Showing papers on "Yeast published in 1985"

Transformation of yeasts of the genus pichia

Abstract: 57 Process for transforming yeast strains of the genus Pichia is disclosed. Novel yeast strains of the genus Pichia which can be transformed with recombinant DNA material are also disclosed. In addition, a method for isolating functional genes and other functional DNA sequences from yeast strains of the genus Pichia is described.

Heterologous protein secretion from yeast

Abstract: Secretion of calf prochymosin from yeast yields fully activable zymogen while production in the yeast cytoplasm yields insoluble, unactivable enzyme with aberrant disulfide bonding. Factors that in...

Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis.

Abstract: Xylose reductase from the xylose-fermenting yeast Pichia stipitis was purified to electrophoretic and spectral homogeneity via ion-exchange, affinity and high-performance gel chromatography. The enzyme was active with various aldose substrates, such as DL-glyceraldehyde, L-arabinose, D-xylose, D-ribose, D-galactose and D-glucose. Hence the xylose reductase of Pichia stipitis is an aldose reductase (EC 1.1.1.21). Unlike all aldose reductases characterized so far, the enzyme from this yeast was active with both NADPH and NADH as coenzyme. The activity with NADH was approx. 70% of that with NADPH for the various aldose substrates. NADP+ was a potent inhibitor of both the NADPH- and NADH-linked xylose reduction, whereas NAD+ showed strong inhibition only with the NADH-linked reaction. These results are discussed in the context of the possible use of Pichia stipitis and similar yeasts for the anaerobic conversion of xylose into ethanol.

Expression, Glycosylation, and Secretion of an Aspergillus Glucoamylase by Saccharomyces cerevisiae

Abstract: A strain of Saccharomyces cerevisiae capable of simultaneous hydrolysis and fermentation of highly polymerized starch oligosaccharides was constructed. The Aspergillus awamori glucoamylase enzyme, form GAI, was expressed in Saccharomyces cerevisiae by means of the promoter and termination regions from a yeast enolase gene. Yeast transformed with plasmids containing an intron-free recombinant glucoamylase gene efficiently secreted glucoamylase into the medium, permitting growth of the transformants on starch as the sole carbon source. The natural leader sequence of the precursor of glucoamylase (preglucoamylase) was processed correctly by yeast, and the secreted enzyme was glycosylated through both N- and O-linkages at levels comparable to the native Aspergillus enzyme. The data provide evidence for the utility of yeast as an organism for the production, glycosylation, and secretion of heterologous proteins.

Kinetics of ethanol inhibition in alcohol fermentation.

Abstract: The inhibitory effect of ethanol on yeast growth and fermentation has been studied for the strain Saccharomyces cerevisiae ATCC No. 4126 under anaerobic batch conditions. The results obtained reveal that there is no striking difference between the response of growth and ethanol fermentation. Two kinetic models are also proposed to describe the kinetic pattern of ethanol inhibition on the specific rates of growth and ethanol fermentation: microi/micro0 = 1 - (P/Pm)alpha (for growth) nui/nu0 = 1 - (P/P'm)beta (for ethanol production). The maximum allowable ethanol concentration above which cells do not grow was predicted to be 112 g/L. The ethanol-producing capability of the cells was completely inhibited at 115 g/L ethanol. The proposed models appear to accurately represent the experimental data obtained in this study and the literature data.

Effects of beta-pinene on yeast membrane functions.

Abstract: The effects of beta-pinene on yeast cells were studied. This terpene inhibited respiration with glucose or ethanol as the substrate. The inhibition depended on the ratio of the terpene to the amount of yeast cells; for a fixed concentration of pinene, inhibition decreased as the amount of yeast cells increased. Pinene also inhibited the pumping of protons and K+ transport, but this inhibition was more marked with with ethanol than with glucose as the substrate, indicating the mitochondrial localization of the inhibition. The studies on isolated mitochondria showed a series of effects, starting with the disappearance of the respiratory control and deenergization of the organelles and followed by an inhibition of respiration at higher concentrations of the terpene. The effect on respiration could be localized to the cytochrome b region of the electron transport chain. No effect could be detected on the activity of ATPase. The effects can be ascribed to a localization of pinene on membranes which was also accompanied by a decrease in the fluorescence polarization of diphenyl hexatriene, probably meaning an increase in the fluidity of the membrane, localized preferentially to the mitochondria.

Growth of Natural Yeast Flora during the Fermentation of Inoculated Wines.

Abstract: The growth of yeasts that occur naturally in grape juice was quantitatively examined during the fermentation of four wines that had been inoculated with Saccharomyces cerevisiae. Although S. cerevisiae dominated the wine fermentations, there was significant growth of the natural species Kloeckera apiculata, Candida stellata, Candida colliculosa, Candida pulcherrima, and Hansenula anomala.

Function and autoregulation of yeast copperthionein.

Abstract: The CUP1 gene of yeast encodes a small, metallothionein-like protein that binds to and is inducible by copper A gene replacement experiment shows that this protein protects cells against copper poisoning but is dispensable for normal cellular growth and development throughout the yeast life cycle The transcription of CUP1 is negatively autoregulated This feedback mechanism, which is mediated through upstream control sequences, may play an important role in heavy metal homeostasis

The synthesis and in vivo assembly of functional antibodies in yeast

Abstract: The yeast Saccharomyces cerevisiae can synthesize, process and secrete higher eukaryotic proteins1–5. We have investigated the expression of immunoglobulin chains in yeast and demonstrate here (1) the synthesis, processing and secretion of light and heavy chains, (2) the glycosylation of heavy chain, (3) the intracellular localization of these foreign proteins by immunofluorescence, and (4) the detection of functional antibodies in cells co-expressing both chains. This may provide the basis of a microbial fermentation process for the production of monoclonal antibodies. The co-expression of light and heavy chains in Escherichia coli has been reported but functional antibodies were not assembled in vivo6,7. Furthermore, only low-level assembly of these chains was found in vitro.

Mammalian and yeast ras gene products: biological function in their heterologous systems

Abstract: Activated versions of ras genes have been found in various types of malignant tumors. The normal versions of these genes are found in organisms as diverse as mammals and yeasts. Yeast cells that lack their functional ras genes, RASSC-1 and RASSC-2, are ordinarily nonviable. They have now been shown to remain viable if they carry a mammalian rasH gene. In addition, yeast-mammalian hybrid genes and a deletion mutant yeast RASSC-1 gene were shown to induce morphologic transformation of mouse NIH 3T3 cells when the genes had a point mutation analogous to one that increases the transforming activity of mammalian ras genes. The results establish the functional relevance of the yeast system to the genetics and biochemistry of cellular transformation induced by mammalian ras genes.

Extracellular polymer of Candida albicans: isolation, analysis and role in adhesion.

Abstract: SUMMARY: Extracellular polymeric material (EP) was isolated from culture supernatants of Candida albicans grown on carbon sources (50 mm-glucose, 500 mm-sucrose or 500 mm-galactose) known to promote yeast adhesion to different extents. Galactose-grown yeasts, which are the most adherent, produced more EP than sucrose-grown organisms, particularly after incubation for 5 d, while glucose-grown yeasts (the least adherent) gave the lowest yield. EP produced on all three carbon sources was of similar composition and contained carbohydrate (65 to 82%; mannose with some glucose), protein (7%), phosphorus (0·5%) and glucosamine (1·5%). Serological studies indicated that these EP preparations were immunologically identical but that galactose-grown yeasts had more antigenic determinants than sucrose-grown organisms while glucose-grown yeasts had the fewest determinants. Antigenic differences were apparent between EP preparations of some strains of C. albicans. Pretreatment of acrylic strips with EP to form a polymeric coating promoted yeast adhesion to the acrylic surface, but similar pretreatment of buccal epithelial cells with EP inhibited subsequent yeast adhesion. These results indicate that EP originates from the cell surface of C. albicans and that it contains the surface component(s), probably mannoprotein in nature, responsible for yeast adhesion.

Nucleotide sequence of the yeast ILV2 gene which encodes acetolactate synthase

Abstract: We have determined the nucleotide sequence of the yeast ILV2 gene which codes for the amino acid biosynthetic enzyme acetolactate synthase (ALS). ALS has recently been shown to be the target in bacteria, yeast and plants, of the potent new herbicide sulfometuron methyl. The coding sequence for the ILV2 polypeptide contains 2061 base pairs. Comparison of deduced amino acid sequences indicates considerable conservation between the yeast protein and the large subunits of the E. coli ALS II and ALS III isozymes. A major distinction between the three proteins is the presence of an additional 90 amino acids at the amino terminal of the yeast protein. The amino acid sequence in this region shows similarities to yeast mitochondrial transit sequences and may function as such, since yeast ALS is localized in the mitochondria. Consensus sequences for initiation and termination of transcription that are consistent with the ends of the ILV2 mRNA, as well as general amino acid control regulatory sequences have been identified.

Ethanol fermentation of crude acid hydrolyzate of cellulose using high-level yeast inocula

Abstract: High-level yeast inocula was investigated as a means of overcoming the toxicity problem in ethanol fermentation of acid hydrolyzate of wood cellulose. When the inoculum level exceeded 10(8) initial cells/mL, 50% of the yeast cells survived the initial cell death period during which furfural and HMF were depleted. The fermentation thus proceeded to completion by virtue of cell regrowth. The specific ethanol productivity in batch fermentation on the basis of viable cells was comparable to that of pure glucose fermentation. Continuous fermentation with cell recycle was superior to batch fermentation in that there was no overall cell decline and the ethanol yield was substantially higher. The maximum ethanol productivity in continuous fermentation was 4.9 g/L h and it occurred at a dilution rate of 0.24 hr(-1).

A novel method for transformation of intact yeast cells by electroinjection of plasmid DNA

Abstract: It was found that plasmid DNA (YEp13) can be introduced into intact yeast cells by electric field pulses. The frequency of transformation by this electroinjection method depend upon the initial electric field intensity, the capacitance of the electric discharge capacitor, and the number of pulses applied. A maximum number of transformants (90±20/μgDNA) was obtained by three successive pulses with an initial intensity of 5 KV/cm and with a capacitance of 1 μF. The present electroinjection method is simple, and transformants can be obtained within 2 to 3 days after transformation treatment.

Enhanced yeast transcription employing hybrid promoter region constructs

Abstract: ENHANCED YEAST TRANSCRIPTION EMPLOYING HYBRID PROMOTER REGION CONSTRUCTS ABSTRACT OF THE DISCLOSUREYeast promoters of glycolytic enzymes are modified by isolating a fragment encompassing the RNApolymerase binding site and joining to the 5' end of this fragment a DNA sequence providing for enhanced inducible or constitutive transcription of a structural gene. Constructs are prepared for efficient expression of foreign genes in yeast. Yeast strains 2150-2-3(pCl/1GAPSOD) and AB110(pCl/1GAPATi9), producing human .alpha.1-antitrypsin and superoxide dismutase, were deposited at the A.T.C.C. on May 9, 1984 and given Accession Nos. 20708 and 20709, respectively; and 2150-2-3(GAP5), 2150-2-3(Pyk5) and 2150-2-3(PHO5GAP1), expressing Hepatitis B surface anti-gen, were deposited at the A.T.C.C. on May 9, 1984 and given Accession Nos. 20705, 20706 and 20707, respective-ly.

Mutations affecting the enzymes involved in the utilization of 4-aminobutyric acid as nitrogen source by the yeast Saccharomyces cerevisiae.

Abstract: We present genetic evidence for the enzymes 4-aminobutyrate: 2-oxoglutarate aminotransferase (EC 2.6.1.19) and succinate-semialdehyde dehydrogenase [NAD(P)+] (EC 1.2.1.16) constituting the functional pathway for the utilization of 4-aminobutyric acid as a nitrogen source by Saccharomyces cerevisiae. We show that the pathway is induced by 4-aminobutyric acid and that the presence of the pathway enzymes probably requires the integrity of a positive control element.

Multiple chemical forms of hepatitis B surface antigen produced in yeast.

Abstract: Hepatitis B surface antigen (HBsAg) has been extracted from yeast cells that produce HBsAg. These cells contain the gene for surface antigen carried on a plasmid that replicates in the cells. Analysis of the yeast-derived HBsAg by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis shows that the antigen that is initially released from yeast cells is a high molecular weight aggregate of the fundamental Mr 25,000 subunit. Unlike HBsAg derived from human plasma, the yeast antigen is held together by noncovalent interactions and can be dissociated in 2% NaDodSO4 without the use of reducing agents. During in vitro purification of the yeast antigen, some disulfide bonds form spontaneously between the antigen subunits, resulting in a particle composed of a mixture of monomers and disulfide-bonded dimers. Treatment with 3 M thiocyanate converts the 20-nm particles into a fully disulfide-bonded form that is not disrupted in NaDodSO4 unless a reducing agent is added. This disulfide-bonded particle resembles the naturally occurring, plasma-derived surface antigen particle, and the in vitro formed particle has been used to prepare a vaccine for humans against hepatitis B virus infection.

A quantitative screening of some xylose-fermenting yeast isolates

Abstract: A quantitative screening procedure for xylose fermentation was conducted on 56 yeast isolates. Several of the isolates were found to be markedly superior toC. shehatae CSIR-Y492, one of the better xylose-fermenting yeasts identified thus far. The outstanding isolate was a strain ofPichia stipitis which had an ethanol yield coefficient of 0.45 from xylose and which produced no detectable amounts of xylitol.