Evolution Reveals a Glutathione-Dependent Mechanism of 3-Hydroxypropionic Acid Detoxification

Biologically produced 3-hydroxypropionic acid (3HP) is a potential source for sustainable acrylates and can also find direct use as monomer in the production of biodegradable polymers. For industrial-scale production, high titer, rate and yield are essential; thus there is a need for robust cell factories tolerant to high concentration of 3HP, preferably at low pH. Through adaptive laboratory evolution we selected S. cerevisiae strains with improved tolerance to 3HP at pH 3.5. Genome sequencing of three independent clones identified single-nucleotide changes in the SFA1 gene encoding S-(hydroxymethyl)glutathione dehydrogenase. Introduction of the mutated SFA1 alleles or overexpression of any of the SFA1 alleles in a sfa1Δ strain enabled growth in the presence of above 40 g/L 3HP. We further found that aldehyde dehydrogenase (ALD6), S-formylglutathione hydrolase (YJL068C) and glutathione play a role in 3HP detoxicification. Addition of glutathione relieved growth inhibition by 3HP for several yeast species and for E. coli; but glutathione could not enable growth of a S. cerevisiae sfa1Δ strain. Based on our findings we propose a 3-hydroxypropionic aldehyde-mediated mechanism underlying 3HP toxicity as well as a glutathione-dependent route for detoxification of 3-hydroxypropionic aldehyde (reuterin). The identified molecular response to 3HP and reuterin may well be a general mechanism for handling resistance to organic acids and aldehydes by living cells.