Influence of plasmid origin and promoter strength in fermentations of recombinant yeast

The effects of plasmid promoter strength and origin of replication on cloned gene expression in recombinant Saccharomyces cerevisiae have been studied in batch and continuous culture. The plasmids employed contain the Escherichia coli lacZ gene under the control of yeast promoters regulated by the galactose regulatory circuit. The synthesis of beta-galactosidase was therefore induced by the addition of galactose. The initial induction transients in batch culture were compared for strains containing plasmids with 2mu and ARS1 origins. As expected, cloned gene product synthesis was much lower with the ARS1 plasmid: average beta-galactosidase specific activity was an order of magnitude below that with the 2mu-based plasmid. This was primarily due to the low plasmid stability of 7.5% when the plasmid origin of replication was the ARS1 element. The influence of plasmid promoter strength was studied using the yeast GAL1, GAL10, and hybrid GAL10-CYC1 promoters. The rate of increase in beta-galactosidase specific activity after induction in batch culture was 3-5 times higher with the GAL1 promoter. Growth rate under induced conditions, however, was 15% lower than in the absence of lacZ expression for this promoter system. The influence of plasmid promoter strength on induction behavior and cloned gene expression was also studied in continuous fermentations. Higher beta-galactosidase production and lower biomass concentration and plasmid stability were observed for the strain bearing the plasmid with the stronger GAL1 promoter. Despite the decrease in biomass concentration and plasmid stability, overall productivity in continuous culture using the GAL1 promoter was three times that obtained with the GAL10-CYC1 promoter.     

Development of a LAC4 promoter-based gratuitous induction system in Kluyveromyces lactis

A gratuitous induction system based on the strong, indigenous LAC4 promoter was developed for Kluyveromyces lactis. To prevent consumption of the inducer galactose, a strain with a gal1-209 mutation was employed; this mutation disables the galactokinase function but retains the regulatory function for induction. The Escherichia coli lacZ gene (encoding beta-galactosidase) is functional in K. lactis and was used as the reporter gene downstream of the LAC4 promoter on a multicopy plasmid. The gal1-209 strain exhibited several unexpected phenomena, including partial consumption of the inducer galactose (although at a much slower rate relative to GAL1 strains) and growth inhibition at high concentrations of galactose. These unusual characteristics, however, did not prevent the successful construction of a strong gratuitous induction system. Due to the low rate of inducer consumption for the gratuitous strain, very low concentrations of galactose (1:20 galactose:glucose) resulted in high-level induction. Under these conditions, beta-galactosidase specific and volumetric activities were 4.2- and 5.5-fold higher, respectively, than those for the "GAL1" nongratuitous strain. This research demonstrated the improved productivity possible via LAC4 promoter-based gratuitous induction (and thus a more stable inducer concentration). The effects of various carbon source concentrations on growth and induction were also determined.     

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.     

Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture

A gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed-batch culture. The Escherichia coli lacZ gene (beta-galactosidase; intracellular) and MFalpha1 leader-BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K. lactis LAC4 promotor, inserted into partial-pKD1 plasmids, and transformed into a ga1-209 K. lactis strain. To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures. A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both beta-galactosidase and secreted BPTI. This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same. In shake-flask culture, biomass concentration, beta-galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively. Fed-batch culture (with a high glucose concentration and a low galactose [inducer] concentration feed) resulted in a 6.5-fold increase in biomass, a 23-fold increase in beta-galactosidase activity, and a 3-fold increase in BPTI secreted activity. The results demonstrate the success of gratuitous induction during high-cell-density fed-batch culture of K. lactis. A very low concentration of galactose feed was sufficient for a high production level.     

Analysis of two-stage recombinant bacterial fermentations using a structured kinetic model

A structured kinetic model has been employed to analyze the performance of a two-stage continuous fermentation of a recombinant Escherichia coli. Separating the cell growth phase from the gene expression phase in two fermentors minimizes the growth rate difference between the recombinant cells and the plasmid-free cells in the first fermentor, thereby increasing the plasmid stability. The plasmid-harboring cells from the first fermentor are continuously fed into the second fermentor, in which the foreign protein synthesis is turned on by the addition of the inducer. Consequently, the recombinant cells experience an immediate reduction in growth rates as soon as they enter the second stage and then recover to synthesize the foreign protein. To analyze the fermentation performance contributed by these cells with different intracellular foreign protein levels and growth rates, a novel method for determining the residence time distribution of the growing cells in the second stage has been formulated. Combined with this method, the structured kinetic model for recombinant bacterial cells is used to predict the plasmid stability and foreign productivity at various operation conditions, such as induction strength and dilution rates. This model can provide us with thorough understanding of the characteristics of the two-stage fermentations, and is useful for the development of large scale continuous cultures of recombinant bacteria.     

Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: II. Two-stage continuous cultures and model simulations

A two-stage culture strategy was studied for continuous high-level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid-bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid-bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two-stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two-stage continuous cultures. (c) 1993 John Wiley & Sons, Inc.     

Two-stage cultivation of recombinant Saccharomyces cerevisiae to enhance plasmid stability under non-selective conditions: experimental study and modeling

A leucine auxotroph strain of Saccharomyces cerevisiae was used to study plasmid stability and expression using a recombinant plasmid, which contained a foreign gene for firefly luciferase (luc). This recombinant yeast was tested in a series of continuous cultures in semi-defined media with varying concentrations of yeast extract in order to study its effect on stability. While the biomass concentration and luciferase activity increased with increasing concentrations of yeast extract, the plasmid stability declined. An analysis of the growth rates showed that the recombinants enjoyed a growth rate advantage over the plasmid-free cells at critically low yeast extract concentrations, possibly due to leucine starvation in the media. A two-stage cultivation strategy was designed in order to create a yeast extract limited environment so that plasmid-free cells could not grow and overtake the recombinant cells. The cells were cultivated in selective media in the first stage, and then transferred continuously to the second stage where the media was enriched by feeding yeast extract. The feed rate was kept low in order to ensure yeast extract and hence leucine starvation, thereby selecting against the plasmid-free cells. This strategy resulted in a stable existence of recombinant cells, which stabilized around 60% at steady state during the tested period of cultivation. The complex nitrogen feed helped in increasing the cell density and volumetric activity by approximately 9 and 18-fold respectively with respect to that achieved in minimal medium. The experimental data was used to formulate a mathematical model to predict cell growth and plasmid stability in two-stage cultivation, which correctly explained the experimental data.     

Yeast reporter system for rapid determination of estrogenic activity

An in vitro test system for the determination of estrogens, xeno- and phytoestrogens, based on the activation of human estrogen receptor-alpha, has been examined for ability in monitoring environmental estrogens. The system consists of an expression plasmid for the human estrogen receptor-alpha and a reporter plasmid containing the lacZ gene under the control of the vitellogenin hormone response element. These plasmids have been transformed into S. cerevisae. Cultivation of yeast in the presence of estrogenic substances leads to activation of the estrogen receptor and induces the expression of the reporter lacZ. beta-Galactosidase activity of the translated gene lacZ is a measure of the estrogenic activity of a compound. First, the selectivity of the system was compared to data available in the literature. Then the sensitivity of the system was checked. The detection limit is 0.1 ng 17-beta estradiol or an equivalent activity per liter, if a sample can be concentrated 1000-fold. The system has been further characterized by selected compounds with known and unknown estrogenic activity.     

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.     

Persistence and expression of the plasmid pBR322 inEscherichia coli K12 cultured in complex medium

Summary The stability and gene expression of plasmid pBR322 in a chemostat with complex non-selective medium at different dilution rates were studied. It was observed that pBR322 was eventually lost from the population after a long lag period. The rate of plasmid loss decreases with decreasing dilution rate. This result is different from those obtained with cells grown in defined medium, where plasmid loss was observed to decrease with increasing dilution rate. In addition, it was observed that the -lactamase activity per ml per optical density of cell culture, independent of the dilution rates, increases with time and reaches a maximum around 4.5 units after 100 hrs of continuous culture.     

Controlled expression and purification of human immune interferon from high-cell-density fermentations of Saccharomyces cerevisiae

Conditions for high-cell-density fermentations of Saccharomyces cerevisiae strains producing recombinant-DNA-derived proteins were established. Strains producing human immune interferon (IFN-gamma) from the constitutive PGK promoter failed to grow to high cell densities and exhibited low plasmid stability. Regulated expression of IFN-gamma was obtained in similar strains by employing a hybrid yeast GPD promoter that was subject to carbon source regulation due to the presence of regulatory DNA sequences from the yeast GAL 1,10 intergenic region. IFN-gamma expression programmed by this vector was low during growth on glucose and was induced by galactose. Previously defined fermentation conditions employing glucose as a carbon source were applied to this strain, resulting in high ceil densities with higher plasmid stability. Various methods of galactose induction of IFN-gamma expression in high-cell-density fermentations were investigated. Optimal conditions resulted in a 2000-fold induction and production of 2 g IFN-gamma/L fermentation culture.     

Anaerobic and aerobic continuous cultures of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>: comparison of plasmid stability and<Emphasis Type="Italic"> EXG1</Emphasis> gene expression

Two bioreactor continuous cultures, at anaerobic and aerobic conditions, were carried out using a recombinant Saccharomyces cerevisiae strain that over-expresses the homologous gene EXG1. This recombinant system was used to study the effect of dissolved oxygen concentration on plasmid stability and gene over-expression. Bioreactor cultures were operated at two dilution rates (0.14 and 0.03 h^–1) to investigate the effect of other process parameters on EXG1 expression. Both cultures suffered severe plasmid instability during the first 16 generations. Segregational plasmid loss rate for the aerobic culture was two-fold that of the anaerobic operation. In spite of this fact, exo--glucanase activity at aerobic conditions was 12-fold that of the anaerobic culture. This maximal activity (30 U ml^–1) was attained at the lowest dilution rate when biomass reached its greatest value and glucose concentration was zero.     

Construction and characterization of a thermostable whole-cell chitinolytic enzyme using yeast surface display

To develop a novel yeast whole-cell biocatalyst by yeast surface display technology that can hydrolyze chitin, the chitinaseC gene from Serratia marcescens AS1.1652 strain was cloned and subcloned into the yeast surface display plasmid pYD1, and the recombinant plasmid pYD1/SmchiC was electroporated into Saccharomyces cerevisiae EBY100 cell. Aga2p-SmChiC fusion protein was expressed and anchored on the yeast cell surface by induction with galactose, which was verified by indirect immunofluorescence and Western blotting. The chitinolytic activity of the yeast whole-cell biocatalyst or partially purified enzyme was detected by agar plate clear zone test, SDS-PAGE zymography and dinitrosalicylic acid method. The results showed that the chitinaseC gene from S. marcescens AS1.1652 strain was successfully cloned and expressed on the yeast cell surface, Aga2p-SmChiC fusion protein with molecular weight (67 kDa) was determined. Tests on the effect of temperature and pH on enzyme activity and stability revealed that the yeast whole-cell biocatalyst and partially purified enzyme possessed both thermal stability and activity, and even maintained some activity under acidic and weakly alkaline conditions. The optimum reaction temperature and pH value were set at 52 °C and 5.0, respectively. Yeast surface display technology succeeded in preparing a yeast whole-cell biocatalyst with chitinolytic activity, and the utilization of chitin could benefit from this process of enzyme preparation.     

Evaluation of pKD1-based plasmid systems for heterologous protein production in Kluyveromyces lactis

The stability of pKD1-based vectors was evaluated during the synthesis of intracellular and extracellular gene products in the yeast Kluyveromyces lactis. The Escherichia coli lacZ and MFalpha1 leader-BPTI (bovine pancreatic trypsin inhibitor) cassettes were placed under the control of the inducible K. lactis LAC4 promoter and inserted into the pKD1-based plasmids. To induce gene expression while maintaining inducer level, a gratuitous gal1-209 K. lactis strain was employed. Selective medium containing 5 g glucose/l and 0.5 g galactose (inducer)/l allowed optimum expression and secretion of heterologous products without a significant effect on the growth of the recombinant cells. During long-term sequential batch cultures (60 generations), plasmid instability was mainly the result of structural instability. The expression and secretion of BPTI resulted in greater structural instability relative to the intracellular beta-galactosidase. For both products, vectors carrying the pKD1 replication origin and the cis-acting stability locus (partial-pKD1 vectors) were more stable than vectors carrying the full pKD1 sequence (full-pKD1 vectors). However, after 55 generations, the beta-galactosidase and BPTI activities were still higher with the full-pKD1 vectors. This was due to the significantly higher initial beta-galactosidase and BPTI activities for the full-pKD1 vectors (approximately 85% and 47% higher, respectively) relative to the partial-pKDI vectors. Southern blots confirmed that these increases were due to the higher copy number of the vectors carrying the full pKD1 sequence. In contrast to our previously reported results for the secretion of invertase, full-pKD1 vectors were preferred for the expression/secretion of beta-galactosidase and BPTI for at least 55 generations. Due to their structural stability, partial-pKD1 vectors will be advantageous for very long cultivation times.     

Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae

We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24(s) monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous culture. The hepatitis B virus S gene, which encodes the p24(s) monomer, is transcribed under the control of the GAL 10p on a chimeric 2-mum plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host-cell-derived components. Steady states were evaluated in a range from 0.015 h(-1) to washout (0.143 h(-1)). Both p24(s) monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24(s) monomer to HBsAg particle, estimated at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-mum plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h(-1). Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h(-1)), even though HBsAg expression was maximal. Total p24(s) monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h(-1). (c) 1996 John Wiley & Sons, Inc.