Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPE1, and TKL1 in Saccharomyces cerevisiae.

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, suggesting that furfural tolerance is a complex process. We focused on four mutants, zwf1, gnd1, rpe1, and tkl1, which 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.

Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran.

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C6H8O3 and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.

The dynamin-related GTPase, Mgm1p, is an intermembrane space protein required for maintenance of fusion competent mitochondria.

Mutations in the dynamin-related GTPase, Mgm1p, have been shown to cause mitochondrial aggregation and mitochondrial DNA loss in Saccharomyces cerevisiae cells, but Mgm1p's exact role in mitochondrial maintenance is unclear. To study the primary function of MGM1, we characterized new temperature sensitive MGM1 alleles. Examination of mitochondrial morphology in mgm1 cells indicates that fragmentation of mitochondrial reticuli is the primary phenotype associated with loss of MGM1 function, with secondary aggregation of mitochondrial fragments. This mgm1 phenotype is identical to that observed in cells with a conditional mutation in FZO1, which encodes a transmembrane GTPase required for mitochondrial fusion, raising the possibility that Mgm1p is also required for fusion. Consistent with this idea, mitochondrial fusion is blocked in mgm1 cells during mating, and deletion of DNM1, which encodes a dynamin-related GTPase required for mitochondrial fission, blocks mitochondrial fragmentation in mgm1 cells. However, in contrast to fzo1 cells, deletion of DNM1 in mgm1 cells restores mitochondrial fusion during mating. This last observation indicates that despite the phenotypic similarities observed between mgm1 and fzo1 cells, MGM1 does not play a direct role in mitochondrial fusion. Although Mgm1p was recently reported to localize to the mitochondrial outer membrane, our studies indicate that Mgm1p is localized to the mitochondrial intermembrane space. Based on our localization data and Mgm1p's structural homology to dynamin, we postulate that it functions in inner membrane remodeling events. In this context, the observed mgm1 phenotypes suggest that inner and outer membrane fission is coupled and that loss of MGM1 function may stimulate Dnm1p-dependent outer membrane fission, resulting in the formation of mitochondrial fragments that are structurally incompetent for fusion.

Purification and properties of gammagamma-enolase from pig brain.

Isoelectric focusing revealed three enolase isoforms in pig brain, which were designated as alphaalpha-(pI = 6.5), alphagamma- (pI = 5.6), and gammagamma-enolase (pI = 5.2). The pI of purified gammagamma-enolase was also 5.2. The gammagamma-enolase isoform of enolase was purified from pig brain by a purification protocol involving heating to 55 degrees C for 3 min, acetone precipitation, ammonium sulfate precipitation (40%-80%), DEAE Sephadex ion-exchange chromatography (pH 6.2), and Sephadex G200 gel filtration. The final specific activity was 82 units/mg protein. As with other vertebrate enolases, gammagamma-enolase from pig proved to be a dimer with a native mass of 85 kDa and a subunit mass of 45 kDa. The pH optimum for the reaction in the glycolytic direction is 7.2. The Km values for 2-PGA, PEP, and Mg2+ were determined to be 0.05, 0.25, and 0.50 mM, respectively, similar to Km values of other vertebrate enolases. The amino acid composition of pig gammagamma-enolase, as determined by amino acid analysis, shows strong similarity to the compositions of gammagamma-enolases from rat, human, and mouse, as determined from their amino acid sequences. Despite the differences seen with some residues, and considering the ways that the compositions were obtained, it is assumed that pig gammagamma-enolase is more similar than the composition data would indicate. Moreover, it is likely that the sequences of pig gammagamma-enolase and the other gammagamma-enolases are almost identical. Li+ proved to be a noncompetitive inhibitor with either 2-PGA or Mg2+ as the variable substrate. This enolase crystallized in the monoclinic space group P2, or P2(1). An Rsymm <5% was obtained for data between 50 and 3.65 A, but was a disappointing 30% for data between 3.65 and 3.10 A, indicating crystal disorder.