Biotechnology - Y.O. Sazykin 2006

General Biotechnology
Molecular mechanisms of intracellular regulation and their application in biotechnological production
Transport of substances across cellular membrane structures and its regulation

The most general component of The Cell envelope, common to microorganisms, plants, and animals, is the cytoplasmic membrane—a bilayer phospholipid subcellular Structure embedded with functionally diverse Proteins. In microorganisms (as well as in plants), a Cell wall is located outside the cytoplasmic membrane. This rigid polymer consists of Cellulose in higher plants, peptidoglycan in eubacteria and actinomycetes, and layers of Chitin, glucan, and mannoprotein in Fungi (i.e., different polymers).

In gram-negative Bacteria, In addition to the cytoplasmic membrane, there is another membrane known as the outer membrane, as it is located outside The cell wall. The outer membrane has a structure distinct from the cytoplasmic membrane, which is referred to as the inner membrane when an outer membrane is present. The outer membrane is asymmetric; its outer leaflet facing the environment consists mainly of lipopolysaccharides, whose molecules are coordinated by magnesium ions, while the inner leaflet facing the cell wall consists of Phospholipids. The outer membrane is traversed by porin proteins, whose trimers form transmembrane aqueous channels.

The space between the outer and inner membranes, which houses the cell wall, has a gel-like structure and is called the periplasmic space.

Animal Cells possess only a cytoplasmic membrane. Since the cytoplasmic membrane is common to all cells, it follows that the regulatory systems for transporting essential substances from the environment into the cell, as well as for expelling waste products from the cell into the environment, are inherently associated with the cytoplasmic membrane. The cell wall plays no significant role either in The transport of low-molecular-weight metabolites or in The regulation of this process. However, the outer membrane of gram-negative bacteria and, especially, the periplasmic space contain A number of Enzymes involved in the transport of low-molecular-weight compounds.

Transport processes across the cell envelope are subdivided into passive diffusion, Facilitated Diffusion, and Active Transport.

During passive diffusion—that is, along a concentration gradient, when the concentration in the environment is higher than that inside the cell—Water, Hydrocarbons, oxygen, nitrogen, and hydrogen molecules penetrate the cell.

In the case of facilitated diffusion, substances required by the cell are transported from the environment into the cell with the help of permeases, a specific Class of membrane-bound proteins. The transported substance reacts with a permease on the outer surface of the membrane and is released inside the cell after crossing the membrane. During facilitated diffusion, the substance entering the cell moves along its concentration gradient. This process requires no Energy Expenditure, just like passive diffusion.

During active Transport of substances into the cell, which requires energy expenditure, the movement of the transported substance can occur against a concentration gradient. Consequently, the concentration of a compound accumulating inside the cell can exceed its environmental concentration by hundreds or thousands of times. This happens because when the membrane carrier faces the exterior of the membrane, it exhibits high Specificity for its substrate, whereas once it faces the cell interior, its affinity drops sharply due to the dissociation of the substrate-carrier complex. If energy-yielding reactions are blocked—for example, by enzyme poisons that react with the Functional groups of the protein moiety—active transport of low-molecular-weight substances into the cell ceases.

The source of energy for active transport is very frequently the transmembrane Electrochemical Potential of hydrogen ions. Carriers possessing binding sites for protons and substrate molecules utilize the Membrane Potential to transport hydrogen ions and nutrients into the cell. Upon binding a proton, the carrier increases its affinity for the substrate. After releasing the proton at the inner surface of the membrane, the carrier decreases its affinity for the substrate. Thus, this mechanism actually achieves the cotransport of two substrates in the same direction, a process known as “symport.” If the carrier transports only a single substrate, the term “uniport” is used. Finally, the mechanism by which a single carrier transports two substrates in opposite directions is called “antiport.”

ATP-dependent active transport systems utilize ATP energy. Such systems include proteins located in the periplasmic space that exhibit high affinity for various metabolites, such as Peptides, Amino Acids, sugars, etc. They prevent certain metabolites from leaking out of the Cytoplasm into the environment. With their assistance, specific nutritional compounds from the environment accumulate in the periplasmic space.

When discussing The active transport of substances into the cell, one must mention group translocation systems. Their operation results in the transport of substances—such as CARBOHYDRATES—into the cell in the form of phosphate esters, yielding an intracellular concentration far exceeding that of the environment.

Phosphotransferases involved in the operation of such systems also trigger a number of other intracellular reactions. In general, the transport of many substrates into the cell is subject to regulation both at the level of The Biosynthesis of these system components and through the functional activity of already synthesized components.

Of independent interest is the export of excess metabolic products from the cell, notably defense enzymes, Antibiotics, and exoenzymes that enable the utilization of polymers found in the environment. Low-molecular-weight substances can be exported via passive or facilitated diffusion. However, energy-dependent systems also exist. Biotechnology professionals—including those working in recombinant protein production—are particularly interested in The problem of exporting proteins synthesized in the cytoplasm, including foreign target proteins. In this case, the biotechnologist follows the path of mimicking evolutionarily established mechanisms for secreting such proteins, much like extracellular enzymes.

When a protein destined to cross the cytoplasmic membrane and exit the cell into the environment is synthesized, first, its synthesis takes place on Ribosomes associated with the cytoplasmic face of the cytoplasmic membrane; second, the polypeptide chain leaving the ribosome contains a signal or leader peptide (15–30 amino acid residues) at its N-terminal end. This additional segment in the polypeptide chain is temporarily required for exporting the extracellular protein and possesses specific features:

• a terminal amino acid residue that is positively charged (which facilitates interaction with the negatively charged membrane surface);

• an extended stretch of hydrophobic amino acid residues that facilitates passage through the lipid layers of the membrane;

• the presence of a specific site for the action of a so-called signal protease (or peptidase)—a membrane enzyme that catalyzes the Cleavage of the leader peptide from the main polypeptide chain after it has fulfilled its function as a guide, escorting the new protein molecule from the cell into the environment.

Thus, a recombinant protein—for example, a species-specific human protein hormone synthesized in a microbial cell without a native leader sequence—can be engineered using genetic techniques to include a leader sequence, provided that it can serve as a substrate for the microbial signal peptidase. This gives rise to The Challenge of designing hybrid (chimeric) proteins featuring a leader sequence derived, for example, from extracellular penicillinase.

Of course, the problem of foreign protein export is not solved solely and straightforwardly by attaching a leader sequence, as many other factors influence protein excretion.

In Conclusion of this Brief Overview of cellular transport issues, we should pay special attention to Mutations that can affect transport enzyme systems, carrier molecules, and the Structural components of the cytoplasmic and outer membranes. This provides the biotechnologist with a rich array of mutants exhibiting highly diverse modifications in PHYSIOLOGICAL AND BIOCHEMICAL properties.

The Specific features of transport systems in certain biological objects determine their ability to impact the environment in a manner desired by humans.

For instance, a number of microorganisms of the genus Pseudomonas, as well as mutants purposely derived from natural cultures, detoxify A wide variety of chemical compounds (such as cyclic hydrocarbons and others) whose release into the environment disrupts ecosystems across vast geographic regions. Moreover, the ability of these microorganisms to neutralize pollutants is driven not only by a battery of enzymes that break down xenobiotic substances, but also by the unique characteristics of their transport systems, starting with the oscillation regime of outer membrane porin channels.