EA032051B1 - Method for identification of a bacterial gene composition involved in synthesis and metabolism of different neuroactive compounds in microbiomes and individual bacterial genomes - Google Patents

Abstract

The invention relates to a method for identifying bacterial genes involved in the synthesis and metabolism of neuroactive compounds in microbiomes and individual bacterial genomes, in particular in the human intestinal microbiome. Can be used in medicine, medical microbiology and neurology. The method uses a set of 45 oligonucleotide pairs (primers) for PCR analysis of total genomic DNA. Primers were selected for ten key genes encoding enzymes involved in the synthesis and metabolism of neuroactive compounds: serotonin, dopamine, histamine, melatonin, gamma-aminobutyric acid, tyramine, spermidine, butyric acid, the gas transducer NO, identified in the 27 dominant bacteria species in the genomes of bacteria. person The developed primer pairs can be used to separately search for selective genes of neuroactive compounds in individual bacterial genomes and to monitor the whole microbiome. The proposed sets of pairs of primers can be used as diagnostic systems for microbiota research in normal and pathological conditions. Primers are designed in such a way that they can be used to determine the level of transcripts of individual genes using real-time PCR analysis.

Description

The invention relates to methods for identifying bacterial genes of neuroactive compounds in human intestinal metagenomes and in individual bacterial genomes.

In recent decades, numerous scientific and clinical studies of the human microbial community have been carried out. It was found that intestinal microbiota plays a key role in maintaining overall body homeostasis and human health. The total genome of gut bacteria microbiomes includes 400 thousand genes, which is 12 times the human genome [Eckburg P.B. et al. Diversity of the human intestinal microbial flora. Science. 2005; 308: 1635-1638]. The structure and composition of the intestinal microbiota reflect the natural selection of both: the microbe and humans through a complex of competitive and symbiotic relationships and depend on a large number of factors. Intestinal microbiota is also considered an endocrine organ for its ability to produce hundreds if not thousands of humoral agents [Brown J. M. Et al. The gut microbial endocrine organ: bacterially-derived signals driving cardiometabolic diseases. Annu Rev Med. 2015. 66: 343-359]. In a healthy person, the intestinal microbiota is balanced and supports the general homeostasis of the body. Changes in biodiversity and the high temporary instability of microbiota are characteristic of a number of gastrointestinal diseases, metabolic diseases, neurological diseases and other pathologies [Young V.B. The intestinal microbiota in health and disease. Opin Gastroenterol. 2012.28: 63-69]. At present, the effect of intestinal microbiota on the functioning of the brain-intestinal axis is considered, which mediates bidirectional communication between the central nervous system and the enteric nervous system of the intestine, which extends it to the microbiota-intestine-brain (OMCM) axis. This axis represents an already complex network of communications between the intestine itself, intestinal microbiota and the brain and covers the functions of the immune, endocrine, nervous systems and nonspecific natural immunity [Mayer E.A. Gut feelings: the emerging biology of gutbrain communication. Nat. Rev. Neurosci. 2011.12: 453 - 466; Bravo J. A. et al. Communication between gastrointestinal bacteria and the nervous system. Current Opinion in Pharmacology. 2012.12: 667-672; Stilling R.M. et al. Microbes do have a significant impact on epigenetic regulation in the host's gut epithelium and immune system, Microbial genes, brain & behavior-epigenetic regulation of the gut-brain axis. Genes Brain Behav. 2014.13: 69-86]. Through a complex system of interactive and symbiotic signaling between the host and microorganisms, the intestinal microbiota is involved in the regulation of neurochemical and neurometabolic processes and has an impact on many fundamental aspects of the central nervous system, including responses to stress, growth and survival of neurons, plasticity, receptor expression and neurotransmission [ Heijtz rd et al. Normal gut microbiota modulates brain development and behavior. Proc. Natl. Acad Sci. 2011.108: 3047-3052; Tillisch K. The effects of gut microbiota on CNS function in humans. Gut Microbes. 2014.5 (3): 404-410]. An important mechanism of communication between the intestinal microbiota and the central nervous system is realized through the interaction of low molecular weight metabolites of bacteria that make up the normal microbiocenosis with sensitive (sensory) nerve endings in the synapse with intestinal mucosal epithelial cells by conducting visceral messages from the intestine to the brain [De Vadder F. et al. Microbiota-generated metabolites promote metabolic benefits via gut-brain neural circuits. Cell. 2014. 156: 84-96]. A sufficient amount of neurotransmitters and other molecules of microbial origin have the potential to modulate the activity of the vagus nerve - the main nerve that connects the ENS to the central nervous system, and subsequently affect brain function. Also, neuromodulators secreted by bacteria stimulate specific intestinal epithelial cells to release molecules that modulate neurotransmission in the enteric nervous system, subsequently affecting the brain and human behavior. [Grenham S. Et al. Brain-gut-microbe communication in health and disease. Front Physiol. 2011; 2: 1-15; Khanna S. et al. A clinician's primer on the role of the microbiome in human health and disease. Mayo Clin. Proc. 2014 89: 107-114; Yano J.M. et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell. 2015. 161: 264-276]. The ability of some representatives of the human intestinal microbiota to synthesize and secrete various neuroactive substances, including neurotransmitters and hormone-like compounds, is known. The study of this ability and the relationship between microbiota and the central nervous system is a new field of science - microbial endocrinology. [Lyte M. The role of microbial endocrinology in infectious disease. J. Endocrinol. 1993. 137: 343-345]. In a number of bacteria, the ability to synthesize serotonin, dopamine, norepinephrine, acetylcholine and other choline, histamine and other amines [Roshchina V.V. Evolutionary considerations of neurotransmitters in microbial, plant and animal cells. In:

Lyte M., Freestone P.P., editors. Microbial endocrinology, interkingdom signaling in infectious disease and health. New York: Springer. 2010. P. 17-52; Clarke G. et al. Gut microbiota: the neglected endocrine organ. Mol. Endocrinol. 2014.28: 1221-1238; Lyte M. et al. Microbial Endocrinology: The Microbiota-Gut-Brain Axis in Health and Disease. Book Series: Advances in Experimental Medicine and Biology, Subseries: Microbial Endocrinology. 2014.817: 436; Oleskin A.V. et al. Role of neuromediators in the functioning of the human microbiota: business talks among microorganisms and the microbiota-host dialogue. Microbiolog. 2016. 85 (1): 325]. Important commensals of the intestinal microbiota of bifidobacteria and lactobacilli are able to metabolize glutamate (the most common excitatory neurotransmitter) into gamma-aminobutyric acid [Barrett E. et al. γ-Aminobutyric acid production by culturable bacteria from the human intestine. J. Appl Microbiol. 2012 113 (2): 411-417; Yunes R. A. et al. GABA production and structure of

- 1 032051 gadB / gadC genes in Lactobacillus and Bifidobacterium strains from human microbiota, Anaerobe. 2016.42: 197-204]. In addition to neurotransmitters, bacteria in the intestine are capable of producing a number of biologically active compounds that have different effects on the nervous system. For example, it was shown that some bacteria are able to produce polyamine - spermidine, which is able to exert a beneficial effect on memory [Eisenberg T. et al. Induction of autophagy by spermidine promotes longevity. Nat Cell Biol. 2009.11 (11): 1305-1314; Gupta V.K. et al. Restoring polyamines protects from age-induced memory impairment in an autophagy dependent manner. Nat Neurosc 2013].

Intestinal bacteria can produce a neurotransmitter, tyramine. Lactobacillus brevis and Enterococcus species are capable of decarboxylating tyrosine to tyramine [Lucas P. et al. The tyrosine decarboxylase operon of Lactobacillus brevis IOEB 9809: characterization and conservation in tyramine-producing bacteria. FEMS Microbiol. Lett. 2003. 229: 65-71]. Amino acids of microbial origin are involved in the formation of nerve cells, improve the conductivity and excitability of neurons [Sharon G. et al. Specialized metabolites from the microbiome in health and disease. Cell Metabolism. 2014.20: 719-730]. Among the producers of microbial amino acid neurotransmitters, the most important are lactobacilli and bifidobacteria, which are valuable probiotics [Oleskin A.V. et al. Role of neuromediators in the functioning of the human microbiota: business talks among microorganisms and the microbiota-host dialogue. Microbiolog. 2016. 85 (1): 3-25].

Nitric oxide formed as a result of bacterial metabolism plays an important role in intercellular signal transmission, being a gas transmitter. Participates in the regulation of motor activity of the intestine and actively affects nerve conduction in the intestine [CulottaD.E. et al. NO news is good news. Science. 1992. 258: 1862-1865]. Intestinal bacteria are a key source of short chain fatty acids (SCFAs) such as butyric, propionic, acetic and other acids. Although these substances are not classical neuroactive compounds, they act on the functioning of neurons in a different way. The most active is butyric acid, which acts as a powerful inhibitor of deacetylases and affects the expression of the neurotrophic factor in the hippocampus [Davie J.R. Inhibition of histone deacetylase activity by butyrate. J. Nutr. 2003.133: 2485-2493].

Various animal models have repeatedly shown the effect of microorganisms on anxiety, depression, and various aspects of cognitive function [Desbonnet L. et al .. The probiotic Bifidobacteria infantis: An assessment of potential antidepressant properties in the rat. J. Psych. Research. 2009. 43: 164-174; Perez-Burgos A. et al. Psychoactive bacteria Lactobacillus rhamnosus (JB-1) elicits rapid frequency facilitation in vagal afferents. Am J. Physiol Gastrointest Liver Physiol. 2013. 304: 211 -220]. The effect of taking a probiotic mixture (Lactobacillus helveticus and Bifidobacterium longum) for 30 days on behavioral manifestations such as anxiety and depression was tested on a group of healthy volunteers in a clinic setting. Mental conditions were observed in test patients [Messaoudi et al. Beneficial psychological effects of a probiotic formulation Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 in healthy human volunteers. Gut Microbes. 2011.2 (4): 256261].

Changes in the composition and number of symbiotic microbiota can be accompanied by impaired development and brain functions responsible for behavioral processes. At the same time, neuropsychiatric and behavioral changes can lead to a stable or reversible modification of the structure of the intestinal microbiota and, as a result, to a disruption in its production of the corresponding hormones and mediators that regulate the activity of certain brain regions [StillingR.M. et al. Microbes do have a significant impact on epigenetic regulation in the host's gut epithelium and immune system, Microbial genes, brain & behavior-epigenetic regulation of the gut-brain axis. Genes Brain Behav. 2014.13: 69-86].

To study and monitor the state of the intestinal microbiota, a complex of modern approaches based on metagenomic, proteomic, metabolic and bioinformatic technologies is currently being used. Intestinal microbiome (the totality of genes of all microorganisms of the intestinal microbiota) is studied using high-tech sequestration methods. Using genetic platforms such as GS FLX (Roche), HiSeq 2000 (Illumina), SOLiD ™ 4 System (Applied Biosystems), in-depth studies of the microbiome are carried out based on the analysis of sequenced 16S rRNA genes and other genes of microorganisms, which allows to obtain a more holistic picture of the interaction of representatives of the intestinal microbiocenosis with the host organism. An important goal of studying intestinal microbiome is to study the dynamics of changes in the composition of the intestinal microbiota in normal and pathological conditions, as well as to search for associations with the development of various conditions and diseases. New technologies for the metagenomic analysis of the human microbiome have created enormous prerequisites and great expectations in the study of the relationship of microbiota-intestines-brain. To date, a huge amount of data has been collected from metagenomic studies of the intestinal microbiota of people under normal conditions and with various pathologies living in different regions of the world, which is available in various information repositories, at the National Center for Biotechnology Information (NCBI) and in other resources. The catalog of genes of the microbiome of the intestine of an adult has been created [Qin J. et al. A human gut microbial gene cataloque established by HMP Consortium. Nuture 2010. 464: 59-67]. The primary intestinal microbiota of children was studied and the gene catalog of the intestinal microbiome of a child was formed [Gritz E.C. et al. The human neonatal gut microbiome; a brief review. Frontiers in Pediatrics. 2015.3 (17):

1-12]. However, separate accessible information on bacterial genes encoding neuroactive compounds has not yet been presented as part of intestinal metagenomes.

The objective of the invention is to develop a method for rapid molecular genetic identification in the studied microbiomes and bacterial genomes of key functional bacterial genes encoding enzymes involved in the synthesis and metabolism of a number of neuroactive compounds: serotonin, dopamine, histamine, melatonin, gamma-aminobutyric acid, tyramine, spermide , butyric acid, gas transmitter NO.

The claimed invention provides a developed set of primers selected for ten key genes of neuroactive compounds detected in the genomes of the dominant bacteria of the human intestinal microbiota for PCR analysis of the total genomic DNA of the microbiome.

The proposed oligonucleotides and gene identification method have advantages over the use of expensive methods for sequencing the total genomic DNA of bacteria and further metagenomic analysis using various bioinformatics programs and can be used in any modern clinical laboratory that has DNA amplification devices. The proposed methodological approach allows monitoring the metabolic potential of the human intestinal microbiota both in normal conditions and in pathologies associated with neuropsychiatric disorders.

The technical result of the invention is the ability to quickly and accurately carry out real-time PCR, real-time PCR analysis of the quantitative content of bacterial genes involved in the synthesis and metabolism of neuroactive compounds in the studied microbiome. Also, the technical result is simplicity in execution, reliability, the need for a small amount of test material.

The indicated technical result is achieved due to the fact that a method for identifying in microbiomes and individual bacterial genomes a composition of bacterial genes involved in the synthesis and metabolism of various neuroactive compounds is proposed, and characterized in that bacterial genomic DNA is amplified using sets of 45 pairs of oligonucleotides developed presented in table. 4, matched to ten key genes of neuroactive compounds shown in table. 1 identified in the genomes of 27 species of bacteria presented in table. 5, and the PCR products are analyzed on an agarose gel, the size of the obtained product, indicated in table. 4, determined using a DNA marker.

1. The selection of the dominant bacteria of the intestinal microbiota of a person.

In recent years, a set of modern approaches based on metagenomic, metabolic, and bioinformatics technologies has been used to study and monitor the state of intestinal microbiota. By analyzing a large number of metagenomes of the intestinal microbiota from various healthy people performed in various laboratories of the world, a significant phylogenetic diversity was revealed in the composition of the intestinal microbiota, however, with the dominance of a number of phyla, genera and even bacterial species [Arumugam M. et al. Enterotypes of the human gut microbiome. Nature. 2011.473: 174-180; Schloissnig S. et al. Genomic variation landscape of the human gut microbiome. Nature. 2013.493: 45-50]. It is assumed that the metabolites and structural components of the dominant bacteria can have a significant effect on both the human immune and nervous systems. We selected 32 bacterial species belonging to the dominant fila: Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, the most represented in the intestinal microbiota of a healthy person. Further, a list of bacterial species was compiled that prevailed in the intestinal microbiota of a healthy adult and was more often found in the primary intestinal microbiota of a healthy child in the first year of life. The bacterial genomes of these species available in the NCBI database were used to search for genes encoding enzymes involved in the synthesis and metabolism of neuroactive compounds.

2. Creating a catalog of bacterial genes of neuroactive compounds.

The search for key bacterial enzymes involved in the synthesis and degradation of neuroactive compounds was carried out using a wide analysis of the available scientific literature and analysis of the metabolic pathways of known neuroactive compounds (neurotransmitters) presented in the KEGG PATHWAY database http://www.genome.jp/kegg/pathway .html, Using the NCBI database, a catalog of 59 bacterial genes whose products are involved in the synthesis and metabolism of neuroactive compounds has been created. The 10 most important genes encoding proteins involved in the synthesis and metabolism were selected from this catalog: serotonin, dopamine, histamine, gamma-aminobutyric acid, neurotransmitter amino acids, spermidine, short chain fatty acids, NO gas transmitter (Table 1 (Example 1)) .

3. The selection of representatives of bacterial genera of the intestinal microbiota containing the largest number of key genes of neuroactive compounds.

To select bacterial genera containing the largest number of genes involved in the synthesis and metabolism of neuroactive compounds, a metagenomic analysis of the intestinal microbiota of a healthy person was carried out. The analysis was performed using the NEUROHUNTER program

- 3 032051 (https://github.com/Alexey-Kovtun/Neurohunter/tree/master) and a catalog of orthologs of selected genes of neuroactive compounds developed by the inventors.

At the first stage, the collected metagen was analyzed. For this, the genomes of 508 bacteria isolated from the microbiota of the intestines of a healthy person were taken from the database of the Human Microbiome Project consortium (http://hmpdacc.org/). Of all the available contigs (22342), a common metagen was compiled. Using the NEUROHUNTER program, this metagen was analyzed with a minimum identity threshold setting of 60%. Such a threshold makes it possible to determine homologous amino acid sequences in the genomes of bacteria of the same genus. As a result of the analysis, 57 of the 59 analyzed genes were identified. Only two genes encoding the phenylalanine-4-hydroxylase and tyrosine hydroxylase enzymes were not found.

At the second stage, 148 metagenomes from the database of the Human Microbiome Project consortium (http://hmpdacc.org/) were analyzed. The analysis was also carried out using the NEUROHUNTER program with a minimum identity threshold setting of 60%. As a result of the analysis, 52 of the 59 analyzed genes were identified. Unable to identify genes encoding enzymes involved in the synthesis of dopamine, serotonin, melatonin, tyramine and NO. The generalized result of metagenomic analysis is given in table. 2.

Using the program used, the genus of bacteria in which the studied genes were found most often was also determined (Figure 1). Eight genera of bacteria were selected from them: Bacteroides, Bifidobacterium, Clostridium, Escherichia, Eubacterium, Enterococcus, Lactobacillus, Streptococcus (Table 3), the representatives of which searched for ten key genes for neuroactive compounds.

4. Development of oligonucleotides for key genes involved in the synthesis and metabolism of neuroactive compounds detected in various types of bacteria of the human intestinal microbiota.

Sequencing of bacterial genomes, both of individual strains and of the microbiome (metagenome), is an expensive procedure. Therefore, to reduce the cost of the analysis of intestinal metagenomes, we propose the use of the PCR analysis method. To search for genes of neuroactive compounds in the studied DNA samples using this method, we developed specific oligonucleotides (primers) matched to the genes of bacteria of various species. Using the NCBI database, in the available genomes of the most common bacterial species of the selected eight genera (table 3), a search was made for the 10 most important genes of neuroactive compounds (table 1). These are the genes encoding dopa decarboxylase, tyrosine decarboxylase, tyrosine hydroxylase, histidine decarboxylase, glutamate decarboxylase, monoamine oxidase, butyryl CoA dehydrogenase, spermidine synthase, nitric oxide synthase nitroacetone serotonone acetone nitroacetone. All genes identified in the genomes of various bacteria are listed in Table 4. To search for orthologs for the genes, the Microbial Nucleotide BLAST (blastn) program was used. It turned out that some genes, orthologs from different strains belonging to the same species, contain nucleotide polymorphisms, therefore, oligonucleotides were selected to their conservative sites. Specific primers were selected using the Primer3Plus program (ht1p: //www.bioinformatics.nl/cgi-bin/primer3plus/primer3manager.cgi) taking into account the size of the formed fragment and the annealing temperature. The product formed by the primers should not exceed 250 bp, which is important for using these primers in real-time PCR analysis of microbiota cDNA. For all primers, the annealing temperature was 58 °. A total of 50 pairs of primers were developed (Table 4), matched to genes from the genomes of 27 bacterial species (Table 6) of the dominant genera. Primers were grouped into five sets (Table 5). Three sets include primer pairs for different genes and two sets contain primer pairs matched to one gene. The bioinformatic validation of the quality of the developed primers was carried out using the catalog of the genes of the human intestinal metagenomes (http://meta.genomics.cn/meta/home). The nucleotide sequences of the primers were combined into a fasta file, which included 90 nucleotide sequences, including reverse complementary ones. Further, using the bowtie2 program, primer sequences were mapped to the nucleotide sequences of genes from the catalog. Primers were mapped to nucleotide sequences of similar genes.

The implementation of the invention

Identification of the selected 10 genes (Table 1) involved in the synthesis and metabolism of various neuroactive compounds, both in the studied intestinal metagenomes and in individual bacterial genomes, is carried out by PCR analysis of the studied DNA using the developed primer pairs (table 4).

To quickly isolate bacterial total genomic DNA from stool samples, use the commercial QIAamp® Fast DNA Stool Mini kit (Qiagen, Germany), following the manufacturer's instructions. Isolation of genomic DNA from the analyzed strain of bacteria is carried out in accordance with the manufacturer's instructions for the commercial Bacterial Genomic DNA Isolation Kit (Qiagen, Promega, Norgen).

Further, PCR analysis of the studied DNA samples is carried out using special kits for PCR analysis on a device - an amplifier. The composition of the mixture for PCR (per 100 μl): 10 μl

- 4 032051

10x PCR buffer (10X High Fidelity PCR Buffer with 15 mM MgCl ₂ ), 10 μl of a mixture of 2mM dNTPs, 5 μl of DMSO, 0.3 μg of genomic DNA and 1 μl (1.25 i) of the enzyme. Oligonucleotide primers in pairs are added at a concentration of 20 pmol per 100 μl of the mixture. Parameters for PCR reaction: 95 ° C for 5 min (cell lysis and denaturation of genomic DNA); then 35 cycles of amplification - 94 ° C - 1 min (denaturation), 58 ° C for 50 s (annealing of oligonucleotides), 72 ° C - 40 s, (completion (extension) of the chain); final elongation of fragments at 72 ° C for 10 min, storage at 4 ° C.

The amplification results are taken into account by analyzing the amplification products of the test samples by electrophoresis in 1% agarose gel. After amplification is completed, 1/5 of the 6X DNA Loading Dye (Fermentas) solution is carefully added under oil to the tubes with the test samples after amplification, mixed, and 10 μl of the obtained sample is added to the wells of the agarose gel. Electrophoresis is carried out in a chamber for horizontal electrophoresis. The results of electrophoresis are taken into account in ultraviolet light with a wavelength of 254 nm on a transilluminator. As a control of the size of the obtained fragment, a DNA marker GeneRuler ™ 50+ bp was used. (Fermentas).

Evaluation of the results

The result of PCR analysis of the studied DNA samples using the developed primers is the presence of the PCR product of the required size (Table 4) for each pair of primers detected by electrophoresis in 1% agarose gel.

Example 1. The formation of a catalog of bacterial genes and their products involved in the synthesis and metabolism of neuroactive compounds.

Using the available scientific literature and databases KEGGPATHWAY http://www.genome.jp/kegg/pathway.html, the metabolic pathways for the synthesis and degradation of various neuroactive substances in bacteria were analyzed and the key enzymes involved in these processes were selected. And with the help of the NCBI database, a catalog was created, which included 59 genes encoding these enzymes. The most important genes encoding proteins involved in the synthesis and metabolism were selected from this list of genes: serotonin, dopamine, histamine, melatonin, gamma-aminobutyric acid, tyramine, spermidine, butyric acid, NO gas transmitter (Table 1).

Example 2. The selection of the dominant genera of bacteria of the intestinal microbiota containing the largest number of key genes of neuroactive compounds.

To select the dominant genera of bacteria, published data from a metagenomic analysis of the intestinal microbiota of healthy people, performed in various laboratories of the world, were used. Basically, they relied on work on the study of the species biodiversity of the intestinal microbiota of a healthy person [Arumugam M, et al. Enterotypes of the human gut microbiome. Nature. 2011; 473: 174-180; Schloissnig S. et al. Genomic variation landscape of the human gut microbiome. Nature. 2013; 493: 45-50] and where a large sample of the analyzed metagenomes was used (more than 260 metagenomes). As a result of the analysis, 32 genera of bacteria belonging to the dominant fila were selected: Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria. Next, bacteria of the intestinal microbiota containing the largest number of key genes of neuroactive compounds were selected. For this, a metagenomic analysis of the intestinal microbiota of healthy people was carried out. The analysis was performed using the NEUROHUNTER program (https://github.com/Alexey-Kovtun/ Neurohunter / tree / master) and the created catalog of orthologs for the selected genes of neuroactive compounds developed by the inventors. At the first stage, the collected metagen was analyzed. For this, the genomes of 508 bacteria isolated from the microbiota of the intestines of a healthy person were taken from the database of the Human Microbiome Project consortium (http://hmpdacc.org/). Of all the available contigs (22342), a common metagen was compiled. Using the developed program, this metagenome was analyzed with a minimum identity threshold setting of 60%. Such a threshold allows the determination of homologous amino acid sequences in the genomes of bacteria of the same genus. As a result of the analysis, 57 of the 59 analyzed genes were identified. Only two genes encoding the phenylalanine-4-hydroxylase and tyrosine hydroxylase enzymes were not found.

At the second stage, 148 metagenomes from the database of the Human Microbiome Project consortium (http://hmpdacc.org/) were analyzed. The analysis was also carried out by the NEUROHUNTER program with a minimum identity threshold setting of 60%. As a result of the analysis, 52 of the 59 analyzed genes were identified. Unable to identify genes encoding enzymes involved in the synthesis of dopamine, serotonin, melatonin, tyramine and NO. The generalized result of metagenomic analysis is given in table. 2.

Using the program used, the genus of bacteria in which the studied genes were found most often was determined (Figure 1). Of these, we selected eight dominant bacteria: Bacteroides, Bifidobacterium, Clostridium, Escherichia, Eubacterium, Enterococcus, Lactobacillus, Streptococcus (table 3).

Example 3. The development of oligonucleotides for key genes involved in the synthesis and metabolism of neuroactive compounds detected in various types of bacteria of the intestinal microbiota.

Using the NCBI database, in the available genomes of the most common bacterial species

- 5 032051 selected eight genera (table 3), a search was made for 10 key genes involved in the synthesis and metabolism of neuroactive compounds (table 1). These are genes encoding dopa decarboxylase, tyrosine decarboxylase, tyrosine hydroxylase, histidine decarboxylase, glutamate decarboxylase, monoamine oxidase, butyryl CoA dehydrogenase, spermidine synthase, nitric oxide synthase nitrate, and nitric oxide synthase. All genes identified in the genomes of bacteria belonging to 27 different species (table 5) are shown in table. 4. To search for orthologs for the genes, the Microbial Nucleotide BLAST program (blastn) was used. It turned out that some orthologs from different strains belonging to the same species contained nucleotide polymorphisms, therefore, oligonucleotides (primers) were selected to their conservative sites. Specific primers were selected using the Primer3Plus program (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3manager .cgi) taking into account the size of the formed fragment and the annealing temperature. The product formed by the primers should not exceed 250 bp, which is important when using primers for PCR analysis of microbiota cDNA in real time. For all primers, the annealing temperature was 58 °. A total of 45 pairs of primers were developed, including direct and reverse oligonucleotides, which are listed in table. 4. A separate list of nucleotide sequences of all PCR products formed by the primers is attached.

Primers were grouped into six sets (Table 6). Each set includes primers for genes from different genomes, but encoding a single enzyme. Three sets include primer pairs for different genes and two sets contain primer pairs matched to one gene.

PCR analysis of the studied DNA samples using the developed primers is carried out using special kits for PCR analysis on a device - an amplifier. Composition of the mixture for PCR (per 100 μl): 10 μl of 10x PCR buffer (10X High Fidelity PCR Buffer with 15 mM MgCl ₂ ), 10 μl of a mixture of 2mM dNTPs, 5 μl of DMSO, 0.3 μg of genomic DNA and 1 μl (1.25 i) of the enzyme. Oligonucleotide primers are added at a concentration of 20 pmol per 100 μl of the mixture. Parameters for PCR reaction: 95 ° C for 5 min (cell lysis and denaturation of genomic DNA); then 35 cycles of amplification - 94 ° С - 1 min (denaturation), 58 ° С for 0.5 min (annealing of oligonucleotides), 72 ° С - 40 s. (completion (elongation) of the chain); final elongation of fragments at 72 ° C for 10 min, storage at 4 ° C. The amplification results are taken into account by analyzing the amplification products of the test samples by electrophoresis in 1% agarose gel. After amplification is completed, 1/5 of the 6X DNA Loading Dye (Fermentas) solution is carefully added under oil to the tubes with the test samples after amplification, mixed, and 10 μl of the obtained sample is added to the wells of the agarose gel. Electrophoresis is carried out in a chamber for horizontal electrophoresis. The results of electrophoresis are taken into account in ultraviolet light with a wavelength of 254 nm on a transilluminator. As a control of the size of the obtained fragment, a DNA marker GeneRuler ™ 50+ bp was used. (Fermentas).

Example 4. Bioinformatics validation of designed primers.

The bioinformatic validation of the quality of the developed primers was carried out using the human intestinal metagenome gene catalog http://meta.genomics.cn/meta/home). The nucleotide sequences of the primers were combined into a fasta file, which included 90 sequences, including reverse complementary ones. Further, using the bowtie2 program, primer sequences were mapped to the nucleotide sequences of genes from the catalog. Primers were mapped to nucleotide sequences of similar genes.

a) Tables.

Table 1. List of key enzymes encoded by bacterial genes involved in the synthesis and metabolism of neuroactive compounds

No. Encoded protein Function Neuroactive compound Biogenic amines and their precursors 1 T irosin hydroxylase Dopamine synthesis Dopamine 2 Dofa decarboxylase Synthesis of Dopamine and Serotonin Dopamine, serotonin 3 Monoamine oxidase Metabolism of dopamine, norepinephrine and serotonin Dopamine, norepinephrine, serotonin 4 G lutamate decarboxylase GABA synthesis GABA 5 G istidine decarboxylase Histamine synthesis Histamine 6 Serotonin Nacetyltransferase Serotonin metabolism for subsequent melatonin synthesis Melatonin 7 Spermidine synthase Spermidine synthesis Spermidine Fatty acid 8 ¹ 1 Butyryl CoA dehydrogenase | Synthesis of butyric acid | Butyric acid G nitrogen transmitter 9 | Nitric oxide synthase | NO synthesis | NO Neurotransmitter Amino Acids 10 | Tyrosine decarboxylase | Tyramine synthesis | Tyramine

- 6 032051

Table 2. The distribution of genes for 59 enzymes in terms of their frequency in the collected metagenome and 148 metagenomes from the database

> 90% ¹ 148 metagenomes & assembled metagenome 10-90% 148 Metagenomes & Metagenome Collected <10% 148 metagenomes & collected metagenome Are absent G lutamate decarboxylase, γ-aminobutyrate antiporter, Serotonin Nacetyl transferase, Phospholipase A2, Phosphotransacetylase, Butyryl-CoAdehydrogenase, H lutathione peroxide aza, Catalase, Superoxide dismutase, Hololglycine hydrolase, Spermidine synthase, Aminodeoscorizim synthase, subunit 1, Aminodeoscopic Monoamine oxidase, histidine decarboxylase, Atsetilserotonin Ometiltransferaza, 2,3dioksigenaza-Tryptophan, kynurenine 3-monooxygenase Hinolinatfosforiboziltransferaza, linoleic acid isomerase, synthetic route BI ^2, a synthetic route B2, B6 synthesis pathway, synthesis pathway of B7 ³ Dopadekarboksilaza ^4, nitric oxide synthase ⁴ kynureninase ^4, Histamine Nmetiltransferaza ^4/3 subunit of the nicotinic acetylcholine receptor Propionyl-CoA synthetase, Tirozindekarboksilaza ^4, glutathione reductase, glutathione-8-transferase, transcriptional activator tenA, Operonovy biotin repressor ⁴ Phenylalanine-4-hydroxylase, tyrosine hydroxylase synthase, subunit 2

¹ - enzyme found in> 90% of 148 metagenomes,

- the complete synthesis pathway was found, with the exception of the transcriptional activator tenA, ³ - the full synthesis pathway was found, with the exception of the operon biotin repressor, ⁴ - the enzyme was found only in the assembled metagenome.

Table 3. List of bacterial genera for which the largest number of genes of neuroactive compounds were detected in the studied metagenomes

Phil Kind Bacteroidetes Bacteroides Firmicutes Clostridium Enterococcus Eubacterium Lactobacillus Streptococcus Ruminococcus Actinobacteria Bifidobacterium Proteobacteria Esherichia

- 7 032051

Table 4. Oligonucleotides for searching in the genomes and metagenomes of bacterial genes involved in the synthesis and metabolism of neuroactive compounds

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CGCCAGATACAAAGAC.ACCA

AGCGC GTGTAC TCCATCAGT

T AC TGC AASASAASSTSAS-S

GC TGCTIGGAGC GTAAGAAC ‘

CGGTGACGGAGGTGTATCA

CTGAAGAGGCTGA TAG ACC 4

TCATCC AGCAACACCATACC

C GGACTTCAAAC CCAATACC

CGTGAAAGGAAGCATTTAC GG

GCTGGCACGCAATACAGTTA

CTCCGT AATCCTGCCGTTG

GGC GATATGCAGTCACACCT

CGAGC GGGTGC ATATTTAC T '

CT GGAAGGGAATGGTGATGT '

CGlGGAGATTGACCAGAAGA

C GATTGAAGCС CC GACTAACT

TGC GTGCTCIATTGGCGTAT.

caaggaaccatctgcgacaa

CGCTAAGAGAACGAATGGAG i

GCTTC TTGTAAGTGAGCATGC

GCTG TGC AAGTG ^ AGGATGTL '

CC GTTGTTC GTGGGTATTC T

TGAACTC AC TGC C GATT C TG

C-CTTGGTCGAGATCGTGAAT

AATCC. C.TGAC GAAC AAC GAG '

TAGCG-A4GAT GGTTC CGTTC

GAGC GCAC AAAAGAAGTAGC

GATGC TGGTGTATGGGAAAC

CATCGGAACGTC CAC CATC

CAAGAAC ACCACC AGC ATCC

GCAC C TGATGACGAACACTT. ·

GATAGCCAGCC GAAATAAC G

C AAGGAACC ATCTGC GAGAA

ATGCCTGCTCTATTGGC GT A

GTAGCT TI GAT GGGC GAT TG

GCTCCC GTTACAAATGTTC C

TTGC.GCC TGCATGTAGATAG

GTCAAGGACGTGAACGACAC

ACTGGAACGTGAGC AAGGAG

GCTT PBX ICGTG1IGAIGTGG (opsuoro ’reverse)

AATC CTGGCGGTATC TTCGT '

T-AAGGCC-TCGTIATC 7GTC G

GAC GAGTGAC-TTCGG-CAAG

AACAGCC AGTAGCCTGAC G

I € GGACA1 acGGAGGGAC G

ATCGAAGTCCA

AATGGGCTGTTGCC AT AATC T

CACAGTGGTTGTGGGCTGTA

ACCaGCGACTACAGCAGGI.

CCTTCATTGCCTACGITTCC

GGGG AAGTCTATIGTCAGC /

TC ACCGACA.AAACGGTGTAG

GC AGATTGTC GAGATAC. "

C GC AAATGGAACC TGTAC G

C TCIGAACGTCTGC GTC TG

CTGTTCTGCTGGGCAATACC

AGGC.GTTGTAC ATCGTC C AG

CAC GTTCGACCTGATGACC.

GCAGCAGAITGAGCAGTACG

- 8 032051

Table 5. Dominant species of bacteria of the intestinal microbiota containing genes involved in the synthesis and metabolism of neuroactive compounds

No. Kinds Kind Phil 1 2 3 4 B. fragilis B. vulgatus B.ovatus B. pectinophilus Bacteroides Bacteroidetes 5 6 7 C. sporogenes C. leptum C. asparagiforme Clostridium Firmicutes 8 9 E. faecium E. faecalis Enterococcus 10 eleven 12 E. rectale E. eligens E. hallii Eubacterium thirteen 14 fifteen L. plantarum L. brevis L. reuteri Lactobacillus 16 S. thermophilus Streptococcus 17 S. epidermidis Staphylococcus 18 R. torques Ruminococcus 19 20 21 22 23 24 25 26 B. catenulatum B. adolescentis B. dentium B. angulatum B. longum B. breve B. pseudocatenulatum B. animalis subsp. lactis Bifidobacterium Actinobacteria 27 E. coll Esherichia Proteobacteria

Table 6. Primer sets

Dial number Enzyme The number of pairs of primers by genes the general 1 Dofa decarboxylase Tyrosine hydroxylase G istidine decarboxylase 2 3 3 8 2 Serotonin Nacetyltransferase / Arylalkylamine N Acetyltransferase Monoamine oxidase 6 3 9 3 Tyrosine decarboxylase 7 7 4 Glutamate decarboxylase 10 10 5 Spermidine synthase Butyryl CoA Dehydrogenase Nitric oxide synthase 6 3 2 eleven

- 9 032051

The list of nucleotide sequences of the gene regions corresponding to the PCR product, and the localization of oligonucleotides on them:

Gene encoding dopa decarboxylase:

Staphylococcus epidermidis

FP->

CAGGTCCAGC ATCACGTCTA TCATGGCTTG GAGACATTTT AACAACAGCA

AATAATATCC ATGCCTCTAA TTTTGCCAAT GCGACGCTGC CAATTAATAT

101 TGAGCGTAAT TTAATTAACT ACTTGGTAGG AAAAATCGGT TATGAGATCA

151 AACCGGCCGG TGGTGTCTTT GTATCTGGCG

RP

Escherichia coli

FP->

CTGCACTCCC TTTCAGCTTC CAGTGCTTCA CTGTAGGACG CATCATCAAT

TTTCGTGCCT TTGCTTTCGG CATAGCGTCG TAATTTATTT GCGTCAGCTT

101 CATTAAGTTC TAAAAGATGA TCGCGCAAAA GACCGATAAG CCTGCTTTO

151 TATATCTCAT AKTACAGCCC ACAACCACTG TG

RP tyrosine decarboxylase:

Lactobacillus plantarum

FP- *

TGCCTGCTCT ATTGGCGTAT AACTATGCCA TGTTATGGAA TGGTAATAAC

GTGGCTTATG AATCTTCACC AGCAACGTCG CAAATGGAAG AAGAAGTTGG

101 CCAAGAATTT GCCCGATTAA TGGGTTATGA CTATGGCTGG GGCCACA7TG

151 TCGCAGATGG TTCCTTG

RP

Lactobacillus brevis

FP ^

CAAGGAACCA TCTGCGACAk TGTGGCCCCA CCCATAGTCA TAACCCATTA

ACCGAGCAAA TTCTTGGCCA ACTTCTTCTT CCATTTGCGA CGTTGCTGGC

101 GAAGATTCAT AAGCCACGTT ATTACCATTC CATAACATGG CATAGTTATA

151 CGCCAATAGA GCAGGCAT

- RP

Enterococcus faecium

FP

CGTGAAAGGA AGCATTTACG GTAATGAATT CTTAACTTCA CATACTGACT

TTGCTATCCC AGACTATGGC AACAGCCCAT TGCAATTCGT GAACCAACTA

101 GGATTCTCAG ATGAAGAATG GAACCGTGCC GGAAAAGTAA CTGTATTGCG

151 TGCCAGC

RP

Enterococcus faecalis

FP

CGACAAAGAA ATCGAAGTCC LSACATTAAC TCATCCAGAC TTCAACATGG

TTGACTATGT GTTTAAAGAA AAAGGCAACG ATGATTTAGT AGCAATGAAC

101 AAATTAAACC ATGATGTATA TGACTATGCA TCATACGTTA AAGGAAATAT

151 TTACAACAAC GAATTCATTA CTTCACATAC TGACTTTGCT ATTCCJGL47T

201 ATGGCAACAG CCCATT

RP

Clostridium sporogenes

FP

GTAGCTTTGA TGGGCGATTG GTTAGTAAGT ACATTTGATC AAAATGCCAC

AGGCAGAGAT AATTCCAGTG CAGCAAAGCT TGAGGAAGAA ACCATAAATT

101 TATTAAAGCA GCTTTTGGGG TTGCCGGAAA CATATTCGGG AACATTTGTA

151 ACGGGAGC

RP

Streptococcus thermophilus

FP- *

GCGCGACGTG ATTTCATACT TCGCGACATA CGTCTTTGAA AAAGGTGCAG

ATATTCCTGC ATTATTGGGT GCATATATTC TTGAAGGTTC AAAAGCTGGC

101 GCAACTGCTG CATCAGTATG GGCTGCTCAT AAGACATTAC CACTGAACGT

151 TACTGGCTAT GGAAAGTTAG TCGGGGCTTC AATCG

RP

Bifidobacterium catenulatum

FP

TGCGTAGGCG TTACAACTTC CCATTCCGGC TGGTTCCGAA GCATGGACTC

CGTCTGTTGG GCTAGGTCGA TGCCGTGGGT CACCATATCA CTGATCTGAT

- 10 032051

101 CCGTCCCCAA CGTCTGCAGG GTGAGCCATA ATTTCAGGCT GCGCGCGGGG

151 CGGGTCAGTT CAATGCCCCA TTCCCAATAG ETGATTTCTC CGTTCTCTGC

201 g

RP tyrosine hydroxylase:

Bacteroides fragilis

FP- "

CCAACATAGT CGTTCCATTG CCATGACATT GCTATGGTAT ACACGCACAT

AATCCTCATG AACAGGGTAG AGAGCCTGAG CTCTTTGTAC AGCCTTGATA

101 AATAATTTCC CCATATTCTT GAAAAAATTG ACAATGTGTT GATTAAGAAG

151 CCAAGGAACA TGGCAAAATA AATCGTGAAT CATATCAGGA AAAAGAGAAA

201 AATCGTCATA TTGAGATGGA CGAAGGCATG

RP

E. faecium

FP- "

CGCTAAGAGA ACGAATCCAC TATTTTGATG ATATACACAC TAAAATCTAT

GAAGCACGTA CAAATCACGA AGACGAAAGC AAATTAGGAT TAGCTCAACA

101 ACGAATATTT GAATGGCTGA ATACGAATAC TGAGCAAGAA CCGTTAAGAG

151 CAATTAGTAA TCTTTTTGAA TTTGGAACGA AGCATTGGTT TAACGCTGTA

201 GAAAAGGATG CTGACTTACA AGAAGC "- RP

E. coli

FP- "

ACTGGAACGT GAGCAAGGAG ACTTAAAAAT CTATCTGTCT AGCATTGAGC

GTTCTATAGA GAGCCTAGAG GATAAAGTTA ATCGTATAAA GAATGAGG /

101 ATTCATGATA TTAAGGACGA TATATCCTTC TTAAAATCTT GGTTGAAAAX

151 TGTTGGTAAA ATTGCCACAT CAACACGAGA TAAGC “- RP histidine decarboxylase:

B. fragilis

FP- "

TGTAGCGAAG GATCGTTGTG AGAAGAACGA TGCGTATCTC GGGCAAGTCA

ACCTGATGAA AGCCTCGTCT TTTTGCGGAC AGAATGGAGC CATCTGGGGA

101 TTTGACCTGG CGATGCACGA TGACATTGCC AAAAGGAAAG AGATGCCTAT

151 CTACATGCAG GCGCAA <- RP

Lactobacillus reuteri

FP- *

ATTGAACCGC CTGGGATAGA ATCAACCGGC ATTGAAACGT AAGGTGCTAC

GGTAATTGCA TTACCAATTT GACCTGGTTC CATCATTGCA TGAGCAAAGC

101 CGATCCATGA ACCCTTGAAT GAGGTATTAG CATCTTGACC ACACTCAGCA

151 ATTGACTTAG CAATTGCCTT ACGACGACCC TTAAGGTATT CAATTAATTC

201 ATCTTCATTA GGTGTTTCCC ATACACCAGC ATC <- RP monoamine oxidase:

Bifidobacterium longum

FP->

CAAGAACACC ACCAGCATCC CCACCCTCGG CAATCCGCCG ATCGTGCCGG

ACTGCTGGAT CTACGTGCAG GACCCGGGCT ACAAGGTCGG ACGTCTGCAG

101 ATCTTCAACA ACTGGAGCCC GTACCTGGTC AAGGACGTGG ACGACACCGT

151 GTGGATCGGT CTCGAGTACT TCTGCGAGGA GGGCGACTCG TTCTGGAAC4

201 TGAGCGAGGA GGATGTCAC <- RP

Bifidobacterium breve

FP

TCGAAGTATG CAGGGTAGGC CTTCTTCACG CGTTCACGGT GGGAGTCGAG

GACCTCATCC GGGCCATTGA TCACGCGCAT GCGGGTGAGC TCGGAAATGG

101 CGAACTTGGT GGCGTCTTCC TCACTCATGT TCCAGAAGGA ATCACCCTCT

151 TCGCAGAAGT ACTCCAGACC AACCCACACG GTGTCGTTCA CGTCCTTGAC

- RP

E. coli

FP->

TCATCCAGCA ACACCATACC GTGGGCGTCT TTAATGGGTT GCCAGGAGAG

CAGTTTGTTG TTTTGCAGAT CCACCACCGC TTCGATGATA TGTTTGCCGT

101 CGAGCATAAT GACGTCGGCT TTGCGCGGCT GGTCAACCGG TTTGTTTTCC

151 AGCGCAAACG CCCAGACAGC TTCTTTATCT GGCGGTAGCA GGGAGATCTC

201 AGTAAAACGG GTATTGGGTT TGAAGTCCG

- 11 032051

RP glutamate decarboxylase:

Bacteroides vulgates

FP ^

GCGGTTTTTCA GGTTGTTTTGG GAAAAGTTCG CCCAACTTTG GCAAATTGAA

ATGCGCCAAG TACCTCTCAC CTTGGACAAA ACCACTCTGG ACCCCGAAGA

101 AGCACTGAAG ATGTGTGACG AGAACACCAT CTGCATCGTC CCCATCCAAG

151 GTGTGACATG GACAGGTTTG AACGATGACG TGGAAGCGTT GGACAAAGCA

201 CTGGATGCCT ACA

RP

Bacteroides ovatus

FP ^

AATCCCTGAC GAACAACGAC ACGCATCACT ACATAATCTT CCAGTTTAGA

AGGCAATGTA TAAGCAGGAA CCATCCAACC ATGTTGAGAT AGTTTGTCTT

101 GCAAATCATA CAAAGTCCAT TTGGCGCTCT TTGCATATTC CGGTTTCAGA

151 TACCAGATGA ATAGCGGGTT CACAACATCT TCTGAGTAGT TAACGA4CGG

201 AACCATCTTG GCTA <- RP

C. sporogenes

FP ^

GAGCGCACAA AAGAAGTAGC TATGTATATT TCAAAAGAAC TTGAAAACAC

GGGATTATTC AGTATATATA ATGATGGCAG TAATCTTCCT ATAGTTTGCT

101 ATAAACTAAA AGAACAATCT AAAGTGAAGT GGAATCTTTA TGATTTAGCA

151 GATAGACTAG CTATGAAAGG TTGGCAAATA CCTGCTTACC CCCT

RP

E. faecium

FP ^

GCTCTGCAAG TGAGGATGTT GACTTGCCTA AATATAAGAA TCCATTGAAC

CACGAATTGC TTATCAATTA GTACAAGACG AGATGTTGGA TGAAGGAAAT

101 GCGCGATTAA ACTTAGCTAC TTTTTGTCAA ACGTATATGG AACCTGAAGC

151 AGTGAAATTG ATGACCCAAA CGTTAGAAAA AAATGCAATT GATAAATCAG 201 AATACCCACG AACAACGG

RP

L. brevis

FP- *

TGAACTCACT GCCGATTCTG ATCGCGAATG GACCCTCTAC GATTTATCCG

ATCGGTTATT AATGAAGGGC TGGCAGGTTC CCACCTATCC CTTACCAAAA

101 AACATGACGG ACCGCGTTAT TCAACGGATC GTGGTTCGGG CTGACTTTGG

151 TATGAGTATG GCCCACGACT TTATTGATGA TCTAACCCAA GCCATTCACG

201 ATCTCGACCA AGC

RP

L. plantarum

FP->

GCACCTGATG ACGAACACTT TACGGGTACC TCTACGATTG GCTCCTCTGA

AGCTTGTATG TTAGGCGGTT TAGCAATGAA ATTCGCCTGG CGTAAACGCG

101 CTCAAGCGGC AGGTTTAGAT CTGAATGCCC ATCGACCTAA CCXCGTTATT

151 TCGGCTGGCT PBX

RP

Bifidobacterium adolescentis

FP- "

ACTTCTCCCA GCACACCTGC ACGGCGCTGC TCATCACCAG ATTCGGATGA

TCGGTCGGCA GGTCGGCCTT TTCGCGGGCT TCCTTCCAAC GGCGCTTCAA

101 CGCGAGTCCG CCCAGCATGC AAGCTTCCGA CGAGCCGL4TG GTGGAGGTTC

151 CGATG "- RP

Bifidobacterium dentium

FP->

AGCGCGTGTA CTCCATCAGT ACCTCCGGAC ACAAGTACGG TCTGGTCTAC

CCGGGCCTGG GCTGGGTGGT ATGGCGTGAG ACCGCCGACC TGCCGGAAAG

101 CCTGATCTTC AAGGTGAGCT ATCTGGGCGG CGAAATGCCG ACCTTTGCGC

151 TGAACTTCTC CCGCCCCGGC GCTCAGGTGC TGTTGCAGTA <- RP

Bifidobacterium angulatum

FP

TGCAGATTGT CGAGATACGC TACGCAGGCC TTGAGATCCT TAAGGAACGA

TTCGGCCAGA TCATGGCTGA ACCCGTTGCG CACCACGATG CGTTGCACGG

101 TCACATCGGT CAGATCGACC GGCATCGGAT ATGCCGGCAC CAGCCATCCC

151 TTCATGCGCA GGCGATCCTG CAGATCGZ4C AGGTTCCATT TGCG * - RP

- 12 032051

E. coli

FP- *

CTCTGAACGT CTGCGTCTGC GCGGCTGGCA GGTTCCGGCC TTCACTCTCG

GCGGTGAAGC CACCGACATC GTGGTGATGC GCATTATGTG TCGTCGCGGC

101 TTCGAAATGG ACTTTGCTGA ACTGTTGCTG GAAGACTACA AAGCCTCCCT

151 GAAATATCTC AGCGATCACC CGAAACTGCA GGGTATTGCC CAGCAGAACA 201 G

RP butyryl CoA dehydrogenase:

B. longum

FP->

AGGCGTTCTA CATCGTCCAG CAGGGCATAG CGGACGCGGC CACCGTGGAC

GACGTGATGA AGTACAGCCT CGGCCGCCGC TGGAACCTGG TCGGCCCGAT

101 CGCCAGCATC GATCTCGGCG GACTCGACGT GTTCTACAAC ATCAGCACCT

151 ACCTGTTCGA CGACA <- RP

B. breve

FP->

CACGTTCGAC CTGATGACCA G4ATCGGCAA GAAACCGGCG AAACTCAACA

AGGAATCGCT CGGATTCATC GGCAACCGAC TGCAGATGGC GGTGCTGCGC

101 GAGGAATTCC ATATCATTCA GGAAGGCATC GCGGACGCGG CCACCGTGGA

151 TGACGTG / IFG AAGTACAGCC TCGGC

RP

Bifidobacterium pseudocatenulatum

FP- "

ACTTCGACTA GCGGCATCAG ATGCGCAGGA TTCCAGAAAT GAGCCACCAC

AAAGCGTTCC GGGTGCTTCA GCACAGATTG GATTGCGGTT GGGCCCAATC

101 CGGAAGTGTT TGTCGCAAAG ATCGCATCGA CCGGCGCGTA CTGCTCAATC

151 TGCTGC <- RP spermidine synthase:

Bacteroides pectinophilus

FP->

GACGAGTGAG TTCGGCAAGG TGCTTGTCCT TGACGGCGAG CTTATGATTA

CACAGAAGGA TGAATTTATC TATCATGAGA TGATTACGCA TGTGCCGATG

101 GCCGTGCATC CGCATGTCAG GGATGTGCTT GTGATAGGTG CGGGTGATGG

151 CGGAACTATC AGGG <- RP

Eubacterium rectale

FP

AACAGCCAGT AGCCTGACGG ACAGGTTGGA ATATGTGCCT GATATACCCT

GCTTATAGGA AATGATTTAT ACACCTTTCT GTGCATTTTC CGGCACTCAA

101 GCTCATCCTC ATCATAAAAA GGACTGCCGT GCTGGTAAAC CATGATTCCA

151 TCATCCTTTA GTGCCTTATA ACAGCTTCCG TAAAACTCTT TTGTGAAA4G

201 TCCCTCCGTA TGTCCGA <- RP

Eubacterium eligens

FP->

CCTGCTTGGA GCGTAAGAAC CTTAATGCAT CTGAATTATA AATGTGCACT

CTCTCATCTT CTAATCCTGA TGCCGTCTCT GGAAAATACT CCCTGCACAC

101 ATCAACAAAC ATGTTGTCCT GTTCAACTAT GTCAATATTT TCGATATTGT

151 CATACTGCAA CAGTTCCTTT GATACACCTC CGTCACCG

RP

Clostridium leptum

FP1 CGAGCGGGTG CATATTTACT ATGAGGATGG ATTGAAGTTT ATCCGCTCCA

AGGAAAACGA GTACGACCTG ATTATCGTGG ATTCCACCGA TCCCTTCGGC

101 CCCGGAGAGG GCCTGTTCAC CAAGGAGTTT TACGGCAACT GCTATAAGGC

151 GCTGAAAGAG GACGGCATCA T

RP

B. longum

FP1 CTGGAAGGGA ATGGTGATGT CCTGATACGC ATCGACCATG ATCACGTCGT

ACTTGTCATG CGAGGCGGCG AGCCAGGCTC GGCCGTCATA CGTGGTCACG

101 GGGATGTCGG CGGGCTCATC GAAGTATTCG CCGGCCAGAT CGGTGATCTT

151 CTGGTCAATC TCCACG <- RP

- 13 032051

E. coli

FP- *

AATCCTGGCG GTATCTTCGT CGCACAAAAC GGCGTCTGCT TTTTACAGCA

GGAAGAAGCC ATCGACAGCC ATCGCAAACT CAGCCATTAC TTCAGCGACG

101 TTGGCTTTTA TCAGGCGGCG ATCCCGACCT ATTACGGCGG TATCATGACT

151 TTTGCATGGG CGACAGATAA CGACGCCTTA

RP nitric oxide synthase:

E. faecalis

FP->

ACCAGCGACT ACACCAGGTY CTGTGACAGT AATCCAACCT AGACCAGCAA

ACACAATATC CGTTTTTTCT TTCACGGAAA ATTCAAAGCG CACTAATTCA

101 GGAAACTCAG CGACTTCATC GGCACGTGGG GGCTGCAATA AGCCACCAAC

151 ATGTTTTTCA TAAAAAGCAT CGGCGGTTGC TGTTTTTGTT CGGTGTAAAT

201 TTAAGTCATT GGAAACGTAG GCAATGAAGG <- RP

E. faecium

FP- "

GGGGAAGTCT ATTGTCAGCG TTGTTTCAGA TTACGCCATT ATAATGAGAT

CCAAGATGTC TCATTGACGG ATGACGACTT TTTGCGCTTA CTGAATGAAT

101 TAGGGCAAAA AGATGCATTG ATCGTCAATG TGGTGGATAT TTTTGACTTC

151 AATGGTTCAC TAATCCCAGG ACTACACCGT TTTGTCGGTG A <- RP serotonin N-acetyltransferase / arylalkylamine N-acetyltransferase:

Ruminococcus torques

FP->

CCGAAGAGAT GATGAAGCAG CGGGCTCTTA AGATTCGGGA ATTGTTTTTT

GTGGCGGTTG ACAGAAAAAG CGGTGAAGTG GCAGGTTTTT TGAATGGTCT

101 GGCAACGGAC GGGGATTGTT TGAAAGATGA ATTTTTCAGG AATGCAGACT

151 TGCATGATCC GACAGGTGAT AACGTGATGA TACTCGGTCT TGCAGTTTTG

201 CCGAAGTACC GGGGAATCGG AGTGGCATCA G <- RP

Eubacterium hallii

FP1 CCATTTCTGG CTGATGTGTG AGGAAGAGAA AGTAATTGCG TTTGTAGATG

GAATGGTAAC GGATGAAAAA AATCTGACAG ATGAAATGTA TGAGAAGGCT

101 TGCCTTCATG ATGAAAAGGG AGCGTGGCAG ATGATTTTCG GAGTAAATAC

151 ATTGCCGAAA TATCGAAGAC AGGGCTATGC GGAAAAGCTG CTTCGCTACG

201 CAATCAATG

RP

Clostridium asparagiforme

FP1 CCATTTCTGG CTGATGTGTG AGGAAGAGAA AGTAATTGCG TTTGTAGATG

GAATGGTAAC GGATGAAAAA AATCTGACAG ATGAAATGTA TGAGAAGGCT

101 TGCCTTCATG ATGAAAAGGG AGCGTGGCAG ATGATTTTCG GAGTAAATAC

151 ATTGCCGAAA TATCGAAGAC AGGGCTATGC GGAAAAGCTG CTTCGCTACG

201 CAATCAATG + - RP

Bifidobacterium, animalis subsp. lactis

FP1 AGATGATACT CGGCGTCAAC ACCGCACCCA TGTACCAGCA TCGTGGCTGT

GCCTCGTTCC TTATGCGCCA CGTGATTCTC GACAGCGCGC TTTCGCACAG

101 GCACGGCATC GTGCTCGCCT GCAAGGAGAA AATGTTGCCA TTCTACAGCT

151 CGTTCGGGTT CATTGACGAG GGCCGCAGCC AATCCGTGCA CGGCAATGCG

201 GTGTGGCACC AGATGCGTCT GCCATTCACC ACCT

- RP

B. pseudocatenulatum

FP1 GACGACCAAC GAAAAGGACT TGGCCGACGT CATGTATGAG GATGTTTCCA

TGCATGACGA GCAGGGTGAT TGGCAGATGA TTTTCGGCGT GGACGTGGCT

101 CCCGTCTACC AGCATCGCGG TTGCGCCAGC TATCTGCTGC GTCGTGTGAT

151 CCTTGATTCG ACGATTGCCG GACGCAAAGG CATTGTGCTG ACTTGTAAGG

201 AACGTCTTGT CGGATTTTAC GC

RP

S. thermophilus

FP1 GCCTCCTTGA CTAGCATTAG GAGTTACCTT GTAGAATAAG TCATCTGTAA

GATATCGTTC CGAAATCACA GGTCCTTCGA TATAATCAGC TACAGTGCCA

101 TCAATTTCAG CAATTAGGAA AGTATCGCAA ATTGTCTCTA AACGCTCTGC

151 AAGAGCCTCT GGTGTAGCAG CCTCTTCAG + - RP

FP - Forward Primer - direct primer; RP - Reverse Primer - reverse primer.

- 14 032051

Claims (1)

CLAIM The method of identification in microbiomes and individual bacterial genomes of the totality of bacterial genes involved in the synthesis and metabolism of neuroactive compounds, characterized in that bacterial genomic DNA is amplified using the sets of developed 45 pairs of oligonucleotides shown in Table 1 for identification. 4, matched to ten key genes of neuroactive compounds shown in table. 1 identified in the genomes of 27 species of bacteria presented in table. 5, and the PCR products are analyzed on an agarose gel, the size of the obtained product, indicated in table. 4, determined using a DNA marker. Genes distribution of neuroactive compounds among bacteria of the dominant genera according to the metagenomic analysis of the intestinal microbiome of healthy people