By combining 13C-based metabolic flux and global gene expression analysis in microaerobic, glucose-limited chemostats, we revealed key features of yeasts' normal response to furfural. While low concentrations of furfural (<15 mM) can be reduced by NADH-dependent oxireductases, the main physiological response to higher concentrations of furfural (>15 mM) was increased in pentose phosphate pathway flux to provide sufficient NADPH for the reduction of furfural. In wild-type yeast, upregulated expression of several genes coding for NADPH-dependent oxireductases indicates their involvement in the reduction of furfural. Moreover, our transcript data show that genes for iron transmembrane transport are upregulated in response to furfural. By comparing the transcript levels of an evolved, furfural-resistant strain to those of its parent, we demonstrated the key molecular mechanism of the evolved furfural resistance to be the upregulation of the NADPH-dependent dehydrogenase ADH7 and the uncharacterized ORF YKL071W.
Previous chemostat studies showed that the cofactor for furfural reduction is NADH, but only at low concentrations of furfural, around 6 mM (20
). These strains were also much less resistant than the semi-industrial strain TMB3400 used here, such that washout already occurred at 8.6 mM furfural. These results are consistent with our flux data at the furfural concentration of 15 mM, where the NADH supply also seems to suffice to quantitatively reduce furfural. At 25 and 36 mM furfural, however, our redox balance data show clearly that the in vivo available NADH would not suffice for furfural reduction. Therefore, we suggest the following model. Upon furfural exposure, yeast preferentially ceases glycerol production as a first means to free NADH to carry out the reduction (40
). As this source is exhausted, pentose phosphate pathway activity is increased as a second mechanism to deliver more reduction equivalents in the form of NADPH. Consistent with this model, most of the upregulated genes in our data set at high furfural challenge have a preference for NADPH, whereas previously identified NADH-dependent genes (ADH1
) were not upregulated. These enzymes are possibly already fully expressed at 15 mM furfural. Our finding is consistent with the study of Gorsich et al. (17
) that demonstrated that a knockout of pentose phosphate pathway genes leads to an increased sensitivity at a high concentration of 50 mM furfural on agar plates. The overexpression of ZWF1
provides no growth advantage at low concentrations of furfural but enables growth at otherwise lethal concentrations of 50 mM furfural in liquid culture.
Lin and coworkers found that in batch cultures with a very high concentration of 177 mM furfural, the yeast response consists of an increase in enzymes catalyzing the reaction of the tricarboxylic acid cycle and upregulation of mainly NADH-dependent dehydrogenases and ADH6
as a NADPH-dependent dehydrogenase (27
). The conditions were fundamentally different from our chemostat experiments for the following two main reasons. First, the yeast was cultivated under aerobic conditions, which would in principle allow an increase in tricarboxylic acid activity. Second, cell growth stopped completely after the addition of furfural, which strongly affects metabolic activity (43
). Furthermore, since pentose phosphate pathway activity is regulated mainly at the posttranslational level, a potential increase could not be detected by their method.
We attribute the increased resistance of our evolved strain to the observed strong overexpression of ADH7 and YKL071W as well as to the generally higher levels of a further four oxireductases. The higher expression of these genes leads to the demonstrated higher maximal reduction capacity that ensures growth at high concentrations of furfural. By separate, plasmid-based overexpression of ADH7 and YKL071W in the parent strain, we demonstrate the relevance of both events in furfural resistance. Neither of them alone, however, could fully restore the observed resistance level of our evolved strain. We contribute this difference mainly to the 50% higher in vitro reduction activity of the evolved strain compared to only 30% higher in vitro reduction activity of the plasmid-based overexpression strains. Moreover, several further dehydrogenase genes are upregulated upon furfural increase in the evolved strain, cumulating in a higher total reduction activity, which might allow the strain to attain a higher level of resistance.
The inheritance pattern of the resistant trait indicates a single genome alteration that would be causal for the observed phenotype. Since several dehydrogenases were upregulated and neither ADH7 nor YKL071W overexpression alone was sufficient to restore the evolved phenotype, we speculate that a regulatory mutation is the basis for the altered expression level of these genes that conjointly lead to furfural resistance.