(436n) Identification of Organic Acid Tolerance Genes in E. Coli for Biorefinery Applications

Synthesis of building block chemicals at high titers can be toxic to host microorganisms. We have developed Mixed Library Parallel Gene Trait Mapping (ML-PGTM) as a technique that identifies the effect of specific genes on the expression of a specific bacterial phenotype. We have utilized this method to select for organic acid tolerance. Selections using 3-hydroxypropionic acid (3-HP) were carried out in both minimal media and buffered minimal media systems. Genes identified conferred both acid tolerance and resistance to 3-HP and 3-hydroxypropionaldehyde (3-HPA), a toxic intermediate formed during 3-HP production. We have identified one loci with putative functions involving oxidative stress for which increased copy confers increased 3-HP tolerance. Confirmations of increased tolerance were carried out by determining minimum inhibitory concentrations and constructing growth curves of selected transformants in the presence of 3-HP and other organic acids. A 50% increase in tolerance was observed for 3-HP when compared with wild type E. coli.