoic acid (3-HP). 3-HP is a building block chemical that has the potential to become a renewable alternative to petrochemicals through biorefining. Metabolic pathways of recombinant E. coli have been discovered that can produce 3-HP at low titers, but indicate that increased titers of 3-HP significantly inhibit cell growth. In general, organic acids enter the cell and dissociate. The resultant protons increase internal pH, while the anions react in various metabolic pathways. For this project, genomic libraries were constructed using the pEZSeq vector and growth curves were used to analyze tolerance levels. Increases in specific growth rate were observed for all selected clones grown in 10 g/L 3-HP, buffered to pH 7 in minimal media. Plasmid DNA sequencing revealed multiple genes that contributed to stress tolerance. When media was enriched with amino acids, clones expressed a shortened lag phase. Growth curve results for media supplemented with amino acids provide support for the mechanism of pHi maintenance via amino acid decarboxylation. The genes identified through this work can be integrated into future E. coli strains to be used for the viable industrial production of 3-HP.