Vol 60, No 3 (2024)

Methionine biosynthesis in most microorganisms proceeds in two alternative ways. Each pathway is catalyzed by independent enzymes and is tightly regulated by methionine. The transulfurylation pathway involves the formation of a cystathionine, and cysteine acts as a source of sulfur. The enzymes of this metabolic pathway are characterized in detail. The direct sulfhydrylation pathway involves the synthesis of homocysteine with the participation of an inorganic sulfur source directly from O-acetylhomoserine and is predominant in most classes of bacteria. The subject of this review is the properties and functioning of one of the least studied enzymes of the direct sulfhydrylation pathway – O-acetylhomoserine sulfhydrylase. A deep understanding of the mechanisms controlling the substrate and reaction specificity of O-acetylhomoserine sulfhydrylase is a necessary step in the rational redesign of the enzyme in order to create a promising catalyst for the synthesis s of methionine and its derivatives, as well as, in combination with crystallographic data, for the development of new antimicrobial compounds based on effective enzyme inhibitors.

Using previously engineered adipate-secreting Escherichia MG1655 lacI^Q, ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, P_L-SD_φ10-atoB, P_trc-ideal-4-SD_φ10-fadB, ∆fadE, P_L-SD_φ10-tesB, ∆yciA, P_trc-ideal-4-SD_φ10-fabI, P_L-SD_φ10-paaJ, ∆aceBAK, ∆glcB as a core strain, the derivatives capable of enhanced synthesis of the target compound from glucose via the reversed fatty acid β-oxidation pathway were obtained. The respective effect was achieved due to the intensification of the tricarboxylic acid cycle in the cells. Prevention of multiple cycle turnovers, resulting from the inactivation of succinate dehydrogenase, had no pronounced effect on the formation of adipic acid by the recombinant. Upon the cycle intensification due to enhancing anaplerotic oxaloacetic acetic acid formation from phosphoenolpyruvate, resulting from the increased expression of the native ppc gene, the synthesis of adipic acid rose 1.2-fold to ~390 μM. Enabling the formation of oxaloacetate from pyruvic acid, by introducing in the cells of heterologous Bacillus subtilis pyruvate carboxylase, resulted in a 1.5-fold intensification of the cycle, concomitantly with the proportional increase in adipic acid secretion to ~496 μM. Subsequent inactivation of sdhAB genes in the strain increased the secretion of the target compound only slightly and adipic acid titer reached ~520 μM. The obtained data indicated a direct dependence of the efficiency of adipic acid synthesis by the engineered strains on the degree of intensification of the tricarboxylic acid cycle.

The processes of biocatalytic synthesis of cefamandole and cefazoline, as well as four “chimeric” cephalosporins carrying functional groups of these antibiotics in the C3 or C7 position of β-lactam, were carried out using immobilized cephalosporin-acid synthetase under mild standard conditions. A higher efficiency of biocatalytic acylation of β-lactams with a 1(H)-tetrazolylacetic acid residue compared to acylation with almond acid residue was demonstrated. The chemical structure of the obtained compounds was confirmed using the HPLC–MS method. The possibility of using directly reaction mixtures for evaluating the antibacterial activity of synthesized compounds without isolating the target products was demonstrated. The activity of the obtained cephalosporins against twelve microorganisms belonging to the genera Enterococcus, Acinetobacter, Serratia, Pseudomonas, Staphylococcus and Escherichia was evaluated by diffusion into agar. The activity of synthesized “chimeric” cephalosporins against four microorganisms was found: Escherichia coli VKPM B-6695, Staphylococcus aureus VKPM B-6646, Staphylococcus aureus VKPM B-8171 and Staphylococcus epidermidis VKPM B-12635.

A significant variation in the relative representation of copies of bacterial genes of dye-decolorizing DyP peroxidases typical for the genus Shewanella and a number of other microorganisms was found in the bottom sediments of freshwater reservoirs. It was found that the specific rate of decolorization of crystal violet in a laboratory bioelectrochemical system by a mixed culture of bottom sediments, which showed the highest representation of DyP genes, depended on the method of electrical stimulation of the external circuit and the concentration of the dye. After an increase in the concentration of more than 20 microns, the maximum speed was achieved in the presence of an ionistor polarly connected to the external electrical circuit of the bioelectrochemical system and amounted to 3.23 ± 0.11 μM/h, while with the opposite polarity connection, a minimum value of 2.07 ± 0.08 μM/h was observed. In the case of an open circuit and a resistor, similar indicators occurred – 2.88 ± 0.09 and 2.67 ± 0.12 μM/h, respectively. When analyzing the decolorization products, a consistent decrease in the maxima of the absorption bands of the dye was noted, indicating its more complete degradation by mixed culture. The results may be of interest for the development of methods to improve the efficiency of bioelectrochemical methods of environmental biotechnology, by electrostimulation of the external circuit.

The article presents the results of studies of intensive culture of a new species of bentoplanktonic diatom N. shiloi (Lee, Reimer et McEnery) Round, Hallsteinsen et Paasche 1999 for the Black Sea. The features of the process of isolating the species into an algologically pure culture, as well as the morphological and taxonomic characteristics of the strain in light and electron scanning microscopes are described in detail. The biochemical and production characteristics of the strain were studied, as well as the ability to accumulate fucoxanthin (Fx) and polyunsaturated fatty acids (PUFA) in laboratory conditions. In the exponential growth phase, the specific culture growth rate was µ=0.8 1/day, and the maximum productivity P = 0.46 g dry weight /(L day). The accumulation of PUFAs in the biomass of N. shiloi reached 67.39 mg/g dry weight of algae. The Fx concentration in the biomass at the beginning of the stationary growth phase was 10 mg/g dry weight. The fairly high rate of Fx biosynthesis in microalgae cells, as well as the composition of fatty acids of the Black Sea strain, make it possible to classify N. shiloi as a promising object in biotechnology.