TOLERANCE TO FURFURAL-INDUCED STRESS IS ASSOCIATED WITH PENTOSE PHOSPHATE PATHWAY GENES ZWF1, GND1, RPE1,AND TKL1 IN SACCHAROMYCES CEREVISIAE

Authors: Gorsich, Steven; Dien, Bruce; Nichols, Nancy; Slininger, Patricia - Pat; Liu, Zonglin; Skory, Christopher - Chris

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/12/2005
Publication Date: 10/13/2005
Citation: Gorsich, S.W., Dien, B.S., Nichols, N.N., Slininger, P.J., Liu, Z., Skory, C.D. 2005. Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPEL, and TKL1 in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 71(3):339-349. DOI: 10.1007/s00253-005-0142-3

Interpretive Summary: The interest in agriculture as an energy producer has significantly increased as the need for alternative and environmentally friendly energy sources becomes apparent. Large scale industrial production of fuel ethanol by fermentation of agricultural products began during the 1970’s in Brazil, the United States, and Canada. A less expensive alternative is to use lignocellulosic biomass such as agricultural residues, municipal solid wastes and pulp and paper industry wastes. Approximately 50 billion gallons of ethanol could be produced annually from current biomass wastes in the U.S. However, one serious obstacle for efficient conversion of biomass to ethanol is the availability of fermentative microorganisms (yeast and bacteria) that can tolerate industrial fermentation inhibitors. In efforts to understand the physiological effect of these inhibitors at the genetic level 62 yeast genes were identified that function in inhibitor tolerance. These genes represent a broad range of physiological processes thus illustrating the complexity of this problem. This is the first comprehensive identification of fermentation inhibitor tolerant genes and will lead to a better understanding of inhibitor tolerance during industrial fermentation. Moreover, by engineering these genes into industrial yeast strains we hope to improve inhibitor tolerance, growth, and ethanol yield – thus making the process of converting biomass to fuel ethanol more cost-effective.

Technical Abstract: Engineering yeast to be more tolerant to fermentation inhibitors, furfural and 5-hydroxymethylfurfural (HMF), will lead to more efficient lignocellulose to ethanol bioconversion. To identify target genes involved in furfural tolerance, a Saccharomyces cerevisiae gene disruption library was screened for mutants with growth deficiencies in the presence of furfural. It was hypothesized that overexpression of these genes would provide a growth benefit in the presence of furfural. Sixty-two mutants were identified whose corresponding genes function in a wide spectrum of physiological pathways, demonstrating the complexity of furfural tolerance. We focused on four mutants, zwf1, gnd1, rpe1, and tkl1 that represent genes encoding pentose phosphate pathway (PPP) enzymes. At various concentrations of furfural and HMF, a clear association with higher sensitivity to these inhibitors was demonstrated in these mutants. PPP mutants were inefficient at reducing furfural to the less toxic furfuryl alcohol, which we propose is a result of an overall decreased abundance of reducing equivalents or to NADPH’s role in stress tolerance. Overexpression of ZWF1 in S. cerevisiae allowed growth at furfural concentrations that are normally toxic. These results demonstrate a strong relationship between PPP genes and furfural tolerance and provide additional putative target genes involved in furfural tolerance.