Fundamentals of Biochemical Engineering Part 1 - Bailey J., Ollis D. 1989

Molecular genetics and regulatory systems
Recombinant DNA technology
Genetic engineering using other host cells

E. coli is the Organism of choice for the vast majority of cloning and Introduction/32.html">Genetic Engineering projects, largely because it has been studied in the greatest detail at THE MOLECULAR LEVEL. At the same time, E. coli is virtually unknown in the industrial biotechnology sector. As a Gram-negative bacterium, its outer membrane contains toxic lipopolysaccharides (E. coli Sepsis is fatal in 50% of cases) and it does not naturally secrete Proteins into the culture medium. Furthermore, being a prokaryote, E. coli lacks the machinery for splicing and Post-translational protein modification characteristic of Eukaryotic Cells. For these and several other reasons, developing cloning and expression systems in organisms other than E. coli is of considerable interest. In all such cases, however, It is important to keep in mind the following factors that can hinder foreign Gene Expression:

1. Degradation of foreign DNA or RNA by host Cell Nucleases.

2. Failure of the vector Replication mechanism.

3. Low activity of the promoter or METABOLISM/31.html">Transcription terminator.

4. Incomplete mRNA splicing.

5. Inefficient Translation.

6. Proteolytic degradation.

In addition, an efficient method is required to ensure high-yield introduction of the vector into the host cell.

Cloning and gene expression Methods have been established for several bacterial species. Among these, Bacillus subtilis is arguably the best studied. It is a Gram-positive, non-pathogenic, non-parasitic microorganism that was used in the industrial Fermentation sector for The production of various Enzymes and Polypeptide Antibiotics long before the dawn of genetic engineering. B. subtilis secretes some of its proteins directly into the medium. In genetic engineering, this trait is particularly advantageous because secreted proteins are usually free of contamination by large amounts of closely related intracellular proteins. Moreover, protein secretion into the medium theoretically allows for much higher concentrations of the synthesized product compared to intracellular accumulation. (What is the upper limit of intracellular protein per unit volume of culture broth?)

A number of Plasmids and Bacteriophages can be cloned in B. subtilis. Transformation, Transduction, and protoplast fusion methods can be used to introduce foreign DNA into B. subtilis. Although the successful expression of several mammalian proteins—including Insulin and interferons—has been achieved in B. subtilis, the recovery of foreign proteins from this host remained problematic up to the time this chapter was written.

Cloning technology is also applicable to several strains of Pseudomonas and Streptomyces. By combining genetic engineering with traditional mutagenesis and Selection, researchers have generated Pseudomonas strains with entirely novel metabolic pathways; for instance, these strains can grow on normally toxic chlorinated Hydrocarbons as their sole carbon source. The immense industrial importance of various Streptomyces species has driven intensive research into applying genetic engineering techniques to these microorganisms. The main objective is to isolate strains with higher yields of commercially valuable enzymes or those capable of synthesizing novel semi-synthetic or hybrid antibiotics.

These latter Examples merit a somewhat more detailed Structure/133.html">Discussion, as they mark our first encounter with metabolic engineering. In general, genetic engineering allows not only for the synthesis of proteins of direct interest, but also for the introduction of specific enzymes, regulatory proteins, permeases, and virtually any other protein into living cells. In this way, we can confer entirely novel enzymatic, regulatory, or transport activities upon a cell—activities that would be extremely unlikely to occur naturally or to be obtained through random mutagenesis. Consequently, we are now able to rationally and purposefully redesign and reconstruct specific segments of a cell's metabolic network. Looking ahead, this is precisely the direction in which genetic engineering will primarily evolve. For now, progress is hindered by a shortage of detailed data on specific critical metabolic pathways and their rate-limiting steps. Moreover, our understanding of overall Metabolic Regulation is clearly insufficient to predict how altering one pathway will affect the flux through others. Finally, the expression of numerous novel genes in recombinant cells calls for The Development of innovative genetic engineering techniques.

In recent years, major breakthroughs have been achieved in the GENETIC ENGINEERING OF eukaryotic cells. The Yeast Saccharomyces cerevisiae has been studied most intensively. This microorganism boasts a robust genetic system capable of directly expressing both certain eukaryotic and several prokaryotic genes, alongside The ability to take up pure DNA via transformation. Yeast also carries out at least some typical eukaryotic post-translational modifications and secretes certain proteins into the culture medium. For example, recombinant S. cerevisiae strains produce the hepatitis B surface antigen, which appears indistinguishable in its degree of glycosylation and aggregation from the antigen found in patients suffering from the disease. Genetically modified yeast also synthesizes and secretes human immune interferon (IFN-γ) into the medium.

Gene cloning and expression in mammalian cells have thus far been accomplished primarily using vectors derived from SV40 virus. The Genome of this virus (a covalently closed circular DNA molecule) can replicate in mammalian cells both autonomously and after integration into the host cell chromosome. Expression of the cloned gene is driven by SV40 promoters and an origin of replication that function efficiently in mammalian cells.

Nevertheless, the fast-growing E. coli bacterium remains the most convenient organism for DNA Cloning and identification, as the majority of genetic engineering protocols are optimized for it. Therefore, when the ultimate goal is multiple rounds of vector replication followed by GENE EXPRESSION IN another organism, it is often advantageous to employ a so-called shuttle vector capable of replicating in both E. coli and the alternative host. Obviously, such a shuttle vector must possess two origins of replication—one for each host organism. Fig. 6.28 illustrates a shuttle vector designed for the production of immune interferon (IFN-γ) in monkey cells. In this construct, a 342-base-pair fragment containing the SV40 origin of replication and late promoter is joined to a cDNA fragment comprising the pre-IFN-γ coding sequence, as well as a pBR322 fragment containing the ampicillin resistance gene and the E. coli origin of replication. Transfection of COS-7 monkey cells with pure viral DNA yielded an IFN-γ activity of approximately 50–100 units per ml of culture fluid after 3–4 days. This example illustrates the distinct advantages of using genetically modified mammalian host cells for the synthesis of mammalian proteins, particularly when all stages of Biosynthesis and secretion must faithfully reproduce the in vivo pathways and transport mechanisms as closely as possible.

Significant progress is also being made in introducing foreign genes into whole plants and animals and achieving their subsequent expression. Although analyzing the methodology of these efforts goes beyond The Scope of this book, their potential significance for humanity is self-evident. These groundbreaking scientific and technical achievements have simultaneously sparked widespread debate (including in the literature) regarding the potential adverse impacts of biotechnology on society [24].

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FIG. 6.28. Diagram of a shuttle vector for E. coli and monkey cells, designed for the expression and secretion of immune interferon in monkey cells [23].