We report the identification of a novel small open reading frame in Escherichia coli. The sORF (called iroK) encodes a 21 amino cid peptide, which when translated confers a 133% (ca. 20g/L) increase in resistance to 3-hydroxypropionic acid. We show that iroK conferred tolerance is additive to previously identified tolerance mechanisms involving relief of inhibited metabolism, yet does not involve altered 3-HP transport. This result demonstrates the continued surprises that microbial genomes hold and emphasize the importance of comprehensive discovery methods in future strain and metabolic engineering efforts. Biotechnol. Bioeng. 2012; 109:13471352. (C) 2011 Wiley Periodicals, Inc.