Alcoholic fermentation by ‘non‐fermentative’ yeasts

Improvement of stress tolerance in microorganisms applied in industrial fermentations for the production of ethanol is of major interest (26, 34). Based on screens for ethanol sensitivity/tolerance in Saccharomyces cerevisiae (12, 16-18, 35, 37, 40), it appears that this trait in yeast is possibly controlled by several genes acting in concert. Using global transcription machinery engineering (gTME), a tool to reprogram gene transcription for eliciting new phenotypes important for technological applications, Alper et al. (2) found mutants of S. cerevisiae with improved glucose/ethanol tolerance. In that work, mutated versions of the SPT15 gene, which codes for the TATA-binding protein, were generated by random in vitro mutagenesis and expressed in the laboratory strain BY4741. The authors identified one dominant allele, SPT15-300, which corresponds to the three amino acid changes F177S, Y195H, and K218R, that conferred increased tolerance of the yeast to ethanol (2). Although extensive analyses, such as transcriptional profiling and deleting and overexpressing individual genes, were carried out, a particular pathway or a genetic network responsible for the observed growth gain of the SPT15-300-expressing strain could not be identified (2). During our attempts to analyze the effect of the mutant SPT15-300 alleles in various yeast species of industrial importance, we discovered that the described improvement of growth in the presence of ethanol of the standard laboratory strain BY4741, the strain used by Alper et al. (2), is associated with improved uptake and/or utilization of leucine on media containing small amounts of leucine. MATERIALS AND METHODSStrains, media, and molecular procedures. The Saccharomyces strains investigated in this study were S. cerevisiae strains BY4741 (MATa his3⌬D1 leu2⌬0 met15⌬0 ura3⌬0) (5), in which the LEU2 gene is completely deleted (obtained from Euroscarf, Frankfurt, Germany), and Y55 (23) 2), and YSC lacking leucine (prepared as described for YSCϪUra, with Qbiogene CSM-LEU [2]). SC media were buffered (pH 5.5) with 1% (wt/vol) succinic acid and 0.6% (wt/vol) NaOH. SC and YSC media were supplemented with glucose and/or ethanol as indicated. S. cerevisiae strains were incubated at 20 or 30°C (as indicated), and S. bayanus and S. pastorianus were cultivated at 20°C. Saccharomyces species were transformed by use of the lithium acetate method (3).Escherichia coli strain DH5␣ (Invitrogen A/S, Taastrup, Denmark) was used for plasmid selection/propagation and cultivated as described previously (31).

Section: Resultssupporting

confidence: 64%

Impaired Uptake and/or Utilization of Leucine bySaccharomyces cerevisiaeIs Suppressed by theSPT15-300Allele of the TATA-Binding Protein Gene

Baerends

Qiu

Rasmussen

et al. 2009

“…Both approaches resulted in strains growing on xylose and fermenting it into ethanol. Although expression of XR and XDH resulted in rapid fermentation of xylose, NADPH/NAD cofactor imbalance under anaerobic conditions led to considerable accumulation of xylitol (6,14,15,30,32). However, employing XI instead of XR/XDH avoids cofactor imbalance and xylitol accumulation, as D-xylose is converted directly into D-xylulose without a redox reaction being involved.…”

mentioning

confidence: 99%

Functional Expression of a Bacterial Xylose Isomerase in Saccharomyces cerevisiae

Brat

Boles

Wiedemann

2009

255

196

In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like D-xylose and L-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize D-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

“…The ability to ferment sugars to ethanol is widespread among yeasts (18). In principle, this property seems to indicate that the yeasts are capable of anaerobic free-energy transduction and, therefore, of growth in the absence of oxygen.…”

mentioning

confidence: 99%

Oxygen Requirements of the Food Spoilage Yeast Zygosaccharomyces bailii in Synthetic and Complex Media

Rodrigues

Côrte‐Real

Leão

et al. 2001

Self Cite

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.