Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/23/2002
Publication Date: N/A
Technical Abstract: Biomass has great potential as a renewable substrate for microbial fermentations to produce value added products. Acid hydrolysis of biomass yields sugar monomers, but also results in formation of organic acids, phenolics, and furans that inhibit microbial growth. Furfural is a significant microbial inhibitor formed during biomass hydrolysis. However, ,some bacteria use furfural as a carbon source, suggesting bioremediation a an approach for inhibitor abatement. Pseudomonas putida converts furfural to 2-oxoglutarate via 2-furoic acid and several CoA intermediates, but the genes for furfural degradation have not been described. To identify the genes, two independent transposon mutants of P. putida that did not grow on furfural or 2-furoic acid were constructed. Both mutants grew at wild-type rates on succinate and 4-hydroxybenzoate, indicating that the mutations are specific to furfural degradation. Marker rescue was used to clone the mutated DNA and identify the insertion site for both strains. For one mutant, the flanking nucleotide sequence was similar only to genes of unknown functions. Sequence from the second mutant revealed strong similarity to numerous aromatic decarboxylase genes. This mutation was crossed back into the wild-type strain to confirm involvement of the cloned DNA in furoic acid utilization. The mutant phenotype suggests a role for the putative decarboxylase gene or a neighboring gene in furoic acid metabolism, although there is no decarboxylation step in the proposed pathway. Both mutants gave rise to revertants that grew at least three-fold slower on furoic acid compared to the wild type. Both revertants retained the transposon insertion, indicating that related enzymes may compensate for the lost functions in the mutants.