Identification and transcriptional profiling of Pseudomonas putida genes involved in furoic acid metabolism

Authors: Nichols, Nancy; Mertens, Jeffrey

Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2008
Publication Date: 5/1/2008
Citation: Nichols, N.N., Mertens, J.A. 2008. Identification and transcriptional profiling of Pseudomonas putida genes involved in furoic acid metabolism. FEMS Microbiology Letters. 284:52-57.

Interpretive Summary: To meet the U.S. national goal of producing significant amounts of biofuels; agricultural biomass, waste residues, etc., must be used, in addition to corn, as production feedstocks. During the process of obtaining usable sugars from biomass feedstocks, compounds termed furans often form as byproducts. The byproducts are toxic to many microbes, including the fermenting microbes used to produce important products such as fuel ethanol. Left untreated, furans can cause the fermentation to fail. Even though furan compounds are fermentation inhibitors, some bacteria and fungi can metabolize furans. It may be possible to exploit that natural ability by transferring the genes for furan metabolism into fermenting microbes. This would allow the microbe to detoxify inhibitory byproducts and carry on with fermentation of sugars. We identified genes involved in furan metabolism in the bacterium Pseudomonas putida and demonstrated that the genes are turned on in response to the presence of furans in the growth environment. This work provides a genetic basis for understanding furan metabolism, which is needed to engineer a fermenting microbe that has better inhibitor tolerance.

Technical Abstract: Furfural (2-furaldehyde) is a furan formed by dehydration of pentose sugars. Pseudomonas putida Fu1 metabolizes furfural through a pathway involving conversion to 2-oxoglutarate, via 2-furoic acid and Coenzyme A intermediates. To identify genes involved in furan metabolism, two P. putida transposon mutants were isolated that had impaired growth on furfural and furoic acid. Both strains grew at wild-type rates on succinate and 4-hydroxybenzoate, indicating the mutations specifically affect furan degradation rather than growth in general. DNA flanking the transposon insertion site was cloned from both mutants. The transposons disrupted a LysR-family regulatory gene in mutant PSF2 and a GcvR-type regulatory gene in PSF9. Expression of several open reading frames in the proximity of the regulatory genes was induced during growth of P. putida on furoic acid. Real-time quantitative reverse transcriptase-PCR analysis of P. putida Fu1 RNA demonstrated increased transcription, in response to furoic acid, of 10-fold (a putative permease gene) to greater than 1,000-fold (a putative decarboxylase gene). Disruption of two of the genes demonstrated that both are important for growth on furoic acid. The LysR family gene appears to act positively, and the GcvR-family gene negatively, in regulating expression of neighboring genes.