Physiological aspects of growth and recombinant DNA stability inSaccharomyces cerevisiae

Despite the fact that plasmid stability in the yeastSaccharomyces cerevisiae is influenced by both genetical and physiological parameters most attention has been focussed on the former. Physiological factors affecting the stability of plasmids have been poorly characterized despite the need for such information in order to optimize the use ofS. cerevisiae as a host for recombinant protein production processes. The physiology of wild typeS. cerevisiae differs considerably when grown using different cultivation techniques. A limited amount of phenomenological data has been reported concerning plasmid instability effects under these different conditions and in this article these have been collected together with the intention of providing an overview to instability effects and to try and propose reasons as to how the physiological response to different growth conditions can be manifested as stability/instability effects.     

Efficient expression and secretion of Aspergillus niger RH5344 polygalacturonase in Saccharomyces cerevisiae

An Aspergillus niger endopolygalacturonase (EC 3.2.1.15) cDNA was expressed in the yeast Saccharomyces cerevisiae. Secretion of the protein into the growth medium was efficiently directed by the fungal leader sequence, and processing occurred at the same site as in Aspergillus. The expression level was significantly enhanced by using a short version of the yeast ADHI promoter. An additional increase in the yield of heterologous protein was due to a higher plasmid stability and a rise in plasmid copy number. This was achieved by deleting most of the bacterial sequences from the expression vector. The yeast-derived enzyme showed the same enzymatic and biochemical properties as the fungal polygalacturonase, such as substrate specificity, pH and temperature optima and pI value. The yeast-derived enzyme, however, showed a higher degree of glycosylation and exhibited a more pronounced temperature stability than the fungal enzyme.     

Profiling of external metabolites during production of hantavirus nucleocapsid protein with recombinant Saccharomyces cerevisiae

Recombinant strains of Saccharomyces cerevisiae, producing hantavirus Puumala nucleocapsid protein for diagnostics and as a candidate vaccine were analyzed for uptake and excretion of intermediary metabolites during process optimization studies of fed-batch bioreactor cultures. Concentrations of glucose, maltose, galactose, pyruvate, acetaldehyde, ethanol, acetate, succinate and formaldehyde (used as a selection agent) were measured in the culture medium in order to find a metabolite pattern, indicative for the physiological state of the producer culture. When the inducer galactose was employed as a growth substrate, the metabolite profile of recombinant yeast cells was different from those of the non-recombinant original strain which excreted considerable amounts of metabolites with this substrate. In contrast, galactose-induced heterologous gene expression was indicated by the absence of excreted intermediary metabolites, except succinate. A model strain expressing a GFP fusion of hantavirus nucleocapsid protein differed in the excretion of metabolites from strains without GFP. In addition, the influence of alkali ions, employed for pH control is also demonstrated.     

Production of hantavirus Puumala nucleocapsid protein in Saccharomyces cerevisiae for vaccine and diagnostics

The production of hantavirus Puumala nucleocapsid (N) protein for potential applications as a vaccine and for diagnostic purposes was investigated with Saccharomyces cerevisiae as a recombinant host. The N protein gene and the hexahistidine tagged N (h-N) protein gene were expressed intracellular from a 2-microm plasmid vectors under the control of a fused galactose inducible GAL10-PYK promoter. For monitoring the recombinant gene expression, a h-N and a GFP fusion protein was used. Different cultivation strategies and growth media compositions were tested in shake flasks and a 5 l bioreactor. When using defined YNB growth medium, we found the biomass yield to be unsatisfactorily low. Higher concentrated YNB medium, promoted cell growth but showed a pronounced inhibitory effect on heterologous gene expression. This phenomenon could not be attributed to plasmid losses, as we could demonstrate high stability of the vector under the applied cultivation conditions. Supplementation of YNB medium with extracts of plant origin resulted in increased biomass yields with concomitant high expression levels of the recombinant gene. The modified medium was used for fed-batch cultivations where basic metabolic features as well as growth parameters were determined in addition to recombinant gene expression. The maximal volumetric yield of N protein was 316 mg l(-1), the respective yield of h-N protein was 284 mg l(-1). Our study provides a basis for large-scale production of hantavirus vaccines, which satisfies economic efficiency as well as biosafety regulations for human applications.     

A simple structured model for biomass and extracellular enzyme production with recombinant Saccharomyces cerevisiae YPB-G

A simple structured model is proposed for simulating batch cultivation data on growth, substrate utilization, and heterologous enzyme production of recombinant Saccharomyces cerevisiae YPB-G. The enzyme is a fusion protein displaying α-amylase and glucoamylase activities. Cell growth is modulated mainly by intracellular substrate and ethanol concentrations. Intracellular substrate concentration is evaluated by means of the extracellular substrate and biomass concentrations. Extracellular α-amylase and glucoamylase activities are taken to depend on biomass concentration. The nine parameters of the proposed model are determined using nonlinear estimation techniques, and the model is validated against experiments not used in parameter determination. The model developed simulates glucose consumption, cell mass, α-amylase and glucoamylase production in a batch system. Simulation and experimental results are found to be in good agreement. Journal of Industrial Microbiology & Biotechnology (2002) 29, 111–116 doi:10.1038/sj.jim.7000281 Received 07 January 2002/ Accepted in revised form 22 May 2002     

GOlf Complements A Gpa1 Null Mutation in Olf Saccharomyces Cerevisiae and Functionally Couples to the Ste2 Pheromone Receptor

Abstract

We have produced a plasmid designed for the expression of heterologous G protein α subunits in the yeast Saccharomyces cerevisiae Introduction of these genes is by simple cassette replacement using unique restriction sites, and their expression is controlled by the regulatory sequences of the S. cerevisiae GPA1 gene. Levels of expression are therefore suitable for interaction of these heterologous proteins with elements of the yeast pheromone response pathway. We believe that this plasmid will facilitate the coupling of more members of the seven transmembrane domain superfamily of receptors, through their native G protein α subunit, to the yeast pheromone response pathway.

The plasmid pRGP, is a stable centromeric shuttle vector with a HIS3-selectable marker. We have demonstrated that production of GPA1 from this plasmid functionally complements a gpal- null mutation. A similar response is obtained when an alternative G protein a subunit, G_olf, is introduced using pRGP. We believe that this is the first example of a heterologous G protein shown to couple to a yeast pheromone receptor.     

The Presence of Actinidin (Cysteine Protease) and Recombinant Plasmids Carrying the Actinidin Gene Influence the Growth of Saccharomyces Cerevisiae

Actinidin is a protease found abundantly in the fruit of Actinidia chinensis or Kiwi fruit. The overproduction of this protein in microorganisms, especially using the yeast Saccharomyces cerevisiae, would be economically valuable as it would simplify the extraction and purification of the protein. It was observed, however, that the yeast growth rate was reduced by the presence of externally supplied actinidin in the growth medium. It was also observed that actinidin present in the yeast growth medium was partially degraded. Several actinidin-encoding gene variants have been cloned in a yeast expression and secretion vector. It was observed that different actinidin gene constructions influenced the growth rate of S. cerevisiae in complete media. Recombinant plasmids carrying only the mature actinidin-encoding DNA sequences reduced yeast transformability significantly and had the least stability. The results thus suggest that the presence of a recombinant plasmid carrying a gene of a potentially toxic protein may result in a deleterious effect on the host cell.     

Optimization of 1,2,4‐butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance

1,2,4‐Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4‐butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2‐ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2‐keto‐3‐deoxy‐xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2‐ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.     

Heterologous expression of a gene of Magnaporthe oryzae chrysovirus 1 strain A disrupts growth of the human pathogenic fungus Cryptococcus neoformans

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1‐A) is the causal agent of growth repression and attenuated virulence (hypovirulence) of the rice blast fungus, M. oryzae. We have previously reported that heterologous expression of MoCV1‐A ORF4 in Saccharomyces cerevisiae results in growth defects, a large central vacuole and other cytological changes. In this study, the effects of open reading frame (ORF) 4 expression in Cryptococcus neoformans, a human pathogenic fungus responsible for severe opportunistic infection, were investigated. Cells expressing the ORF4 gene in C. neoformans showed remarkably enlarged vacuoles, nuclear diffusion and a reduced growth rate. In addition, expression of ORF4 apparently suppressed formation of the capsule that surrounds the entire cell wall, which is one of the most important components of expression of virulence. After 5‐fluoroorotic acid treatment of ORF4‐expressing cells to remove the plasmid carrying the ORF4 gene, the resultant plasmid‐free cells recovered normal morphology and growth, indicating that heterologous expression of the MoCV1‐A ORF4 gene induces negative effects in C. neoformans. These data suggest that the ORF4 product is a candidate for a pharmaceutical protein to control disease caused by C. neoformans.     

Data analysis of plasmid stability in continuous culture of recombinantSaccharomyces cerevisiae

Summary A simple method using non-linear regression is developed to analyse experimental data from plasmid stability studies of recombinantSaccharomyces cerevisiae grown in continuous cultures with non-selective and selective media. This method simultaneously provides quantitative information on the probability of plasmid loss due to segregation during cell division and the specific growth rates of plasmid-containing and plasmid-free cells at particular dilution rates. The method is applied to a set of experimental data. The three-parameter model, together with the estimated parameter values, provides a good fit to the experimental data.     

Testing plasmid stability of Escherichia coli using the Continuously Operated Shaken BIOreactor System

Plasmids are common vectors to genetically manipulate Escherichia coli or other microorganisms. They are easy to use and considerable experience has accumulated on their application in heterologous protein production. However, plasmids can be lost during cell growth, if no selection pressure like, e.g., antibiotics is used, hampering the production of the desired protein and endangering the economic success of a biotechnological production process. Thus, in this study the Continuously Operated Shaken BIOreactor System (COSBIOS) is applied as a tool for fast parallel testing of strain stability and operation conditions and to evaluate measures to counter such plasmid loss. In specific, by applying various ampicillin concentrations, the lowest effective ampicillin dosage is investigated to secure plasmid stability while lowering adverse ecological effects. A significant difference was found in the growth rates of plasmid‐bearing and plasmid‐free cells. The undesired plasmid‐free cells grew 30% faster than the desired plasmid‐bearing cells. During the testing of plasmid stability without antibiotics, the population fraction of plasmid‐bearing cells rapidly decreased in continuous culture to zero within the first 48 h. An initial single dosage of ampicillin did not prevent plasmid loss. By contrast, a continuous application of a low dosage of 10 µg/mL ampicillin in the feed medium maintained plasmid stability in the culture. Consequently, the COSBIOS is an apt reactor system for measuring plasmid stability and evaluating methods to enhance this stability. Hence, decreased production of heterologous protein can be prevented. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1418–1425, 2016     

Heterologous interferons synthesis in yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The HuIFNA16, HuIFNB1, and BoIFNG genes encoding human α16, β-interferons and bovine γ-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed. There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the “inclusion bodies.” The treatment of human β-interferon with endoglycosidase H showed that protein was expressed in glycosylated and unglycosylated forms. On the strength of these data, the hypothesis was suggested that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features of its metabolism.     

From shake flasks to bioreactors: survival of E. coli cells harboring pGST-hPTH through auto-induction by controlling initial content of yeast extract

A high content of yeast extract in complex media can cause auto-induction of phage T7 RNA polymerase and the consequent expression of recombinant protein in Escherichia coli BL21(DE3) during long-term cultivation. Our study demonstrated that the auto-induction of recombinant protein varied in different vectors harboring heterologous genes. Trx, GST, and their fusion proteins such as GST–human parathyroid hormone (hPTH), expressed by pET32a (+), were easily auto-induced by media containing a high content of yeast extract; however, rtPA was not easily auto-induced when using pET22b (+), although both pET systems were under the control of T7lac promoter. Furthermore, the auto-induction of GST–hPTH may start within 1–2 h after inoculation in bioreactors, which is a deficiency in the scale-up from shake flasks to bioreactors. Our results indicated that too much yeast extract in bioreactor cultivations may be responsible for the early auto-induction of target proteins and consequent loss of cell viability and plasmid instability. To achieve a satisfactory yield, host cells with both high cell viability and plasmid stability were necessary for the starter cultures in shake flasks and pre-induction cultures in bioreactors. This could be achieved simply by controlling the initial content of yeast extract and its subsequent supplementation.     

A Novel Culture Method for High Level Production of Heterologous Protein in Saccharomyces cerevisiae

A high level production system for heterologous protein by cold culture of yeast transformants at 15°C was developed. The yeast transformants, carrying a plasmid containing cDNA for Aspergillus oryzae α-amylase (Taka-amylase A) or human lysozyme synthetic DNA, were cultivated in a selective medium for 1 or 2 days until full growth at 30°C. The yeast cells were harvested by centrifugation from the culture fluid and then were transferred to YPD medium. These inoculated broths were incubated for 2 days at 15°C and then for another 2 days at 30°C. By the cold culture method described above, higher amounts of Taka-amylase A (28.6 mg/liter) and human lysozyme (6.1 mg/liter) were produced by the yeast transformants compared to those by conventional methods.

Heterologous protein productions using YEp, YCp, and YIp types of yeast expression vectors with ADH1 or GAPDH promoter by the cold culture method showed effective productivity of about 2-fold compared to those by the conventional method of culture at 30°C. The high level production of heterologous protein by this method was not specific to the S. cerevisiae strains examined.     

Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full‐length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde‐3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β‐1,4‐endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.     

Oxidative stress-resistance assay for screening yeast strains overproducing heterologous proteins

Many natural proteins have been developed into drugs and produced for direct application. Identifying improved hosts to achieve high-level heterologous protein production is a challenge in the study of heterologous protein expression in recombinant yeast. In this study, a novel high-throughput assay to screen such overproducing Saccharomyces cerevisiae strains was systematically developed. The protocol designed was based on screening host strain derivatives with increased superoxide dismutase dependent resistance to oxidative stress. Yeast cells transformed with recombinant plasmid carrying SOD1 gene as a reporter responded exquisitely to oxidative stress induced by elevated concentrations of paraquat. Improved yeast strains resulting from screening clones subjected to genome shuffling through selective pressure argue for a more effective screening system compared with traditonal selection. Moreover, this approach can be employed in general biochemical analysis without utilization of flow cytometry or well plate reader. Therefore, it is expected that the high-throughput assay would make superior strains producing heterologous proteins.     

Production of the Artemisinin Precursor Amorpha-4,11-diene by Engineered Saccharomyces cerevisiae

The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN·PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced 600 and 100 μg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days’ batch cultivation. Revisions requested 14 November 2005; Revisions received 17 January 2006     

Mathematical model for aerobic culture of a recombinant yeast

A mathematical model was formulated to simulate cell growth, plasmid loss and recombinant protein production during the aerobic culture of a recombinant yeast S. cerevisiae. Model development was based on three simplified metabolic events in the yeast: glucose fermentation, glucose oxidation and ethanol oxidation. Cell growth was expressed as a composite of these metabolic events. Their contributions to the total specific growth rate depended on the activities of the pacemaker enzyme pools of the individual pathways. The pacemaker enzyme pools were regulated by the specific glucose uptake rate. The effect of substrate concentrations on the specific growth rate was described by a modified Monod equation. It was assumed that recombinant protein formation is only associated with oxidative pathways. Plasmid loss kinetics was formulated based on segregational instability during cell division by assuming constant probability of plasmid loss. Experiments on batch fermentation of recombinant S. cerevisiae C468/pGAC9 (ATCC 20690), which expresses Aspergillus awamori glucoamylase gene and secretes glucoamylase into the extracellular medium, were carried out in an airlift bioreactor in order to evaluate the proposed model. The model successfully predicted the dynamics of cell growth, glucose consumption, ethanol metabolism, glucoamylase production and plasmid instability. Excellent agreement between model simulations and our experimental data was achieved. Using published experimental data, model agreement was also found for other recombinant yeast strains. In general, the proposed model appears to be useful for the design, scale-up, control and optimization of recombinant yeast bioprocesses.     

Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding

High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability, and has excellent recombinant DNA tools. We demonstrate here that this yeast exhibits three additional characteristics that are desirable in a eukaryotic expression host. First, expression in yeast significantly improves the solubility of proteins that are expressed but insoluble in E. coli. The expression and solubility of 83 Leishmania major ORFs were compared in S. cerevisiae and in E. coli, with the result that 42 of the 64 ORFs with good expression and poor solubility in E. coli are highly soluble in S. cerevisiae. Second, the yield and purity of heterologous proteins expressed in yeast is sufficient for structural analysis, as demonstrated with both small scale purifications of 21 highly expressed proteins and large scale purifications of 2 proteins, which yield highly homogeneous preparations. Third, protein expression can be improved by altering codon usage, based on the observation that a codon-optimized construct of one ORF yields three-fold more protein. Thus, these results provide direct verification that high level expression and purification of heterologous proteins in S. cerevisiae is feasible and likely to improve expression of proteins whose solubility in E. coli is poor.     

Selection of optimum expression system for production of kringle fragment of human apolipoprotein(a) in<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

RecombinantSaccharomyces cerevisiae expression systems were developed to produce a novel human anti-angiogenic protein called LK8, an 86 amino-acid kringle fragment protein with three disulfide linkages. Galactose-inducible LK8 expression plasmid was constructed, and LK8 production levels by fourS. cerevisiae strains were compared in order to select an optimal host strain.S. cerevisiae 2805 was the most efficient among the strains tested. Elevating the LK8 gene copy number through multiple integration using δ-sequences as target sites resulted in more than a two-fold increase in the LK8 production level compared with the plasmid-based expression system. The maximum LK8 protein concentration of 25 mg/L was obtained from batch cultivation of the yeast transformant that harbors 16 copies of the LK8 gene. In conclusion, the strain integrated with the multiple LK8 gene secreted the protein with relatively high yield, although, the increased LK8 gene dosage over 11 copies did not lead to further enhancement in batch cultivations.