RU2405833C2 - Method for microbiologial synthesis of purine nucleoside of 5'-aminoimidazole-4-carboxamide riboside (aicar) and bacillus subtilis strain - producer of aicar - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method for synthesis of purine nucleoside 5'-aminoimidazole-4-carboxamide riboside (AICAR) and a Bacillus subtilis strain VKPM V-10167 - producer of AICAR. AICAR is obtained by culturing modified Bacillus subtilis bacteria in a suitable culturing medium. The modified bacteria used are Bacillus subtilis bacteria, obtained through successive introduction of mutations purR, purH::Em^R, PurΔ_L and P_rpsF-prs.

EFFECT: disclosed invention widens the range of methods for microbiological synthesis of AICAR and enables to engineering of the strain which produces AICAR for realising this method.

2 cl, 1 dwg, 2 tbl, 11 ex

Description

The claimed group of inventions relates to biotechnology and is a method of microbiological synthesis of 5'-aminoimidazole-4-carboxamidriboside (AICAR), as well as a bacterial strain Bacillus subtilis (B. subtilis) - AICAR producer for implementing this method.

AICAR is a natural analogue of adenosine purine nucleoside and replaces it in a number of biochemical processes. AIKAR phosphorylation occurs in animal and human cells with the formation of its phosphorylated form (AIKAR-F), which is an analogue of adenosine 5'-monophosphate (AMP) and capable of activating the enzyme AMP-activated protein kinase (AMPK) - a regulator of carbohydrate and lipid metabolism in eukaryotes (Eur. J. Biochem., 229, 558-565 (1995)). Due to the ability to activate the enzyme AMPK, AICAR preparations have a wide therapeutic potential, normalizing carbohydrate and lipid metabolism, limiting cell proliferation. Long-term use of AICAR effectively affects the components of the metabolic syndrome (Trends Pharmacol. Sci., 26, 69-76 (2005)). AICAR alleviates the effects of hypoxia, reduces the risk of myocardial infarction after coronary artery bypass surgery (US 20040072138), reduces lipid synthesis and provokes their oxidation, which is used to treat obesity and decrease insulin resistance (US 20030212034, Diabetes, 51, 2199-2206 (2002) ) AICAR has been shown to be effective in preventing type 2 diabetes (Diabetes, 54, 928-934 (2005)). AICAR specifically induces apoptosis and is effective as an anti-cancer drug in the treatment of chronic and acute leukemia (US 20050233987, Molecular Cancer 6:46, 1-12, (2007), J. Biol. Chem., 280, 39582-39593 (2005)).

AICAR purine nucleoside is a derivative of purine metabolism, it is found in the cells of any organisms. The phosphorylated form of AICAR (nucleotide AICAR-F) is a direct precursor of the purine nucleotide inosine-5'-monophosphate (IMP) and, therefore, adenyl and guanyl nucleotides (Figure 1).

The conversion of AICAR to IMP consists in the intercalation of the C1 residue into the purine component of AICAR-F and the closure of the purine ring. The process of conversion of AICAR-F into IMP is universal and in the cells of microorganisms is under the control of the purH gene. The corresponding PurH enzyme is a two-domain protein sequentially catalyzing AICAR-F formylation and purine cyclization.

Information on AICAR producer strains and methods for its microbiological synthesis is available (US 3238110, CN 1710065). The method of microbiological synthesis of AICAR according to patent US 3238110 will be considered as the closest analogue of the proposed method, and B. subtilis ATCC strain No. 15116 (US 3238110), capable of producing AICAR, will be considered as the closest analogue of the claimed strain. Strain B. subtilis ATCC No. 15116 obtained by mutagenesis and is a conditional auxotroph for purines. The microbiological synthesis method developed on the basis of this strain allows to obtain up to 3.18 g / l AICAR on a medium with glucose.

The method of genetic construction of the producer strain AICAR and the corresponding method of microbiological synthesis of AICAR based on it are not currently known.

The task of the claimed group of inventions is to expand the arsenal of methods for microbiological synthesis of AICAR and the construction of a producer strain of AICAR for the implementation of this method. The inventive method allows to increase the level of synthesis of AICAR.

The problem is solved by

- development of a method of microbiological synthesis of AICAR by culturing in a suitable nutrient medium modified bacteria of the genus Bacillus, combining mutations that lead to the weakening or termination of expression of the purH gene that controls the conversion of AICAR-F into IMP, with mutations that provide an increased level of purine nucleotide biosynthesis, and

- designing a bacterial strain Bacillus subtilis AM25-41 - producer AICAR.

The inventive strain is deposited in the All-Russian collection of industrial microorganisms and has a registration number VKPM B-10167. It was obtained on the basis of the parent strain B. subtilis VNIIgenetika-304 (VKPM B-2116) by genetic engineering and contains a mutation purH :: Em ^R , which disrupts the expression of the purH gene, a mutation in the purR gene encoding a purine nucleotide biosynthesis repressor, deletion of the terminator transcription in the leader region of the pur operon and an additional copy of the prs gene responsible for the synthesis of phosphoribosyl pyrophosphate (FRPF) under the control of the promoter of the rpsF gene encoding the synthesis of ribosomal S6 protein in the chromosome.

SUMMARY OF THE INVENTION

AICAR producer strain

The inventive method is based on the use of AICAR as a producer of bacteria, characterized by an increased ability to biosynthesis of purine nucleotides, for example, such as representatives of the genus Bacillus, producing riboflavin. Strain B.subtilis VNII Genetics 304 (VKPM B-2116) (hereinafter B.subtilis 304) contains a ribO mutation in the leader region of a rib operon, as well as a mutation in the ribC gene encoding the synthesis of the riboflavin kinase enzyme. According to the transcriptome analysis, the strain of B. subtilis 304 is characterized by increased (8-10 times) expression of the main operon of purine biosynthesis (pur operon) and presumably contains a mutation in the purR gene encoding the global pur operon repressor protein. In addition, this strain is characterized by a weakened growth on ribose and gluconate as the only carbon sources, indicating a decreased activity of the transketolase enzyme (tkt gene).

Another example of a strain of the genus Bacillus, reconstructed into a producer strain in the claimed group of inventions, is a strain of B. subtilis Mu8u5u6 (leu, met, purF), obtained from prof. P.Nigarda (Denmark). PurR :: neo cassette from B.subtilis strain LCC28 (J. Bacteriol. 179: 2540-2550) is introduced into the genome of this strain by transformation in order to inactivate the purR gene encoding the transcriptional repressor of genes involved in purine biosynthesis.

To enhance the expression of the pur operon, the negative regulation of this operon is removed from the so-called sensory RNA (G-box), which is encoded by the leader region of the pur operon and causes premature termination of transcription of structural genes (Cell. 113: 577-586, 2003). To this end, deletions are added to the genome of the parental strains that remove the transcription terminator in the leader region of the pur operon.

The inventive method of microbiological synthesis of AICAR is based on the interruption of the enhanced biosynthesis of purine nucleotides at the stage of conversion of the AICAR-F nucleotide to inosine monophosphate (IMP) due to the weakening or absence of expression of the purH gene encoding the AICAR transformylase protein (5'-phosphoribosyl-4-carboxamide 5-aminocamide transformylase) [EC 2.1.2.3] and IMP cyclohydrolase [EC 3.5.4.10]. The AICAR-F nucleotide is dephosphorylated in the producer cells by endogenous phosphatase to form the AICAR nucleoside, which is released into the culture medium (see drawing). The nucleotide sequence of the purH gene and the amino acid sequence of the PurH protein are shown in sequence listing No. 1 (SEQ ID NO: 1).

The term “AICAR-F transformylase-IMP cyclohydrolase activity” means the ability to catalyze the transfer reaction of formyl from 10-formyl tetrahydrofolate to AICAR-F followed by the formation of the purine ring of the resulting inosine-5'-monophosphate (IMP). The activity of AICAR-F transformylase - IMP cyclohydrolases is determined using AICAR-F and (6-R) N10-formyl tetrahydrofolate (Gene, 106: 197-205, 1991) as a substrate or by the method of complementation of mutations (J. Biol. Chem. 264: 21239-46, 1989).

The terms purH gene expression “absent” or “attenuated” mean that the enzymatic activity of the PurH protein is completely absent or decreased (“attenuated”) as a result of genetic modification of the purH gene (deletion of the entire gene or its part, shift of the reading frame of the gene, introduction of missense / nonsense mutations (s) or modifications of regions adjacent to the gene that include sequences that control gene expression, such as promoters, enhancers, attenuators, ribosome binding sites, etc.).

A key step in the construction of the AICAR producer strain is the inactivation of the purH gene. The purH gene is inactivated by integrating the erythromycin resistance cassette (Em ^R ) into the chromosome of the parent strains, in particular Bacillus subtilis 304 and B. subtilis Mu8u5u6 purR :: neo.

To increase the level of AICAR biosynthesis, mutations that increase the level of FRPF, a key precursor of purine biosynthesis, are introduced into the producer strain along with mutations that enhance the expression of the pur operon. To this end, substitution of the structural prs gene responsible for the FGFR synthesis is performed under the control of the strong promoter of the rpsF gene encoding the synthesis of ribosomal protein S6 using the integrative vector pDG268. The rpsF gene promoter is first cloned into this vector, and then this promoter is docked with the prs structural gene. The hybrid plasmid pDG268 (Cell 111: 747-756, 2002) P _rpsF -prs is integrated into the chromosomal locus amyE of B.subtilis AM25-15 and Mu8u5u6-9 strains (derived from the parental B.subtilis strains of All-Russian Research Institute of Genetics 304 and B.subtilis Mu8u5u6) and receive AICAR producing strains AM25-41 and Mu8u5u6-14.

The method in General

The inventive method is as follows.

A strain of bacteria of the genus Bacillus - producer AICAR is cultured on a nutrient medium (synthetic or natural). Since the AICAR producer has a mutation in the purH gene, purine bases (hypoxanthine, adenine, guanine) and / or their corresponding ribonucleosides must be present in the medium.

Glucose or sucrose, various organic acids, alcohols (ethanol and glycerin) are used as carbon sources. Inorganic ammonium salts (ammonia, ammonium sulfate), other nitrogen compounds (amines) or natural nitrogen sources (peptone, soybean hydrolyzate, microorganism fermentolizate) are used as a nitrogen source. Potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride and the like are used as mineral additives, and thiamine, yeast extract, etc. are used as vitamins.

The cultivation is carried out under aerobic conditions at a temperature in the range from 20 to 40 ° C, preferably in the range from 30 to 38 ° C. The pH of the medium is maintained in the range from 5 to 9, preferably from 6.5 to 7.2. The pH of the medium is regulated by ammonia, calcium carbonate, various acids, bases and buffer solutions. Cultivation for 1 to 5 days leads to the accumulation of the target product - AICAR in the culture medium. The method allows to accumulate AICAR both in the process of growth of the culture, and after building up the biomass. The concentration of AICAR in the culture fluid is determined by thin layer chromatography (Krasikov V.D. Fundamentals of planar chromatography. St. Petersburg: Khimizdat, p.231, 2005). The inventive engineered strain of Bacillus subtilis VKPM B-10167 is a producer of AICAR and has the following properties.

Cultural and morphological features

Spore-forming gram-positive bacillus with rounded ends and an edge arrangement of spores. On LB agar (Miller J. 1979. Experiments in molecular genetics. World) at 37 ° C on the second day forms colonies with an uneven edge 3-4 mm in diameter, and on Spitzizen’s minimal medium (J. Bacterial. 81: 741-746 , 1961) - the same colonies with a diameter of 2-3 mm.

Physiological and biochemical characteristics

The culture of the claimed strain is capable of fermenting saccharides such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trialose and starch hydrolyzate; alcohols such as glycerin, mannitol and sorbitol; organic acids such as gluconic, fumaric, citric and succinic acids, and the like.

Optimal cultivation conditions

Temperature is from 30 to 38 ° C.

pH of the medium is 6.5 to 7.2.

The composition of the fermentation medium (wt.%):

Glucose 3.0 KH ₂ PO ₄ 0.6 K ₂ HPO ₄ 1.4 MgSO ₄ 0.04 FeSO ₄ · 7H ₂ O 0.001 MnCl ₂ 0.001 (NH ₄ ) ₂ SO ₄ 0.2 CaCO ₃ 3.0 Casein hydrolysis 0.2 Yeast extract 1.0 Water Rest

pH 7.0.

Storage

The strain is stored in lyophilized form or in pairs of liquid nitrogen.

Stability

The strain is stable, does not lose the ability to synthesize AICAR after 10 transfers in a complete medium.

Contains a mutation purH :: Em ^R leading to impaired purH gene expression, mutations in the purR gene encoding a purine nucleotide biosynthesis repressor, deletion of the transcription terminator in the leader region of the pur operon, and an additional copy of the prs gene responsible for the synthesis of phosphoribosyl pyrophosphate (FRPF) under control the rpsF gene promoter encoding the synthesis of ribosomal S6 protein in the chromosome.

When cultured in accordance with the claimed method, the strain AM25-41 (VKPM B-10167) is able to synthesize on a medium with glucose up to 3.5 g / l AICAR.

The claimed group of inventions is illustrated by the following figures of the graphic image.

Example 1. Inactivation of the purH gene in the parent strain B.subtilis VNIIgenetika-304.

PurH gene inactivation is carried out in several stages. At the first stage, a vector is selected with a cassette suitable for the inactivation of the purH gene containing the determinant of antibiotic resistance. Plasmid pBSII KS (Nucleic Acids Res., 17: 9494-9498, 1989), which is a standard vector capable of replication in E. coli cells, but not in B. subtilis, and contains the gene for resistance to ampicillin.

The erythromycin resistance gene (Em ^R ) is cloned from the plasmid genome from plasmid pKS1 (FEMS Microbiol. Letters, 245: 315-319, 2005), which is a two-replicon vector capable of replication in both B. subtilis and E cells .coli, and contains erythromycin and kanamycin resistance genes (Figure 3).

Using the pKS1 plasmid and primers Er1 (SEQ ID NO: 2) and Er2 (SEQ ID NO: 3) as an DNA template, the Em ^R gene is amplified. The resulting PCR product is 892 bp in size. purified on an agarose gel, digested with restriction enzymes PstI and HindIII and cloned in the pBSII KS vector at the PstI and HindIII sites.

Then, fragments of the purine operon of B.subtilis flanking the purH gene are amplified with the chromosomal DNA of B.subtilis strain 168 trpC2 (American Journal of Botany. 34: 345-348, 1947). To amplify a 652 bp operon fragment containing the purN gene located in front of the purH gene, PurH1 primers (SEQ ID NO: 4) containing the SacII site and PurH2 (SEQ ID NO: 5) containing the Pst1 site are used. PurH3 primers (SEQ ID NO: 6) containing the HindIII site and PurH4 (SEQ ID NO: 7) containing the XhoI site were used to amplify the 862 bp fragment of the 862 bp operon containing the purD gene located after the purH gene. Cloning of these fragments into the pBSII KS vector yields a plasmid in which the purH gene is replaced by the Em ^R cassette and flanked by the nucleotide sequence of the purN and purD genes.

From this plasmid, PCR amplification of the fragment was carried out using primers PurH1 and PurH4, and the resulting fragment was 2406 bp in size. transform into the cells of the recipient B.subtilis 304 and with selection of recombinants Em ^R. The phenotype Pur ^- in the obtained Em ^R transformants indicates the integration of the Em ^R cassette into the purH gene. The result is a strain of B.subtilis AM25-11 with the genotype purH :: Em ^R.

Example 2. Inactivation of the pur R gene in B.subtilis Mu8u5u6 strain.

In order to inactivate the purR gene encoding the transcriptional repressor of genes involved in purine biosynthesis, the purR :: neo cassette containing the neomycin resistance determinant in the purR gene from B.subtilis LCC28 strain (J. Bacteriol. 179: 2540-2550). As a result, a B. subtilis Mu8u5u6-1 strain with the genotype purR :: neo was obtained, in which the PurR repressor protein was inactivated.

Example 3. Inactivation of the purH gene in B.subtilis strain Mu8u5u6-1.

The procedure is carried out as in example 1, but for another strain B.subtilis Mu8u5u6-1 purR :: neo. The result is a strain of B.subtilis Mu8u5u6-3, with the genotype purH :: Em ^R.

Example 4. Obtaining a deletion (PurΔ _L ) in the leader region of the pur operon using PCR amplification.

According to available data, the leader region of the B. optron pur operon encodes sensory RNA, which, interacting with purine derivatives, inhibits the expression of the operon structural genes due to the formation of a transcription terminator. To eliminate this negative regulation, strain AM25-11 obtained a deletion of that part of the leader region that encodes sensory RNA. Deletion in the leader region was obtained by PCR. First, PCR amplification of two fragments of size 650 and 780 bp is carried out. involving primer pairs P _L1 (SEQ ID NO: 8) and P _L2 (SEQ ID NO: 9) and P _L3 (SEQ ID NO: 10) and P _L4 (SEQ ID NO: 11), respectively. Since the primers P _L2 and P _L3 contain overlapping nucleotide sequences (indicated in bold), annealing the resulting fragments to each other and subsequent amplification using flanking primers P _L1 and P _L4 leads to the formation of a 94 bp leader region deletion, including the terminator transcription. The inclusion of a PurΔ _L deletion is confirmed by the production of a shorter PCR fragment using the control primers P _L5 (SEQ ID NO: 12) and P _L6 (SEQ ID NO: 13). It is important to emphasize that the resulting deletion mutant PurΔ _L retains the intact pur operon promoter. As a result, a PCR fragment containing deletions (PurΔ _L ) in the leader region of the pur operon was obtained.

Example 5. Obtaining a deletion (ΔpurE) in the genome of strain B.subtilis 304.

To integrate the obtained in the PCR fragment (Example 4), a deletion (ΔpurE) is first obtained in the genome of strain 304, overlapping the entire leader region of the pur operon and simultaneously the first structural gene of this purE operon, which causes the need for purines in this strain when it grows by minimal environment.

Obtaining such a deletion is carried out using the plasmid pKS1 described above.

To this end, DNA fragments flanking the leader region of the pur operon on the B.subtilis chromosome are amplified. To amplify a fragment of 830 bp containing the distal region of the yebG gene, which is located before the pur operon promoter, primers are used: PurE1 (SEQ ID NO: 14) containing the SacII site, and PurE2 (SEQ ID NO: 15), containing the PstI site. For amplification of the purE gene deleted from the 5 ′ end of the gene, primers are used: PurE3 (SEQ ID NO: 16) containing the HindIII site, and PurE4 (SEQ ID NO: 17) containing the XhoI site. The size of the obtained PCR fragment was 890 bp Cloning of these fragments into pKS1 vector resulted in a plasmid in which the leader region of the pur operon, including the main promoter (PL), and the proximal region of the purE gene are replaced by the Km ^R cassette and flanked by the nucleotide sequence of the distal region of the yebG gene and the more distal region of the purE gene.

From this plasmid, PCR amplification of the fragment was carried out using primers PurE1 and PurE4 and the resulting fragment 3091 bp in size. transform into the cells of the recipient B.subtilis 304, as well as the control strain B.subtilis 168 (with unmodified purine metabolism), followed by selection of Km ^R recombinants. The phenotype Pur ^- in the obtained Km ^R transformants indicates the integration of the Km ^R cassette into the leader region of the pur operon. The obtained Km ^R transformants were checked by PCR amplification with the control primers PurE5 (SEQ ID NO: 18) and PurE6 (SEQ ID NO: 19) to confirm the integration of the Km ^R cassette into the leader region of the pur operon. Thus, isogenic strains of B. subtilis AM25-13 ΔpurE and B. subtilis 168 ΔpurE were obtained.

Example 6. Transfer of a deletion of PurΔ _L to strain AM25-13 ΔpurE.

To transfer the PurΔL deletion, the obtained recipients AM25-13 ΔpurE and B.subtilis 168 ΔpurE are transformed (Example 5) by a PCR fragment amplified using primers P _L1 and P _L4 from the matrix of the previously obtained mutant (Example 4) containing a transcription termination deletion in leader region of the pur operon (PurΔ _L ). Pur ⁺ recombinants were selected in these crosses on minimal Spitsaisen medium without purine source. The inclusion of the PurΔ _L deletion in Pur ⁺ recombinants was confirmed by the production of a shorter PCR fragment using the control primers P _L5 and P _L6 . As a result, strains AM25-15 PurΔ _L and B. subtilis 168 PurΔ _{L were} obtained containing a deletion of the transcription terminator in the leader region of the pur operon.

Example 7. Transfer of a deletion of PurΔ _L to B.subtilis strain Mu8u5u6.

Transfer of deletions is carried out, as in examples 4, 5, 6, but for strain B.subtilis Mu8u5u6. The result is a strain Mu8u5u6-9 having the genotype purR :: neo purH :: Em ^R PurΔ _L.

Example 8. Assessment of the level of expression of purine biosynthesis genes in the obtained strains.

To evaluate the expression level of the pur operon against the background of the inactivated purR gene and deletion of the PurΔ _L transcription terminator in the leader region of the pur operon, transcription fuses of the promoter of this operon with the lacZ reference gene were obtained by integrating the expression plasmid pMutin2 (Microbiology 144: 3097-3104, 1998) in purH gene.

Plasmid pMutin2 contains the lacZ reference gene without its own promoter, the HindIII-EcoRI-NotI-SacII-BamHI polylinker region for cloning B.subtilis genetic material, and the erythromycin resistance marker (Em ^R ). Since the plasmid pMutin2 is not capable of autonomous replication in bacillus cells, its integration into the B.subtilis chromosome is carried out by cloning into a polylinker region of any fragment of B.subtilis chromosomal DNA with subsequent selection of Em ^R transformants.

As a result of this integration, the formation of the transcriptional fuse of the target gene with the lacZ reference gene occurs, which allows us to evaluate the level of expression of the target gene by determining the activity of β-galactosidase. Moreover, the transcription terminator is located at the distal end of the inserted plasmid, followed by the P _spac promoter under the control of the lactose operator, which allows the expression of distally located genes to be _induced using isopropylthiogalactoside (IPTG). To assess the expression level of the pur operon promoter, the proH proximal region of the purH gene is cloned by PCR amplification of a fragment of this gene using primers PurH7 (SEQ ID NO: 20) and PurH8 (SEQ ID NO: 21). The resulting PCR fragment of 316 bp clone into the HindIII-BamHI sites of the plasmid pMutin2. The selected plasmid pMutin2 with the purH gene insert is transformed, followed by selection of recombinants Em ^{R of the} initial B.subtilis 304 strains (selected strain AM25-21) and B.subtilis Mu8u5u6 (selected strain Mu8u5u6-17), as well as the strains of AM25- obtained in the previous step 15 PurΔ _L (strain AM25-32 selected), 168 PurΔ _L (strain 168-1 PurΔ _L selected) and strain Mu8u5u6-9 (strain Mu8u5u6-19 selected) that contain a deletion of the transcription terminator in the leader region of the pur operon. In addition, the plasmid pMutin2 with the purH gene insert was integrated into the chromosome of the B.subtilis168 strain (purR ⁺ ) used as a control (strain 168-1 was selected). Phenotype Pur ^- from the transformants indicates the integration of a recombinant plasmid into pMutin2 purH gene.

The resulting strains and their genotype are presented in Table 1.

As follows from the data presented in Table 1, the introduction of a deletion of PurΔ _L (strain 168-1 PurΔ _L ) leads to a 10-fold increase in the activity of the pur operon promoter compared to that of the control strain 168-1 (purR ⁺ ). The presence of the inactivated purR gene in the genome of the Mu8u5u6-17 strain leads to an approximately 25-fold increase in pur operon expression. An even higher level of activity is observed in strain AM25-21, a producer of riboflavin, which implies that the purR gene is already inactivated in the genome of strain AM25 and, in addition, this strain probably carries some additional mutations that increase the expression of pur operon . Finally, from the data in Table 1 it follows that the introduction of a PurΔ _L deletion into the genome of strains AM25-32 and Mu8u5u6-19 leads to a further increase in the activity of β-galactosidase (3200-3600 units), which exceeds the basal level of pur operon expression in the control strain 168 more than 300 times. This allows us to conclude that the combination of a mutation in the regulatory gene of purR and a deletion of PurΔ _L in the leader region leads to the maximum expression of the pur operon, and, consequently, quite efficient AICAR production when genetic damage is introduced into the purH gene.

Example 9. Enhanced expression of the prs gene of B.subtilis AM25-32 strains.

To enhance the expression of the prs gene responsible for FGF synthesis, it is substituted under the control of the strong promoter of the rpsF gene encoding the synthesis of S6 ribosomal protein using the integrative vector pDG268 (Mironov AS et al. 2002, Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria Cell, v. 111, p. 747-756). The pDG268 vector contains an expression cassette that includes a polylinker for cloning promoters, a lacZ reference gene without its own promoter, and a chloramphenicol resistance gene (Cm ^R ).

The cassette is flanked by fragments of the amyE gene, which allows integration of the vector with the cloned fragment into the amyE locus on the B.subtilis chromosome.

At the first stage, a DNA fragment containing the promoter region of the rpsF gene was generated by PCR from B.subtilis 168 chromosome using Rps1 primers (SEQ ID NO: 22) and Rps2 (SEQ ID NO: 23). The resulting PCR fragment is treated with restriction endonucleases EcoR1 and BamHI and cloned into the corresponding sites of the plasmid pDG268 treated with the same restrictase. The traditionally used recipient strain of E. coli TG1 is transformed with a ligase mixture (Sambrook J., Fritsch E.F., Maniatis T. 1989. Molecular cloning: a laboratory manual / 1 st and 2nd ed. NY / Cold Spring Harbor Laboratory). The selection of transformants carrying the insert of the desired fragment is carried out on an indicator medium containing ampicillin at a concentration of 120 μg / ml and X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (transformant colonies are blue in color ) (Sambrook J., Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual / 1st and 2nd ed. NY / Cold Spring Harbor Laboratory). The presence of the corresponding insert was confirmed by PCR using Rps1 and Rps2 primers.

At the next stage, the cloned promoter of the rpsF gene is docked with the structural gene prs encoding the FRPF synthesis. For this purpose, PCR amplification of the full-sized structural gene prs is carried out, including the ribosome binding site (SD sequence) using the primers flanking this gene, Prs1 (SEQ ID NO: 24) and Prs2 (SEQ ID NO: 25), which contain restriction enzyme recognition sites BamHI and NotI respectively. The obtained PCR fragment is treated with BamHI and NotI restriction endonucleases and cloned into the corresponding sites of the plasmid pDG268 treated with the same restrictase. The presence of the corresponding insert was confirmed by PCR using primers Prs1 and Prs2.

The integration of the obtained P _rpsF -prs hybrid fragments in the vector pDG268 into the amylase locus of the recipient B.subtilis AM25-32 and B.subtilis 168 PurΔ strains (Example 8) was carried out by transformation on LB medium containing 25 μg / ml chloramphenicol. As a result, the inventive strain B.subtilis AM25-41 of the genotype purH :: Em ^R purR PurΔ _L P _rpsF -prs was obtained.

Example 10. Enhanced expression of the prs gene in B.subtilis strain Mu8u5u6-19.

The expression of the prs gene in B.subtilis Mu8u5u6-19 strain was enhanced as in Example 9, but the amylase locus of B.subtilis Mu8u5u6-19 strain was used as a recipient for the integration of hybrid fragments of P _rpsF -prs in the vector pDG268. As a result, a B. subtilis Mu8u5u6-23 strain of genotype purH :: Em ^R purR PurΔ _L P _rpsF -prs was obtained.

Example 11. The level of AICAR production in the claimed and other obtained strains of B.subtilis.

The inventive strain B.subtilis AM25-41 (VKPM B-10167) and other obtained strains with the inactivated purH gene and various combinations of mutations are tested for the ability to synthesize AICAR in the culture fluid (Table 2). To prepare seed, the inoculum of each strain was seeded in 20 × 200 mm tubes containing 5 ml of LB medium and cultured at 34 ° С for 18 h. Then, 0.5 ml of the obtained culture was inoculated in 4.5 ml of fermentation medium in 20 × tubes 200 mm and cultivated at 34 ° C for 48 hours on a rotary shaker (250 rpm).

After cultivation, the amounts of purine derivatives accumulated in the medium are determined by thin layer chromatography. The results are presented in Table 2.

As follows from Table 2, the presence of a combination of mutations purR purH :: Em ^R PurΔ _L P _rpsF -prs leads to the maximum accumulation of AICAR in the culture fluid of the claimed strain B.subtilis AM25-41 (VKPM B-10167) and strain B.subtilis Mu8u5u6-14 (up to 3.3-3.5 g / l).

Thus, the developed method for the microbiological synthesis of AICAR is based on the use of bacteria of the genus Bacillus as producer strains modified by sequentially introducing mutations: purR, purH :: Em ^R , PurΔ _L , P _rpsF -prs. Modification of producer strains was carried out by phased genetic construction aimed at enhancing the metabolic flow of AICAR precursors from glucose to ribose and FGF, activation of gene expression of purine operon.

The high level of homology of the genetic apparatus and the universality of biochemical processes in various representatives of the genus Bacillus are the basis for extrapolating the developed scheme for obtaining AICAR producer strains to other species of this genus.

Claims (2)

1. The method of microbiological synthesis of purine nucleoside 5'-aminoimidazole-4-carboxamidriboside (AICAR) by culturing modified Bacillus subtilis bacteria in a suitable nutrient medium, characterized in that Bacillus subtilis bacteria obtained by sequentially introducing purR, purH mutations are used as modified bacteria :: Em ^R , PurΔ _L and P _rpsF -prs. 2. The bacterial strain Bacillus subtilis VKPM B-10167 - producer of purine nucleoside 5'-aminoimidazole-4-carboxamidriboside (AICAR) for implementing the method according to claim 1.