Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Industrial Protein Synthesis Using Recombinant Microorganisms
Typical Large-Scale Fermentation Systems

Recombinant microorganisms are widely used to obtain various protein products used in medicine (e.g., Insulin), as well as serving as "factories" for The production of commercially valuable metabolites (e.g., Antibiotics). Proteins are synthesized most intensively from the mid-exponential phase to its completion, while metabolites are produced during the deceleration phase and stationary phase. All of this must be considered when selecting parameters for large-scale Fermentation processes.

Optimizing the Synthesis of the desired product is a major scientific challenge. When it comes to proteins, this is typically addressed using cloned genes under the control of strong regulated promoters. Initially, it was thought that constitutive expression of the cloned Gene would be sufficient to obtain the required amount of product. However, experience has shown that continuous METABOLISM/31.html">Transcription and Translation of a cloned gene depletes all The Cell's energy resources and slows its growth. To coordinate the expression of the cloned gene with a specific growth phase, an induction mechanism can be used. To achieve this, Cells are first grown under optimal conditions to a relatively high density, and then transcription is induced either by changing the Temperature or by adding a chemical inducer to the medium, depending on The Nature of the promoter (for example, isopropyl-ß-thiogalactopyranoside).

Two-stage fermentation in a large bioreactor (>100 L) faces certain difficulties, as it is technically very challenging to rapidly raise the temperature (typically from 30 to 42 °C) in a large volume or to ensure rapid and uniform Distribution of a chemical inducer. This problem can be solved by using two interconnected bioreactors (two-stage fermentation): cells are grown in one, and induction is carried out in the other. This allows the growth and induction processes to be optimized separately and increases The amount of product synthesized per unit of time.

Two-stage fermentation in tandem airlift bioreactors. E. coli strain NM989, carrying the T4 DNA ligase gene under the transcriptional control of the p^L promoter and the temperature-sensitive repressor cI, was grown and induced in a two-stage airlift bioreactor (Fig. 16.5). The DNA ligase gene was integrated into the chromosomal DNA, which eliminated all Problems associated with plasmid instability during long-term fermentation. Cells were grown at 30 °C in an airlift bioreactor with external recirculation and a working volume of 10 L. Under these conditions, the DNA ligase gene was not expressed. For induction at 42 °C, an airlift bioreactor with external recirculation and a working volume of about 5 L was used. The bioreactors were connected by a tube with a pump that ensured a continuous feed of the suspension from the first bioreactor to the second. The cell suspension, having reached a certain density, was removed from the induction bioreactor and subjected to further Processing.

The maximum specific growth rate of the culture (μ_mах) was approximately 0.66 h^-1 in the first bioreactor and 0.54 h^-1 In the second, corresponding to doubling times of 63 and 77 min. Fresh medium was continuously added to the growth fermenter at a rate of 2 L/h, and an equal volume of suspension was withdrawn from the induction fermenter. Since the working volumes of the bioreactors differed, the cells spent approximately 5 h in the growth bioreactor and 2 h in the induction bioreactor. This difference in residence time was necessary to optimize cell number, product yield, and DNA ligase stability. The residence time of the cells in the different reactors can be varied by changing their relative working volumes and the volume of nutrients supplied to the first bioreactor.

The airlift fermenter with double external recirculation used in this study (Fig. 16.5) simplified the Regulation of the relative working volumes of the fermenters and increased The flexibility of the system (providing different growth conditions for different populations of recombinant cells). In the synthesis of DNA ligase, the best results were obtained with a flow rate of approximately 33 mL of cell suspension per minute from the first bioreactor to the second. This is equivalent to only 0.67% of the volume of the induction bioreactor, ensuring an almost instantaneous temperature rise of the entire incoming suspension from 30 to 42 °C. To maintain the growth of the cells in the second bioreactor in the exponential phase, the required amount of nutrients was continuously added in a concentrated form. This prevented the degradation of DNA ligase by Proteolytic Enzymes, which are typically synthesized by cells during the deceleration and stationary phases.

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Fig. 16.5. A two-stage airlift bioreactor system used for the temperature induction of protein product synthesis. From the fermenter in which cultivation is carried out at 30 °C (left), cells enter the fermenter at 42 °C (right), where induction occurs. Both bioreactors have a double external recirculation loop equipped with Valves. By adjusting the valves, the working volume can be optimized for the given conditions.

When grown in such a two-stage continuous bioreactor, the culture of the strain

E. coli NM989 can reach a density of 4 g (dry weight) per 1 L, and T4 DNA ligase can account for up to 4% of the total cellular protein, which corresponds to approximately 25,000 units of enzymatic activity per 1 g (dry weight). In general, using the described approach, it is possible to synthesize approximately 100,000 units of enzymatic activity per 1 L of culture, i.e., up to 4,800,000 units per day. Given that after Enzyme Purification the activity level decreases to 20% and that the cost per unit of activity is approximately 0.25 dollars, we find that enzyme worth 240,000 dollars can be synthesized daily. Although we have not accounted for all the costs of protein production itself, it is clear that the profit from the sale of valuable products obtained by continuous fermentation in small- or medium-scale bioreactors significantly exceeds the costs.

Two-stage fermentation in a single mechanically stirred bioreactor

The tripartite recombinant protein AGßgal, used in immunological assays, was synthesized on an industrial scale in a single mechanically stirred bioreactor. The gene for this protein was engineered to contain segments encoding five immunoglobulin G (IgG) binding sites of Staphylococcus aureus protein A, two IgG binding sites of Streptococcus strain G148 protein G, and E. coli β-galactosidase. It was under the control of the bacteriophage λ p^R promoter (regulated in the same manner as the p^L promoter) and was integrated into a plasmid carrying an ampicillin resistance gene; this construct was used to transform E. coli cells. The strain harboring the plasmid with AGßgal DNA contained a second plasmid carrying the gene for the temperature-sensitive repressor protein cI and a kanamycin resistance gene. A 5 L culture was grown at 30 °C in the presence of ampicillin and kanamycin to ensure the maintenance of both Plasmids, and was then used as an inoculum to initiate antibiotic-free culture growth at 30 °C in a Reactor with a working volume of 45 L. The cell suspension from the 45 L fermenter, in turn, served as inoculum for cultivation in a 600 L bioreactor, where the cells continued to grow at 30 °C without antibiotics (Fig. 16.6). (Generally, to reduce process costs, antibiotics are not added to the medium during large-scale cultivation.) Once the culture density in the 600 L bioreactor reached approximately 4 g/L, the temperature was raised from 30 to 40 °C to induce the expression of the AGßgal protein gene. Raising the temperature under these conditions took about an hour. Induction was performed at 40 °C rather than 42 °C because, at the lower temperature, the same amount of AGßgal protein was synthesized, but the cells could grow for a longer period. In other words, at the lower induction temperature (40 °C), the yield of the protein product was higher.

Fig. 16.6. Schematic representation of the industrial synthesis of AGßgal protein. The volume of the culture medium at each stage is indicated in parentheses. It accounts for 60 to 75% of the volume of the respective bioreactors (fermenters).

The specific activity of the AGßgal protein increased for 2 h after THE START OF induction and then declined. This was likely due to the synthesis of proteases by cells entering the deceleration or stationary phases. Furthermore, approximately 50% of the cells grown for 4 h at 40 °C lost the plasmid. Nevertheless, even under these conditions, after 4 h of cultivation at 40 °C, the AGßgal protein accounted for approximately 20% of the total dry cell weight. Considering all of this, it may not be necessary to integrate the genes for the AGßgal protein and the cI repressor into the chromosomal DNA of the E. coli host cell to increase product yield.

Batch fermentation and fed-batch fermentation

In some cases, to achieve high culture density and obtain large amounts of product, it is sufficient to carry out fermentation in a conventional batch mode. In one experiment, a plasmid carrying a fusion protein gene, one component of which was the insulin B peptide, was placed under the control of the E. coli trp promoter and introduced into a trp^- E. coli strain; the transformed cells were cultured in media with varying Tryptophan content. At high concentrations of tryptophan, the fusion protein was not synthesized; subsequent depletion of tryptophan from the medium by the growing cells led to the Induction of the target Protein Synthesis. Adding tryptophan to the medium increased both biomass and synthesized protein yields, and The Use of fed-batch fermenters further enhanced this effect (Table 16.1).