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Applied and Environmental Microbiology, October 2009, p. 6132-6141, Vol. 75, No. 19
0099-2240/09/$08.00+0     doi:10.1128/AEM.01187-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Furfural Inhibits Growth by Limiting Sulfur Assimilation in Ethanologenic Escherichia coli Strain LY180{triangledown}

Elliot N. Miller,1 Laura R. Jarboe,1 Peter C. Turner,1 Priti Pharkya,2 Lorraine P. Yomano,1 Sean W. York,1 David Nunn,3 K. T. Shanmugam,1 and Lonnie O. Ingram1*

Department of Microbiology and Cell Science, Box 110700, University of Florida, Gainesville, Florida 32611,1 Genomatica, Inc., 10520 Wateridge Circle, San Diego, California 92121,2 Verenium Corporation, 4955 Directors Place, San Diego, California 921213

Received 8 June 2009/ Accepted 7 August 2009

A wide variety of commercial products can be potentially made from monomeric sugars produced by the dilute acid hydrolysis of lignocellulosic biomass. However, this process is accompanied by side products such as furfural that hinder microbial growth and fermentation. To investigate the mechanism of furfural inhibition, mRNA microarrays of an ethanologenic strain of Escherichia coli (LY180) were compared immediately prior to and 15 min after a moderate furfural challenge. Expression of genes and regulators associated with the biosynthesis of cysteine and methionine was increased by furfural, consistent with a limitation of these critical metabolites. This was in contrast to a general stringent response and decreased expression of many other biosynthetic genes. Of the 20 amino acids individually tested as supplements (100 µM each), cysteine and methionine were the most effective in increasing furfural tolerance with serine (precursor of cysteine), histidine, and arginine of lesser benefit. Supplementation with other reduced sulfur sources such as D-cysteine and thiosulfate also increased furfural tolerance. In contrast, supplementation with taurine, a sulfur source that requires 3 molecules of NADPH for sulfur assimilation, was of no benefit. Furfural tolerance was also increased by inserting a plasmid encoding pntAB, a cytoplasmic NADH/NADPH transhydrogenase. Based on these results, a model is proposed for the inhibition of growth in which the reduction of furfural by YqhD, an enzyme with a low Km for NADPH, depletes NADPH sufficiently to limit the assimilation of sulfur into amino acids (cysteine and methionine) by CysIJ (sulfite reductase).

* Corresponding author. Mailing address: Department of Microbiology and Cell Science, 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 14 August 2009.

Applied and Environmental Microbiology, October 2009, p. 6132-6141, Vol. 75, No. 19
0099-2240/09/$08.00+0     doi:10.1128/AEM.01187-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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