Influence of the composition of the fusion medium on the yield of electrofused yeast hybrids

Abstract Electrofusion between cells of yeast strains with different genetic markers in isotonic sorbitol solutions leads to high yields of hybrids when 0.1 mM Ca²⁺ and 0.5 mM Mg²⁺ salts are aded. On average, 1000–2000 hybrids are obtained when electrofusion is performed (in a helical chamber) compared to a yield of about 40–120 in the absence of these bivalent cations. A further increase in yield can be achieved by the addition of 1 mg/ml albumin, which results in up to 4000 hybrids per experimental run. The entire fusion process leads to very reproducible results in the presence of these substances.     

Flux analysis of recombinant Saccharomyces cerevisiae YPB-G utilizing starch for optimal ethanol production

Genetically modified Saccharomyces cerevisiae strain (YPB-G) which secretes a bifunctional fusion protein that contains both Bacillus subtilis -amylase and Aspergillus awamori glucoamylase activities was used for the direct conversion of starch into ethanol. Starch was either supplied initially to different nutrient media or added instantaneously to the reactor at various discrete time instants (pulse feeding). Stoichiometric modeling was used to investigate the effects of initial substrate concentration and growth rate of the recombinant yeast culture on ethanol production. Reaction stoichiometries describing both the anabolism and catabolism of the microorganism were used as an input to flux balance analysis (FBA), the preferred metabolic modeling approach since the constructed stoichiometric network was underdetermined. Experiments for batch and fed-batch systems at different substrate concentrations were analyzed theoretically in terms of flux distributions using ethanol production rate as the maximization criteria. Calculated ethanol rates were in agreement with experimental measurements, suggesting that this recombinant microorganism is sufficiently evolved to optimize its ethanol production. The function of the main pathways of yeast metabolism (PPP, EMP, TCA) are discussed together with the node analyses of glucose-6-P and pyruvate branch points. Theoretical node analysis revealed that if the split ratio in G6P branch point is changed by genetic manipulations, the ethanol yield would be affected considerably.     

Use of live Saccharomyces cerevisiae cells as a biological response modifier in experimental infections

Abstract A short-term oral administration of live Saccharomyces cerevisiae cells, strain Sillix Hansen DSM 1883, resulted in enhanced resistance of mice toward infections with K. pneumoniae, S. pneumoniae and S. pyogenes A produced by intranasal inoculation. Yeast pre-treatment also increased the efficacy of antibiotic therapy in bacterial infections and of antiviral drugs in viral infections. Yeast treatment of animals stimulated phagocytosis, activated the complement system and induced interferon which are likely to represent the main mechanisms of action whereby pretreatment of mice with live S. cerevisiae cells increases resistance to infection. It is concluded that preventive administration of live Saccharomyces cerevisiae cells should be used for increasing resistance to bacterial infections, in particular of the respiratory tract, or to viral infections, as well as an adjunct to antibiotic and antiviral drug therapy.     

Effects of glucose, tetrapyrroles and protein kinase C activators on cell proliferation in cultures of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source. Cell proliferation was followed by colony counts on agar medium. Cells in the glycerol-supplemented medium divided less than once in 10 days. When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 × 10⁶ cells ml⁻¹ Addition of either of the protein kinase C activators oleoyl-acetylglycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium. However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h. Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S. cerevisiae : one operating through a protein kinase C system and another through a guanylate cyclase system.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Purification and characterization of α-glucosidases produced by Saccharomyces in response to three distinct maltose genes

α-Glucosidases or maltases (EC 3.2.1.20) were purified to electrophoretic homogeneity from a respective strain of Sacchromyces cerevisiae which carries a single MAL gene, either MALα, MALβ or MALγ, using gluconate-Sepharose affinity chromography and isoelectrofocusing. Of these maltases, two types of maltase were obtained from the MALγ strain, the pI values of which were 5.6 and 5.9. From the MALα and MALβ strain was obtained only one type of maltase with the pI at 5.6 which was identical to one of the maltases from the MALγ strain. These four maltases possessed the same properties, except for pI. They were monomers with molecular weights of between 66 000 and 67 000. With regard to the substrate specificity, they hydrolyzed maltose and sucrose exclusively but not α-methulglucoside nor maltooligosaccharide. They did not differ in immunological properties.     

Enantioselective hydrolysis of esters of secondary alcohols using lyophilized Bakers'' yeast

A number of acetoxycarboxylic acid esters were hydrolysed enantioselectively by Saccharomyces cerevisiae Hansen leading to chiral hydroxycarboxylic acid esters of high optical purity. The scope and limitations of this method with respect to the substitutional pattern of substrates were investigated. Subcellular localization of the hydrolytic activity on the plasma membrane led to the assumption that unspecific carboxyl esterases are responsible for hydrolysis of this type of substrate. Comparative experiments using viable cells and lyophilized cells as source of enzyme revealed the latter to be superior with respect to enantioselection and ease of handling.     

Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis

Summary Insertion of the transposable element Ty at the ADH4 locus results in increased levels of a new alcohol dehydrogenase (ADH) activity in Saccharomyces cerevisiae. The DNA sequence of this locus has been determined. It contains a long open reading frame which is not homologous to the other ADH isozymes that have been characterized in S. cerevisiae nor does it show obvious homology to Drosophila ADH. The hypothetical ADH does, however, show strong homology to the sequence of an iron-activated ADH from the bacterium Zymomonas mobilis. Thus ADH4 appears to encode an ADH structural gene which, along with the Zymomonas enzyme, may define a new family of alcohol dehydrogenases.Now The Plant Cell Research Institute, Inc., 6560 Trinity Court, Dublin, CA 94568, USA     

Factors encoded by and affecting the holding stability of yeast plasmid pSR1

Summary A 2 m DNA-like plasmid, pSR1, isolated from a strain of Zygosaccharomyces rouxii has three coding frames, P, S and R. Insertional inactivation of R completely abolished the intramolecular recombination, and the defect was complemented by an intact R frame on a coexistent plasmid molecule. The P and S regions were also transactive and important, but not essential, for the stable maintenance of the plasmid molecules. Insertional disruption of the P frame suggested that it produces a protein factor. Similar insertional disruption of the S frame affected the plasmid stability in Z. rouxii and Saccharomyces cerevisiae hosts differently, depending on whether the inserted DNA fragment was a short 8 bp SalI linker or a long (2.2 kb) DNA fragment. Results strongly suggested that the S region encodes two factors, one RNA and the other a protein, and that the S protein is compatible with a sprecific hostfactor in Z. rouxii, but not in S. cerevisiae. In addition, a cis-acting locus, Z, was found at a site in the plasmid molecule where no distinct open reading frames were located. No long direct repeats or inverted repeats were observed in the Z region, such as are found in the REP3 locus of 2 m DNA.     

Mutations that increase the mitotic stability of minichromosomes in yeast: characterization of RAR1

Summary In an attempt to identify proteins involved in the initiation of DNA replication, we have isolated a series of Saccharomyces cerevisiae mutants in which the function of putative replication origins is affected. The phenotype of these Rar^- (regulation of autonomous replication) mutants is to increase the mitotic stability of plasmids whose replication is dependent on weak ARS elements. These mutations are generally recessive and complementation analysis shows that mutations in several genes may improve the ability of weak ARS elements to function. One mutation (rar1-1) also confers temperature-sensitive growth, and thus an essential gene is affected. We have determined the DNA sequence of the RAR1 gene, which reveals an open reading frame for a 48.5 kDa protein. The RAR1 gene is linked to rna1 on chromosome XIII.