Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae autoselection strains with mutations in the ura3, fur1, and urid-k genes have been obtained through a sequential isolation procedure. This autoselection system is an extension of one described by Loison et al. The mutations effectively block both the pyrimidine biosynthetic and salvage pathways and in combination are lethal to the host. Therefore, a plasmidencoded URA3 gene is essential for cell viability regardless of the growth conditions, and complex (traditionally nonselective) media can be employed without the risk of plasmid loss. The effects of medium enrichment on growth and cloned gene product synthesis were examined in batch culture for two autoselection strains. The plasmid gene product beta-galactosidase was under the control of the yeast GAL1 promoter, and two methods of induction were employed; one strain was induced via temperature shift while the other was induced by galactose addition. Three nutrient media were investigated: a lean selective medium (SD), a richer semidefined medium (SDC), and a rich complex medium (YPD). The results demonstrated the improvements in cloned gene productivity possible when the growth medium is enriched, with up to 10-fold increases in beta-galactosidase productivity observed. Plasmid instability and mutation reversion were not problems for the autoselection strains, even in uracil-containing medium. Short-term plasmid stabilities were approximately 90% in all three media tested. During continuous culture of the autoselection temperature-sensitive strain, long-term plasmid stability was excellent and beta-galactosidase expression remained high after more than 25 residence times under inducing conditions. In contrast, both beta-galactosidase specific activity and plasmid stability decreased linearly with time for an analogous nonautoselection strain. The introduced fur1 and uridk mutations were very stable; after more than 50 generations of growth in complex medium, stability values of 99-100% were measured. (c) 1993 Wiley & Sons, Inc.     

Increased protein productivity from immobilized recombinant yeast

The Saccharomyces cerevisiae strain Mc16/p520 has an unstable plasmid, p520, which directs production of a wheat alpha-amylase. The effects of immobilizing this microorganism on the plasmid stability and the specific productivity of the secreted alpha-amylase were investigated. Small gelatin beads were used as the support in both fluidized and packed bed configurations, and the yeast cells were attached by covalent cross-linking with glutaraldehyde. These data were then compared to those for nonimmobilized, suspension cells.Plasmid stability was increased for the immobilized cells during continuous culture at dilution rates both above and below washout. Continuous suspension cultures were not stable and rapidly lost the plasmid. Immobilization caused an increase in specific and volumetric productivity during continuous culture, with a packed bed design resulting in the highest specific productivity.     

Increasing yeast secretion of heterologous proteins by regulating expression rates and post-secretory loss

Heterologous protein secretion involves the coupled processes of protein synthesis, protein folding, and secretory trafficking. A more complete understanding of how these processes interrelate could help direct optimization of secretion systems. Here we provide a detailed study regarding the dynamics of heterologous protein secretion from yeast in terms of intracellular protein levels, secreted protein levels, and unfolded protein response (UPR). Three different protein expression induction temperatures (20, 30, and 37 degrees C) were investigated as a means to modulate expression rates and thus cellular responses. Inducing at 20 degrees C yielded the slowest initial secretion rate, but the highest absolute level of product. Correspondingly, the level and the rate of both intracellular protein accumulation and unfolded protein response (UPR) activation were also the lowest at 20 degrees C. In addition, secretion ceased after approximately 22 h at 30 and 37 degrees C, respectively, while it was continuous until nutrient depletion at 20 degrees C. Maxima in secretion levels were observed that were a result of the additive effects of secretion cessation and post-secretory protein loss. The post-secretory loss of protein did not appear to result from solution phase proteolysis or aggregation, but required the presence of yeast cells. Refeeding of both yeast nitrogen base and casamino acids successfully prevented the post-secretory loss of protein at both high (37 degrees C) and low (20 degrees C) temperatures, and further increased secretion levels 1.5-fold at 20 degrees C where the secretory pathway was still functioning. Taken together, these findings suggest that there exists an appropriate balance between protein synthesis, processing and secretion rates required for secretion optimization.     

Metabolic pathway analysis of a recombinant yeast for rational strain development

Elementary mode analysis has been used to study a metabolic pathway model of a recombinant Saccharomyces cerevisiae system that was genetically engineered to produce the bacterial storage compound poly-beta-hydroxybutyrate (PHB). The model includes biochemical reactions from the intermediary metabolism and takes into account cellular compartmentalization as well as the reversibility/irreversibility of the reactions. The reaction network connects the production and/or consumption of eight external metabolites including glucose, acetate, glycerol, ethanol, PHB, CO(2), succinate, and adenosine triphosphate (ATP). Elementary mode analysis of the wild-type S. cerevisiae system reveals 241 unique reaction combinations that balance the eight external metabolites. When the recombinant PHB pathway is included, and when the reaction model is altered to simulate the experimental conditions when PHB accumulates, the analysis reveals 20 unique elementary modes. Of these 20 modes, 7 produce PHB with the optimal mode having a theoretical PHB carbon yield of 0.67. Elementary mode analysis was also used to analyze the possible effects of biochemical network modifications and altered culturing conditions. When the natively absent ATP citrate-lyase activity is added to the recombinant reaction network, the number of unique modes increases from 20 to 496, with 314 of these modes producing PHB. With this topological modification, the maximum theoretical PHB carbon yield increases from 0.67 to 0.83. Adding a transhydrogenase reaction to the model also improves the theoretical conversion of substrate into PHB. The recombinant system with the transhydrogenase reaction but without the ATP citrate-lyase reaction has an increase in PHB carbon yield from 0.67 to 0.71. When the model includes both the ATP citrate-lyase reaction and the transhydrogenase reaction, the maximum theoretical carbon yield increases to 0.84. The reaction model was also used to explore the possibility of producing PHB under anaerobic conditions. In the absence of oxygen, the recombinant reaction network possesses two elementary modes capable of producing PHB. Interestingly, both modes also produce ethanol. Elementary mode analysis provides a means of deconstructing complex metabolic networks into their basic functional units. This information can be used for analyzing existing pathways and for the rational design of further modifications that could improve the system's conversion of substrate into product.     

Construction of a recombinant autolytic wine yeast strain overexpressing the csc1‐1 allele

During the aging step of sparkling wines and wines aged on lees, yeast cells kept in contact with the wine finally die and undergo autolysis, releasing cellular compounds with a positive effect on the wine quality. In view of the interest of autolysis for wine properties, biotechnologists have tried to improve autolytic yield during winemaking. In this work we used genetic engineering techniques to construct an autolytic industrial strain by expressing the csc1‐1 allele from the RDN1 locus. The expression of this mutant allele, that causes a “constitutive in autophagy phenotype,” resulted in accelerated autolysis of the recombinant strain. Although autophagic phenotype due to csc1‐1 expression has been reported to require the mutant allele in multicopy, autolytic acceleration was achieved by expressing only one or two copies of the gene under the control of the constitutive promotor pTDH3. The acceleration of autolysis together with the unaltered fermentative capacity, strongly supported the overexpression of csc1‐1 allele as a strategy to obtain wines with aged‐like properties in a shortened time. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Construction of a recombinant yeast strain converting xylose and glucose to ethanol

Candida shehatae gene xyll and Pichia stipitis gene xyl2,encoding xylose reductase (XR) and xylitol dehydrogenase (XD) respectively,were amplified by PCR.The genes xyl1 and xyl2 were placed under the control of promoter GAL in vector pYES2 to construct the recombinant expression vector pYES2-PI2.Subsequently the vector pYES2-P12 was transformed into S.cerevisiae YS58 by LiAc to produce the recombinant yeast YS58-12.The alcoholic ferment indicated that the recombinant yeast YS58-12 could convert xylose to ethanol with the xylose consumption rate of 81.3%.     

Plasmid stability in recombinant Saccharomyces cerevisiae

Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.     

Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast

In eukaryotes, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). Many heterologous proteins are retained in the ER due to suboptimal folding conditions. We previously reported that heterologous secretion of Pyrococcus furiosus beta-glucosidase in Saccharomyces cerevisiae resulted in the accumulation of a large fraction of inactive beta-glucosidase in the ER. In this work, we determine the effect of introducing additional genes of ER-resident yeast proteins, Kar2p (binding protein [BiP]) and protein disulfide isomerase (PDI), on relieving this bottleneck. Single-copy expression of BiP and PDI worked synergistically to improve secretion by reverse similar 60%. In an effort to optimize BiP and PDI interactions, we created a library of beta-glucosidase expression strains that incorporated four combinations of constitutively or inducibly-expressed BiP and PDI genes integrated to random gene copynumbers in the yeast chromosome. Approximately 15% of the transformants screened had secretion level improvements higher than that seen with single BiP/PDI gene overexpression, and the highest secreting strain had threefold higher beta-glucosidase levels than the control. Nineteen of the improved strains were re-examined for beta-glucosidase secretion as well as BiP and PDI levels. Within the improved transformants BiP and PDI levels ranged sevenfold and tenfold over the control, respectively. Interestingly, increasing BiP levels decreased beta-glucosidase secretion, whereas increasing PDI levels increased beta-glucosidase secretion. The action of PDI was unexpected because beta-glucosidase is not a disulfide-bonded protein. We suggest that PDI may be acting in a chaperone-like capacity or possibly creating mixed disulfides with the beta-glucosidase's lone cysteine residue during the folding and assembly process.     

The STT3 protein is a component of the yeast oligosaccharyltransferase complex

N-linked protein glycosylation is an essential process in eukaryotic cells. In the central reaction, the oligosaccharyltransferase (OTase) catalyzes the transfer of the oligosaccharide Glc₃Man₉GlcNAc₂ from dolicholpyrophosphate onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum. The product of the essential gene STT3 is required for OTase activity in vivo, but is not present in highly purified OTase preparations. Using affinity purification of a tagged Stt3 protein, we now demonstrate that other components of the OTase complex, namely Ost1p, Wbp1p and Swp1p, specifically co-purify with the Stt3 protein. In addition, different conditional stt3 alleles can be suppressed by overexpression of either OST3 and OST4, which encode small components of the OTase complex. These genetic and biochemical data show that the highly conserved Stt3p is a component of the oligosaccharyltransferase complex. Received: 3 June 1997 / Accepted: 29 July 1997     

Properties of endogenous β-glucosidase of a Saccharomyces cerevisiae strain isolated from Sicilian musts and wines

Three hundred sixty-one yeast strains (80 of which ascribable to Saccharomyces cerevisiae) were isolated from Sicilian musts and wines with the purpose of looking for β-glucosidase (βG, EC 3.2.1.21) activity. Of these, the AL 41 strain had highest endogenous βG activity and was identified as belonging to the species S. cerevisiae by biochemical and molecular methods. This enzyme was subsequently characterized. It had optimum effect at pH 3.5–4.0, whilst optimum temperature was 20 °C, compatible with typical wine-cellar conditions; it was not inhibited by ethanol, at concentrations of 12–14%, or fructose and glucose. The βG was also characterised in terms of the kinetic parameters K_m (2.55 mM) and V_max (1.71 U mg⁻¹ of protein). Finally, it remained stable for at least 35 days in model solutions of must and wine.     

Stability studies of recombinant Saccharomyces cerevisiae in the presence of varying selection pressure

A recombinant yeast plasmid carrying the Ieu2 gene for auxotrophic complementation and a reporter gene for beta-galactosidase under the control of Gal10 promoter was studied in Saccharomyces cerevisiae. Growth, product formation, and plasmid stability were studied in defined, semi-defined, and complex media. The biomass concentration and specific activity were higher in complex medium than in defined medium, which was selective for the growth of plasmid-containing cells, leading to a 10-fold increase in volumetric activity. However, plasmid instability was very high in complex media with 50% plasmid-free cells emerging in the culture within 75 h of cultivation. In order to control instability, the growth rates of the plasmid-containing and plasmid-free cells were determined in semi-defined media, which consisted of defined medium supplemented with different concentrations of yeast extract. Below a critical concentration of yeast extract (0.05 g/L), the plasmid-containing cells had a growth rate advantage over the plasmid-free cells. This was possibly because, at this concentration of yeast extract, the availability of leucine became the rate-determining factor in the specific growth rate of plasmid-free cells. A feeding strategy was designed which maintained a low concentration of the residual yeast extract in the medium and thus continuously provided the plasmid-containing cells with a competitive advantage over the plasmid-free cells. This resulted in high stability as well as high cell density under non-selective conditions, which led to a 10-fold increase in the volumetric activity compared to that achieved in defined selective media. A simple mathematical model was formulated to verify the experimental data. The important state variables and process parameters, i.e., biomass concentration, beta-galactosidase expression, sucrose consumption, yeast extract consumption, and specific growth rates of the two cell populations, were evaluated. These variables and parameters along with the differential equations based on material balances as well as the experimental results obtained were used in a mathematical model for the fed-batch cultivation. These correctly verified the experimental data and clearly illustrated the concept behind the success of the fed-batch strategy under yeast extract starvation.     

Amplification of a gene for metallothionein by tandem repeat in a strain of cadmium-resistant yeast cells

Abstract In a cadmium-resistant strain of Saccharomyces cerevisiae , cells are protected against cadmium toxicity by the production of large amounts of cadmium-binding metallothionein, as occurs similarly in a copper-resistant strain. The apoprotein of the metallothionein is encoded by the CUP1 gene on chromosome VIII. The CUP1 gene is present as 8–10 copies in the cadmium-resistant strain as a result of tandem repeat of a 2.0-kb fragment of DNA that includes CUP1 , while the wild-type strain contains only a single copy of CUP1 . In the cadmium-resistant strain, some evidence for elongation of chromosome VIII with variations in length (maximum to 200 kb) was obtained. However, the elongation was not due to the tandem repeats of the CUP1 -containing region.     

Stimulation of dehydrogenase synthesis by cadmium in a cadmium-resistant strain of Saccharomyces cerevisiae

The activity of dehydrogenase in Saccharomyces cerevisiae was estimated by reduction of 2,3,5-triphenyltetrazolium chloride. By the adaptation of yeast to cadmium, the high activity of dehydrogenase was observed. Furthermore, the activity of dehydrogenase in Cd-resistant cells was increased by growing in medium containing CdSO₄. However, the activity of dehydrogenase was inhibited by the addition of CdSO₄ to the reaction mixture. The activity of dehydrogenase in Cd-sensitive cells was increased slightly by incubation with low concentrations of CdSO₄.High activity of dehydrogenase in Cd-resistant cells was completely negated by the addition of cycloheximide to the incubation medium. The increase of dehydrogenase activity is due partly to de novo synthesis of protein.     

Unconventional protein secretion: an evolving mechanism

The process by which proteins are secreted without entering the classical endoplasmic reticulum (ER)–Golgi complex pathway, in eukaryotic cells, is conveniently called unconventional protein secretion. Recent studies on one such protein called Acb1 have revealed a number of components involved in its secretion. Interestingly, conditions that promote the secretion of Acb1 trigger the biogenesis of a new compartment called CUPS (Compartment for Unconventional Protein Secretion). CUPS form near the ER exit site but lack ER‐specific proteins. Other proteins that share some of the features common with the secretion of Acb1 are interleukin‐1β and tissue transglutaminase. Here I will review recent advances made in the field and propose a new model for unconventional protein secretion.     

Amplification of synthesis and secretion of haemolysin using a run-away plasmid in Escherichia coli

Molin and co-workers have described the construction of a ‘run-away’ plasmid, pOU71 which could be useful for the amplification of cloned genes at high temperature when the plasmid replicates to high copy number.In this paper we describe the kinetics of synthesis of a plasmid-coded gene product, β-lactamase, concomitant with pOU71 amplification at 42°C. Maximum amplification was obtained by shifting a culture growing at 30–42°C for 60 min resulting in a 70- to 80-fold amplification for the β-lactamase gene product when the culture was returned to 30°C.The haemolytic determinant LE2001 from an Escherichia coli strain of human origin was cloned into plasmid pOU71 giving rise to plasmid pLG570. Using an identical amplification procedure a 20-fold amplification of the synthesis and secretion of haemolysin was achieved.     

Identification and characterization of a yeast homolog of U1 snRNP-specific protein C.

U1C is one of the three human U1 small nuclear ribonucleoprotein (snRNP)-specific proteins and is important for efficient complex formation between U1 snRNP and the pre-mRNA 5' splice site. We identified a hypothetical open reading frame in Saccharomyces cerevisiae as the yeast homolog of the human U1C protein. The gene is essential, and its product, YU1C, is associated with U1 snRNP. YU1C depletion gives rise to normal levels of U1 snRNP and does not have any detectable effect on U1 snRNP assembly. YU1C depletion and YU1C ts mutants affect pre-mRNA splicing in vivo, and extracts from these strains form low levels of commitment complexes and spliceosomes in vitro. These experiments indicate a role for YU1C in snRNP function. Structure probing with RNases shows that only the U1 snRNA 5' arm is hypersensitive to RNase I digestion when YU1C is depleted. Similar results were obtained with YU1C ts mutants, indicating that U1C contributes to a proper 5' arm structure prior to its base pairing interaction with the pre-mRNA 5' splice site.     

High resolution NMR analysis of the seven transmembrane domains of a heptahelical receptor in organic-aqueous medium

The NMR properties of seven peptides representing the transmembrane domains of the alpha-factor receptor from Saccharomyces cerevisiae were examined in trifluoroethanol/water (4:1) at 10 to 55 degrees C. The parameters extracted indicated all peptides were helical in this membrane mimetic solvent. Using chemical shift indices as the criterion, helicity varied from 64 to 83%. The helical residues in the peptides corresponded to the region predicted to cross the hydrocarbon interior of the bilayer. A study of a truncated 25-residue peptide corresponding to domain 2 gave evidence that the helix extended all the way to the N-terminus of this peptide, indicating that sequence and not chain end effects are very important in helix termination for our model peptides. Both nuclear Overhauser effect spectroscopy (NOESY) connectivities and chemical shift indices revealed significant perturbations around prolyl residues in the helices formed by transmembrane domains 6 and 7. Molecular models of the transmembrane domains indicate that helices for domains 6 and 7 are severely kinked at these prolyl residues. The helix perturbation around proline 258 in transmembrane domain 6 correlates with mutations that cause phenotypic changes in this receptor.