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Applied and Environmental Microbiology, July 2009, p. 4315-4323, Vol. 75, No. 13
0099-2240/09/$08.00+0     doi:10.1128/AEM.00567-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Silencing of NADPH-Dependent Oxidoreductase Genes (yqhD and dkgA) in Furfural-Resistant Ethanologenic Escherichia coli{triangledown}

E. N. Miller, L. R. Jarboe, L. P. Yomano, S. W. York, K. T. Shanmugam, and L. O. Ingram*

Department of Microbiology and Cell Science, Box 110700, University of Florida, Gainesville, Florida 32611

Received 9 March 2009/ Accepted 1 May 2009

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 Km values for NADPH (8 µM and 23 µM, 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 Km for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural.

* Corresponding author. Mailing address: Department of Microbiology and Cell Science, P.O. Box 110700, University of Florida, Gainesville, FL 32611. Phone: (352) 392-8176. Fax: (352) 392-5922. E-mail: Ingram{at}ufl.edu

{triangledown} Published ahead of print on 8 May 2009.

Applied and Environmental Microbiology, July 2009, p. 4315-4323, Vol. 75, No. 13
0099-2240/09/$08.00+0     doi:10.1128/AEM.00567-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Miller, E. N., Jarboe, L. R., Turner, P. C., Pharkya, P., Yomano, L. P., York, S. W., Nunn, D., Shanmugam, K. T., Ingram, L. O. (2009). Furfural Inhibits Growth by Limiting Sulfur Assimilation in Ethanologenic Escherichia coli Strain LY180. Appl. Environ. Microbiol. 75: 6132-6141 [Abstract] [Full Text]  


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