BIOCHEMISTRY FOR TEACHERS - F. F. BOYECHKO - 1985

MAJOR CHEMICAL COMPONENTS OF CELLS

PROTEIN BIOSYNTHESIS

Protein Synthesis is one of the most complex and critical challenges in modern natural science. Its exploration and study are closely intertwined with the most pressing issues in biological science: elucidating the laws of heredity and variation, regulating the GROWTH AND DEVELOPMENT of organisms, uncovering the ROOT causes of numerous Hereditary diseases, and developing Methods for their Prevention and Treatment.

The first hypothesis regarding protein synthesis was put forward by O. Ya. Danilevsky as early as 1886. Known as the reverse proteolysis hypothesis, it suggested The formation of protein-like substances (plasteins) through the action of gastric juice Enzymes on a concentrated solution of peptones—the products of Protein Hydrolysis. However, as established later, this pathway of protein formation plays no significant role in the living Organism. Although subsequent hypotheses were proposed, none could fully uncover The Mechanism of protein synthesis.

Several decades ago, the scientific literature put forward the idea of protein molecule Replication on some sort of «template», marking the first attempts to link protein synthesis with such vital cellular Biopolymers as Nucleic Acids. Thus, the template theory of protein synthesis began to take shape gradually, playing a pivotal role in unravelling this fundamental problem.

The works of Soviet scientists A. M. Belozersky and A. S. Spirin, alongside foreign researchers such as J. Watson, F. Crick, S. Ochoa, and M. Nirenberg, were of paramount importance in clarifying the mechanisms by which nucleic acids participate in protein synthesis.

It is well known that Proteins synthesized within an organism differ primarily in their chemical nature and The sequence of amino acid residues in their polypeptide chains—in other words, their Primary Structure. The genetic blueprint for any given protein is encoded in DNA AS A specific sequence of nucleotide residues within a polynucleotide chain. Since DNA resides in The Nucleus while METABOLISM/35.html">Protein Biosynthesis takes place on Ribosomes, DNA transmits its genetic instructions via mRNA, which is synthesized on a specific region (Gene) of one of the DNA nucleotide strands.

The transmission of this information relies on THE PRINCIPLE OF complementarity. In the synthesized mRNA, The nucleotide sequence corresponds directly to that of one of the DNA polynucleotide strands, with the sole exception that thymidine NUCLEOTIDES are replaced by uridine nucleotides in mRNA. The process of copying this information from DNA to mRNA is known as Transcription.

Having received the instructions from DNA, mRNA leaves the nucleus and migrates to the ribosomes, where it directs protein synthesis. In essence, mRNA serves as a template upon which proteins are assembled, with their primary structure dictated by the code inherited from DNA. The process of translating the genetic message—recorded as a specific sequence of nucleotides in the mRNA molecule—into the language of Amino acid sequences in protein molecules is called translation.

Consequently, the flow of information from DNA to protein synthesis can be represented by the following scheme:

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Protein synthesis utilizes activated forms of Amino Acids linked to their respective tRNAs, which transport them to the site of protein biosynthesis—the ribosomes. The process of conjugating amino acids with tRNA, mediated by the enzyme aminoacyl-tRNA synthetase, is often referred to as recognition (from the English word recognize).

According to current understanding, protein biosynthesis occurs via a series of complex biochemical processes involving nucleic acids, various enzyme systems, Metal Ions, and numerous other factors. It encompasses three main stages: transcription, recognition, and translation.

Activation of Amino Acids and formation of aminoacyl-tRNA (recognition). Amino Acid Activation takes place in the Cell Cytoplasm with the participation of energy-rich ATP and enzymes known as Aminoacyl-tRNA synthetases (aminoacyl synthetases). Each amino acid has its own specific enzyme within The Cell. Academician V. A. Engelhardt termed these enzymes codases. Magnesium ions are required for them to exhibit maximum activity, while ions of other divalent metals—specifically manganese, cobalt, and calcium—exhibit a somewhat weaker activating effect. The amino acid activation reaction can be represented by the following scheme:

This reaction occurs On the surface of the catalyzing enzyme; the resulting aminoacyl adenylate does not pass into solution but remains complexed with the enzyme. Within the aminoacyl adenylate molecule, the amino acid residue is joined to the ATP residue by a high-energy (macroergic) bond, which enhances the reactivity of the amino acid.

At the next stage, the aminoacyl adenylate-enzyme complex interacts with a tRNA specific to that particular amino acid. This results in The transfer of the aminoacyl group from the aminoacyl adenylate to the tRNA, forming a new complex—aminoacyl-tRNA—while AMP and the enzyme are released.

The amino acid residue attaches to the third carbon atom of the ribose of the terminal tRNA nucleotide while preserving the high-energy bond. This reaction is catalyzed by the same enzyme responsible for amino acid activation—aminoacyl-tRNA synthetase. The resulting enzyme molecule possesses two specific sites that enable it to recognize, on the one hand, «its» Amino Acid and, on the other, «its» tRNA.

Translation is The conversion of the nucleotide sequence of mRNA into the Amino Acid Sequence of a polypeptide chain during protein synthesis. This phase of protein synthesis occurs on ribosomes and consists of consecutive stages: initiation, elongation, and termination. The translation process has been studied in greatest detail in bacterial Cells.

It is well established that ribosomes of bacterial cells, much like those of higher organisms, are ribonucleoprotein complexes composed of proteins and ribosomal Ribonucleic Acids (rRNA) synthesized on DNA templates in the nucleoli. Ribosome sizes are characterized by sedimentation coefficients expressed in Svedberg units (S). The larger the particles, the faster they sediment during ultracentrifugation and the higher their sedimentation coefficient. Prokaryotes are characterized by ribosomes with a sedimentation coefficient of 70S, whereas eukaryotes feature 80S ribosomes.

Each ribosome consists of two subunits—a small and a large one—into which it can dissociate under certain conditions. Prokaryotes typically feature a small 30S and a large 50S subunit, while eukaryotes possess 40S and 60S subunits, respectively.

The ability of ribosomes to dissociate into subunits and subsequently reassociate is of vital importance to the process of protein synthesis.

Initiation. This stage of protein synthesis has been thoroughly investigated using Escherichia coli Bacteria as a model. Studies of bacterial cell proteins revealed that N-formylmethionine frequently serves as the N-terminal amino acid, meaning protein synthesis always begins with Methionine. It was established that in bacterial cells, alongside the regular tRNA^met which delivers methionine to ribosomes, there exists another tRNA—the so-called initiator tRNA—which transports formylmethionine (tRNA^met). Both tRNAs

recognize the AUG triplet in the mRNA structure, whereas tRNA^met delivers methionine to the triplets located in the internal regions of mRNA, while tRNA^fmet delivers it to the triplet positioned at the 5'-end of the mRNA polynucleotide chain. The Role of formylmethionine is that it contains a bound (formylated) amino group, allowing the first peptide bond to form solely through the carboxyl group, thus ensuring that polypeptide synthesis proceeds in the N- to C-terminal direction. Upon completion of the Polypeptide chain synthesis, the formyl group or the entire formylmethionine is cleaved off, and the amino acid following methionine becomes the N-terminal residue.

It has been established that protein synthesis initiation requires the following components: mRNA, tRNA_f^met, GTP, 30S and 50S ribosomal subunits, and initiation protein factors IF-1, IF-2, and IF-3. First, with the participation of factors IF-3 and IF-2 and in the presence of GTP, the 30S subunit interacts with formylmethionyl-tRNA. Then, this complex, in the presence of factor IF-1, binds to mRNA to form the initiation complex. The latter interacts with the 50S ribosomal subunit. This process releases the initiation factors and results in the hydrolysis of GTP into GDP and H₃PO₄. Under these conditions, a functionally active ribosome (70S ribosome-mRNA-tRNA) is formed.

A functionally active ribosome contains two sites: the aminoacyl (A-site) and the peptidyl (P-site).

Formylmethionyl-tRNA first enters the A-site and is subsequently transferred to the P-site. This frees up the A-site, making it ready to accept the next (second) aminoacyl-tRNA. This marks the end of the initiation process.

Elongation (the growth of the polypeptide chain) also occurs in several steps. The first step involves delivering the next aminoacyl-tRNA to the A-site of the ribosome and binding it to the corresponding mRNA codon located adjacent to the initiation codon (AUG). GTP and elongation factors EF-Tu and EF-Ts participate in this process.

At the next stage of elongation, the first peptide bond is formed in the A-site of denominators where the delivered aminoacyl-tRNA resides. tRNA_f^met is transferred here from the P-site, and the first peptide bond is formed through the interaction of the formylmethionine carboxyl group with the amino group of the delivered amino acid carried by the aminoacyl-tRNA. Under these conditions, peptidyl-tRNA is formed and tRNA is released:

The released tRNA is located in the P-site, while the peptidyl-tRNA is in the A-site of the ribosome.

In the third step of elongation, the peptidyl-tRNA is relocated (translocated) to the P-site. Concurrently, the free tRNA is removed from the P-site, and the mRNA shifts by the length of one codon. The A-site is thus vacated and can accept the next (third) amino acid.

Termination (the completion of polypeptide chain synthesis) occurs when specific signals appear in the Introduction/21.html">RNA Structure, namely the UAA, UAG, and UGA triplets. They are frequently referred to as nonsense, terminator, or stop triplets.

The termination process involves factors RF₁ and RF₂, which catalyze the Cleavage of the synthesized polypeptide chains from the ribosome. Factor RF₁ responds to the appearance of UAG and UAA; RF₂ responds to the UAA and UGA codons.

In eukaryotes, termination takes place with the participation of a single factor, RF, the action of which requires GTP.

The termination process consists of several steps. First, the polypeptide chain from the ribosome's A-site, where the final peptide bond is formed, is relocated to the P-site. Next, the ester bond between the C-terminal amino acid and its tRNA is cleaved, and the protein leaves the ribosome. The ribosome-mRNA-tRNA complex dissociates. The ribosome breaks down into subunits. The liberated ribonucleic acids can presumably be reused in subsequent cycles of protein synthesis. This process is quite complex and not yet fully understood.

It has been calculated that the synthesis of a typical protein consisting of approximately 150–200 amino acid residues takes 1–3 minutes on ribosomes. Consequently, each ribosomal cycle—which extends the protein's polypeptide chain by a single amino acid residue—takes a fraction of a second. Elucidating the fundamental Stages of Protein Synthesis represents a major achievement in biology at THE MOLECULAR LEVEL. The genetic role of nucleic acids has been established experimentally, The Essence of The Genetic Code based on the molecular structure of DNA has been uncovered, and this has concretized The Nature of Mutations, which form The basis of the evolution and Variability of Living systems.

MOLECULAR MECHANISMS OF Protein Biosynthesis Specificity.

Genetic Code

As noted previously, protein synthesis occurs on ribosomes via an enzymatic pathway in accordance with the information encoded within the DNA Structure. DNA itself acts as the template or matrix that programs the specific Primary structure of the protein being synthesized.

The question arises: how does the sequence of four different nucleotides that make up the Structure of Nucleic Acids determine the sequence of 20 amino acid residues in a protein? How was the genetic code for protein synthesis deciphered?

Using a 4-letter alphabet of nitrogenous bases, one can construct 4 single-letter words—a singlet code, 16 two-letter words (4×4=16)—a doublet code, or 64 three-letter words (4×4×4=64)—a triplet code (codon). For 20 amino acids, 16 two-letter words are insufficient, whereas 64 three-letter words are more than enough.

In 1954, a code word consisting of three nitrogenous bases—the triplet code—was proposed. However, it was necessary to determine which nucleotides and in what order within the DNA (mRNA) molecule correspond to the various amino acids. These studies were conducted by M. Nirenberg and H. Khorana, for which they were awarded the Nobel Prize.

The scientists obtained a synthetic RNA polymer—polyuridylic acid. This obtained RNA was added to a system where protein synthesis was taking place. Consequently, polyphenylalanine was synthesized, while the other 19 amino acids were not incorporated into the polynucleotide chain. Thus, it was proven that three uridylic residues (UUU) encode the incorporation of the amino acid phenylalanine into the polypeptide chain of the protein molecule. Subsequently, The codon composition for all Other Amino Acids was elucidated. For example, if the codon GGG is encountered in the nucleic acid structure, it ensures the incorporation of glutamic acid into the polypeptide chain, AAA codes for Lysine, CCC for Proline, and so on (Table 8).

As seen from the data presented in Table 8, Amino acids are encoded by multiple codons. It should be emphasized that out of the 64 codons, 61 specify the sequence of amino acid residues in the polypeptide chain, with the codons GUG (valine) and AUG (methionine) also serving as initiation (start) codons. Three other codons—UAG, UAA, and UGA—are termed nonsense codons and function as signals for the termination of polypeptide chain synthesis.

Table 8. The Genetic Code

Analyzing the table of the genetic code, one can observe that the first two nucleotides in a codon are more crucial than the third. Some researchers believe that replacing the third nucleotide in a significant portion of codons does not affect their ability to encode the corresponding amino acid. For instance, Alanine is coded by the following nucleotides: GCU, GCC, GCA, and GCG, which differ from one another only at the third nucleotide position.

Given that the first two nucleotides in triplets exhibit high specificity, English physicist F. Crick developed The Wobble Hypothesis in 1965, which implies that the first two nucleotides play the primary role in codon recognition by the anticodon. They strictly adhere to the complementary base-pairing principle. As for the third nucleotide of the codon, it can non-specifically interact with more than one type of anticodon nucleotide. All of this helps enhance the stability of Genetic information in the event of DNA damage.

Some Features of the genetic code. It should be noted that the genetic code is apparently universal. It is identical across all organisms—bacteria, plants, and animals. However, some organisms show a preference for certain triplets over others. The universality of the code suggests that it emerged at early stages in the evolution of living systems and has remained largely unchanged throughout evolutionary history.

A characteristic feature of the code is its degeneracy, as evidenced by the fact that two or more triplets can encode a single amino acid. Nevertheless, the code is not ambiguous: no codon, with the exception of the initiation codons (GUG and AUG), codes for more than one amino acid.

In most cases, code degeneracy results in the presence of multiple tRNAs for a single amino acid, which differ from one another in their anticodons.

Another key feature of the code is that it is non-overlapping, meaning that the nucleotides comprising a given triplet do not participate in forming adjacent triplets. Once information from one triplet is read, the reading mechanism shifts forward by exactly three nucleotides.

A characteristic feature of the code is also its continuity. Each triplet encoding a specific amino acid lies adjacent to the next without any intervening spacer regions (factors).

A rather important feature of the code is its unidirectionality. During protein synthesis, codons are translated in a single direction, starting from the first nucleotide base. The first nitrogenous base is located near the 5' nucleotide end, and the last near the 3' end, meaning that information is transcribed in the 5'→3' direction.

In recent years, data have emerged indicating certain deviations from the generally accepted features of the genetic code. For instance, studies on the nucleotide sequence of human Mitochondrial DNA have shown that its genetic code is overall similar to the previously known one. At the same time, four codons were found to have changed their meaning: the UGA codon specifies Tryptophan, AUA specifies methionine, and the AGA and AGG codons have become termination signals. In Yeast Mitochondria, all four leucine codons starting with CU have shifted to Threonine. Consequently, leucine is left with only two codons, while threonine has six. Thus, the existence of two different codes has been discovered within a single human cell. These findings apparently prove that the code has undergone certain minor evolutionary changes, and code universality has its nuances.

The non-overlapping feature of the code is also subject to certain adjustments. Experiments with Viruses and certain bacteria have established that the same DNA segment can encode several different proteins through a reading frame shift principle. For example, given a code with a specific nucleotide sequence such as UAGAUGCGC, reading it from the first letter yields the triplets UAG, AUG, CGC, whereas starting from the second letter yields entirely different triplets—AGA, UGC, GCA, and so on. Furthermore, it has been established that for certain proteins, a portion of the nucleotide sequence is shared: when a given nucleotide sequence ends for one protein, it may continue for another. These data indicate that genetic transcription is subject to specific control mechanisms that ensure the Selection of the correct reading frame.

A number of new facts have been established showing that eukaryotic genes contain non-coding regions; in other words, genes in higher organisms are not continuous but are constructed from separate segments interrupted by other nucleotide sequences. The gaps between gene segments vary widely, ranging from 10 to 20,000 Base Pairs.

Regulation of Protein Synthesis

A prerequisite for the existence of living systems and the maintenance of their vital Functions is the presence of a coordinated regulatory system for key physiological processes. This primarily applies to protein synthesis, as the optimal ratio between the quantity and quality of various proteins plays a crucial role in sustaining vital processes in both unicellular and Multicellular Organisms. Enzyme proteins regulate metabolism, mediate adaptation to changing internal and external environmental conditions, and govern ontogeny and the differentiation of cells, Organs, and systems. Therefore, controlling the synthesis of precisely those enzyme proteins required by the cell under specific conditions and in accordance with their functions is of paramount importance. Through a long historical course of evolution, living organisms—regardless of their level of Organization—have developed a complex, coordinated mechanism for regulating protein synthesis that ensures a constant Qualitative and quantitative protein composition.

Elucidating the essence of the mechanism regulating protein synthesis is a highly complex problem that remains fully unresolved. A large group of Soviet and foreign geneticists, biochemists, and biologists worked on this problem for decades. A logical development of these important studies was THE CONCEPT OF protein synthesis regulation formulated in 1961 by Nobel laureates, the French scientists F. Jacob and J. Monod.

The concept proposed by F. Jacob and J. Monod has gained widespread recognition and is currently universally accepted. According to this concept, The regulation of protein synthesis occurs at the DNA level, where molecules consist of specific functional regions—genes grouped according to their functions. One group of these functional regions is called structural genes, or cistrons, which contain information for synthesizing specific polypeptide chains of a protein. They direct the synthesis of mRNA molecules, which then travel to ribosomes to serve as templates during protein synthesis. mRNA synthesis—the reading of genetic information—begins at a functional DNA region called the promoter, which acts as the initiation point for its synthesis.

Another group of functional DNA regions consists of regulatory genes, which control The activity of structural genes by switching them on and off. Regulatory genes include the operator gene, which is located directly adjacent to a group of structural genes, and the regulator gene, situated at some distance from them.

Operator genes act as a trigger mechanism that, depending on conditions, permits or inhibits mRNA synthesis on structural DNA genes. It is believed that the operator gene is localized at the terminal segment of cistrons and apparently serves as the starting point for the action of DNA-dependent RNA polymerase.

Operator genes, together with groups of structural genes, form coordinated functional blocks known as operons, each responsible for the interrelated synthesis of a series of specific proteins; thus, the Operon is the unit of transcription. If the operator gene is inactive, the entire operon becomes inactive, inhibiting the synthesis of mRNA and, consequently, enzyme proteins. The activity of the operator is regulated by the regulator gene. Since the regulator gene and the structural GENES OF THE operon are located on different regions of DNA, the communication between them is mediated by repressor proteins. These proteins are synthesized on ribosomes within the nucleus using a specific mRNA template transcribed from the regulator gene.

The repressor protein derives its name from its ability to inhibit (repress) the activity of the operator gene, placing it in the "off" position and thereby halting the functioning of the entire group of structural genes. Repressors have an affinity for the operator and the capacity to reversibly bind to it. Additionally, repressors can specifically bind to certain low-molecular-weight substances known as Inducers or effectors.

Under normal physiological conditions, repressors can exist in active and passive states. The transition from one state to the other is regulated by intracellular metabolic products or substances entering from the external environment.

Depending on the state of the repressor, inducible and repressible systems of gene regulation in protein synthesis are distinguished.

In an inducible regulatory system, the repressor produced by the regulator gene is in an active state. Its effect on the operator gene blocks operon activity and halts the synthesis of mRNA and specific enzyme proteins. Their synthesis can only be resumed when products appear in the cell that require these enzymes for their utilization. The repressor binds to these products, termed inducers, and loses its ability to control the operator gene, thereby restoring mRNA synthesis.

It is believed that the inducer, by binding to the repressor protein, induces A change in its tertiary structure, causing it to lose its ability to bind to the substrate—the regulator gene. Meanwhile, the operator, freed from the control of the regulator gene, becomes active and triggers the block of structural genes responsible for synthesizing the mRNA required to produce specific enzymes. Since these enzymes are directed toward utilizing the product (inducer), the repressor remains passive until the complete breakdown of the inducer is accomplished by the enzymes. Following this, the repressor is released, transitions into an active state, and blocks the operon, terminating the synthesis of mRNA that encodes the primary structure of those enzymes. Fig. 17 illustrates the MAIN STAGES OF the inducible system of protein synthesis regulation.

Fig. 17. Scheme of protein synthesis regulation via induction (according to T. T. Berezov and B. F. Korovkin).

RG — regulator gene; P — promoter; OG — operator gene.

An example of inducible regulation of protein synthesis is the Synthesis of the enzyme galactosidase in Escherichia coli cells, which catalyzes the hydrolysis of milk sugar (lactose) into glucose and galactose. Bacterial strains grown on glucose cannot thrive if transferred to a medium containing lactose instead of glucose until they synthesize the specific enzymes required to utilize this substrate and use it as an energy source.

Lactose entering the medium acts as an inducer that binds to the repressor protein and blocks its attachment to the operator gene. The operator and structural genes are thereby released, initiating the synthesis of mRNA, which encodes the primary STRUCTURE OF THE galactosidase enzyme and drives its Synthesis on Ribosomes.

In addition to gene induction, cells also exhibit gene repression. This process is particularly common in various synthesis pathways, where the levels of certain enzymes drop significantly as the concentrations of the End products of the catalyzed reactions increase. In a repressible system of protein synthesis regulation, the repressor—synthesized on nuclear ribosomes—remains in a passive state and cannot

suppress the activity of the operator gene, thus leaving the operon—where mRNA synthesis takes place—uncontrolled. The transition of the repressor to an active state, the blocking of the operon, and the cessation of mRNA synthesis are triggered by a co-repressor substance. The end products of the synthetic pathway, or one of these products, serve as co-repressors. Evidence suggests that in the synthesis of Amino acid metabolism enzymes, the co-repressor may not be a free amino acid (the end product of the biosynthetic reaction), but rather its complex with tRNA (aminoacyl-tRNA).

Blocking of the operator gene ceases when the co-repressor is exhausted. Consequently, in the absence of the activator (co-repressor), the repressor returns to its normal passive state, releasing the operator gene and the group of structural genes, which resumes the synthesis of enzyme proteins. The diagram of the repressible protein synthesis regulation system is shown below (Fig. 18).

The concept of protein synthesis regulation proposed by F. Jacob and J. Monod applies to lower organisms—prokaryotes. The regulatory mechanisms in highly organized organisms, or eukaryotes, are significantly more complex. A notable contribution to this field was made by the Soviet scientist G. P. Georgiev. According to his hypothesis, genes in higher organisms are also grouped into operons, but their organization is more complex than in microorganisms. The main difference is that an operon possesses multiple operator genes, each interacting with a distinct repressor. Blocking any operator hinders the progression of DNA-dependent RNA polymerase. As a result, mRNA synthesis on the structural genes slows down or stops entirely.

Fig. 18. Diagram of protein synthesis regulation by repression (after T. T. Berezov and B. F. Korovkin).

Letter designations are the same as in Fig. 17.

Because there are multiple operators, operon activity is influenced by various factors. For instance, one operator may be blocked when the concentration of a specific hormone rises, another when a certain metabolic product accumulates in the cell, and so on. Furthermore, the same operator gene may be part of different operons, causing these operons to respond to the identical factor. This indicates that a single factor can regulate multiple operons simultaneously, while the activity of a single operon may depend on a combination of different factors. This creates a remarkably precise yet flexible system of self-regulation.

It should be noted that the mechanisms governing the Specificity of protein biosynthesis, as well as the regulation of protein synthesis, are not yet fully understood.

Genetic Engineering and Protein Biosynthesis

Successful research in genetic engineering holds not only theoretical significance but also immense practical importance, particularly for the biological synthesis of a range of essential proteins. Using specific techniques (such as mRNA-directed synthesis via Reverse Transcriptase or standard chemical synthesis), scientists isolate the gene that dictates the synthesis of a desired protein. This gene is then integrated into a suitable system that allows it to rapidly replicate. Such systems, commonly referred to vectors, often utilize Plasmids—autonomous structures that carry their genetic information as small circular DNA molecules, found in bacterial cells alongside the bacterial chromosome. Besides plasmids, other systems such as Viruses and Phages can also be employed.

Plasmids containing the integrated gene (recombinant DNA) are introduced into bacterial cells, thereby granting them The ability to synthesize substances novel to them.

The hormone Insulin is a prime example of such a vital protein. Insulin deficiency in The Human Body leads to diabetes, a severe and widespread disease. Animal-derived insulin, extracted from the Pancreas of cattle or pigs, is currently used for its treatment.

However, it has been found that animal insulin is ineffective or causes allergic reactions in some individuals due to structural differences between Human and Animal insulin. Consequently, a need arose to produce human insulin. Extracting it purely through chemical synthesis is economically unviable.

Genetic engineering has provided a more cost-effective method for insulin production. Bacterial strains carrying recombinant DNA capable of producing human proinsulin have been successfully developed, from which insulin can be easily obtained. The Challenge of industrial insulin production has thus been solved, and the drug is currently undergoing clinical trials.

Interferon, a universal antiviral agent, represents the second major focus of genetic engineering. It is based on a protein produced by body cells in response to viral infection. Studies of interferon's properties have shown that it exhibits high species specificity; therefore, only human interferon can be used for human treatment.

Currently, interferon is primarily extracted from donor Blood Leukocytes. However, this extraction method is quite expensive and fails to fully meet the demand. Consequently, intensive efforts are underway to develop novel production methods, particularly through genetic engineering. The Development of an interferon-producing strain has been successfully completed, which will soon enable the manufacture of the drug in sufficient quantities.

Recently, genetic engineering has yielded another crucial protein: somatotropin. This human Growth Hormone consists of 131 amino acid residues and is used in the treatment of dwarfism, bone fractures, Burns, and a range of other medical conditions.

Thus, genetic engineering inaugurates a novel technology (biotechnology) for synthesizing vital biological substances, particularly proteins. This makes it possible to significantly reduce costs and broaden the production range of Hormones, enzymes, and other protein Pharmaceuticals utilized in medicine and agriculture, in accordance with the objectives set by the 26th Congress of the CPSU in the Guidelines for the Economic and Social Development of the USSR for 1981–1985 and the Period up to 1990.

Increasing Protein Production: A Global Challenge

Today, ensuring that the planet's population has access to wholesome food products—and above all, essential dietary components such as proteins—has acquired critical importance. A shortage of feed proteins is observed worldwide, and the demand for animal proteins is met, on average, only by 40–50%. Protein deficiency is felt most acutely in economically backward and developing countries.

Furthermore, There is a deficit of feed protein for livestock farming.

Driven by population growth, the reduction of arable land due to urbanization and industrialization, ocean pollution, and various other factors, the protein deficit is projected to escalate. Consequently, the search for novel ways and methods to scale up The production of protein substances stands as one of the most critical global challenges. Promising approaches in this regard include industrial protein production Methods based on cultivating lower organisms—Yeasts, bacteria, Fungi, and Algae—on non-food raw material sources. These methods hold the greatest potential among all strategies for resolving the global protein crisis.

The viability of Single-Cell Protein production is driven by the remarkably high biomass doubling rate of microorganisms compared to plants or animals, alongside their high protein content. Certain bacteria are known to double their cell count every 5–8 minutes, with a protein content in their biomass reaching up to 70 %.

Furthermore, microorganisms can be cultivated uniformly year-round, eliminating poor harvest years, epizootics, plant diseases, and other agricultural risks. Microbiological production requires minimal land area while making efficient use of fertilizers and Water. Microorganisms adapt readily to novel environmental conditions, and their biomass composition is easily managed through technological and breeding controls. Crucially, when properly organized, microbial protein production does not harm natural biocenoses.

Various species of yeasts are most commonly employed in microbiological manufacturing. Since ancient times, bread and wine have been produced using precisely these organisms. Yeasts were utilized in the initial modern, large-scale facilities for single-cell protein production both domestically and abroad. Their primary advantages over other microorganisms include high resistance to contamination, ease of Separation from the growth medium due to larger cell sizes, the ability to thrive on relatively simple media, and the capacity to readily assimilate diverse carbon and nitrogen sources. Additionally, yeast-derived biomass offers high nutritional quality and a pleasant aroma.

Recently, there has been a trend toward broadening the taxonomic range of microorganisms utilized for protein production. Specifically, various bacterial species have begun to be employed for this purpose. This shift is primarily due to the high growth rates and superior protein and methionine content characteristic of bacterial strains compared to other organisms.

Industrial cultivation of both lower fungi (such as *Penicillium*) and higher fungi is also utilized to obtain protein biomass. The advantages of fungi include the relative ease of biomass separation, the ability of certain species to degrade Lignin, and a low nucleic acid content in their biomass—ranging from 1,5 to 2,8 %.

Algae are likewise employed for protein production. Their biomass is characterized by a high protein content (up to 70 %) and significant biological value. Like higher plants, algae can be cultivated autotrophically via Photosynthesis. Compared to traditional plants, algae contain a greater proportion of dry matter and protein. However, establishing optimal conditions for photosynthetic algae cultivation requires supplying carbon dioxide (IV) and light, along with specialized equipment for biomass agitation and other operational needs. All of this entails relatively high capital investment. Consequently, it remains more economically viable to cultivate heterotrophic organisms on various carbon-containing substrates.

To date, only a fraction of existing microorganisms in nature have been studied for protein production. The scientific literature increasingly highlights novel cultures capable of intensive PROTEIN SYNTHESIS AND the utilization of specific substrates.

It is worth noting that natural reserves of raw Materials for microbiological processes are quite substantial, given that microorganisms can utilize most naturally occurring carbon compounds. However, for the process to be economically viable, the feedstock must be inexpensive, readily available, and supplied in sufficient quantities.

In its initial stages, single-cell protein production relied exclusively on waste streams from specific industries, primarily food Processing.

Toward the late 20th century, microorganisms capable of growing on petroleum Hydrocarbons were discovered. Extensive research in this field was conducted by the Soviet scientist V. O. Tausson in the early 20th century; however, it was only in the 1960s that the true industrial potential of this feedstock was established. For instance, just 2 % of the world's total oil extraction would be sufficient to produce 25–30 million tons of yeast protein—an amount capable of providing a complete, balanced diet for 2 billion people throughout the year.

The yeast Candida guilliermondii is frequently employed for the microbiological synthesis of protein. These organisms thrive and propagate on normal alkanes with carbon chain lengths ranging from C₁₁ to C₂₄. Kinetic studies of hydrocarbon utilization by these microorganisms have demonstrated that they most readily assimilate normal alkanes with a chain length of C₁₁—C₁₄. Hydrocarbons with chain lengths of C₁₅—C₁₈ occupy an intermediate position, whereas higher-molecular-weight hydrocarbons are assimilated with greater difficulty. The scientific foundations for producing protein substances from petroleum hydrocarbons were developed by a team of Soviet scientists led by Academics M. D. Yerusalimsky and G. K. Skryabin, for which they were awarded the USSR State Prize.

Using purified hydrocarbons with microorganisms makes it possible to obtain feed protein without the biomass purification stage.

Unrefined petroleum feedstocks, such as petroleum distillates, are also used to cultivate microorganisms. Under such conditions, the resulting biomass undergoes thorough purification.

In recent years, both domestically and abroad, technologies for the microbiological synthesis of protein using gaseous hydrocarbons have been developed. Due to their volatility, residual unused hydrocarbons are easily removed from the biomass.

Currently, microbial protein is produced using microorganisms grown on alcohols (methanol, ethanol), organic acids, ketones, aldehydes, esters, and Other Compounds collectively known as oxidized hydrocarbons, or oxidates. Researchers consider alcohols to be the most suitable feedstock for cultivating microorganisms, as they are readily soluble in water and easily washed out of the finished product.

Recently, considerable attention has been drawn to the virtually limitless sources of Cellulose-containing substrates. The annual global increase in plant biomass is 155 billion tons. Processing a portion of this biomass generates hundreds of millions of tons of waste. For instance, only 50% of the raw material is utilized when processing wood into cellulose for papermaking. There is also a significant amount of other non-food plant raw materials, such as rice, sunflower, and buckwheat husks, grapevine trimmings, sunflower stalks and heads, straw, and others. Their breakdown via hydrolysis produces simple sugars, or monoses. Such hydrolyzates are used for microbial protein synthesis. Research is currently underway to identify microorganisms capable of intensively accumulating biomass using cellulose hydrolyzates and other complex CARBOHYDRATES.

Since reserves of organic matter are not inexhaustible, developing the technological foundations for producing protein using autotrophic bacteria—which do not require Organic compounds for their development—is becoming critically important. Photosynthetic microorganisms hold significant promise in this regard.

It has been proven that yeasts, bacteria, and algae can serve as a valuable source of protein, Vitamins, and minerals for humans. However, The amount of nucleic acids introduced into the diet along with single-cell protein should not exceed 2 g per day. Consuming larger quantities of these acids in Human Nutrition can contribute to polyarthritis and the formation of uric acid stones in the Urinary Tract. To reduce the nucleic acid content in the biomass, it is treated with alkalis, specific enzymes, or through the activation of intracellular Nucleases, which breaks down nucleic acids into low-molecular-weight products that can be easily removed from the biomass if necessary.

In many countries around the world, single-cell protein is used to enrich a variety of food products, including sausages, cheese, bread, and canned goods. For example, biochemists in Poland have developed a technology for producing foods containing 3–5% dried food yeast. In Ukraine, a microbiological method for producing food protein has been proposed, which is close to chicken meat in its biological value. In Switzerland, structured single-cell protein is used to manufacture meat substitutes, while in Japan, microbial protein is widely utilized in the production of various food seasonings.

It should be emphasized that, in addition to microbiological protein production, other methods are used to extract proteins from non-food plant materials, such as tree leaves, inedible grains, and beans. Leaves are considered to have the greatest protein reserves, with a content ranging from 2 to 5 tons per hectare per year. However, extracting proteins from leaves is energy-intensive. Research on extracting protein from cottonseed is currently underway in the former USSR.

The production of Proteinogenic Amino Acids, particularly essential ones (see p. 51), is of great importance for solving the food protein problem. Adding them to plant proteins, which are characterized by a low content of Essential Amino Acids, significantly increases their biological value. For instance, adding 0.4% lysine to wheat flour raises its biological value by at least 50%. In poultry and pig farming, soybean meal used in feed is enriched with methionine, which promotes better protein utilization and positively impacts livestock productivity.

If amino acids are added to diets with insufficient protein content, the growth rate of piglets increases by 20–25%, and by 10–15% under normal protein levels compared to the control group. This approach reduces feed and protein consumption per unit of output. Calculations show that adding 1 kg of lysine per ton of mixed feed yields an additional 60 kg of pork or poultry meat and saves up to 180 kg of feed.

Thus, it can be noted that humanity has now entered the era of industrial protein production—the most crucial and scarce food product. Success in this field will help provide Earth's population with adequate dietary protein and increase its content in animal diets.