Biotechnology - Yu.O. Sazykin 2006

General Biotechnology
Molecular mechanisms of intracellular regulation and their application in biotechnological production
Regulation of the assimilation of nitrogen-containing compounds

It is well known that nitrogen is quantitatively the dominant gas in the Earth's atmosphere; however, the evolution of life on our planet did not take the direct route of its assimilation by the Cells of mammals, plants, and most microorganisms. Atmospheric nitrogen can be utilized only by nodule Bacteria developing in the rhizosphere of legumes and by certain free-living nitrogen fixers, which in turn leads to the presence of ammonium salts and nitrogen oxides in the soil.

Many microorganisms assimilate organic nitrogen compounds, yet among the wide variety of such compounds, ammonium chloride and ammonium sulfate are assimilated most easily and rapidly.

Central to the synthesis of nitrogen-containing substances are metabolic reactions involving amino group Donors: glutamate, glutamine, and aspartate:

Class="center">Глутамат + NH₃ + АТФ → Глутамин + АДФ + Ф

Глутамин + а-Кетоглутарат + НАДФН → 2-Глутамат + НАДФН⁺

Глутамат + Оксалоацетат → Аспартат + а-Кетоглутарат

where Ф stands for inorganic phosphorus.

Using Glutamine Synthetase—a crucial initial enzyme of Nitrogen METABOLISM that catalyzes The conversion of ammonia into an amide group (the first reaction)—we can observe the full complexity of regulatory interactions within a highly branched metabolic pathway. The amide group of glutamine serves as a nitrogen source in The Biosynthesis of six compounds: two aromatic Amino Acids (Tryptophan and Histidine), as well as AMP, CTP, glucosamine-6-phosphate, and carbamoyl phosphate. Experiments on E. coli cells have demonstrated that any of the end products, when present at a saturating concentration (relative to the microorganism's requirements), can act via feedback inhibition. However, the inhibition caused in this case is incomplete. At the same time, as the number of metabolites (at saturating concentrations) increases, additivity manifests itself; that is, Enzyme Inhibition intensifies almost to the complete cessation of its activity, a phenomenon known as cumulative feedback inhibition. It has also been established that all inhibitory metabolites of glutamine synthetase bind to the enzyme at distinct, specific sites. Consequently, their binding does not interfere with one another, which is of fundamental importance for The regulation of glutamine synthetase activity.

However, another mechanism of regulation exists for this complex enzyme, which consists of 12 subunits (each with a Molecular Weight of 12 kDa). On the one hand, if a microorganism culture finds itself in an environment extremely poor in carbon and Energy Sources, but with an excess of ammonium and glutamine ions, The activity of glutamine synthetase drops sharply due to a more radical mechanism. Each of the 12 subunits is adenylylated, covalently binding a single AMP residue. The enzyme's activity practically vanishes, which favors the microorganism's survival. On the other hand, when the microorganism is transferred to a medium depleted of readily available nitrogen sources, glutamine synthetase is deadenylylated and "mobilizes" its activity.

In some microorganisms, the regulatory system of glutamine synthetase is further complicated by the fact that the enzyme exists in two isoforms: in one isoform, the regulatory system involves adenylylation-deadenylylation, whereas in the other, it involves reversible inactivation by excess ammonia.

Along with the aforementioned mechanisms regulating glutamine synthetase activity, there is yet another intracellular regulation mechanism that allows certain microorganisms to cope with nitrogen starvation in the nutrient medium by enhancing the expression of the glutamine synthetase Gene.

Enzymes involved in the assimilation of nitrogen-containing compounds are, in most cases, inducible and subject to the laws of nitrogen catabolite repression, which manifests after these compounds are converted into glutamine. During the synthesis of Glycine and Alanine, the a-amino group of glutamine is utilized with the participation of transaminases. Here, The Cell's nitrogen status is determined by The ratio of glutamine to a-ketoglutarate. Furthermore, studies on various Eukaryotic cells have shown that a deficiency of carbon and energy sources in the nutrient medium leads to noticeable proteolytic degradation of the enzymes involved in utilizing nitrogen-containing compounds.

The regulation of nitrogen assimilation in eukaryotes—primarily Fungi, including Yeasts—shares much in common with prokaryotes, though certain differences exist. For instance, fungi preferentially utilize ammonium salts and glutamine as nitrogen sources.

The biotechnologist's task involves intensifying the biosynthesis reactions of glutamine, glutamate, and aspartate, which is achieved through Genetic Methods or the optimization of nutrient media. When producing a range of primary metabolites for which these substances serve as precursors, the intensification of these reactions has a favorable effect on production processes. It is well known that primary metabolites form The basis of diverse classes of pharmaceutical agents. Moreover, since secondary metabolites are typically modifications of primary ones, enhancing the biosynthesis of the latter positively impacts the biosynthesis of secondary metabolites as well, such as many Antibiotics, particularly those containing peptide structures.