The use of omics technologies in creating LBP and postbiotics based on the Limosilactobacillus fermentum U-21.

In recent years, there has been an increasing tendency to create drugs based on certain commensal bacteria of the human microbiota and their ingredients, primarily focusing on live biotherapeutics (LBPs) and postbiotics. The creation of such drugs, termed pharmacobiotics, necessitates an understanding of their mechanisms of action and the identification of pharmacologically active ingredients that determine their target properties. Typically, these are complexes of biologically active substances synthesized by specific strains, promoted as LBPs or postbiotics (including vesicles): proteins, enzymes, low molecular weight metabolites, small RNAs, etc. This study employs omics technologies, including genomics, proteomics, and metabolomics, to explore the potential of Limosilactobacillus fermentum U-21 for innovative LBP and postbiotic formulations targeting neuroinflammatory processes. Proteomic techniques identified and quantified proteins expressed by L. fermentum U-21, highlighting their functional attributes and potential applications. Key identified proteins include ATP-dependent Clp protease (ClpL), chaperone protein DnaK, protein GrpE, thioredoxin reductase, LysM peptidoglycan-binding domain-containing protein, and NlpC/P60 domain-containing protein, which have roles in disaggregase, antioxidant, and immunomodulatory activities. Metabolomic analysis provided insights into small-molecule metabolites produced during fermentation, revealing compounds with anti-neuroinflammatory activity. Significant metabolites produced by L. fermentum U-21 include GABA (γ-aminobutyric acid), niacin, aucubin, and scyllo-inositol. GABA was found to stabilize neuronal activity, potentially counteracting neurodegenerative processes. Niacin, essential for optimal nervous system function, was detected in vesicles and culture fluid, and it modulates cytokine production, maintaining immune homeostasis. Aucubin, an iridoid glycoside usually secreted by plants, was identified as having antioxidant properties, addressing issues of bioavailability for therapeutic use. Scyllo-inositol, identified in vesicles, acts as a chemical chaperone, reducing abnormal protein clumps linked to neurodegenerative diseases. These findings demonstrate the capability of L. fermentum U-21 to produce bioactive substances that could be harnessed in the development of pharmacobiotics for neurodegenerative diseases, contributing to their immunomodulatory, anti-neuroinflammatory, and neuromodulatory activities. Data of the HPLC-MS/MS analysis are available via ProteomeXchange with identifier PXD050857.

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry.

In the 1980s, Escherichia coli was the preferred host for heterologous protein expression owing to its capacity for rapid growth in complex media; well-studied genetics; rapid and direct transformation with foreign DNA; and easily scalable fermentation. Despite the relative ease of use of E. coli for achieving the high expression of many recombinant proteins, for some proteins, e.g., membrane proteins or proteins of eukaryotic origin, this approach can be rather ineffective. Another microorganism long-used and popular as an expression system is baker's yeast, Saccharomyces cerevisiae. In spite of a number of obvious advantages of these yeasts as host cells, there are some limitations on their use as expression systems, for example, inefficient secretion, misfolding, hyperglycosylation, and aberrant proteolytic processing of proteins. Over the past decade, nontraditional yeast species have been adapted to the role of alternative hosts for the production of recombinant proteins, e.g., Komagataella phaffii, Yarrowia lipolytica, and Schizosaccharomyces pombe. These yeast species' several physiological characteristics (that are different from those of S. cerevisiae), such as faster growth on cheap carbon sources and higher secretion capacity, make them practical alternative hosts for biotechnological purposes. Currently, the K. phaffii-based expression system is one of the most popular for the production of heterologous proteins. Along with the low secretion of endogenous proteins, K. phaffii efficiently produces and secretes heterologous proteins in high yields, thereby reducing the cost of purifying the latter. This review will discuss practical approaches and technological solutions for the efficient expression of recombinant proteins in K. phaffii, mainly based on the example of enzymes used for the feed industry.

The Molecular Aspects of Functional Activity of Macrophage-Activating Factor GcMAF.

Group-specific component macrophage-activating factor (GcMAF) is the vitamin D3-binding protein (DBP) deglycosylated at Thr420. The protein is believed to exhibit a wide range of therapeutic properties associated with the activation of macrophagal immunity. An original method for GcMAF production, DBP conversion to GcMAF, and the analysis of the activating potency of GcMAF was developed in this study. Data unveiling the molecular causes of macrophage activation were obtained. GcMAF was found to interact with three CLEC10A derivatives having molecular weights of 29 kDa, 63 kDa, and 65 kDa. GcMAF interacts with high-molecular-weight derivatives via Ca2+-dependent receptor engagement. Binding to the 65 kDa or 63 kDa derivative determines the pro- and anti-inflammatory direction of cytokine mRNA expression: 65 kDa-pro-inflammatory (TNF-α, IL-1ß) and 63 kDa-anti-inflammatory (TGF-ß, IL-10). No Ca2+ ions are required for the interaction with the canonical 29 kDa CLEC10A. Both forms, DBP protein and GcMAF, bind to the 29 kDa CLEC10A. This interaction is characterized by the stochastic mRNA synthesis of the analyzed cytokines. Ex vivo experiments have demonstrated that when there is an excess of GcMAF ligand, CLEC10A forms aggregate, and the mRNA synthesis of analyzed cytokines is inhibited. A schematic diagram of the presumable mechanism of interaction between the CLEC10A derivatives and GcMAF is provided. The principles and elements of standardizing the GcMAF preparation are elaborated.

Accurate noise-robust classification of Bacillus species from MALDI-TOF MS spectra using a denoising autoencoder.

Bacillus strains are ubiquitous in the environment and are widely used in the microbiological industry as valuable enzyme sources, as well as in agriculture to stimulate plant growth. The Bacillus genus comprises several closely related groups of species. The rapid classification of these remains challenging using existing methods. Techniques based on MALDI-TOF MS data analysis hold significant promise for fast and precise microbial strains classification at both the genus and species levels. In previous work, we proposed a geometric approach to Bacillus strain classification based on mass spectra analysis via the centroid method (CM). One limitation of such methods is the noise in MS spectra. In this study, we used a denoising autoencoder (DAE) to improve bacteria classification accuracy under noisy MS spectra conditions. We employed a denoising autoencoder approach to convert noisy MS spectra into latent variables representing molecular patterns in the original MS data, and the Random Forest method to classify bacterial strains by latent variables. Comparison of the DAE-RF with the CM method using the artificially noisy test samples showed that DAE-RF offers higher noise robustness. Hence, the DAE-RF method could be utilized for noise-robust, fast, and neat classification of Bacillus species according to MALDI-TOF MS data.

A GC-MS Chemotaxonomic Study on Lipophilic Compounds in the Bark of S. aucuparia subsp. sibirica Trees from the Population Growing in Akademgorodok, Novosibirsk (Russia).

Determination of chemotypes and of their role in the polymorphism of populations is an important field in the research on secondary metabolites of plants. In the present study, by gas chromatography coupled with mass spectrometry, the composition of bark extracts from rowan S. aucuparia subsp. sibirica was determined for 16 trees growing within Akademgorodok of Novosibirsk, with bark samples collected both in winter and summer. Among 101 fully or partially identified metabolites, there are alkanes, alkenes, linear alcohols, fatty acids and their derivatives, phenols and their derivatives, prunasin and its parent and derivative compounds, polyprenes and their derivatives, cyclic diterpenes, and phytosterols. These compounds were grouped according to their biosynthesis pathways. Cluster analysis revealed two groups among the bark samples collected in winter and three groups among bark samples collected in summer. The key determinants of this clustering are the biosynthesis of metabolites via the cyanogenic pathway (especially potentially toxic prunasin) and their formation via the phytosterol pathway (especially potentially pharmacologically useful lupeol). It follows from the results that the presence of chemotypes having sharply different profiles of metabolites in a population from a small geographic area invalidates the practice of general sampling to obtain averaged data when a population is described. From the standpoint of possible industrial use or plant selection based on metabolomic data, it is possible to select specific sets of samples containing a minimal amount of potentially toxic compounds and the largest amount of potentially useful substances.

Heterologous Expression of Extracellular Proteinase pAsPs of Aspergillus pseudotamarii in Komagataella phaffii.

Neutral protease pAsPs gene was obtained by sequence optimization of NpI protease from Aspergillus pseudotamarii. pAsPs was for the first time integrated in the genome of yeast strain Komagataella phaffii T07, and then produced in a 5 L bioreactor with an enzyme yield of 150,800 U/mL of culture liquid towards casein. The specific activity of the pAsPs was 7,657,000 U/mg toward casein, 2320 U/mg toward hemoglobin, and 25,344 U/mg toward azocasein per 1 mg of the protein. The enzyme was found to be inhibited by Cu2+. Optimal activity pH was shown in the range of pH 6.5-8.0, and optimal temperature-50-60 °C. The molecular mass of the recombinant protease pAsPs was shown to be 67.5 kDa. Mass-spectrometric analysis confirmed the identity of the amino acid sequence of the obtained pAsPs preparation with the predicted sequence, with 17% coverage and protein score 288. Thus, the novel neutral protease pAsPs is a promising candidate for large-scale use in manufacturing, including the food industry.

Specific Features of the Proteomic Response of Thermophilic Bacterium Geobacillus icigianus to Terahertz Irradiation.

Studying the effects of terahertz (THz) radiation on the proteome of temperature-sensitive organisms is limited by a number of significant technical difficulties, one of which is maintaining an optimal temperature range to avoid thermal shock as much as possible. In the case of extremophilic species with an increased temperature tolerance, it is easier to isolate the effects of THz radiation directly. We studied the proteomic response to terahertz radiation of the thermophilic Geobacillus icigianus, persisting under wide temperature fluctuations with a 60 °C optimum. The experiments were performed with a terahertz free-electron laser (FEL) from the Siberian Center for Synchrotron and Terahertz Radiation, designed and employed by the Institute of Nuclear Physics of the SB of the RAS. A G. icigianus culture in LB medium was THz-irradiated for 15 min with 0.23 W/cm2 and 130 µm, using a specially designed cuvette. The life cycle of this bacterium proceeds under conditions of wide temperature and osmotic fluctuations, which makes its enzyme systems stress-resistant. The expression of several proteins was shown to change immediately after fifteen minutes of irradiation and after ten minutes of incubation at the end of exposure. The metabolic systems of electron transport, regulation of transcription and translation, cell growth and chemotaxis, synthesis of peptidoglycan, riboflavin, NADH, FAD and pyridoxal phosphate cofactors, Krebs cycle, ATP synthesis, chaperone and protease activity, and DNA repair, including methylated DNA, take part in the fast response to THz radiation. When the response developed after incubation, the systems of the cell's anti-stress defense, chemotaxis, and, partially, cell growth were restored, but the respiration and energy metabolism, biosynthesis of riboflavin, cofactors, peptidoglycan, and translation system components remained affected and the amino acid metabolism system was involved.

Heterologous Expression of Xylanase xAor from Aspergillus oryzae in Komagataella phaffii T07

Xylanases (EC 3.2.1.8) hydrolyze the hemicellulose of plant cell walls. Xylanases are used in the food and paper industries and for bioconversion of lignocellulose to biofuel. In this work, the producer-strain with four copies of the xAor xylanase gene was organized in two tandem copies for optimal expression in Komagataella phaffii T07 yeast. The secreted 35 kDa xylanase was purified from culture medium by gel filtration on Sephadex G-25 and anion exchange chromatography on DEAE-Sepharose 6HF. Tryptic peptides of the recombinant enzyme were analyzed by liquid chromatography-tandem mass spectrometry where the amino acid sequence corresponded to Protein Accession # O94163 for Endo-1,4-beta-xylanase from Aspergillus oryzae RIB40. The recombinant xylanase was produced in a bioreactor where the secreted enzyme hydrolyzed oat xylane with an activity of 258240 IU/mL. High activity in the culture medium suggested xylanase could be used for industrial applications without being purified or concentrated. The pH optimum for xylanase xAor was 7.5, though the enzyme was active from pH 2.5 to pH 10. Xylanase was active at temperatures from 35 °C to 85 °C with a maximum at 60 °C. In conclusion, this protocol yields soluble, secreted xylanase suitable for industrial scale production.

Diversity and Metabolism of Microbial Communities in a Hypersaline Lake along a Geochemical Gradient.

In the south of western Siberia (Russia), there are many unique and unexplored soda, saline, and freshwater lakes. In this study, the results are presented on microbial diversity, its metabolic potential, and their relation with a set of geochemical parameters for a hypersaline lake ecosystem in the Novosibirsk region (Oblast). The metagenomic approach used in this work allowed us to determine the composition and structure of a floating microbial community, the upper layer of silt, and the strata of bottom sediments in a natural saline lake via two bioinformatic approaches, whose results are in good agreement with each other. In the floating microbial community and in the upper layers of the bottom sediment, bacteria of the Proteobacteria (Gammaproteobacteria), Cyanobacteria, and Bacteroidetes phyla were found to predominate. The lower layers were dominated by Proteobacteria (mainly Deltaproteobacteria), Gemmatimonadetes, Firmicutes, and Archaea. Metabolic pathways were reconstructed to investigate the metabolic potential of the microbial communities and other hypothetical roles of the microbial communities in the biogeochemical cycle. Relations between different taxa of microorganisms were identified, as was their potential role in biogeochemical transformations of C, N, and S in a comparative structural analysis that included various ecological niches.

E. coli aggregation and impaired cell division after terahertz irradiation.

In this study we demonstrated that exposure of Escherichia coli (E. coli) to terahertz (THz) radiation resulted in a change in the activities of the tdcABCDEFGR and matA-F genes (signs of cell aggregation), gene yjjQ (signs of suppression of cell motility), dicABCF, FtsZ, and minCDE genes (signs of suppression of cell division), sfmACDHF genes (signs of adhesin synthesis), yjbEFGH and gfcA genes (signs of cell envelope stabilization). Moreover, THz radiation induced E. coli csg operon genes of amyloid biosynthesis. Electron microscopy revealed that the irradiated bacteria underwent increased aggregation; 20% of them formed bundle-like structures consisting of two to four pili clumped together. This could be the result of changes in the adhesive properties of the pili. We also found aberrations in cell wall structure in the middle part of the bacterial cell; these aberrations impaired the cell at the initial stages of division and resulted in accumulation of long rod-like cells. Overall, THz radiation was shown to have adverse effects on bacterial populations resulting in cells with abnormal morphology.

Metagenomics data of microbial communities in bacterial mats and bottom sediments in water bodies within the Kurai Mercury Province (Gorny Altai, Russia).

Below is data on the microbial diversity in bottom sediments and microbial mats in water bodies within the Kurai Mercury Province (Ulagan District, Aktash village, Gorny Altai). A database on the geochemical features of water bodies in the study area is presented. Data was obtained using 16 s rRNA amplicon directed metagenomic sequencing on Illumina MiSeq. The raw sequence data used for analysis is available in NCBI under the Sequence Read Archive (SRA) with BioProject No. PRJNA670076 and SRA accession numbers SRX9316205, SRX9316207, SRX9316208, SRX9316209.

Fluorescent bacterial biosensor E. coli/pTdcR-TurboYFP sensitive to terahertz radiation.

A fluorescent biosensor E. coli/pTdcR-TurboYFP sensitive to terahertz (THz) radiation was developed via transformation of Escherichia coli (E. coli) cells with plasmid, in which the promotor of the tdcR gene controls the expression of yellow fluorescent protein TurboYFP. The biosensor was exposed to THz radiation in various vessels and nutrient media. The threshold and dynamics of fluorescence were found to depend on irradiation conditions. Heat shock or chemical stress yielded the absence of fluorescence induction. The biosensor is applicable to studying influence of THz radiation on the activity of tdcR promotor that is involved in the transport and metabolism of threonine and serine in E. coli.

Metagenomics data of microbial communities of natural organic matter from the dispersion train of sulfide tailings.

Below is data on the microbial diversity of natural organic matter from the Dispersion Train of Sulfide Tailings (northern Salaire Ridge, southwestern Siberia, Russia, Ursk Village). Data was obtained using 16s rRNA amplicon directed metagenomic sequencing on Illumina MiSeq. The raw sequence data used for analysis is available in NCBI under the Sequence Read Archive (SRA) with BioProject No. PRJNA670045 and SRA accession number SRX9314152, SRX9314376. The data sequences of the 16s rRNA gene are presented at the links MW142408-MW142413, MW142414-MW142447.

Metagenomics dataset used to characterize microbiome in water and sediments of the lake Solenoe (Novosibirsk region, Russia).

This is data on the microbial diversity in the floating cyanobacterial community and sediment samples from the lake Solenoe (Novosibirsk region, Russia) obtained by metagenomic methods. Such a detailed data of the microbial diversity of the Novosibirsk oblast lake ecosystem was carried out for the first time. The purpose of our work was to reveal microbial taxonomic diversity and abundance, metabolic pathways and new enzyme findings the studied lake ecosystem using the next-generation sequencing (NGS) technology and metagenomic analysis. The data was obtained using metagenomics DNA whole genome sequencing (WGS) on Illumina NextSeq and NovaSeq. The raw sequence data used for analysis is available in NCBI under the Sequence Read Archive (SRA) with the BioProjects and SRA accession numbers: PRJNA493912 (SRR7943696), PRJNA493952 (SRR7943839) and PRJNA661775 (SRR12601635, SRR12601634, SRR12601633) corresponding to floating cyanobacterial community and sediment layers samples, respectively.

Young «oil site¼ of the Uzon Caldera as a habitat for unique microbial life.

BACKGROUND: The Uzon Caldera is one of the places on our planet with unique geological, ecological, and microbiological characteristics. Uzon oil is the youngest on Earth. Uzon oil has unique composition, with low proportion of heavy fractions and relatively high content of saturated hydrocarbons. Microbial communities of the «oil site¼ have a diverse composition and live at high temperatures (up to 97 °C), significant oscillations of Eh and pH, and high content of sulfur, sulfides, arsenic, antimony, and mercury in water and rocks. RESULTS: The study analyzed the composition, structure and unique genetics characteristics of the microbial communities of the oil site, analyzed the metabolic pathways in the communities. Metabolic pathways of hydrocarbon degradation by microorganisms have been found. The study found statistically significant relationships between geochemical parameters, taxonomic composition and the completeness of metabolic pathways. It was demonstrated that geochemical parameters determine the structure and metabolic potential of microbial communities. CONCLUSIONS: There were statistically significant relationships between geochemical parameters, taxonomic composition, and the completeness of metabolic pathways. It was demonstrated that geochemical parameters define the structure and metabolic potential of microbial communities. Metabolic pathways of hydrocarbon oxidation was found to prevail in the studied communities, which corroborates the hypothesis on abiogenic synthesis of Uzon hydrothermal petroleum.

Study on the effects of terahertz radiation on gene networks of Escherichia coli by means of fluorescent biosensors.

Three novel fluorescent biosensors sensitive to terahertz (THz) radiation were developed via transformation of Escherichia coli (E. coli) cells with plasmids, in which a promotor of genes matA, safA, or chbB controls the expression of a fluorescent protein. The biosensors were exposed to THz radiation from two sources: a high-intensity pulsed short-wave free electron laser and a low-intensity continuous long-wave IMPATT-diode-based device. The threshold and dynamics of fluorescence were found to depend on radiation parameters and exposure time. Heat shock or chemical stress yielded the absence of fluorescence induction. The biosensors are evaluated to be suitable for studying influence of THz radiation on the activity of gene networks related with considered gene promoters.

Changes in the proteomic profile of blood serum in coronary atherosclerosis.

BACKGROUND: Our aim was to study changes in the serum proteomic profile in coronary atherosclerosis. METHODS: The study involved two groups of patients: 1) men with coronary heart disease and coronary atherosclerosis (n = 15); 2) control (n = 15): men without coronary heart disease. The object of this study was blood serum. Separation of proteins for the investigation of differences in serum protein components was performed by two-dimensional electrophoresis. Identification of protein fractions was carried out using peptide mass maps by the matrix-assisted laser desorption ionization method. RESULTS: In blood serum samples from patients with coronary atherosclerosis, protein separation in two-dimensional gels with mass-spectrometric identification revealed an increase of some proteins: hemopexin, transthyretin (monomeric form), retinol-binding protein 4, and components of the complement system: C3 (chain B) and C9. There was a decrease of some proteins: kininogen, zinc finger protein 133, and B-cell CLL/lymphoma 6 member B protein. Comparisons between the experimental and control group were carried out in protein fractions where the protein amount differed more than 1.5-fold (p < 0.05). CONCLUSIONS: Proteome profiling of serum revealed a change in the content of kininogen, hemopexin, transthyretin, retinol-binding protein, and proteins of the complement system (C9, and C3) in coronary atherosclerosis. The contribution to the differential expression of a protein was often made by isoforms of the protein, particularly transthyretin. The change in the concentrations of functionally interacting proteins, such as transthyretin and retinol-binding protein, were noted.

Metagenomic data on prokaryotic diversity of Kunashir island geothermal spring.

This is data on the microbial diversity of a geothermal spring located on the banks of the acidic creek of Kunashir Island. Data was obtained using 16s rRNA amplicon directed metagenomic sequencing on Illumina MiSeq. The raw sequence data used for analysis is available in NCBI under the Sequence Read Archive (SRA) with the BioProject No. PRJNA637298, PRJNA637447 and SRA accession number SRP265942, SRP266050. The data sequences of the 16s rRNA gene are presented at the accession numbers MT604934-MT604967, MT604911-MT604921 in NCBI GenBank database.

A Genome-Scale Metabolic Model of 2,3-Butanediol Production by Thermophilic Bacteria Geobacillus icigianus.

The thermophilic strain of the genus Geobacillus, Geobacillus icigianus is a promising bacterial chassis for a wide range of biotechnological applications. In this study, we explored the metabolic potential of Geobacillus icigianus for the production of 2,3-butanediol (2,3-BTD), one of the cost-effective commodity chemicals. Here we present a genome-scale metabolic model iMK1321 for Geobacillus icigianus constructed using an auto-generating pipeline with consequent thorough manual curation. The model contains 1321 genes and includes 1676 reactions and 1589 metabolites, representing the most-complete and publicly available model of the genus Geobacillus. The developed model provides new insights into thermophilic bacterial metabolism and highlights new strategies for biotechnological applications of the strain. Our analysis suggests that Geobacillus icigianus has a potential for 2,3-butanediol production from a variety of utilized carbon sources, including glycerine, a common byproduct of biofuel production. We identified a set of solutions for enhancing 2,3-BTD production, including cultivation under anaerobic or microaerophilic conditions and decreasing the TCA flux to succinate via reducing citrate synthase activity. Both in silico predicted metabolic alternatives have been previously experimentally verified for closely related strains including the genus Bacillus.

Dissecting DISC regulation via pharmacological targeting of caspase-8/c-FLIPL heterodimer.

Pharmacological targeting via small molecule-based chemical probes has recently acquired an emerging importance as a valuable tool to delineate molecular mechanisms. Induction of apoptosis via CD95/Fas and TRAIL-R1/2 is triggered by the formation of the death-inducing signaling complex (DISC). Caspase-8 activation at the DISC is largely controlled by c-FLIP proteins. However molecular mechanisms of this control have just started to be uncovered. In this study we report the first-in-class chemical probe targeting c-FLIPL in the heterodimer caspase-8/c-FLIPL. This rationally designed small molecule was aimed to imitate the closed conformation of the caspase-8 L2' loop and thereby increase caspase-8 activity after initial processing of the heterodimer. In accordance with in silico predictions, this small molecule enhanced caspase-8 activity at the DISC, CD95L/TRAIL-induced caspase activation, and subsequent apoptosis. The generated computational model provided further evidence for the proposed effects of the small molecule on the heterodimer caspase-8/c-FLIPL. In particular, the model has demonstrated that boosting caspase-8 activity by the small molecule at the early time points after DISC assembly is crucial for promoting apoptosis induction. Taken together, our study allowed to target the heterodimer caspase-8/c-FLIPL and get new insights into molecular mechanisms of its activation.