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

Fundamentals of Molecular Biotechnology
Optimization of Gene Expression Cloned in Prokaryotic Systems
Metabolic Overload

The Introduction of foreign DNA into a Cell and its subsequent expression frequently lead to disruptions in cellular METABOLISM. These disorders are quite diverse and stem from the metabolic burden imposed by the foreign DNA on all cellular processes. Metabolic overload can arise due to several factors.

✵ An increase in plasmid copy number and/or size, along with the corresponding rise in energy required for their Replication and maintenance.

✵ A shortage of dissolved oxygen in the medium, making it impossible to adequately supply both the general metabolic reactions and the expression of plasmid genes.

✵ The overexpression of foreign Proteins, resulting in the depletion of specific aminoacyl-tRNA pools (or even Certain Amino Acids) and/or energy reserves (in the form of ATP and GTP).

✵ Overloading of the export machinery and the consequent mislocalization of vital host proteins caused by the "overproduction" of foreign proteins exported from the Cytoplasm to The Cell membrane or periplasmic space.

✵ Unusual metabolic Properties of the host Organism (such as the high respiratory activity in Azotobacter spp.), rendering it more sensitive to various stresses than standard Cells.

✵ The Direct impact of foreign proteins on host Cell Functioning (for instance, The conversion of crucial, essential precursors into inappropriate and sometimes toxic compounds).

Metabolic overload can trigger a wide range of alterations in host cell physiology and performance. One of the most common consequences is a reduction in Cell Growth Rate following the introduction of foreign DNA. Specifically, plasmid-bearing cells grow more slowly than untransformed cells lacking Plasmids (Table 6.6), a phenomenon frequently accompanied by the loss of the recombinant plasmid. In some cases, metabolic overload exerts selective pressure that causes the deletion of the recombinant Gene or a portion of it from the plasmid.

Class="center">Table 6.6. Effect of plasmid copy number on host cell growth rate¹⁾

¹⁾ Based on data from Seo and Bailey, 1985, Biotechnol. Bioeng. 27: 1668–1674.

²⁾ Plasmids designated as A, B, C, D, and E encode only ß-lactamase and are identical in size.

Because cells growing under metabolic stress lack sufficient energy for normal functioning, highly energy-demanding metabolic processes—such as Nitrogen Fixation or Protein Synthesis—are primarily affected. Furthermore, Cell size and Morphology may change, and excessive extracellular polysaccharide production can occur, causing cells to clump together and complicating microfiltration.

As a consequence of metabolic overload driven by the excessive synthesis of foreign proteins and a shortage of nutrients or building blocks (amino acids), stress response mechanisms may be triggered. In particular, the synthesis of cellular proteinases can be induced, leading to the rapid degradation of the recombinant protein. Amino acid pool depletion can result from the efficient expression not only of cloned target genes, but also of vector genes encoding Antibiotic Resistance markers.

The translational error rate for E. coli ranges from 2∙10^-4 to 2∙10^-3 per cell per generation. However, under conditions of limited specific aminoacyl-tRNAs—which frequently occurs during foreign protein overexpression—the probability of incorporating an incorrect amino acid instead of the scarce one increases significantly. In addition, Translation fidelity is further impaired by a shortage of GTP, an essential component of the proofreading machinery. One study demonstrated that during the hyperproduction of mouse epidermal growth factor in E. coli cells, the frequency of amino acid misincorporation into the recombinant protein increased tenfold. This precludes The Use of the synthesized protein as a therapeutic agent because: 1) the specific activity and Stability of the protein may be much lower than expected; and 2) the presence of "incorrect" amino acids in the molecule can provoke an undesirable immunological reaction upon administration to humans.

Fortunately, with careful experimental design, The impact of metabolic overload can be minimized, recombinant protein yield optimized, and the stability of transformed host cells enhanced. For instance, metabolic burden can be reduced by employing low-copy-number Plasmid Vectors. Even better, vectors can be avoided altogether by integrating the foreign DNA directly into the host chromosome, eliminating the need to maintain plasmid stability. Moreover, the cell does not have to expend resources on synthesizing unnecessary products encoded by antibiotic resistance marker genes. The synthesis of such products—which are typically carried on plasmid vectors alongside target genes—is a major source of metabolic overload. Chromosomal integration is especially valuable when utilizing the recombinant microorganism itself rather than its synthesized product. Utilizing strong yet tightly regulated promoters also helps alleviate metabolic overload. In such cases, Fermentation is conducted in two stages: During the first stage (growth phase), the promoter controlling Transcription of the target gene is switched off, whereas during the Second Stage (induction phase), it is switched on.

If the codon usage of a foreign gene differs significantly from that of the host organism, Structure/149.html">The problem of specific aminoacyl-tRNA scarcity can be addressed by synthesizing part or all of the target gene with a codon bias more closely matching the host. For example, one study showed that The amount of streptavidin produced via the expression of a synthetic gene with a GC content of 54% was 10-fold higher than that obtained from the "natural" gene with a GC content of 69%. Nevertheless, this approach is quite complex and can only be applied in specific cases.

Paradoxically, one way to increase the total amount of foreign protein produced by a recombinant microorganism is to maintain a moderate level of Gene Expression (so that the target product accounts for approximately 5% of the total cellular protein) while maximizing culture cell density. A microbial system with a 5% foreign protein expression level and low metabolic burden, capable of reaching a cell density of 40 g/L (dry weight), proves to be more efficient than a system with a 15% expression level and a density of 10 g/L.

Achieving both a high level of foreign PROTEIN SYNTHESIS AND a high culture density is often hindered by the accumulation of harmful by-products (primarily acetate), which inhibit cell growth and protein synthesis. To mitigate acetate accumulation in a rich medium without impairing growth, glucose uptake can be slowed down by adding its analogue, methyl-α-glucoside. An alternative approach involves using E. coli cells carrying a mutation in the ptsG gene, which encodes enzyme II of the glucose phosphotransferase system. While the maximum cell density of wild-type E. coli cultures reached approximately 10 g/L, the ptsG mutant E. coli culture achieved 15 g/L. Furthermore, the level of ß-lactamase synthesis in mutant cells was 25% higher (per gram of dry weight) than in wild-type cells, resulting in an overall roughly twofold increase in yield. Comparable results can be achieved much more simply and rapidly through Genetic Engineering rather than mutagenesis and Selection. One strategy involved introducing genes encoding acetolactate synthase into E. coli. This enzyme catalyzes The formation of acetolactate from Pyruvate, thereby reducing the amount of acetate produced (Fig. 6.17). The acetolactate synthase genes are introduced into the cells on one plasmid, while the target genes are carried on another plasmid from a different incompatibility group. Transformed cells synthesize significantly less acetate than untransformed ones; instead, they produce acetoin, a compound roughly 50 times less toxic than acetate.

Fig. 6.17. Schematic representation of Glucose metabolism in E. coli cells transformed with a plasmid carrying acetolactate synthase genes.