Biotechnology - Yu.O. Sazykin 2006

General Biotechnology
Biological Objects: Methods for Their Creation and Improvement
Improvement of Biological Objects via Cell Engineering

For many years, mutagenesis and selective breeding have been successfully applied and will continue to be widely used for the improvement of biological objects in the future.

However, significant limitations in achieving biotechnological goals have gradually become apparent. Although the primary path to overcoming these constraints lies in Introduction/32.html">Genetic Engineering Methods, cellular engineering also holds certain Prospects—specifically, the "forced" exchange of chromosomal segments in prokaryotes, or segments and even entire Chromosomes in eukaryotes. This process creates non-natural biological objects from which producers of novel substances or organisms with practically valuable properties can be selected.

The prospects of cellular engineering lie primarily in its potential to generate interspecies and intergeneric hybrid cultures of microorganisms, as well as hybrid Cells between evolutionarily distant Multicellular Organisms. It should be noted upfront that the material concerning The production of hybrids between lymphocytes and tumor cells is discussed in Chapter 10. In this chapter, cellular engineering techniques are examined in relation to microorganisms and in accordance with the objectives set for them as biological objects. Prokaryotic microorganisms (possessing a single chromosome in The Cell) can serve as the simplest example.

To exchange chromosome fragments in prokaryotes, it is first necessary to obtain protoplasts devoid of cell walls from their cells; next, to induce protoplast fusion to form diploids; and then to incubate the resulting diploids for several hours to allow the "breaking" and rejoining of circular chromosomal threads in various combinations. Afterwards, the protoplast suspension is plated onto a solid nutrient medium. During this process, some diploids convert back into haploids—reproduction-competent cells that form distinct colonies. These are subsequently studied, and cultures acquiring novel traits of interest to biotechnologists are selected (Fig. 2).

Thus, The First stage of work involves removing The Cell wall from microorganisms. In prokaryotes—eubacteria and actinomycetes (multicellular Bacteria)—the cell wall consists of a rigid peptidoglycan polymer that maintains cell shape and protects the cytoplasmic membrane from osmotic pressure differentials between the external environment and the Cytoplasm.

Peptidoglycan can be cleaved using Enzymes (peptidoglycan Hydrolases), the best known of which is Lysozyme, which breaks down the polysaccharide chains of peptidoglycan. Chicken egg white serves as the source of lysozyme as a laboratory reagent.

Under normal bacterial cultivation conditions, the enzymatic degradation of the cell wall immediately leads to lysis due to the difference in osmotic pressure, as neither the cytoplasmic nor the outer membrane (in Gram-negative bacteria) can maintain structural integrity. This long-known phenomenon explains, for instance, the lytic effect of penicillin, which disrupts the balance between the action of peptidoglycan synthetases and hydrolases by inhibiting peptidoglycan synthesis. It follows that removing the cell wall while preserving the integrity of the protoplast membrane is only possible by balancing the osmotic pressure inside the cell and in the surrounding medium. Although simple in principle, this approach remained unknown for a long time until J. Lederberg demonstrated its feasibility. Since then, work on the cellular engineering of microorganisms has been based on protoplast formation: to obtain protoplasts, the cell wall is removed via enzymatic Treatment in a "hypertonic" medium containing a 20% sucrose or mannitol solution, and occasionally a 10% sodium chloride solution, depending on the Specific characteristics of the biological object and the goals pursued. The conversion of a cell suspension into a protoplast suspension is typically monitored using Phase-contrast Microscopy.

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Fig. 2. Protoplast formation in prokaryotes:

1 — enzymatic degradation of the cell wall; 2 — protoplast fusion; 3 — chromosome breakage and recombination, diploid formation; 4 — formation of haploids with a recombinant chromosome; 5 — protoplast regeneration

When the biological object belongs to microscopic Fungi (Molds and Yeasts), protoplast isolation typically employs not lysozyme, but a complex enzyme preparation extracted from the digestive tract of the vineyard snail Helix pomatia. This is because the cell wall composition in fungi is more complex than in bacteria, consisting of polymers such as Chitin, glucans, and mannoprotein, each requiring its specific degrading enzyme. The digestive juice of the vineyard snail harvested in early spring—when the snails appear in vineyards before their stomachs are filled with digested grapevine leaves—is known to be the most effective in terms of enzyme assortment and catalytic activity. The crude Histology/2.html">EXTRACT FROM THE snail's digestive tract is freeze-dried (lyophilized) and used as a complex enzyme preparation to obtain protoplasts from fungal cells.

The next stage of work involves combining the Suspensions of two protoplast samples belonging to different strains, different species, or, more rarely, even different genera. Achieving the fusion of two protoplasts of different origins is a rather challenging task. The frequency of fusion increases dramatically upon The addition of polyethylene glycol, which exhibits detergent-like properties. In the case of prokaryotes, the resulting protoplasts possess a double set of chromosomal material—that is, they are protoplasts with two chromosomes. These protoplasts maintain their integrity within a hypertonic medium.

After washing with a hypertonic medium that no longer contains the cell wall-hydrolyzing enzyme preparation, the protoplasts are plated onto a solid nutrient medium. During this process, diploid forms transition into haploid forms in a certain fraction of the protoplasts, resulting in The formation of reproduction-competent normal cells with a cell wall. These cells form colonies on the solid medium. However, while the protoplasts exist in the diploid state, "breaking" and rejoining of chromosomal threads occur in some of them, allowing a fragment of one chromosome to be incorporated into another. Thus, normal cells are formed during protoplast regeneration, some of which possess hybrid chromosomes. Cultures of such cells exhibit novel properties. The production of "hybrid" Antibiotics serves as a prime example.

It is known that among actinomycetes, there are various species that produce glycosidic antibiotics featuring varying aglycones and sugars. For instance, the antibiotic erythromycin possesses a 14-membered macrocyclic aglycone and two sugars (desosamine and cladinose) attached via a glycosidic bond (see p. 110); whereas in anthracycline antibiotics, the aglycone consists of four condensed six-membered carbon rings linked to an aminosugar (see p. 112).

Cellular engineering has enabled the generation of antibiotic producers in which the macrolide aglycone of erythromycin is linked to the carbohydrate moiety characteristic of anthracyclines, and conversely, where the anthracycline aglycone is bound to sugars typical of erythromycin. Similar efforts are underway to produce hybrid beta-lactam antibiotics.

When evaluating the prospects of using the protoplast Procedure in biotechnology, it should be noted that even the fusion of protoplasts belonging to the same population and their subsequent regeneration can result in the genes responsible for target product Biosynthesis ending up in a single haploid in a duplicated state (its chromosome will be enriched with two biosynthetic Gene clusters). In other words, the biosynthetic potential (regarding the target product) of two chromosomes will be concentrated within a single chromosome. Naturally, only a small fraction of the cultures obtained after regeneration will possess this feature. Therefore, post-protoplast formation screening reveals both "plus" and "minus" Variants of the producer compared to the productivity of the initial culture.

The Selection of "plus" variants presents no difficulties; however, maintaining their sustained use in industrial production is far from simple. After several subcultures, their productivity gradually declines, approaching that of the parental culture. The reasons for this phenomenon are not yet fully understood. Evidently, the culture's repair systems drive its biosynthetic parameters back toward natural baselines. Two approaches are proposed to slow down the reversion of "plus" variants to their initial performance levels.

The first approach involves treating "plus" variants with mutagens and selecting mutants with a reduced capacity for returning altered DNA segments to normal. The second approach is associated with the potential use of enzyme engineering, specifically immobilized biological objects. It is possible to immobilize "plus" variant cells—that is, bind them to an insoluble carrier and use them in production without resorting to subculturing for a specified period (ranging from several weeks to several months). Although technically quite challenging, this approach is of considerable interest as a novel trend in biotechnology: combining cellular engineering directly with enzyme engineering in industrial manufacturing.

In Conclusion, protoplasts—the primary object manipulated in cellular engineering—are also widely utilized in genetic engineering. In this context, the exchange of DNA molecules and molecular fragments is achieved much more easily in protoplasts than during the transformation of cells possessing more complex protective envelopes.