Bioreactor studies of heterologous protein production by recombinant yeast

Abstract

Fundamental engineering studies were carried out on heterologous protein production using a recombinant Saccharomyces cerevisiae strain (C468/pGAC9) which expresses Aspergillus awamori glucoamylase gene and secretes glucoamylase into the extracellular medium, as a model system. Performance of a conventional aerobic free-suspension culture was compared to a novel immobilized-cell bioreactor bioprocess, for both bath and continuous conditions.

In the YPG nonselective medium, free-suspension batch cultures showed that cell growth was typically diauxic and glucoamylase secretion was growth-associated. Results of continuous cultures confirmed instability of the model recombinant yeast when growing in this medium. Changes in the fraction of plasmid-bearing cells and glucoamylase activity followed exponential decay patterns during continuous culture. The decay rates of both the plasmid-bearing cell fraction and glucoamylase expression increased with increasing dilution rates. Expressed as a function of cell generation, the decay rates were roughly constant over the dilution rates tested. A novel method is proposed to evaluate the instability parameters. The results indicated that the growth rate difference between plasmid-bearing and plasmid-free cells was negligible. Thus the contribution of preferential growth to apparent plasmid instability was negligible. No significant effect of growth rates on the probability of plasmid loss was observed. The importance of metabolic pathways with regard to the recombinant protein formation was analyzed. Production of glucoamylase was shown to be associated with oxidative growths of the recombinant yeast.

With the information from the experimental results and the literature, a mathematical model was formulated to simulate cell growth, plasmid loss and recombinant protein production. The model development was based on three overall metabolic events in the yeast: glucose fermentation, glucose oxidation and ethanol oxidation. Cell growth was expressed as a composite of these events. Contributions to the total specific growth rate suspended on 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 concentration on the specific growth rate was described by a modified Monod equation. It was assumed that the recombinant protein formation is only associated with oxidative pathways. Plasmid loss kinetics was formulated based on segregational instability during cell division by assuming a constant probability of plasmid loss. When applied to batch and continuous fermentations, the model successfully predicted the dynamics of cell growth (diauxic growth), glucose consumption (Crabtree effect), ethanol metabolism, glucoamylase production and plasmid instability. Good agreement between model simulations and the experimental data was achieved. Using published experimental data, model agreement was also found for other recombinant yeast strains. The proposed model seems to be generally applicable to the design, operation, control and optimization of recombinant yeast bioprocesses.

The novel immobilized-cell-film airlift bioreactor was based on cotton cloth sheets to immobilize the yeast cells by attachment. Continuous culture experiments were performed in it at different dilution rates in the YPG nonselective medium. The immobilized cell systems gave higher glucoamylase concentration (about 60%) and maintained recombinant protein production for longer periods of time (more than 100%) compared with the corresponding free suspension systems. The more stable glucoamylase production was due to a reduced plasmid loss in the immobilized cell system. By operating the immobilized-cell-film bioreactor in repeated bath mode, further reduction of plasmid instability of the recombinant yeast was obtained. An mathematical model was developed to describe the kinetics of plasmid loss and enzyme production decay based on a biofilm concept. The proposed model successfully described the experimental results and provided useful information for better understanding of the stabilizing mechanisms of the immobilized recombinant cells. It may be concluded that a reduced specific growth rate accompanied by an increased plasmid copy number is the basic explanation for the effective enhanced plasmid stability in the immobilized cell system. In the present case, the attached yeast film could be a dynamic reserve of highly concentrated plasmid-bearing cells having a higher plasmid copy number and less segregational instability. The immobilized-cell-film airlift bioreactor design has advantages including maintenance of genetic stability, high cell concentration and cloned gene product productivity, suitability for repeated bath or continuous operations for long periods of time stable reactor operation. Because of its enhanced operational efficiency, it may be useful in the commercial cultivation of recombinant or nonrecombinant yeast cells.