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

Fundamentals of Molecular Biotechnology
Biological Systems Used in Molecular Biotechnology
Prokaryotes and Eukaryotes

The objects of molecular biotechnology are A wide variety of biological systems: microorganisms, insect, plant, and mammalian Cell lines, insect, plant, and mammalian Viruses, and Multicellular Organisms (plants, mice, domestic animals, etc.)—the choice of system depends on the experimental goals. The Nature of the biological system is exceptionally important for the biotechnological process. In many cases, it is the genetically modified, self-replicating biological unit—be it a microorganism, virus, plant, or animal—that serves as the final commercial product. Among the many biological objects used in molecular biotechnology, the primary "workhorses" are the bacterium Escherichia coli, the unicellular Yeast Saccharomyces cerevisiae, and various animal-derived cell lines. All of these play a crucial role in producing Proteins encoded by cloned genes.

In subsequent chapters, we will describe various highly specialized biological systems in detail. In particular, Chapter 7 will examine the "insect virus-insect cell" system, which is used to produce authentic proteins encoded by cloned genes, while Chapter 19 will cover the genetic modification of domestic animals (cows, sheep, pigs). In this chapter, we provide a Brief Overview of the systems most significant to molecular biotechnology, which will also be discussed in later chapters.

All Living organisms are divided into two main groups: PROKARYOTES AND EUKARYOTES. This Classification is based on numerous structural differences, which we will discuss in more detail in subsequent chapters; here, we will highlight only the primary ones: 1) the presence or absence of a nucleus containing chromosomal DNA; 2) the Structure and Chemical composition of The Cell wall; and 3) the presence or absence of subcellular cytoplasmic Organelles. In a Introduction/4.html">Prokaryotic Cell, such as a bacterial cell, the chromosomal DNA is located directly in the Cytoplasm, and The Cell is surrounded by a rigid cell wall, which often contains peptidoglycan but not Chitin or Cellulose; there are no subcellular cytoplasmic organelles in the cell. In a Eukaryotic Cell, There is a nucleus separated from the cytoplasm by a nuclear membrane, and the chromosomal DNA is located within The Nucleus; the cell wall, if present, may contain chitin or cellulose but not peptidoglycan; the cytoplasm contains various subcellular organelles (Mitochondria, Golgi apparatus, METABOLISM/14.html">Chloroplasts in plant Cells) (Fig. 2.1).

Escherichia coli

The bacterium Escherichia coli is one of the most thoroughly studied organisms. Over the past fifty years, comprehensive information has been obtained regarding its genetics, molecular biology, biochemistry, physiology, and general biology. It is a Gram-negative, non-pathogenic, motile rod less than 1 μm in length. Its natural habitat is the human intestine, but it can also be isolated from soil and Water. Due to its ability to reproduce by simple division on media containing only Na⁺, K⁺, Mg²⁺, Ca²⁺, NH₄⁺, Cl^-, HPO₄^2-, and SO₄^2- ions, Trace Elements, and a carbon source (such as glucose), E. coli has become a favorite subject of scientific research. When E. coli is cultured in enriched liquid nutrient media containing Amino Acids, Vitamins, salts, trace elements, and a carbon source, the generation time (i.e., the time between a bacterium's formation and its division) in the logarithmic growth phase at 37 °C is approximately 22 min.

Class="center">

Fig. 2.1. Schematic representation of a prokaryotic bacterial cell (A) and a eukaryotic animal cell (B).

For every living Organism, there is a specific Temperature range optimal for its GROWTH AND REPRODUCTION. At excessively high temperatures, Protein Denaturation and the destruction of other vital cellular components occur, leading to cell death. At low temperatures, biological processes slow down significantly or stop entirely due to structural changes undergone by protein molecules. Based on their preferred temperature ranges, microorganisms can be classified into thermophiles (45 to 90 °C and above), mesophiles (10 to 47 °C), and psychrophiles or psychrotrophs (-5 to 35 °C). Microorganisms that actively reproduce only within a specific temperature range can be useful tools for solving various biotechnological problems. For example, thermophiles often serve as a source of genes encoding thermostable Enzymes used in industrial or laboratory processes, while genetically modified psychrotrophs are used for the biodegradation of toxic wastes in soil and water at low temperatures.

E. coli can be cultured under both aerobic (in the presence of oxygen) and anaerobic (in the absence of oxygen) conditions. However, for optimal production of recombinant proteins, E. coli and other microorganisms are typically grown under aerobic conditions. If the goal of culturing Bacteria in the laboratory is the synthesis and ISOLATION OF A specific protein, the cultures are grown in flasks containing complex liquid nutrient media. To maintain the required temperature and ensure sufficient aeration of the culture medium, the flasks are placed in a water bath or a temperature-controlled room and shaken continuously. While such aeration is sufficient for cell proliferation, it is not always adequate for Protein Synthesis. Cell mass growth and protein production are limited not by the carbon or nitrogen source content in the nutrient medium, but by the dissolved oxygen content, which is approximately nine parts per million at 20 °C. This becomes particularly critical in the industrial production of recombinant proteins using microorganisms. To ensure optimal conditions for maximum protein yield, specialized fermenters are designed and aeration systems are developed.

Table 2.1. Some genetically modified microorganisms used in biotechnology

In addition to E. coli, many other microorganisms are used in molecular biotechnology (Table 2.1). They can be divided into two groups: microorganisms as sources of specific genes, and microorganisms engineered by Genetic Methods to solve specific problems. Examples of specific genes include the Gene encoding thermostable DNA polymerase, which is used in the widely employed Polymerase Chain Reaction (PCR). This gene was isolated from thermophilic bacteria and cloned into E. coli. The second group of microorganisms includes, for example, various strains of Corynebacterium glutamicum that have been genetically modified to increase The production of industrially important amino acids.

Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a non-pathogenic unicellular microorganism with a cell diameter of approximately 5 μm, representing in many respects a eukaryotic counterpart to E. coli. Its genetics, molecular biology, and metabolism have been studied in detail. S. cerevisiae reproduces by budding and grows well on media as simple as those used for E. coli. Its ability to convert sugar into ethanol and carbon dioxide has long been utilized in the production of alcoholic beverages and bread. Currently, more than 1 million tons of S. cerevisiae are consumed worldwide annually. S. cerevisiae yeast is also of great scientific interest. In particular, it serves as a highly convenient model for studying other eukaryotes, including humans, because many of the genes responsible for regulating Cell Division in S. cerevisiae are homologous to those in humans. This discovery facilitated the identification and characterization of human genes responsible for tumor development. The widely used yeast genetic system (yeast artificial chromosome) is an indispensable tool in all human DNA studies. In 1996, the complete nucleotide sequence of the entire set of S. cerevisiae Chromosomes was determined, further enhancing the value of this microorganism for scientific research. This was the first time such work had been completed for a eukaryote.

Eukaryotic proteins synthesized by bacterial cells often require enzymatic modification, which involves attaching low-molecular-weight compounds to the protein molecule—a step that is frequently essential for proper protein function. Unfortunately, E. coli and other prokaryotes are incapable of carrying out these post-translational modifications. Therefore, to obtain fully functional eukaryotic proteins, S. cerevisiae is used, as well as other yeast species: Kluyveromyces lactis, Saccharomyces diastaticus, Schizosaccharomyces pombe, Yarrowia lipolytica, Pichia pastoris, and Hansenula polymorpha. The most efficient producers of fully functional recombinant proteins are P. pastoris and H. polymorpha.