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PubMed2002

Meeting the consumer challenge through genetically customized wine-yeast strains.

Wine producers are facing intensifying competition brought about by a widening gap between wine production and wine consumption, a shift of consumer preferences away from basic commodity wine to top quality wine, and economic globalization. Consequently, they are calling for a total revolution in the 'magical' world of wine. The process of transforming the wine industry from a production- to a market-orientated industry results in an increasing dependence on, amongst others, biotechnological innovation. Market-orientated wine-yeast strains are currently being developed for the cost-competitive production of wine with minimized resource inputs, improved quality and low environmental impact. The emphasis is on the development of Saccharomyces cerevisiae strains with improved fermentation, processing and biopreservation abilities, and capacities for an increase in the wholesomeness and sensory quality of wine.

Consumer Behavior↗

PubMed1995

[Applications of molecular biology in the wine industry].

Population dynamics of natural and inoculated industrial wine fermentations have been studied by using a simple molecular biology technique based on mitochondrial DNA restriction analysis profile. The predominance of the inoculated strain in the inoculated fermentations is obvious. A genetic transformation system has been developed for an industrial wine yeast strain named T73. By using this technique, different fungal hydrolases in this industrial strain have been expressed. Problems and benefits of the application of recombinant DNA techniques in wine yeast strains are also discussed here.

Fermentation↗

PubMedDate not supplied

Establishment of cellulolytic bacteria and development of fermentative activities in the rumen of gnotobiotically-reared lambs receiving the microbial additive Saccharomyces cerevisiae CNCM I-1077.

We studied the effects of a yeast additive used in ruminant nutrition on the establishment of cellulolytic bacteria, on plant cell wall degradation and on digestive functions in the rumen of gnotobiotically-reared lambs. Cellulolytic bacteria inoculated to the lambs tended to become established earlier in the presence of Saccharomyces cerevisiae CNCM I-1077 (SC). In addition, their population was maintained at a higher level, when the physico-chemical conditions of the biotope were altered. In these lambs, specific activities of fibrolytic enzymes were greater, and in sacco degradation of wheat straw tended to increase. In the presence of SC there was a decrease in ruminal ammonia concentration and a higher volatile fatty acid (VFA) concentration when lambs were 20 to 50 days old. These data suggest that this yeast strain may stimulate the development of cellulolytic microflora and enhance microbial activity in the rumen of young ruminants. Such activity could be beneficial in preventing microbial imbalance and a reduction of rumen function efficiency in the case of nutritional transitions. Further studies with conventional animals will soon be performed in order to verify these dings.

Ammonia↗

PubMed2001

The potential of genetic engineering for improving brewing, wine-making and baking yeasts.

The end of the twentieth century was marked by major advances in life technology, particularly in areas related to genetics and more recently genomics. Considerable progress was made in the development of genetically improved yeast strains for the wine, brewing and baking industries. In the last decade, recombinant DNA technology widened the possibilities for introducing new properties. The most remarkable advances, which are discussed in this Mini-Review, are improved process performance, off-flavor elimination, increased formation of by-products, improved hygienic properties or extension of substrate utilization. Although the introduction of this technology into traditional industries is currently limited by public perception, the number of potential applications of genetically modified industrial yeast is likely to increase in the coming years, as our knowledge derived from genomic analyses increases.

Beer↗

PubMed2004

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast: identification of second-site mutations that restore function to a coupling-deficient mutant M3 receptor.

The M(3) muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs). To gain insight into the structural mechanisms governing agonist-mediated M(3) receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M(3) receptors to identify mutant receptors with altered/novel functional properties. Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to Asp-113 in the rat M(3) muscarinic receptor) which is of fundamental importance for receptor activation. As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast. We then subjected the D113N mutant M(3) receptor to PCR-based random mutagenesis followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor. A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D. When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling. Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells). Interestingly, like Asp-113, Arg-165 and Tyr-250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs. Our data suggest a conformational link between the highly conserved Asp-113, Arg-165, and Tyr-250 residues which is critical for receptor activation.

Alleles↗

PubMed2005

Expression and characterization of rubella virus glycoprotein E1 in yeast cells.

OBJECTIVES: To express E1 glycoprotein of rubella virus (RuV) strain JR23 in yeast and develop a diagnostic assay using expressed E1 protein as coating antigen in comparison with other diagnostic assays. METHODS: cDNA of E1 open reading frame of RuV was PCR amplified using plasmid pMD18-T-E1 as template and cloned into plasmid pBluscriptII SK+. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pBluscriptII SK(+)-E1 plasmid DNA was digested by restriction endonuclease EcoR I and Xba I, and a fragment of 1.5 kb was isolated and cloned into a yeast expression pGAPZ(alpha)A, resulting in pGAPZ(alpha)A-E1. After confirmation by sequencing, pGAPZ(alpha)A- E1 was transformed into yeast GS115 cells with LiCl method. E1 protein expression in GS115 was analyzed by SDS-PAGE and Western blot. An indirect ELISA was developed using the recombinant E1 protein as coating antigen for detecting RuV E1 antibodies in 90 serum samples. To compare the specificity, sensitivity and reproducibility of the assay with other methods, the same serum samples were also assayed by RuV culture medium as coating antigen (Jingmei kit and German RECI kit). Statistical analyses were performed to compare the differences among these methods and to determine which coating antigen source, the recombinant E1 protein or RuV-infected culture medium, is more suitable for the assay. RESULTS: A fragment of 1.5 kb, corresponding to the full open reading frame of E1, was PCR amplified and cloned in yeast expression vector. The clone was confirmed by restriction digestion, PCR and sequencing. E1 as a secretive protein was successfully expressed by GS115. Its molecular weight was about 58 kDa. SDS-PAGE showed that the recombinant protein was expressed efficiently and constantly in Pichia pastoris GS115 cells. The expression level reached a peak 48 h after culturing and stabilized thereafter. E1 protein was detected in both supernatant and cells. Western blot showed that the secretive E1 protein in the supernatants could react with both the anti-RuV-positive serum and a monoclonal antibody against E1. However, E1 protein derived from cells could only react with the anti-RuV-positive serum, polyclonal antibody, but not the monoclonal antibody. Compared with the German RECI kit, the sensitivity, specificity, and accordance rate of the assay using recombinant E1 protein as coating antigen were 67.11, 71.43 and 67.78%, respectively, while those of the assay using RuV-infected culture medium as coating antigen were 50, 78.57 and 54.44%, respectively. Compared with the German RECI kit, the sensitivity, specificity, and accordance rate of the ELISA assay using the Jingmei kit were 84.71, 71.43 and 82.22%, respectively. The data indicated that recombinant E1 protein derived from the yeast expression system can serve as a better source than RuV-infected cell medium as coating antigen for detecting antibodies against RuV in the indirect ELISA assay. Statistical analysis of the data generated from two independent experiments using recombinant E1 protein as coating antigen indicated that the assay was very consistent with no statistically significant difference between the two experiments (p > 0.05). 76 out of 90 serum samples were detected positive using the German RECI kit, while 68, 55 and 41 samples were positive using the Jingmei kit, recombinant E1 and RuV-infected cell medium, respectively. Statistical analyses indicated that the positive rates were significantly different among all four assays (p < 0.05) except for one pair (German RECI kit and the Jingmei kit: p > 0.05). Comparing the positive rates obtained from the assay using recombinant E1 and that using RuV-infected cell medium, it seems that the recombinant E1 protein is a better source than RuV culture medium as coating antigen in the indirect ELISA assay for detection of RuV antibody. CONCLUSIONS: The recombinant yeast expression vector of RuV E1 glycoprotein was constructed successfully. The E1 protein as a secretive protein was successfully expressed by GS115 and maintained its antigenicity very well. As coating antigen, the recombinant E1 protein served a better source than RuV culture medium in the indirect ELISA method for the detection of RuV antibody.

Antibodies, Viral↗

PubMed2001

[Genetic control of growth and development of yeast Saccharomyces cerevisiae cells. Phenotypic selection of mutants among strains of the Peterhof genetic collection].

Results of identifying phenotypes intrinsic to mutations of genes that regulate the activity of the signal transduction pathway of RAS-cyclic adenosinemonophosphate in six strains belonging to the Peterhof genetic collection of the yeast Saccharomyces cerevisiae are presented: an increase or decrease in the amount of the accumulated glycogen, resistance or sensitivity to heat shock and nitrogen starvation, and the growth and viability in media containing unfermentable carbon sources (potassium acetate, ethanol, and glycerol) at temperatures 30 and 70 degrees C. Collectively these phenotypic characteristics in five examined yeast strains can be interpreted as indicating disturbances in the activity of the RAS/cAMP pathway. However, the discrepancies revealed between cellular phenotypes in these strains and in the strains with a decreased or increased activity of the RAS/cAMP pathway did not allow them to be assigned to a particular functional activity group. The inconsistencies between phenotypes detected in this study may be prerequisites for the identification of new genes responsible for this signal transduction pathway or new mutations in the known genes that determine other phenotypic combinations.

Cyclic AMP↗

PubMed1968

Control of dimorphism in Mucor by hexoses: inhibition of hyphal morphogenesis.

In anaerobic cultures of Mucor rouxii, morphogenesis was strongly dependent on hexose concentration as well as pCO(2). At low levels of hexose or CO(2), or both, hyphal development occurred; at high levels, the fungus developed as yeast cells. Other dimorphic strains of Mucor responded similarly to hexose and CO(2) but differred in their relative sensitivity to these agents. Glucose was the most effective hexose in eliciting yeast development of M. rouxii; fructose and mannose were next; and galactose was last. The fungus may be grown into shapes covering its entire dimorphic spectrum simply by manipulating the hexose concentration of the medium. Thus, at 0.01% glucose, hyphae were exceedingly long and narrow; at higher sugar concentrations, the hyphae became progressively shorter and wider; finally, at about 8% glucose, almost all cells and their progeny were isodiametric (spherical budding cells). Such yeast development occurred without a manifested requirement for exogenous CO(2). The stimulation of yeast development by hexose is not an artifact due to increased production of metabolic CO(2) (hyphae or yeast cells released metabolic CO(2) at similar rates). Presumably, the effect was caused by some other hexose catabolite which interfered with hyphal morphogenesis (apical growth); deprived of its polarity, the fungus grew into spherical yeastlike shapes. Although 10% glucose inhibited the development of hyphae from germinating spores, it did not prevent the elongation of preformed hyphae. This suggests that hexose inhibits hyphal morphogenesis not by blocking the operation of the enzyme complex responsible for apical growth but by preventing its initiation; such inhibition may be regarded as a repression of hyphal morphogenesis.

Carbon Dioxide↗

PubMed2004

Systematic quantification of gene interactions by phenotypic array analysis.

A phenotypic array method, developed for quantifying cell growth, was applied to the haploid and homozygous diploid yeast deletion strain sets. A growth index was developed to screen for non-additive interacting effects between gene deletion and induced perturbations. From a genome screen for hydroxyurea (HU) chemical-genetic interactions, 298 haploid deletion strains were selected for further analysis. The strength of interactions was quantified using a wide range of HU concentrations affecting reference strain growth. The selectivity of interaction was determined by comparison with drugs targeting other cellular processes. Bio-modules were defined as gene clusters with shared strength and selectivity of interaction profiles. The functions and connectivity of modules involved in processes such as DNA repair, protein secretion and metabolic control were inferred from their respective gene composition. The work provides an example of, and a general experimental framework for, quantitative analysis of gene interaction networks that buffer cell growth.

Cluster Analysis↗

PubMed2003

Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production.

There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; beta- d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone alpha-factor secretion signal (MFalpha1(S)) and the phosphoglycerate-kinase-1 gene promoter (PGK1(P)) and terminator (PGK1(T)). The PGK1(P)- MFalpha1(S)- gox- PGK1(T) cassette (designated GOX1) was introduced into a laboratory strain (Sigma1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8-2.0% less alcohol. This was probably due to the production of d-glucono-delta-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.

Aspergillus niger↗

PubMed2000

Characterization of a new set of mutants deficient in fermentation-induced loss of stress resistance for use in frozen dough applications.

In frozen dough applications a prefermentation period during the preparation of the dough is unavoidable and might also be important to obtain bread with a good texture. A major disadvantage of the prefermentation period is that it is associated with a rapid loss of the freeze resistance of the yeast cells. A major goal for the development of new baker's yeast strains for use in frozen dough applications is the availability of strains that maintain a better freeze resistance during the prefermentation period. We have isolated mutants that retain a better stress resistance during the initiation of fermentation. Some of these showed the same growth rate and fermentation capacity as the wild type cells. These mutants are called 'fil', for deficient infermentation induced loss of stress resistance. First we used laboratory strains and heat stress treatment, given shortly after the initiation of fermentation, as the selection protocol. The first two mutants isolated in this way were affected in the glucose-activation mechanism of the Ras-cAMP pathway. The fil1 mutant had a partially inactivating point mutation in CYR1, the gene encoding adenylate cyclase, while fil2 contained a nonsense mutation in GPR1. GPR1 encodes a member of the G-protein coupled receptor family which acts as a putative glucose receptor for activation of the Ras-cAMP pathway. In a next step we isolated fil mutants directly in industrial strains using repetitive freeze treatment of doughs as selection protocol. Surviving yeast strains were tested individually for maintenance of fermentation capacity after freeze treatment in laboratory conditions and also for the best performing strains in frozen doughs prepared with yeast cultivated on a pilot scale. The most promising mutant, AT25, displayed under all conditions a better maintenance of gassing power during freeze-storage. It was not affected in other commercially important properties and will now be characterised extensively at the biochemical and molecular level.

Fermentation↗

PubMed2001

Influence of aeration on the physiological activity of flor yeasts.

The effect of periodic aeration on the physiological activity of a strain of Saccharomyces cerevisiae yeast during development of velum (flor) and biological aging of Sherry wine of the Fino type was investigated. L-Proline amino acid was the main nitrogen source for yeasts cells during the biological aging, and its exhaustion may be the cause of the production and consumption of other compounds that are involved in the aroma of wines. Aeration was found to increase adenylate energy charge, growth, and viability of the yeast cells. Also, it affected the intracellular redox equilibrium and the consumption and production of compounds including acetoin, acetaldehyde, higher alcohols, ethanol, glycerol, and acetic acid. Acetaldehyde reached its highest level after the second aeration, which coincided with the exhaustion of the nitrogen source in the medium. The enzyme activity of alcohol dehydrogenases I and II decreased immediately after each aeration, subsequently increasing once all of the dissolved oxygen in the wine had been consumed by yeast cells. Aldehyde dehydrogenase activity was detected only after the first aeration, and it may be related to the production and consumption of acetic acid in the wine.

Aging↗

PubMed2001

Cloning and heterologous expression of xylanase from Pichia stipitis in Escherichia coli.

AIMS: The main goal of this study was to characterize the xylanase (xynA) gene from Pichia stipitis NRRL Y-11543. METHODS AND RESULTS: The xylanase gene was cloned into pUC19 in Escherichia coli DH5alphaF' and selected by growth on RBB-xylan. All functional clones contained a recombinant plasmid with an insert of 2.4 kbp, as determined by restriction mapping. The nucleotide sequence of the P. stipitis xylanase gene consisted of 1146 bp and encoded a protein of 381 amino acids with a molecular weight of 43 649 Da. The sequence contained a putative 20-amino acid N-terminal signal sequence and four N-linked glycosylation sites. The Km values for non-glycosylated and glycosylated xylanases were 1.4 mg ml-1 and 4.2 mg ml-1, respectively, and Vmax values were 0.8 and 0.082 micromol min-1 mg-1 protein, respectively. CONCLUSION: Xylanase, a rarely found enzyme in yeast species, has been characterized in detail. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can be used to develop better xylanase-utilizing yeast strains.

Amino Acid Sequence↗

PubMed2005

Kinetics and thermodynamics of ethanol production by a thermotolerant mutant of Saccharomyces cerevisiae in a microprocessor-controlled bioreactor.

AIMS: The present investigation deals with the development of thermotolerant mutant strain of yeast for studying enhanced productivity of ethanol from molasses in a fully controlled bioreactor. METHODS AND RESULTS: The parental culture of Saccharomyces cerevisiae ATCC 26602 was mutated using UV treatment. A single thermotolerant mutant was isolated after extensive screening and optimization, and grown on molasses medium in liquid cultures. The mutant was 1.45-fold improved than its wild parent with respect to ethanol productivity (7.2 g l-1 h-1), product yield (0.44 g ethanol g-1 substrate utilized) and specific ethanol yield (19.0 g ethanol g-1 cells). The improved ethanol productivity was directly correlated with titres of intracellular and extracellular invertase activities. The mutant supported higher volumetric and product yield of ethanol, significantly (P<or=0.05) higher than the parental and other strains. The mutated cultures produced 1.8- and 2.6-fold more extracellular and intracellular invertase productivity, respectively, than that produced by its wild parent at 40 degrees C. Thermodynamic studies revealed that the cell system exerted protection against thermal inactivation during formation of products. CONCLUSIONS: A mutant derivative of Sacchromyces cerevisiae with improved productivity of ethanol and invertases has been obtained, which showed concomitant improvement in thermostability of endogenous metabolism for formation of both ethanol and invertases. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study are of commercial value as the mutant can be used for ethanol production in parts of Pakistan where the temperature may go up to 40 degrees C in April. Ethanol product yield coefficient and volumetric productivity, revealed the hyper-productivity of ethanol from molasses at 40 degrees C, which is not appropriate for wild organism.

Bioreactors↗

PubMed2004

Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions.

Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.

Aquaporins↗

PubMed2003

Enhancing volatile phenol concentrations in wine by expressing various phenolic acid decarboxylase genes in Saccharomyces cerevisiae.

Phenolic acids, which are generally esterified with tartaric acid, are natural constituents of grape must and wine and can be released as free acids (principally p-coumaric, caffeic, and ferulic acids) by certain cinnamoyl esterase activities during the wine-making process. Some of the microorganisms present in grape can metabolize the free phenolic acids into 4-vinyl and 4-ethyl derivatives. These volatile phenols contribute to the aroma of wine. The Saccharomyces cerevisiae phenyl acrylic acid decarboxylase gene (PAD1) is steadily transcribed, but its encoded product, Pad1p, shows low activity. In contrast, the phenolic acid decarboxylase (PADC) from Bacillus subtilis and the p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum display substrate-inducible decarboxylating activity in the presence of phenolic acids. In an attempt to develop wine yeasts with optimized decarboxylation activity on phenolic acids, the padc, pdc, and PAD1 genes were cloned under the control of S. cerevisiae's constitutive phosphoglyceratekinase I gene promoter (PGK1(P)()) and terminator (PGK1(T)()) sequences. These gene constructs were integrated into the URA3 locus of a laboratory strain of S. cerevisiae, Sigma1278b. The overexpression of the two bacterial genes, padc and pdc, in S. cerevisiae showed high enzyme activity. However, this was not the case for PAD1. The padc and pdc genes were also integrated into an industrial wine yeast strain, S. cerevisiae VIN13. As an additional control, both alleles of PAD1 were disrupted in the VIN13 strain. In microvinification trials, all of the laboratory and industrial yeast transformants carrying the padc and pdc gene constructs showed an increase in volatile phenol formation as compared to the untransformed host strains (Sigma1278b and VIN13). This study offers prospects for the development of wine yeast starter strains with optimized decarboxylation activity on phenolic acids and the improvement of wine aroma in the future.

Bacillus subtilis↗

PubMed1986

Effect of elevated temperatures and low levels of trace metals on the growth and phenotypic development of Candida albicans.

A combination of elevated temperatures (within the human febrile range) and trace metal chelation were investigated for their effects on the inhibition of growth and phenotypic development of the dimorphic yeast Candida albicans (strain 3153A). The ability of specific cations to relieve the phenotypic inhibition that occurred also was tested. Elevated temperatures alone (to 41 degrees C) only delayed the timing of the phenotypic development. When compared to the results obtained at 37 degrees C, the recombination of elevated temperature and addition of the trace metal chelator, 1,10-phenanthroline, did not further suppress phenotypic development, but the combination did decrease the viability of C. albicans. When 24 to 48 h stationary phase singlet cells were released into a medium containing 100 microM 1,10-phenanthroline (pH 6.5), supplemental iron (200 microM) alleviated the suppression of mycelium formation at 41 degrees C, whereas under conditions favoring bud formation (pH 4.5), both iron and zinc circumvented suppression and promoted budding. Through studies on the interaction of temperature stress and trace metal availability our data revealed the requirement for iron mycelium formation whereas both iron and zinc may be needed for bud formation.

Candida albicans↗

PubMed1990

A novel screening system for yeast strains capable of secreting tissue plasminogen activator.

We have developed a simple screening procedure that allowed us to identify Saccharomyces cerevisiae strains able to secrete human tissue plasminogen activator (tPA) into the culture medium. The screen can be used to isolate more efficient secretor strains and to look for novel tPA analogs. Employing one of these strains to study the effect of glycosylation on secretion, we show that glycosylation in the catalytic domain of tPA plays an important role in folding and/or secretion of the molecule. Removing this glycosylation site resulted in a 3-5-fold reduction in the level of tPA secretion. We anticipate that this system will prove useful in studying yeast secretory pathway as well as structure-function relationships in the tPA molecule.

Biotechnology↗