High Gravity Fermentation Research Articles

Very high gravity ethanol fermentation by flocculating yeast under redox potential-controlled conditions

BackgroundVery high gravity (VHG) fermentation using medium in excess of 250 g/L sugars for more than 15% (v) ethanol can save energy consumption, not only for ethanol distillation, but also for distillage treatment; however, stuck fermentation with prolonged fermentation time and more sugars unfermented is the biggest challenge. Controlling redox potential (ORP) during VHG fermentation benefits biomass accumulation and improvement of yeast cell viability that is affected by osmotic pressure and ethanol inhibition, enhancing ethanol productivity and yield, the most important techno-economic aspect of fuel ethanol production.ResultsBatch fermentation was performed under different ORP conditions using the flocculating yeast and media containing glucose of 201 ± 3.1, 252 ± 2.9 and 298 ± 3.8 g/L. Compared with ethanol fermentation by non-flocculating yeast, different ORP profiles were observed with the flocculating yeast due to the morphological change associated with the flocculation of yeast cells. When ORP was controlled at −100 mV, ethanol fermentation with the high gravity (HG) media containing glucose of 201 ± 3.1 and 252 ± 2.9 g/L was completed at 32 and 56 h, respectively, producing 93.0 ± 1.3 and 120.0 ± 1.8 g/L ethanol, correspondingly. In contrast, there were 24.0 ± 0.4 and 17.0 ± 0.3 g/L glucose remained unfermented without ORP control. As high as 131.0 ± 1.8 g/L ethanol was produced at 72 h when ORP was controlled at −150 mV for the VHG fermentation with medium containing 298 ± 3.8 g/L glucose, since yeast cell viability was improved more significantly.ConclusionsNo lag phase was observed during ethanol fermentation with the flocculating yeast, and the implementation of ORP control improved ethanol productivity and yield. When ORP was controlled at −150 mV, more reducing power was available for yeast cells to survive, which in turn improved their viability and VHG ethanol fermentation performance. On the other hand, controlling ORP at −100 mV stimulated yeast growth and enhanced ethanol production under the HG conditions. Moreover, the ORP profile detected during ethanol fermentation with the flocculating yeast was less fluctuated, indicating that yeast flocculation could attenuate the ORP fluctuation observed during ethanol fermentation with non-flocculating yeast.

Overexpression of vacuolar H+-ATPase-related genes in bottom-fermenting yeast enhances ethanol tolerance and fermentation rates during high-gravity fermentation

Vacuolar H+-ATPase (V-ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom-fermenting yeast strains, in which the V-ATPase-related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high-sugar medium simulating high-gravity fermentation. Among the strains examined, the DBF2-overexpressing bottom-fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high-gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V-ATPase is a stress target in high-gravity fermentation and suggests that enhancing the V-ATPase activity increases the ethanol tolerance of bottom-fermenting yeast, thereby improving the fermentation rate and cell viability under high-gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling

Impact of dried, creamed and cake supply formats on the genetic variation and ethanol tolerance of three Saccharomyces cerevisiae distilling strains

Scotch whisky fermentations typically employ high-gravity fermentation practices to maximize product formation and to minimize both energy and water inputs. This approach increases ethanol concentrations at the end of fermentation, creating stressful conditions for the yeast. In this work we examined the relative tolerance of four Saccharomyces cerevisiae distilling yeast strains, supplied in dried, creamed, cake or slurry format, to ethanol under CO2-induced anaerobic conditions. The cells were assessed for their capacity to recover and grow on inhibition spot plates and to maintain cell viability in ethanol-dosed suspensions. Variations in ethanol tolerance were observed between strains and between the same strain supplied in different formats. The creamed yeast format typically exhibited a higher tolerance to ethanol. One possible explanation for this observation is that cells surviving the dehydration and rehydration process might incur sub-lethal genome damage. Thus the genetic integrity of the most ethanol-tolerant strain was assessed as a function of supply format (two dried and one creamed). The mitochondrial DNA was examined using mitochondrial restriction fragment length polymorphism and the chromosomal DNA using pulsed field gel electrophoresis and polymerase chain reaction with both ITS and delta-specific primers. In one dried yeast sample, genetic integrity was compromised, highlighting the requirement for yeast intake quality assurance programmes. Copyright © 2012 The Institute of Brewing & Distilling

Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation

BackgroundThe understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance.ResultsA yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased 3 H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation.ConclusionsPDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic fermentation performance for sustainable bio-ethanol production.

English

DNA microarray analysis was used to investigate the expression profile ofSaccharomyces cerevisiae genes in glycolysis pathway, trehalose and steroid biosynthesis and heat shock proteins (HSP) in response to harsh environment under the late stage of very high gravity (VHG) fermentation. The data show that only a few genes (GPM2, PGM1, GAL10 and PGM1) involved in glycolysis pathway and trehalose biosynthesis were up-regulated. Five genes that encode heat shock proteins (HSP26, HSP10, HSP42, HSP78 and HSP82) were up-regulated. Among these five genes, there was a strong expression increase of about 84-fold for HSP26.The results of this study revealed adverse VHG fermentation conditions stress response pattern and suggested interesting information about the mechanisms involved in adaptation of cells to the complex VHG fermentation environment. This identification of genes provides information that will help to genetically modify yeast to further maintain the fermentation fitness and improve its fermentation capacity and process. Key words: DNA microarray, gene expression, yeast, stress, very high gravity (VHG) fermentation.

Transcriptome analysis of Saccharomyces cerevisiae at the late stage of very high gravity (VHG) fermentation

The effect of culture system on growth and economics performance of Orechromis niloticus (Nile tilapia) in concrete tanks was investigated. Four outdoor concrete tanks measuring 2.5 x 2 m was used for the study for 24 weeks culture period. The culture systems included the use of algae only at the stocking rates of 4 fishes/m2 (T1), algae plus commercial feed at the stocking rate of 10 fishes/m2 (T2), and commercial feed only at the stocking rate of 20 fishes/m2 (T3). The tanks were stocked with O. niloticusfingerlings with mean weight of 1.160, 1.030 and 0.255 g for T1, T2 and T3, respectively. Each treatment was replicated twice. Data collected were analyzed using analysis of variance and mean comparison was carried out using Duncan Multiple Range Test (DMRT). All tests were carried out at 5% probability level. The result on growth performance show that the intensive system (T1) was significantly different in some growth parameters (P < 0.05) such as yield (0.115, 0.830 and 1.260 kg/m3 for T1, T2 and T3, respectively), specific growth (1.975, 2.750 and 3.425% for T1, T2 and T3, respectively), survival rate (90, 80 and 91% for T1, T2 and T3, respectively), and feed conversion ratio (1.150 and1.030 for T2 and T3, respectively). Economically, the intensive system was more profitable with a gross margin of N 122, which was 40% more profitable than the extensive system (T1 with Gross margin of N80), and 269% than commercial feed plus algae (T2 with Gross margin of – N 194). The cost of production (incidence of cost) showed that it cost more (N 447) to produce O. niloticus using commercial feed plus algae (T2 ) in concrete tank than the use of algae only (T1) (N 268) and the use of commercial feed only (T3) (N380). Key words: Culture system, growth performance, economic performance,Oreochromic niloticus.

A Novel Strategy to Construct Yeast Saccharomyces cerevisiae Strains for Very High Gravity Fermentation

Very high gravity (VHG) fermentation is aimed to considerably increase both the fermentation rate and the ethanol concentration, thereby reducing capital costs and the risk of bacterial contamination. This process results in critical issues, such as adverse stress factors (ie., osmotic pressure and ethanol inhibition) and high concentrations of metabolic byproducts which are difficult to overcome by a single breeding method. In the present paper, a novel strategy that combines metabolic engineering and genome shuffling to circumvent these limitations and improve the bioethanol production performance of Saccharomyces cerevisiae strains under VHG conditions was developed. First, in strain Z5, which performed better than other widely used industrial strains, the gene GPD2 encoding glycerol 3-phosphate dehydrogenase was deleted, resulting in a mutant (Z5ΔGPD2) with a lower glycerol yield and poor ethanol productivity. Second, strain Z5ΔGPD2 was subjected to three rounds of genome shuffling to improve its VHG fermentation performance, and the best performing strain SZ3-1 was obtained. Results showed that strain SZ3-1 not only produced less glycerol, but also increased the ethanol yield by up to 8% compared with the parent strain Z5. Further analysis suggested that the improved ethanol yield in strain SZ3-1 was mainly contributed by the enhanced ethanol tolerance of the strain. The differences in ethanol tolerance between strains Z5 and SZ3-1 were closely associated with the cell membrane fatty acid compositions and intracellular trehalose concentrations. Finally, genome rearrangements in the optimized strain were confirmed by karyotype analysis. Hence, a combination of genome shuffling and metabolic engineering is an efficient approach for the rapid improvement of yeast strains for desirable industrial phenotypes.

Construction of bottom-fermenting yeasts that over express the ubiquitin ligase RSP5 gene and their effects on high-gravity fermentation

It has been reported that over expression of the ubiquitin ligase RSP5 gene conferred tolerance to various stresses in laboratory yeast Saccharomyces cereivisiae. We constructed strains of bottom-fermenting yeast, natural hybrid, in which the S. cerevisiae(SC)-RSP5 or Saccharomyces bayanus (SB)-RSP5 is highly expressed and used for high-gravity fermentation. The SB-RSP5-overexpressing yeast showed a higher fermentation rate than the parent strain. In contrast, the fermentation rate of the SC-RSP5-overexpressing yeast was slower than the parent strain. These results suggest significant functional differences in brewing between the SC-RSP5 and SB-RSP5 genes. Key words: High-gravity fermentation, RSP5 gene, stress tolerance, bottom-fermenting yeast, natural hybrid.

Viscosity Reduction During Fuel Ethanol Production by Fresh Sweet PotatoFermentation

Fresh sweet potato is one of the major feedstocks for fuel ethanol production in China.However,high-viscosity syrup is the key factor affecting high gravity fermentation of fresh sweet potato.It easily leads to pipe block,which seriously reduces the ethanol fermentation efficiency,influences the industrial production of ethanol with fresh sweet potato and increases the energy consumption.In this paper,the experiments on the viscosity-reducing enzyme and its optimized conditions were carried out,and the results showed that:(1) The cellulase from Sichuan Habio Bioengineering Co.,Ltd was the best enzyme,reducing the viscosity from 1.7×104 mPa.s to 8.8×102 mPa.s,and greatly reduced the cost of ethanol production.(2) The optimal pretreated condition was 110 ℃ for 20 min.(3) The viscosity-reducing enzyme could be applied to different varieties of fresh sweet potatoes,and the viscosity all reduced to less than 1.0×103mPa.s,with the lowest value of 2.7×102mPa.s.The viscosity reduction rates were all more than 95%.(4) Under optimal conditions,the viscosity of fermented sweet potatoes decreased from 1.9×105mPa.s to 2.7×103mPa.s after treated for 2 h by enzyme,and reduced to 790 mPa.s after fermentation of 23 h.The ethanol concentration reached 10.56%(V/V).The results were validated further that the viscosity-reducing enzyme had potential in industrial scale ethanol fermentation with fresh sweet potato.Tab 8,Ref 19

Medium Optimization for Improved Ethanol Production in Very High Gravity Fermentation

An optimal medium (300 g·L −1 initial glucose) comprising 6.3 mmol·L −1 Mg 2+ , 5.0 mmol·L −1 Ca 2+ , 15.0 g·L −1 peptone and 21.5 g·L −1 yeast extract was determined by uniform design to improve very high gravity (VHG) ethanol fermentation, showing over 30% increase in final ethanol (from 13.1% to 17.1%, by volume), 29% decrease in fermentation time (from 84 to 60 h), 80% increase in biomass formation and 26% increase in glucose utilization. Experiments also revealed physiological aspects linked to the fermentation enhancements. Compared to the control, trehalose in the cells grown in optimal fermentation medium increased 17.9-, 2.8-, 1.9-, 1.8- and 1.9-fold at the fermentation time of 12, 24, 36, 48 and 60 h, respectively. Its sharp rise at the early stage of fermentation when there was a considerable osmotic stress suggested that trehalose played an important role in promoting fermentation. Meanwhile, at the identical five fermentation time, the plasma membrane ATPase activity of the cells grown in optimal medium was 2.3, 1.8, 1.6, 1.5 and 1.3 times that of the control, respectively. Their disparities in enzymatic activity became wider when the glucose levels were dramatically changed for ethanol production, suggesting this enzyme also contributed to the fermentation improvements. Thus, medium optimization for VHG ethanol fermentation was found to trigger the increased yeast trehalose accumulation and plasma membrane ATPase activity.

Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination

Genome shuffling is an efficient way to improve complex phenotypes under the control of multiple genes. For the improvement of strain's performance in very high-gravity (VHG) fermentation, we developed a new method of genome shuffling. A diploid ste2/ste2 strain was subjected to EMS (ethyl methanesulfonate) mutagenesis followed by meiotic recombination-mediated genome shuffling. The resulting haploid progenies were intrapopulation sterile and therefore haploid recombinant cells with improved phenotypes were directly selected under selection condition. In VHG fermentation, strain WS1D and WS5D obtained by this approach exhibited remarkably enhanced tolerance to ethanol and osmolarity, increased metabolic rate, and 15.12% and 15.59% increased ethanol yield compared to the starting strain W303D, respectively. These results verified the feasibility of the strain improvement strategy and suggested that it is a powerful and high throughput method for development of Saccharomyces cerevisiae strains with desired phenotypes that is complex and cannot be addressed with rational approaches.

Very high gravity (VHG) ethanolic brewing and fermentation: a research update

There have been numerous developments in ethanol fermentation technology since the beginning of the new millennium as ethanol has become an immediate viable alternative to fast-depleting crude reserves as well as increasing concerns over environmental pollution. Nowadays, although most research efforts are focused on the conversion of cheap cellulosic substrates to ethanol, methods that are cost-competitive with gasoline production are still lacking. At the same time, the ethanol industry has engaged in implementing potential energy-saving, productivity and efficiency-maximizing technologies in existing production methods to become more viable. Very high gravity (VHG) fermentation is an emerging, versatile one among such technologies offering great savings in process water and energy requirements through fermentation of higher concentrations of sugar substrate and, therefore, increased final ethanol concentration in the medium. The technology also allows increased fermentation efficiency, without major alterations to existing facilities, by efficient utilization of fermentor space and elimination of known losses. This comprehensive research update on VHG technology is presented in two main sections, namely VHG brewing, wherein the effects of nutrients supplementation, yeast pitching rate, flavour compound synthesis and foam stability under increased wort gravities are discussed; and VHG bioethanol fermentation studies. In the latter section, aspects related to the role of osmoprotectants and nutrients in yeast stress reduction, substrates utilized/tested so far, including saccharide (glucose, sucrose, molasses, etc.) and starchy materials (wheat, corn, barley, oats, etc.), and mash viscosity issues in VHG bioethanol production are detailed. Thereafter, topics common to both areas such as process optimization studies, mutants and gene level studies, immobilized yeast applications, temperature effect, reserve carbohydrates profile in yeast, and economic aspects are discussed and future prospects are summarized.

Revealing the beneficial effect of protease supplementation to high gravity beer fermentations using "-omics" techniques

BackgroundAddition of sugar syrups to the basic wort is a popular technique to achieve higher gravity in beer fermentations, but it results in dilution of the free amino nitrogen (FAN) content in the medium. The multicomponent protease enzyme Flavourzyme has beneficial effect on the brewer's yeast fermentation performance during high gravity fermentations as it increases the initial FAN value and results in higher FAN uptake, higher specific growth rate, higher ethanol yield and improved flavour profile.ResultsIn the present study, transcriptome and metabolome analysis were used to elucidate the effect on the addition of the multicomponent protease enzyme Flavourzyme and its influence on the metabolism of the brewer's yeast strain Weihenstephan 34/70. The study underlines the importance of sufficient nitrogen availability during the course of beer fermentation. The applied metabolome and transcriptome analysis allowed mapping the effect of the wort sugar composition on the nitrogen uptake.ConclusionBoth the transcriptome and the metabolome analysis revealed that there is a significantly higher impact of protease addition for maltose syrup supplemented fermentations, while addition of glucose syrup to increase the gravity in the wort resulted in increased glucose repression that lead to inhibition of amino acid uptake and hereby inhibited the effect of the protease addition.

Evaluation of value-added components of dried distiller’s grain with solubles from triticale and wheat

This study focused on the detection of value-added co-products in dried distiller’s grain plus soluble (DDGS), a possibility that could open new avenues for further processing and marketing of DDGS and improving economic sustainability of ethanol industry. Varieties of triticale, wheat and two benchmarks, CPS wheat and Pioneer Hi-Bred corn, were fermented using two very high gravity (VHG) fermentation approaches: jet-cooking and raw starch processing (STARGEN fermentation). DDGS from STARGEN fermentation could be promising sources of value-added co-products. Pronghorn triticale DDGS (STARGEN fermentation) had the highest concentration of sterols (3.7 mg/g), phenolic compounds (13.61 mg GAE/g), and β-glucan (2.07%). CDC Ptarmigan DDGS (STARGEN fermentation) had the highest concentration of tocopherols and tocotrienols (107.0 μg/g), 1.93% of β-glucan, and 53.0 mg/g of fatty acids. AC Reed DDGS (STARGEN method) showed 1.97% of β-glucan. This study shows that proper choice of fermentation approach and feedstock for ethanol production could improve commercial quality of DDGS.

Screening and construction of Saccharomyces cerevisiae strains with improved multi-tolerance and bioethanol fermentation performance

In this study, a systemic analysis was initially performed to investigate the relationship between fermentation-related stress tolerances and ethanol yield. Based on the results obtained, two elite Saccharomyces cerevisiae strains, Z8 and Z15, with variant phenotypes were chosen to construct strains with improved multi-stress tolerance by genome shuffling in combination with optimized initial selection. After three rounds of genome shuffling, a shuffled strain, YZ1, which surpasses its parent strains in osmotic, heat, and acid tolerances, was obtained. Ethanol yields of YZ1 were 3.11%, 10.31%, and 10.55% higher than those of its parent strains under regular, increased heat, and high gravity fermentation conditions, respectively. YZ1 was applied to bioethanol production at an industrial scale. Results demonstrated that the variant phenotypes from available yeast strains could be used as parent stock for yeast breeding and that the genome shuffling approach is sufficiently powerful in combining suitable phenotypes in a single strain.

The influence of nitrogen sources on ethanol production by yeast from concentrated sweet sorghum juice

Ethanol production from concentrated sweet sorghum juice was carried out under very high gravity (VHG) fermentation conditions using Saccharomyces cerevisiae yeast strain at 30 °C. Different nitrogen sources (CO(NH2)2 or (NH4)2SO4), with the concentration of 0.8 g nitrogen L−1, were added to promote the ethanol yields and prevent the byproducts formation (no additional phosphorus). With an initial sugar concentration of 300 g L−1, the maximum ethanol concentration and cell number of the fermentation medium with CO(NH2)2 as nitrogen source (135 g L−1 and 5.5 × 108 mL−1, respectively) were higher compared to those of the control one (120 g L−1 and 4.7 × 108 mL−1, respectively). However, no positive effects of (NH4)2SO4 were observed compared with the control. The yield of main byproducts (glycerol and acetic acid) obtained was 0.027 g g−1 of consumed sugar when CO(NH2)2 was used as a nitrogen source, which was clearly lower than those for ammonium-grown culture (0.035 g g−1 consumed sugar) and the control (0.037 g g−1 consumed sugar). The lower values of glycerol and acetic acid yields indicate that CO(NH2)2 succeeds in the efficient balancing of the redox potential and reduces the formation of byproducts during VHG fermentation compared with that when, for example, ammonium salt is the only available nitrogen source. It was concluded that production of high levels of ethanol could be achieved by supplementing urea as nitrogen source during ethanol fermentation from concentrated sweet sorghum juice.

Genetic Characterization of Osmotolerant Fermentative Saccharomyces Yeasts from Tanzania Suitable for Industrial Very High Gravity Fermentation

Naturally occurring yeasts were sought from diverse Tanzanian environments and screened for industrial application. The yeasts were isolated from environments such as traditional brews and wines from various parts of Tanzania. In this regard, a total of thirty yeast isolates were screened for their suitability in Industrial Very High Gravity Fermentation (VHGF). Five of these isolates were able to grow and ferment medium with 40% initial sucrose concentration. Whereby, three were able to grow and ferment medium with 700g/Litre (70% w/w) initial sucrose concentration. One of the three isolates coded LB2 isolated from a traditional Makonde sorghum brew was able to ferment a medium with initial sucrose concentration of 1000g/Litre (100 % w/w) at 30 EsC. On the basis of PCR-RFLP of the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA), all the three most osmotolerant isolates were identified to belong to the Saccharomyces sensu stricto clade. Phylogenetic analysis of the 650bp D1D2 domain of the large subunit 26S rDNA of the isolate LB2 clustered this isolate away from the so far known typical osmotolerant yeasts. The fermentation by LB2 isolate of 100% gravity substrate observed in this work is higher than any other encountered in the literatures reviewed. Key words: Osmotolerant, PCR-RFLP, Saccharomyces, traditional brews, very high gravity fermentation (VHGF).

A novel approach for the improvement of ethanol fermentation by Saccharomyces cerevisiae

Fermentation properties under the control of multiple genes are difficult to alter with traditional methods in Saccharomyces cerevisiae. Here, a novel genome engineering approach is developed to improve ethanol production in very high gravity fermentation with 300 g/L glucose as the carbon source. This strategy involved constructing aneuploid strains on the base of tetraploid cells. The tetraploid strain was constructed by using the plasmid YCplac33-GHK, which harbored the HO gene encoding the site-specific Ho endonucleases. The aneuploid strain, WT4-M, was selected and screened after the tetraploid cells were treated with methyl benzimidazole-2-yl-carbamate to induce loss of mitotic chromosomes. It was found that aneuploid strain WT4-M not only exhibited an increase in ethanol production and osmotic and thermal tolerance, but also an improvement in the sugar-ethanol conversion rate. Notably, WT4-M provided up to 9.8% improvement in ethanol production compared with the control strain. The results demonstrated that the strategy of aneuploidy was valuable for creating yeast strains with better fermentation characteristics.

Mechanisms of ethanol tolerance in Saccharomyces cerevisiae

Saccharomyces cerevisiae is a superb ethanol producer, yet is also sensitive to higher ethanol concentrations especially under high gravity or very high gravity fermentation conditions. Ethanol tolerance is associated with interplay of complex networks at the genome level. Although significant efforts have been made to study ethanol stress response in past decades, mechanisms of ethanol tolerance are not well known. With developments of genome sequencing and genomic technologies, our understanding of yeast biology has been revolutionarily advanced. More evidence of mechanisms of ethanol tolerance have been discovered involving multiple loci, multi-stress, and complex interactions as well as signal transduction pathways and regulatory networks. Transcription dynamics and profiling studies of key gene sets including heat shock proteins provided insight into tolerance mechanisms. A transient gene expression response or a stress response to ethanol does not necessarily lead to ethanol tolerance in yeast. Reprogrammed pathways and interactions of cofactor regeneration and redox balance observed from studies of tolerant yeast demonstrated the significant importance of a time-course study for ethanol tolerance. In this review, we focus on current advances of our understanding for ethanol-tolerance mechanisms of S. cerevisiae including gene expression responses, pathway-based analysis, signal transduction and regulatory networks. A prototype of global system model for mechanisms of ethanol tolerance is presented.

Global transcription engineering of brewer’s yeast enhances the fermentation performance under high-gravity conditions

Global transcription engineering was developed as a tool to reprogram gene transcription for eliciting new phenotypes important for technological applications (Science 2006, 314(5805):1565-1568). A recent report indicated that the beneficial growth advantage of yeast cells expressing the SPT15-300 mutation is the result of enhanced uptake and/or improved utilization of leucine and thus was seen only on defined media with low concentrations of leucine (Appl Environ Microbiol 2009, 75(19):6055-6061). Further investigation towards a leucine-prototrophic strain of industrial lager brewer's yeast indicated that integration one copy of SPT15-300 in SPT15 allele, however, did lead to an increased ethanol tolerance on complex rich medium at high gravity fermentation condition. Under brewing conditions, the SPT15-300 mutant produced 80.78 g/L ethanol from 200 g/L carbohydrates after 384 h, almost twice as much as that of the wild-type strain. The results convinced us that the effect of global regulator modification of yeast is at multi-genes level and is extremely complicated.