Proteomic analysis of responses to osmotic stress in laboratory and sake-brewing strains of Saccharomyces cerevisiae

The difference in responses to osmotic stress between the laboratory and sake-brewing strains of Saccharomyces cerevisiae at the translational level was compared by two-dimensional polyacrylamide gel electrophoresis. Proteins, whose production was significantly changed by the osmotic stress, were identified by peptide mass fingerprinting. In the laboratory strain, translation of Hor2p, the protein responsible for glycerol biosynthesis, and Ald6p, related to acetate biosynthesis, was induced under high osmotic pressure conditions. In addition, production of proteins related to translation and stress response was also changed under this condition. On the other hand, in the sake-brewing strain, translation of Hor2p, Hsp26p, and some stress-related proteins was upregulated. The change in the production of enzymes related to glycolysis and ethanol formation was small; however, the production of enzymes related to glycerol formation increased in both strains. These results suggest that enhancement of glycerol formation due to enhancement of the translation of proteins, such as Hor2p, is required for growth of S. cerevisiae under high osmotic pressure condition. This is the first report on the analysis of responses of a sake-brewing strain to high osmotic pressure stress based on proteomics.     

Xylose utilisation by recombinant strains of Saccharomyces cerevisiae on different carbon sources

Autoselective xylose-utilising strains of Saccharomyces cerevisiae expressing the xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes of Pichia stipitis were constructed by replacing the chromosomal FUR1 gene with a disrupted fur1::LEU2 allele. Anaerobic fermentations with 80 g l⁻¹ d-xylose as substrate showed a twofold higher consumption of xylose in complex medium compared to defined medium. The xylose consumption rate increased a further threefold when 20 g l⁻¹ d-glucose or raffinose was used as co-substrate together with 50 g l⁻¹ d-xylose. Xylose consumption was higher with raffinose as co-substrate than with glucose (85% versus 71%, respectively) after 82 h fermentations. A high initial ethanol concentration and moderate levels of glycerol and acetic acid accompanied glucose as co-substrate, whereas the ethanol concentration gradually increased with raffinose as co-substrate with no glycerol and much less acetic acid formation. Received: 12 March 1999 / Received revision: 31 June 1999 / Accepted: 5 July 1999     

Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains

Background  

Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials.     

Production of natural products through metabolic engineering of Saccharomyces cerevisiae

1. Download : Download high-res image (77KB)
2. Download : Download full-size image

     

Genetic determination of polygalacturonase production in wild-type and laboratory strains of Saccharomyces cerevisiae

The genetic determination of polygalacturonase (PG) production in Saccharomyces cerevisiae was studied by biochemical and classical genetic techniques. Crosses of PG⁺ strains with PG^– strains showed that in the haploid wild-type-derived strain, two structural genes were involved in the production of a hydrolysis halo on plates with polygalacturonic acid. However, in the case of PG⁺ laboratory strain IM1-8b, the phenotype was controlled by only one structural gene although the analysis of PG^– IM1-8b mutants demonstrated the existence of at least two complementation groups. All these genetic results were assessed biochemically by means of cation-exchange chromatography. Two enzymes were separated in the wild-type strain, and only one in the laboratory strain. The three enzymes had different K _m values, molecular masses, and optimal pHs for activity. Received: 24 October 1996 / Accepted: 17 December 1996     

Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae

Laboratory strains of yeast ( Saccharomyces cerevisiae ) based on S288C ferment grape juice relatively poorly. We show that slow fermentation appears to be inherent to this strain, because the original S288C isolate shows fermentation similar to current laboratory isolates. We demonstrate further that some auxotrophic mutations in the laboratory strain show reduced rates of fermentation in grape juice, with lysine auxotrophs particularly impaired compared with isogenic Lys⁺ strains. Supplementing lysine at a 10-fold higher concentration than recommended allowed yeast cultures to reach higher final cell densities and restored the fermentation rate of auxotrophic strains to those of the corresponding wild-type strains. However, even with the additional supplementation, the fermentation rates of S288C strains were still slower than those of a commercial wine yeast strain. Conditions were developed that enable auxotrophic laboratory strains derived from S288C to ferment grape juice to completion with high efficiency on a laboratory scale. Fermentation in media based on grape juice will allow the suite of molecular genetic tools developed for these laboratory strains to be used in investigations of complex ferment characteristics and products.     

Isolation of freeze-tolerant laboratory strains of Saccharomyces cerevisiae from proline-analogue-resistant mutants

Since some amino acids, polyols and sugars in cells are thought to be osmoprotectants, we expected that several amino acids might also contribute to enhancing freeze tolerance in yeast cells. In fact, proline and charged amino acids such as glutamate, arginine and lysine showed a marked cryoprotective activity nearly equivalent to that of glycerol or trehalose, both known as major cryoprotectants for Saccharomyces cerevisiae. To investigate the cryoprotective effect of proline on the freezing stress of yeast, we isolated proline-analogue-resistant mutants derived from a proline-non-utilizing strain of S. cerevisiae. When cultured in liquid minimal medium, many mutants showed a prominent increase, two- to approximately tenfold, in cell viability compared to the parent after freezing in the medium at −20 °C for 1 week. Some of the freeze-tolerant mutants were found to accumulate a higher amount of proline, as well as of glutamate and arginine which are involved in proline metabolism. It was also observed that proline-non-utilizer and the freeze-tolerant mutants were able to grow against osmotic stress. These results suggest that the increased flux in the meta-bolic pathway of specific amino acids such as proline is effective for breeding novel freeze-tolerant yeasts. Received: 6 November 1996 / Accepted: 7 December 1996     

Mechanisms of strontium uptake by laboratory and brewing strains of Saccharomyces cerevisiae.

Laboratory and brewing strains of Saccharomyces cerevisiae were compared for metabolism-independent and -dependent Sr2+ uptake. Cell surface adsorption of Sr2+ to live cells was greater in the brewing than in the laboratory strain examined. However, uptake levels were greater in denatured (dried and ground) S. cerevisiae, and the relative affinities of Sr2+ for the two strains were reversed. Results for the brewing S. cerevisiae strain were similar whether the organism was obtained fresh from brewery waste or after culturing under the same conditions as for the laboratory strain. Reciprocal Langmuir plots of uptake data for live biomass were not linear, whereas those for denatured biomass were. The more complex Sr2+ binding mechanism inferred for live S. cerevisiae was underlined by cation displacement experiments. Sr2+ adsorption to live cells resulted in release of Mg2+, Ca2+, and H+, suggesting a combination of ionic and covalent bonding of Sr2+. In contrast, Mg2+ was the predominant exchangeable cation on denatured biomass, indicating primarily electrostatic attraction of Sr2+. Incubation of live S. cerevisiae in the presence of glucose resulted in a stimulation of Sr2+ uptake. Cell fractionation revealed that this increased Sr2+ uptake was mostly due to sequestration of Sr2+ in the vacuole, although a small increase in cytoplasmic Sr2+ was also evident. No stimulation or inhibition of active H+ efflux resulted from metabolism-dependent Sr2+ accumulation. However, a decline in cytoplasmic, and particularly vacuolar, Mg2+, in comparison with that of cells incubated with Sr2+ in the absence of glucose, was apparent. This was most marked for the laboratory S. cerevisiae strain, which contained higher Mg2+ levels than the brewing strain.     

Mechanisms of strontium uptake by laboratory and brewing strains of Saccharomyces cerevisiae.

Laboratory and brewing strains of Saccharomyces cerevisiae were compared for metabolism-independent and -dependent Sr2+ uptake. Cell surface adsorption of Sr2+ to live cells was greater in the brewing than in the laboratory strain examined. However, uptake levels were greater in denatured (dried and ground) S. cerevisiae, and the relative affinities of Sr2+ for the two strains were reversed. Results for the brewing S. cerevisiae strain were similar whether the organism was obtained fresh from brewery waste or after culturing under the same conditions as for the laboratory strain. Reciprocal Langmuir plots of uptake data for live biomass were not linear, whereas those for denatured biomass were. The more complex Sr2+ binding mechanism inferred for live S. cerevisiae was underlined by cation displacement experiments. Sr2+ adsorption to live cells resulted in release of Mg2+, Ca2+, and H+, suggesting a combination of ionic and covalent bonding of Sr2+. In contrast, Mg2+ was the predominant exchangeable cation on denatured biomass, indicating primarily electrostatic attraction of Sr2+. Incubation of live S. cerevisiae in the presence of glucose resulted in a stimulation of Sr2+ uptake. Cell fractionation revealed that this increased Sr2+ uptake was mostly due to sequestration of Sr2+ in the vacuole, although a small increase in cytoplasmic Sr2+ was also evident. No stimulation or inhibition of active H+ efflux resulted from metabolism-dependent Sr2+ accumulation. However, a decline in cytoplasmic, and particularly vacuolar, Mg2+, in comparison with that of cells incubated with Sr2+ in the absence of glucose, was apparent. This was most marked for the laboratory S. cerevisiae strain, which contained higher Mg2+ levels than the brewing strain.     

Chromosome instability in industrial strains of Saccharomyces cerevisiae batch cultivated under laboratory conditions

Industrial ethanol fermentation is a complex microbiological process to which yeast cells must adapt for survival. One of the mechanisms for adaptation is thought to involve chromosome rearrangements. We found that changes in chromosome banding patterns measured by pulsed-field gel electrophoresis can also be produced in laboratory media under simulated industrial conditions. Based on analysis of their generational variation, we found that these chromosome changes were specific to the genetic backgrounds of the initial strains. We conclude that chromosome rearrangements could be one of the factors involved in yeast cell adaptation to the industrial environment.     

Differences between pectic enzymes produced by laboratory and wild-type strains of Saccharomyces cerevisiae

The laboratory strain of S. cerevisiae, IM1-8b, showed pectolytic activity in the presence of either glucose, fructose, or sucrose as the carbon source, but not with galactose. The enzyme activity was rapidly lost with shaking. The optimum pH and temperature for activity were 4.5 and 45°C, respectively. The enzyme was an endopolygalacturonase, since it preferentially hydrolysed pectate over pectin and decreased the viscosity of a 5% polygalacturonic solution by about 30% in 30min producing oligogalacturonic acid and digalacturonic acid as end-products.     

Comparative metabolic network analysis of two xylose fermenting recombinant Saccharomyces cerevisiae strains

The recombinant xylose fermenting strain Saccharomyces cerevisiae TMB3001 can grow on xylose, but the xylose utilisation rate is low. One important reason for the inefficient fermentation of xylose to ethanol is believed to be the imbalance of redox co-factors. In the present study, a metabolic flux model was constructed for two recombinant S. cerevisiae strains: TMB3001 and CPB.CR4 which in addition to xylose metabolism have a modulated redox metabolism, i.e. ammonia assimilation was shifted from being NADPH to NADH dependent by deletion of gdh1 and over-expression of GDH2. The intracellular fluxes were estimated for both strains in anaerobic continuous cultivations when the growth limiting feed consisted of glucose (2.5 g L-1) and xylose (13 g L-1). The metabolic network analysis with 13C labelled glucose showed that there was a shift in the specific xylose reductase activity towards use of NADH as co-factor rather than NADPH. This shift is beneficial for solving the redox imbalance and it can therefore partly explain the 25% increase in the ethanol yield observed for CPB.CR4. Furthermore, the analysis indicated that the glyoxylate cycle was activated in CPB.CR4.     

Response of Saccharomyces cerevisiae strains to antineoplastic agents

The effect of several antineoplastic agents on Saccharomyces cerevisiae strains has been investigated. Minimum inhibitory concentration (MIC), minimum cytotoxic concentration (MCC) and median effective concentration (EC₅₀) were determined to identify strains with inherent sensitivity to the agents tested. Several strains proved to be sensitive to the antimetabolites 5-fluorouracil and methotrexate as well as to doxorubicin and cis-platine. On the contrary m -amsacrine, procarbazine, vinca alcaloids, melphalan and hydroxyurea were inactive at concentrations up to 400 μg ml ⁻¹. The strain ATCC 2366, the most relatively sensitive to the agents tested, was used for studying the effect of treatment duration and of drug concentration on cell survival. Methotrexate and cis-platine, which according to MIC and MCC tests seemed ineffective for this strain, reduced survival significantly after 6 h of treatment. A correlation of the shape of the survival curves with MIC and MCC values was attempted.     

Transmission of killer activity into laboratory and industrial strains of Saccharomyces cerevisiae by electroinjection

The killer character was electrically introduced into protoplasts of three yeast strains. These were the killer-negative variant of the K1 killer strain Saccharomyces cerevisiae T 158 C (his-); the killer-sensitive laboratory strain S. cerevisiae AH 215 (leu-, his-); and the killer-sensitive industrial strain S. cerevisiae AS 4/H2 (rho-). The killer dsRNA used for electroinjection was isolated from the super-killer strain S. cerevisiae T 158 C. Optimum numbers of transformed cells were obtained after regeneration and selection in appropriate media if the protoplasts were exposed to three exponentially decaying field pulses of 18.2 kV/cm strength and 40 microseconds duration at 4 degrees C. In the case of the killer-negative variant of S. cerevisiae T 158 C the majority of the protoplasts were transformed, whereas in the case of the two other strains the yield of transformed clones was much less. This latter result is expected if the expression of the electroinjected dsRNA was diminished in these two strains. Gel electrophoresis of the dsRNA of the clones of the three strains supported the conclusion that the transformed clones exhibited killer activity. The transformed clones of all three species were stable.