Applied Biochemistry and Microbiology最新文献

In the metabolic engineering of producer strains, strong constitutive promoters are an attractive alternative to inducible promoters, since they do not require the addition of expensive and sometimes toxic inducing substances to the fermentation medium. In this work, we have compared the Yarrowia lipolytica constitutive promoters: pEXP1, pRPS2, pRPL22 (native) and hybRPS2_499, hybRPL22 (synthetic) with the inducible promoter pEYK1-3AB. The synthetic promoters hybRPS2 and hybRPL22 are modified versions of the ribosomal promoters pRPS2 and pRPL22, respectively. Initially, promoter strength was assessed by the expression level of the reporter gene encoding the green fluorescent protein hrGFP. It was shown that the strength of the promoters increased in the following order: pRPS2, pRPL22, pEXP1, hybRPL22, hybRPS2_499, while the level of pEYK1-3AB activity with 1.5% erythritol induction was comparable to the level of unmodified pRPS2 and pRPL22. During heterologous expression of the CarRP-GGPPSs7 genes under the control of the studied promoters, the highest β-carotene production was observed in the recombinant Y. lipolytica strain with the pEXP1 promoter, while the hybRPS2_499 promoter showed the result comparable to the pEYK1-3AB promoter induced by the addition of 1.5% erythritol. Thus, we have shown that in constructing heterologous biosynthetic pathways in Y. lipolytica, a strong constitutive promoter can successfully compete with an inducible variant. However, in our case, the use of the strongest promoter did not lead to the highest yield of the target product. Therefore, rapid identification of the best producer strain requires testing a wide range of promoters of varying strengths.

Ortho-phthalic esters are a new group of secondary plant metabolites. The physiological function of these compounds is unknown. The results of studies of the biological activity of phthalates suggest that one of their functions is protection against phytopathogens. It has been shown that there is a sharp increase in the content of diethylhexyl phthalate at the initial stages of germination of wheat seeds (Triticum aestivum L.). The role of this compound in protecting plants from phytopathogens is discussed.

The use of surface-enhanced Raman spectroscopy (SERS) in immunochromatographic assay (ICA) supports integration of the rapidity, simplicity, and specificity of the immunoassay with high sensitivity and accuracy of SERS registration. An immunochromatographic test system has been developed for detection of the anti-Müllerian hormone (AMH), a marker of ovarian reserve in women of reproductive age. ICA has been implemented in a sandwich format, which facilitates the interaction between the AMH molecules in the tested sample and a pair of monoclonal antibodies that recognise different antigen epitopes. This interaction results in the formation of AMH-labeled antibody complexes (where the antibody is immobilized on the membrane of the immunochromatographic test strip). We have used bimetallic gold–silver core-shell nanoparticles modified with a SERS-active reporter molecule to label antibodies. We have evaluated the size characteristics of the nanoparticles and selected the optimal conditions for their conjugation with antibodies. ICA has been performed under conditions that ensure the achievement of the minimum AMH detection limit. The developed ICA-SERS system has a detection limit of 0.15 ng/mL and a working range of 4.7–29.5 ng/mL satisfying practical diagnostic requirements. The analytical system has been applied successfully to testing blood serum.

One of the promising areas in oncodiagnostics is the development of acoustic sensor systems for the indication of cancer cells, on the surface of which heat shock proteins (HSP) are expressed. The presence of these proteins is an important diagnostic marker for the development of oncology. The paper experimentally demonstrates the possibility of indicating cells, on the surface of which HSP are expressed, using specific antibodies under the conditions of increased conductivity of the measurement environment (conductivity up to 2000 μS/cm) and in the presence of foreign (interfering) cells using an acoustic sensor system based on a piezoelectric resonator with a lateral electric field. The results obtained are promising for the development and adaptation of acoustic sensor systems in the early diagnosis of oncological diseases.

The properties of three mutants of the industrial-scale L-lysine–producing strain Corynebacterium glutamicum VKPM B-11969 were studied. These mutants had one of the anaplerotic pathways for synthesizing lysine precursors, oxaloacetate, blocked due to inactivation of the ppc (encodes phosphoenolpyruvate carboxylase) or pyc (encodes pyruvate carboxylase) genes, or one of the pyruvate synthesis pathways was disrupted due to inactivation of the pyk gene (encodes pyruvate kinase). Inactivation of pyruvate carboxylase, which catalyzes the synthesis of oxaloacetate from pyruvate and CO₂, did not affect L-lysine production. Inactivation of pyruvate kinase, which catalyzes the synthesis of pyruvate from phosphoenolpyruvate, resulted in a decrease in the rate of L-lysine synthesis (by 20–25%). The synthesis of L-lysine decreased to the greatest extent with the inactivation of phosphoenolpyruvate carboxylase, by 30–50% depending on the growing conditions. It was concluded that, in the B-11969 strain, under conditions of active synthesis of L‑lysine, the formation of oxaloacetate occurs predominantly with the participation of phosphoenolpyruvate carboxylase. However, the increase in the expression of the ppc gene in the producer strain led to an increase in the activity of phosphoenolpyruvate carboxylase, but not in the production of L-lysine, which allowed us to assume that in the B-11969 strain the stage of oxaloacetate synthesis is not limiting for the production of lysine.

Rhizosphere biofilm communities exhibit stress resistance, efficient resource use, and species diversity, though lab studies typically focus on mono- and binary cultures. This research aimed to investigate multispecies biofilms of PGPR strains with antibacterial potential for biofertilizer development. Three Pseudomonas strains showing antagonistic activity against rhizobia were identified. Cocultivation of PGPR strains with Rhizobium sp. LPi14 resulted in a synergistic effect, producing a modified multispecies biofilm with enhanced biomass. The cultures demonstrated growth-stimulating activity, and experiments confirmed productive symbiosis between Rhizobium sp. LPi14 and legumes. The obtained results confirm the feasibility of introducing biofilm biofertilizers containing associations of rhizosphere bacteria with PGPR properties and antagonistic activity into agricultural practices.

Oomycetes are a particularly dangerous group of pathogens that significantly reduce both the productivity and the quality of production of agricultural crops. Protective measures using available fungicides are often ineffective against them. One of the simplest and, at the same time, innovative approaches to plant protection from oomycete infection can be considered biological methods, which, if used in a timely manner, can be even more effective than chemical means of protection. Biological control involves the use of beneficial microorganisms that can stop the development of diseases by producing biologically active substances with antibiotic activity, such as antibiotics, biosurfactants, and toxins. In this review, we describe a strategy for sustainable prevention and control of oomycete pathogens using plant-associated microorganisms, including bacteria of the genera Bacillus, Paenibacillus, Streptomyces, Pseudomonas, and Lactobacillus. The mechanisms that determine the protective effects of bioprotective strains in the endo-, phyllo-, and rhizosphere of plants are also considered, which can be used in the development of strategies for constructing phytomicrobiomes.

Uterine leiomyoma (UL) is a benign tumor of monoclonal origin developing from smooth muscle cells and containing varying amounts of fibrous connective tissue. Leiomyomas and myometrium are similar in cellular composition but differ in the cellular transcriptome profiles. A rapid and reliable method for isolating and culturing primary UL cells is described in this paper. Conditions for growing and maintaining a monolayer culture of UL cells in vitro were optimized. The effect of sex steroid hormones, estradiol and progesterone, on UL genetic markers was evaluated. To prevent the loss of driver mutations in the MED12 and HMGA2 genes, the use of the UL cell model cultured in a specialized nutrient medium with the addition of progesterone at the first passage was proposed.