Yeast

doi: 10.1002/yea.320070501pmid: N/A

Janowicz, Zbigniew A.; Melber, Karl; Merckelbach, Armin; Jacobs, Eric; Harford, Nigel; Comberbach, Martin; Hollenberg, Cornelis P.

doi: 10.1002/yea.320070502pmid: 1897310

An expression system has been developed for the methylotrophic yeast Hansenula polymorpha and used to co‐express both the L (preS1‐S2‐S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol‐inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of this H. polymorpha expression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5–8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle‐specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. These H. polymorpha‐derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible glycosylation.

Kinsella, B. T.; Cantwell, B. A.

doi: 10.1002/yea.320070503pmid: 1897311

The α‐glucosidase gene of Candida tsukubaensis is contained within a 3·47 kb BamH1‐Mlu1 fragment which, when introduced into Saccharomyces cerevisiae AH22 on a yeast–Escherichia coli shuttle vector, allows the transformants to utilize maltose as sole carbon source. Thus, the cloned gene confers a dominant selectable phenotype on transformed strains of S. cerevisiae which are otherwise unable to grow in nutrient media containing maltose, dextrin or other α‐1,4‐linked α‐D‐glucopyranosides, specifically hydrolysed by the α‐glucosidase. The cloned enzyme expressed in yeast is secreted into the extracellular medium in a glycosylated form which accounts for up to 60% of the secreted protein and has a molecular size of 70–80 kilodalton (kDa). Deglycosylation of the α‐glucosidase showed that the enzyme is composed of two distinct polypeptides with subunit molecular weights of 63–65 kDa (peptide 1) and 50–52 kDa (peptide 2). An increase in the level of expression of the α‐glucosidase by yeast transformants in selective minimal medium was obtained by using a vector with increased copy number containing the leu2‐d gene as selectable marker. The α‐glucosidase gene promoter functions more effectively than the Gal1–10 promoter in directing α‐glucosidase expression in S. cerevisiae. It also directs the expression of high levels of β‐galactosidase activity in yeast when fused to a promoterless E. coli lacZ gene. Expression of the α‐glucosidase gene under the control of its own promoter is constitutive, orientation dependent and not subject to catabolite repression.

Blagoeva, J.; Stoev, G.; Venkov, P.

doi: 10.1002/yea.320070504pmid: 1897312

The phenotype of VY1160 fragile Saccharomyces cerevisiae mutant is characterized by cell lysis upon transfer to hypotonic solutions and increased permeability of cells growing in osmotically stabilized media. Two mutations, srb1 and ts1, have been identified in VY1160 cells and previous studies have shown that the increased permeability is due to the ts1 mutation which causes a shortening of mannan side‐chains. Here we report that the srb1 mutation, which is the genetic determinant of cell lysis, is responsible for quantitative and structural changes of glucans. Experiments with isogenic determinant of cell lysis, is responsible for quantitative and structural changes of glucans. Experiments with isogenic single mutation strains, genetic studies coupled with quantitative measurements of glucan content per cell, and methylation analysis of glucans provide evidence that srb1 mutation leads to (i) formation of mechanically unstable cell wall network made of insoluble glucan fibrils which are shorter and contain β(1–6) inter‐residue linkages and (ii) insufficient filling of the space between the fibrils due to a shortage of the alkali‐soluble glucan. Although growing exponentially in osmotically stabilized media, the srb1 cells cannot resist an osmotic shock and, hence, burst immediately.

Fellinger, Arthur J.; Verbakel, John M. A.; Veale, Rosemary A.; Sudbery, Peter E.; Bom, Isaac J.; Overbeeke, Nico; Verrips, C. Theo

doi: 10.1002/yea.320070505pmid: 1654681

The methylotrophic yeast Hansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the α‐galactosidase from the plant Cyamopsis tetragonoloba (guar). The yeast/Escherichia coli shuttle expression vector used is based on the origin of replication of the endogenous 2 μm plasmid of Saccharomyces cerevisiae and the LEU2 gene of S. cerevisiae for selection in H. polymorpha. In the expression vector, the α‐galactosidase is controlled by the methanol‐regulated promoter from the methanol oxidase gene, MOX, of H. polymorpha. The signal sequence of SUC2 (invertase) from the yeast S. cerevisiae, was used to ensure secretion of the α‐galactosidase enzyme. After transformation and stabilization, the expression vector was stably integrated in the genome. The active α‐galactosidase enzyme was efficiently secreted (>85%) and after methanol induction, the expression level was 42 mg/l. Amino‐terminal sequencing of the purified α‐galactosidase enzyme synthesized by H. polymorpha showed that the S. cerevisiae invertase signal sequence was correctly processed by H. polymorpha. The secreted α‐galactosidase was glycosylated and had a sugar content of 9·5%. The specific activity of the α‐galactosidase produced by H. polymorpha was 38 U mg−1 compared to 100 U mg−1 for guar α‐galactosidase. Deglycosylation of the H. polymorpha α‐galactosidase restored the specific activity completely.

Berben, Gilbert; Dumont, Jacques; Gilliquet, Véronique; Bolle, Paul‐André; Hilger, François

doi: 10.1002/yea.320070506pmid: 1897313

The YDp plasmids (Yeast Disruption plasmids) are pUC9 vectors bearing a set of yeast gene disruption cassettes, all uniform in structure and differing only in the selectable marker used (HIS3, LEU2, LYS2, TRP1 or URA3). The markers, surrounded by translational termination codons, are embedded in the slightly modified sequence of the pUC9 multiple cloning sites.

Sims, A. P.; Stålbrand, H.; Barnett, J. A.

doi: 10.1002/yea.320070507pmid: N/A

The glucose‐fermenting yeast, Candida utilis cannot use the β‐D‐glucoside, cellobiose, anaerobically, although it is able to do so aerobically. β‐Glucoside transport and hydrolysis and pyruvate decarboxylase activities of this yeast were measured aerobically and anaerobically. β‐Glucoside transport was five‐fold faster aerobically than anaerobically, but there was no corresponding difference in β‐glucosidase activity. Pyruvate decarboxylase activity varied greatly, being synthesized de novo in response to the presence of D‐glucose and anaerobic conditions and about 50% deactivated on the removal of D‐glucose or the addition of air. Activation and deactivation were rapidly reversible. Failure to utilize cellobiose anaerobically, in particular, and the Kluyver effect, in general, probably depends on much reduced glycolytic flux, associated under anaerobic conditions, with (i) lower transport rate, (ii) low substrate affinity of the relevant glycosidase and (iii) deactivation of pyruvate decarboxylase. So, in addition to the complex effects of oxygen, anaerobiosis and specific sugars on induction, repression and derepression, there are fine controls on pyruvate decarboxylase activity, leading to fast activation or deactivation of the enzyme.

Pilkington, Bridget J.; Rose, Anthony H.

doi: 10.1002/yea.320070508pmid: 1897314

Saccharomyces cerevisiae was grown anaerobically in media supplemented with myritoleic 14:1(9c), palmitoleic 16:1(9c), oleic 18:1(9c), linoleic 18:2(9,12c), γ‐linolenic 18:3(9,12,15c) or eicosenoic 20:1(11c) acid. Cells from exponential‐phase cultures contained approximately the same proportions of the major phospholipid classes, namely phosphatidycholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, the greatest differences being detected in cells grown in the presence of 14:1(9c) or 20:1(11c) acids. The extent to which phospholipids from cells were enriched with residues of the exogenously supplied acid varied from 52% in cells grown in the presence of 14:1(9c) acid to 13% in cells grown in media supplemented with 20:1(11c) acid. Analysis of the fatty‐acyl composition of the four major phospholipid classes revealed that the degree of unsaturation varied considerably in three of the classes, while phosphatidylinositol conserved a high degree of saturation. The possible significance of the latter finding in relation to the physiological role of phosphatidylinositol in the plasma membrane is discussed.

Kulakovskaya, T. V.; Matys, S. V.; Okorokov, L. A.

doi: 10.1002/yea.320070509pmid: N/A

Isolated vacuoles of the yeast Saccharomyces pastorianus accumulate citrate, α‐ketoglutarate, malate and guanosine. This accumulation is Mg ATP‐dependent and inhibited by protonophores. The ionophores monensin and A23187 (electroneutral Men+/nH+‐exchange) inhibit guanosine accumulation but fail to block citrate uptake. Mg2+ ions (2 mM) increase the values of both Δ\documentclass{article}\pagestyle{empty}\begin{document}$ \tilde \mu $\end{document}H+ components and stimulate the uptake of all the above compounds. Ca2+ ions (1 mM), hyperpolarizing the yeast vacuolar membrane and dissipating the pH gradient, inhibit guanosine uptake and stimulate that of citrate. It is concluded that guanosine is transported into yeast vacuoles by an H+/guanosine antiporter while citrate, malate and α‐ketoglutarate are translocated by a uniporter(s) at the expense of the membrane potential (positive inside).

Kamiryo, Tatsuyuki; Mito, Naruo; Niki, Toshiro; Suzuki, Takahito

doi: 10.1002/yea.320070510pmid: 1897315

The peroxisomes of the asporogenic yeast Candida tropicalis contain about 20 major polypeptides (PXPs). We have isolated a number of genes encoding them; 11 POX genes encoded independent PXPs and three POY genes were likely to encode three other PXPs. To locate these genes on the chromosomes, chromosomes of C. tropicalis were separated by pulsed‐field gel electrophoresis. Eight chromosomal bands were observed over the range of 1·0 Mbp (band I) to 2·8 Mbp (band VIII); the genome size was estimated to be about 20 Mbp. Southern blot analysis showed that ten genes were on band V, three genes were on band IV, and the other gene was on band VI. Three genes gave hybridization signals of nearly equal intensity on two different chromosomal bands: POX6A and POX8B, on bands V and VII; and POX8A, on bands IV and VI. Ribosomal RNA genes also hybridized to two bands, VI and VII. Most genes assigned to only one band hybridized to two restriction fragments produced by either NotI or SfiI endonuclease. The results suggested that C. tropicalis was diploid and that restriction sites were conserved little between homologues. The three POX genes that were found on two chromosomal bands hybridized to not more than two restriction fragments, implying that the allelic genes were present on different chromosomal bands.