d-Xylulose Fermentation to Ethanol by Saccharomyces cerevisiae

We used commercial bakers' yeast (Saccharomyces cerevisiae) to study the conversion of d-xylulose to ethanol in the presence of d-xylose. The rate of ethanol production increased with an increase in yeast cell density. The optimal temperature for d-xylulose fermentation was 35 degrees C, and the optimal pH range was 4 to 6. The fermentation of d-xylulose by yeast resulted in the production of ethanol as the major product; small amounts of xylitol and glycerol were also produced. The production of xylitol was influenced by pH as well as temperature. High pH values and low temperatures enhanced xylitol production. The rate of d-xylulose fermentation decreased when the production of ethanol yielded concentrations of 4% or more. The slow conversion rate of d-xylulose to ethanol was increased by increasing the yeast cell density. The overall production of ethanol from d-xylulose by yeast cells under optimal conditions was 90% of the theoretical yield.     

Ethanol Tolerance in the Yeast Saccharomyces cerevisiae Is Dependent on Cellular Oleic Acid Content

In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Δ⁹Z-C_16:1) and oleic acid (Δ⁹Z-C_18:1), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C_16:0) and stearic acid (C_18:0), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Δ⁹ and Δ¹¹) and substrate chain-length preferences (i.e., C_16:0 and C_18:0); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Δ¹¹Z-C_18:1), whereas neither Δ¹¹Z-C_16:1 nor palmitoleic acid (Δ⁹Z-C_16:1) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.     

Improved Bioreaction Kinetics for the Simulation of Continuous Ethanol Fermentation by Saccharomyces cerevisiae

A kinetic model for the production of ethanol by Saccharomyces cerevisiae has been developed from semiempirical analysis. The values for the parameters in this model were then determined by nonlinear multiple regression using the data of Bazua and Wilke ( 1977). The final equations were μ=0.427s(1-(p/101.6)^1.95)/(0.245+s), Y_X/p=0.291, and Y_X/s=0.152(1-p/302.3). This model was then used to simulate a continuous stirred tank fermentor (CSTF) and compared to other models using the same experimental data but different kinetics. The equations required to use these kinetics in a CSTF with recycle were then developed. From this simulation, it was found that, for a CSTF with recycle, the best configuration to operate is an external recycle, with a low bleed and recycle ratio.     

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation

At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu²⁺ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu²⁺ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu²⁺ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.     

Engineering a Saccharomyces cerevisiae Wine Yeast That Exhibits Reduced Ethanol Production during Fermentation under Controlled Microoxygenation Conditions

We recently showed that expressing an H₂O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD⁺ reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.     

Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae

Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases in Saccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.     

Antisense-Mediated Inhibition of Acid Trehalase (ATH1) Gene Expression Promotes Ethanol Fermentation and Tolerance in Saccharomyces cerevisiae

Acid trehalase gene (ATH1) expression was decreased using the antisense-RNA technique in Saccharomyces cerevisiae. The 500 bp DNA fragments containing anti-ATH1 gene between +1 and +500 were amplified using PCR and fused to yeast ADH1, CYC1 and ATH1 promoters. Yeast cells harboring the recombinant plasmids had a low activity of acid trehalase and promoted ethanol fermentation compared to the control yeast cells harboring the vector plasmid only. The recombinant yeast had a high viability with 8% (v/v) ethanol.     

Screening of Non- Saccharomyces cerevisiae Strains for Tolerance to Formic Acid in Bioethanol Fermentation

Formic acid is one of the major inhibitory compounds present in hydrolysates derived from lignocellulosic materials, the presence of which can significantly hamper the efficiency of converting available sugars into bioethanol. This study investigated the potential for screening formic acid tolerance in non-Saccharomyces cerevisiae yeast strains, which could be used for the development of advanced generation bioethanol processes. Spot plate and phenotypic microarray methods were used to screen the formic acid tolerance of 7 non-Saccharomyces cerevisiae yeasts. S. kudriavzeii IFO1802 and S. arboricolus 2.3319 displayed a higher formic acid tolerance when compared to other strains in the study. Strain S. arboricolus 2.3319 was selected for further investigation due to its genetic variability among the Saccharomyces species as related to Saccharomyces cerevisiae and availability of two sibling strains: S. arboricolus 2.3317 and 2.3318 in the lab. The tolerance of S. arboricolus strains (2.3317, 2.3318 and 2.3319) to formic acid was further investigated by lab-scale fermentation analysis, and compared with S. cerevisiae NCYC2592. S. arboricolus 2.3319 demonstrated improved formic acid tolerance and a similar bioethanol synthesis capacity to S. cerevisiae NCYC2592, while S. arboricolus 2.3317 and 2.3318 exhibited an overall inferior performance. Metabolite analysis indicated that S. arboricolus strain 2.3319 accumulated comparatively high concentrations of glycerol and glycogen, which may have contributed to its ability to tolerate high levels of formic acid.     

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter⁻¹ acetate during fermentation of 114 g liter⁻¹ glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter⁻¹, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter⁻¹ and raised the ethanol yield to 7% above the wild-type level.     

Kinetics of Ethanol Oxidation Catalyzed by Yeast Alcohol Dehydrogenase in Aqueous Solutions of Sodium Dodecylsulfate

A study has been made of the effect of sodium dodecylsufate (SDS) addition on the oxidation of ethanol catalyzed by yeast alcohol dehydrogenase. Experiments were performed at pH = 8.1 and SDS concentrations employed were below and above the surfactant critical micelle concentration (CMC). The double reciprocal plots obtained in the absence and in the presence of the surfactant were compatible with a sequential bi-bi ordered mechanism. In the presence of the surfactant the initial reaction rates were consistently lower than in pure buffer at all the surfactant concentrations considered (0.5-50 mM). This effect is mainly due to an increase in the dissociation constant of beta-NAD(+) which reaches its maximum value (7,100 +/- 1,700 microM) at the CMC. Above the CMC the effect of the surfactant is mainly due to an increase in the Michaels constants of the alcohol, with values of 41 +/- 1 mM for 15 mM SDS and 50 +/- 1 mM for 50 mM SDS. The catalytic rate constant was found to be practically independent of the presence of the surfactant in the range of concentrations considered (up to 50 mM).     

Ascospore Formation in the Yeast Saccharomyces cerevisiae

Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.     

Atg9 Trafficking in Yeast Saccharomyces cerevisiae

Autophagy can be divided into selective and non-selective modes. This process is considered selective when a precise cargo is specifically and exclusively incorporated into autophagosomes, the double-membrane vesicles that are the hallmark of autophagy. In contrast, during nonselective, bulk autophagy, cytoplasmic components are randomly enwrapped into autophagosomes. To date, approximately 30 autophagy-related genes called ATG have been identified. Sixteen of them compose the general basic machinery catalyzing the formation of double-membrane vesicles in all eukaryotic cells. The rest of them are often not conserved between species and cooperate with the basic Atg proteins during either selective or nonselective autophagy. Atg9 is the only integral membrane component of the conserved Atg machinery and appears to be a crucial organizational element.5 Recent studies in the S. cerevisiae have shown that Atg9 transport is differentially regulated depending on the autophagy mode. In this addendum, we will review and discuss what has recently been unveiled about yeast S. cerevisiae Atg9 trafficking, its modulators and its potential role in double-membrane vesicle biogenesis.

Addendum to:

Atg9 Sorting from Mitochondria is Impaired in Early Secretion and VFT Complex Mutants in Saccharomyces cerevisiae

F. Reggiori and D.J. Klionsky

J Cell Sci 2006: 119:2903-11     

Ethanol tolerance in free and sol-gel immobilised Saccharomyces cerevisiae

The tolerance of sol-gel immobilised and free Saccharomyces cerevisiae to ethanol was studied. The effects of ethanol preincubation time showed that the specific death velocity decreased from 2×10⁵ c.f.u. min^–1 for free cells to 2×10⁴ c.f.u. min^–1 for immobilised cells thus indicating that immobilised yeast was far less sensitive to the ethanol damage. The specific glucose consumption of immobilised and free cells on a per cell basis was 3×10^–12 g cell^–1 h^–1 and 9×10^–12 g cell^–1 h^–1, respectively.     

Glucosylceramide Contained in Koji Mold-Cultured Cereal Confers Membrane and Flavor Modification and Stress Tolerance to Saccharomyces cerevisiae during Coculture Fermentation

In nature, different microorganisms create communities through their physiochemical and metabolic interactions. Many fermenting microbes, such as yeasts, lactic acid bacteria, and acetic acid bacteria, secrete acidic substances and grow faster at acidic pH values. However, on the surface of cereals, the pH is neutral to alkaline. Therefore, in order to grow on cereals, microbes must adapt to the alkaline environment at the initial stage of colonization; such adaptations are also crucial for industrial fermentation. Here, we show that the yeast Saccharomyces cerevisiae, which is incapable of synthesizing glucosylceramide (GlcCer), adapted to alkaline conditions after exposure to GlcCer from koji cereal cultured with Aspergillus kawachii. We also show that various species of GlcCer derived from different plants and fungi similarly conferred alkali tolerance to yeast. Although exogenous ceramide also enhanced the alkali tolerance of yeast, no discernible degradation of GlcCer to ceramide was observed in the yeast culture, suggesting that exogenous GlcCer itself exerted the activity. Exogenous GlcCer also increased ethanol tolerance and modified the flavor profile of the yeast cells by altering the membrane properties. These results indicate that GlcCer from A. kawachii modifies the physiology of the yeast S. cerevisiae and demonstrate a new mechanism for cooperation between microbes in food fermentation.