Increase in Furfural Tolerance in Ethanologenic Escherichia coli LY180 by Plasmid-Based Expression of thyA

Furfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilis YB886,Escherichia coli NC3, andZymomonas mobilis CP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing thethyA gene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in thede novo pathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression ofthyA was no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA inE. coli and to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA inE. coli would be expected to increase the cellular requirement for dTMP. Increased expression ofthyA (E. coli ,B. subtilis , orZ. mobilis ), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.