Comparative analysis of fermentation and enzyme expression profiles among industrial Saccharomyces cerevisiae strains

Applied Microbiology and Biotechnology - Industrial diploid strains of Saccharomyces cerevisiae are selected from natural populations and then domesticated by optimizing the preferred properties...     

Xylose fermentation by Saccharomyces cerevisiae

We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), baker's yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipilis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by baker's yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the non-oxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-1,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae XYL1/XYL2 transformants. Correspondence to: M. Ciriacy     

Maltotriose fermentation by Saccharomyces cerevisiae

Maltotriose, the second most abundant sugar of brewer's wort, is not fermented but is respired by several industrial yeast strains. We have isolated a strain capable of growing on a medium containing maltotriose and the respiratory inhibitor, antimycin A. This strain produced equivalent amounts of ethanol from 20 g l⁻¹ glucose, maltose, or maltotriose. We performed a detailed analysis of the rates of active transport and intracellular hydrolysis of maltotriose by this strain, and by a strain that does not ferment this sugar. The kinetics of sugar hydrolysis by both strains was similar, and our results also indicated that yeast cells do not synthesize a maltotriose-specific α-glucosidase. However, when considering active sugar transport, a different pattern was observed. The maltotriose-fermenting strain showed the same rate of active maltose or maltotriose transport, while the strain that could not ferment maltotriose showed a lower rate of maltotriose transport when compared with the rates of active maltose transport. Thus, our results revealed that transport across the plasma membrane, and not intracellular hydrolysis, is the rate-limiting step for the fermentation of maltotriose by these Saccharomyces cerevisiae cells. Journal of Industrial Microbiology & Biotechnology (2001) 27, 34–38. Received 13 January 2001/ Accepted in revised form 29 May 2001     

Relationship between fermentation capability and fatty acid composition of free and immobilized Saccharomyces cerevisiae

Summary The fatty acid composition of Saccharomyces cerevisiae immobilized by entrapment in calcium-alginate beads or adsorption on sintered glass was compared to that of freely suspended cells under different fermentation conditions. The fermentation product ethanol was found to cause a shift towards saturation in the fatty acid composition under anaerobic conditions. Immobilized cells contained significantly higher percentages of saturated fatty acyl residues, especially of palmitic acid (16:0), and a decreased amount of oleic acid (18:1) compared with free cells. The percentage saturation of total fatty acid composition correlates positively with improved fermentation rates obtained with the immobilized cells. This enhanced saturation of fatty acid composition in immobilized cells may be due to altered osmotic conditions in the microenvironment of the cells.     

Effect of iron ions on the antioxidant enzyme activities in yeast Saccharomyces cerevisiae

A complex of physiological and biochemical indices has been compared in wild and isogenic catalase-deficient strains of Saccharomyces cerevisiae grown on the media with different iron ion concentrations is 2 times higher in cytosolic catalase deficient yeast. Superoxide dismutase activity grown in the medium with 500 microM of ferrous sulphate. Under such conditions, peroxisomal catalase deficient yeast had a 2-fold decreased activity of superoxide dismutase. There is a significant difference between TBA-reactive substances content of the wild and cytosolic catalase deficient strain. It has been suggested that the repletion of iron ions in the growth medium leads to the formation of lipid oxidation products. Catalase prevents TBA-reactive substances formation in the given conditions and plays a protective role.     

Effect of hydrogen peroxide on antioxidant enzyme activities in Saccharomyces cerevisiae is strain-specific

The effect of hydrogen peroxide on the survival and activity of antioxidant and associated enzymes in Saccharomyces cerevisiae has been studied. A difference found in the response of wild-type yeast strains treated with hydrogen peroxide was probably related to the different protective effects of antioxidant enzymes in these strains. Exposure of wild-type YPH250 cells to 0.25 mM H₂O₂ for 30 min increased activities of catalase and superoxide dismutase (SOD) by 3.4-and 2-fold, respectively. However, no activation of catalase in the EG103 strain, as well as of SOD in the YPH98 and EG103 wild strains was detected, which was in parallel to lower survival of these strains under oxidative stress. There is a strong positive correlation (R ² = 0.95) between activities of catalase and SOD in YPH250 cells treated with different concentrations of hydrogen peroxide. It is conceivable that catalase would protect SOD against inactivation caused by oxidative stress and vice versa. Finally, yeast cell treatment with hydrogen peroxide can lead to either a H₂O₂-induced increase in activities of antioxidant and associated enzymes or their decrease depending on the H₂O₂ concentration used or the yeast strain specificity. Published in Russion in Biokhimiya, 2006, Vol. 71, No. 9, pp. 1243–1252.     

Improvement of maltotriose fermentation by Saccharomyces cerevisiae

AIMS: To enhance the fermentation of maltotriose by industrial Saccharomyces cerevisiae strains. METHODS AND RESULTS: The capability to ferment maltotriose by an industrial yeast strain that uses this sugar aerobically was tested in shake flasks containing rich medium. While the presence of maltose in the medium did not improve maltotriose fermentation, enhanced and constitutive expression of the AGT1 permease not only increased the uptake of maltotriose, but allowed efficient maltotriose fermentation by this strain. Supplementation of the growth medium with 20 mmol magnesium l(-1) also increased maltotriose fermentation. CONCLUSIONS: Over expression of the AGT1 permease and magnesium supplementation improved maltotriose fermentation by an industrial yeast strain that respired but did not ferment this sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the roles of the AGT1 permease and nutrients in the fermentation of all sugars present in starch hydrolysates, a highly desirable trait for several industrial yeasts.     

Peptidase activities in Saccharomyces cerevisiae.

At least four distinct aminopeptidase activities and a single dipeptidase activity were found in cell extracts of a leucine-lysine auxotroph of Saccharomyces cerevisiae. The assay for peptidase activity involved polyacrylamide gel electrophoresis followed by an enzyme-coupled activity staining procedure. The aminopeptidases had largely overlapping specificities but could be distinguished from one another by their electrophoretic mobilities and activities toward different peptide substrates. Substrates tested included both free and blocked di- and tripeptides and amino acid derivatives.     

High-cell-density fermentation of recombinant Saccharomyces cerevisiae using glycerol

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 microm plasmid, pYES2 containing a GAL1 promoter for expression of the beta-galactosidase gene), the yeast was grown with glycerol as the substrate by fed-batch fermentation. The feeding strategy was based on an on-line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on-line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed-batch mode with pH control at 5.0 +/- 0.1 was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5-3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.     

Reaction kinetics of d-glucose fermentation by Saccharomyces cerevisiae

Data obtained on the conversion of d-glucose to alcohol using Saccharomyces cerevisiae in batch culture has been analysed kinetically. The effects of different kinetic parameters, e.g. rates of ethanol and biomass formation, rate of d-glucose utilization and variation of pH have been studied. Analysis of data was made on the basis of Michaelis-Menten, Leudeking-Piret and simple kinetics. Unsteady rate behaviour in the lag phase was observed and explained.     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60–3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8–13% vs. 33–47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60-3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8-13% vs. 33-47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

Genetic improvement of Saccharomyces cerevisiae for xylose fermentation

There is considerable interest in recent years in the bioconversion of forestry and agricultural residues into ethanol and value-added chemicals. High ethanol yields from lignocellulosic residues are dependent on efficient use of all the available sugars including glucose and xylose. The well-known fermentative yeast Saccharomyces cerevisiae is the preferred microorganism for ethanol production, but unfortunately, this yeast is unable to ferment xylose. Over the last 15 years, this yeast has been the subject of various research efforts aimed at improving its ability to utilize xylose and ferment it to ethanol. This review examines the research on S. cerevisiae strains that have been genetically modified or adapted to ferment xylose to ethanol. The current state of these efforts and areas where further research is required are identified and discussed.     

Genetic and fermentation properties of the K2 killer yeast, Saccharomyces cerevisiae T206

Saccharomyces cerevisiae T206 K⁺R⁺, a K₂ killer yeast, was differentiated from other NCYC killer strains of S. cerevisiae on the basis of CHEF-karyotyping and mycoviral RNA separations. Genomic DNA of strain T206 was resolved into 13 chromosome bands, ranging from approximately 0.2 to 2.2 Mb. The resident virus in strain T206 yielded L and M RNA species of approximately 5.1 kb and 2.0 kb, respectively. In micro-scale vinifications, strain T206 showed a lethal effect on a K^-R^- mesophilic wine yeast. Metabolite accumulation and toxin activity were measured over a narrow pH range of 3.2 to 3.5. Contrary to known fermentation trends, the challenged fermentations were neither stuck nor protracted although over 70% of the cell population was killed. Toxin-sensitive cells showed cytosolic efflux.     

Ethanol fermentation of mahula (Madhuca latifolia L.) flowers using free and immobilized yeast Saccharomyces cerevisiae

There is a growing interest to find alternate bioresources for production of ethanol, apart from cane/sugar beet molasses and starchy crops like sweet sorghum, cassava and sweet potato. Mahula (Madhuca latifolia L.) is a forest tree abundantly available in the Indian subcontinent and its flowers are very rich in fermentable sugars (28.1-36.3 g 100 g(-1)). Batch fermentation of fresh and 12-month-stored flowers with free (whole cells) and immobilized cells of Saccharomyces cerevisiae (strain CTCRI) was carried out in 2-l Erlenmeyer flasks. The ethanol yields were 193 and 148 g kg(-1) (using free cells) and 205 and 152 g kg(-1) (using immobilized cells) from fresh and 12-month-stored mahula flowers, respectively.