PMID:19429550

Citation	Miller, EN, Jarboe, LR, Yomano, LP, York, SW, Shanmugam, KT and Ingram, LO (2009) Silencing of NADPH-dependent oxidoreductase genes (yqhD and dkgA) in furfural-resistant ethanologenic Escherichia coli. Appl. Environ. Microbiol. 75:4315-23
Abstract	Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low K(m) values for NADPH (8 microM and 23 microM, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low K(m) for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural.
Links	PubMed PMC2704836 Online version:10.1128/AEM.00567-09
Keywords	Alcohol Oxidoreductases/antagonists & inhibitors; Alcohol Oxidoreductases/isolation & purification; Alcohol Oxidoreductases/metabolism; Aldehyde Reductase/antagonists & inhibitors; Aldehyde Reductase/isolation & purification; Aldehyde Reductase/metabolism; Anti-Bacterial Agents/metabolism; Anti-Bacterial Agents/pharmacology; DNA, Bacterial/chemistry; DNA, Bacterial/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/antagonists & inhibitors; Escherichia coli Proteins/isolation & purification; Escherichia coli Proteins/metabolism; Ethanol/metabolism; Furaldehyde/metabolism; Furaldehyde/pharmacology; Gene Deletion; Gene Expression Profiling; Kinetics; Molecular Sequence Data; NADP/metabolism; Oxidation-Reduction; Oxidoreductases/antagonists & inhibitors; Sequence Analysis, DNA
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLX:E2D2R3		GO:0050580: 2,5-didehydrogluconate reductase activity	ECO:0000316:	UniProtKB:E2D2R2	F		Figure 5: when expressed from plasmids, dkgA was found to decrease furfural tolerance. Figure 6: Deletions of dkgA caused an increase in furfural tolerance and a decrease in furfural reductase activity in vivo similar to the results shown in the EMFR9 strain of E. coli. Since deletion of dkgA alone did not lower the in vivo reductase activity or increase furfural tolerance, another gene, yqhD, is presumed to be the more important activity for growth inhibition by low concentrations of furfural. The lowest furfural reductase activity, however, was found after deletion of both genes as seen in Figure 3.	complete CACAO 7933
ECOLX:E2D2R3	enables	GO:0050580: 2,5-didehydrogluconate reductase activity	ECO:0000316: genetic interaction evidence used in manual assertion		F		Seeded From UniProt	Missing: with/from
ECOLX:E2D2R3	enables	GO:0050580: 2,5-didehydrogluconate reductase activity	ECO:0000316: genetic interaction evidence used in manual assertion	UniProtKB:E2D2R2	F		Seeded From UniProt	complete

References

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