Biotechnology - Yu.O. Sazykin 2006

General Biotechnology
Molecular mechanisms of intracellular regulation and their application in biotechnological production
Molecular mechanisms protecting producers against substances with a "suicidal effect"

Biotechnologists developing high-yielding strains of BIOLOGICALLY ACTIVE SUBSTANCES generally face The Challenge of protecting the producer Organism from the high concentrations of the target product it synthesizes. This is especially evident in the creation of superproducers of secondary microbial metabolites. On the one hand, microbial metabolites produced in soil biocenoses often serve as a "weapon in the Struggle for Existence." On the other hand, through Mutations, cellular and Introduction/32.html">Genetic Engineering, and the Selection of specialized nutrient media, Cells can be induced to produce these substances in unnaturally large quantities.

From a biotechnological perspective, Structure/149.html">The problem of protecting producer organisms from the substances they synthesize cannot be viewed solely in the context of Antibiotics. However, antibiotics provide the most striking example of how a Cell shields itself from potentially "suicidal" endogenous compounds that could otherwise cause its death. The mechanisms protecting a producer from its own overproductivity can vary.

In the case of antibiotics, several such mechanisms exist. The specific role of each defense mechanism depends on the mode of biological activity of the particular antibiotic. It is well known that the most widespread group of clinical antibiotics, excluding beta-lactams, consists of bacterial Protein Synthesis Inhibitors operating at the ribosomal level: Tetracyclines, Aminoglycosides, macrolides, and several others. These are produced by actinomycetes—multicellular Bacteria whose ability to withstand high concentrations of their own antibiotics is explained by several factors. The primary factor is the specific nature of their molecular Biosynthesis (specifically, the assembly of the carbon Skeleton), which proceeds with high intensity and reaches a maximum when the producer culture slows its cellular division rate and transitions from the trophophase to the idiophase. In other words, despite the potential ability to halt protein synthesis, the accumulated antibiotic cannot harm its own cells, in which protein synthesis has already nearly ceased.

Other factors observed in certain groups of protein synthesis-inhibiting antibiotics are more specific in nature. Studies on aminoglycoside antibiotics, using the most thoroughly investigated neomycin (produced by Actinomyces fridae), have shown that during or after the assembly of the antibiotic molecule, it undergoes temporary, reversible inactivation.

Experiments with cell-free protein-synthesizing systems demonstrate that the Ribosomes of the neomycin producer are sensitive to the antibiotic. Consequently, the question arises as to how a cell can simultaneously harbor an endogenous inhibitor (neomycin in this case) and functional ribosomes. This can be partly explained by the aforementioned general physiological pattern governing antibiotic biosynthesis: the peak of antibiotic biosynthesis does not coincide in time with the maximum Rate of protein synthesis (the culture's developmental cycle has already been completed).

A specific cellular defense mechanism also exists against neomycin, which can be reversibly inactivated through the phosphorylation of one of the numerous amino or hydroxyl groups within the aminoglycoside structure. ATP serves as the source of the phosphate group transferred to the antibiotic. The reversibility of this inactivation is due to the presence of an alkaline phosphatase localized in The Cell membrane of the producer. During The final stage of antibiotic export into the environment, this enzyme performs reactivation—specifically, dephosphorylation—after which the antibiotic, now in its active state, is released into the medium due to the unidirectional permeability of the membrane.

Moreover, the reactivating enzyme sometimes fails to fully perform its Functions, leading to the secretion of the inactive antibiotic into the medium. By concentrating this medium and treating it with alkaline phosphatase, an additional yield of the antibiotic can be recovered from what appeared to be inactive Fermentation broth.

Interestingly, the Gene encoding this phosphorylating enzyme is located within the neomycin biosynthesis gene cluster, clearly demonstrating the genetic proximity of the producer's protective aminoglycoside phosphotransferase to the Enzymes involved in aminoglycoside biosynthesis. This indicates that the Functions of the cluster's genes are subordinated to a single goal: producing an antibiotic effective against competing microorganisms while remaining harmless to the producer itself.

However, aside from its protective function, the phosphotransferase can also interact with fragments of the assembling aminoglycoside molecule to activate them, thus performing a dual function (acting as a defense factor against a suicidal agent during biosynthesis). In addition to phosphorylation-dephosphorylation, other mechanisms of aminoglycoside inactivation-reactivation exist, such as Acetylation-deacetylation.

Protection against own antibiotics in actinomycetes producing macrolide structures (primarily erythromycin) is mediated by a specific methylase in the cell that methylates a defined adenine residue within the 23 S ribosomal RNA. As a result, the 50 S ribosomal subunit of the erythromycin producer adopts a configuration that prevents the antibiotic from reacting with the peptidyl transferase center of the ribosome, unlike what occurs in eubacteria. In other words, the protein synthesis machinery of the producer is specifically protected against its own antibiotic, supplementing non-specific protection at the physiological level.

The cellular defense mechanisms of tetracycline producers remain poorly understood, and drawing direct analogies with known mechanisms of tetracyclineresistance in pathogenic bacteria is quite difficult.

To characterize the defense system of producers against certain cyclopeptide membrane-active antibiotics (e.g., gramicidin S), the term "compartmentalization" is used. The site of biosynthesis of this antibiotic is isolated from the metabolic Reactions of the producing cell that would otherwise be sensitive to it. The antibiotic molecule (its carbon skeleton) is assembled within multienzyme complexes that ensure an ordered sequence of assembly reactions. The spatial arrangement of enzymes within the complex is coordinated by disulfide and Hydrogen Bonds. These multienzyme complexes are localized at the periphery of the producer cell. Compartmentalization also plays a role in protecting producer cells (typically actinomycetes) when they synthesize DNA-tropic antibiotics (which are used in Cancer Chemotherapy). At the same time, rifamycin family antibiotics, while inhibiting RNA Synthesis in cells by interacting with RNA polymerase, do not affect RNA synthesis within the cells of their own producer.

Interestingly, the problem of protecting the producer from a potential suicidal agent does not arise in the case of the penicillin producer Penicillium chrysogenum, even when the productivity of the fungal strain is increased many thousands of times. Penicillin exerts its antimicrobial effect by inhibiting the synthesis of peptidoglycan in the Bacterial Cell wall. Fungi lack peptidoglycan as a cell wall polymer, and consequently, they also lack the enzyme responsible for peptidoglycan synthesis—D-Alanine transpeptidase—which serves as the target of penicillin action upon contact with a bacterial cell.

Review Questions

1. What is The Effect of feedback inhibition mechanisms on the yield of final pharmaceutical biosynthesis products?

2. How does altering the levels of carbon, nitrogen, and phosphorus sources in the nutrient medium affect antibiotic biosynthesis?

3. What are the mechanisms of Gene Expression regulation and their Applications in biotechnological processes?

4. What is The Role of the guanosine tetraphosphate-mediated Metabolic Regulation system in The biosynthesis of target products?

5. What role does catabolite repression play in the biosynthesis of Pharmaceuticals?

6. What are mutants with altered regulation of Nitrogen METABOLISM, and what are the possibilities for intensifying the Biosynthesis of certain Primary and secondary metabolites and specific enzymes?

7. What are Intracellular Transport and secretion systems for biotechnological products in microorganisms?

8. What are the cellular defense mechanisms when a cell operates as a "superproducer"?

9. How can The activity of industrial microorganism strains be preserved?

10. How are substances transported across cellular membranes?