Method for producing inosine or guanosine

Abstract

Provided is a modified bacterium for producing inosine or guanosine, comprising, compared with an unmodified bacterium, modifications in the expression control sequence of a gene encoding phosphoribosylformylglycinamidine cyclo-ligase (PurM). The modifications of the expression control sequence comprise: a) replacing the 1st nucleotide G before the ATG of the PurM gene ORF with C, the 4th nucleotide A before the ATG with T, the 7th nucleotide G before the ATG with C, and the 10th nucleotide A with T; and b) inserting a P43 promoter before the ATG in the ORF region of the purM gene. Further provided is a method for using the modified bacterium to increase the production of inosine or guanosine.

Description

A method for producing inosine or guanosine

[0001] Priority

[0002] This application claims the benefit of and priority to Chinese Application No. 2024108824234, filed July 2, 2024. The entire contents of which are hereby incorporated by reference in their entirety for all purposes. TECHNICAL FIELD

[0003] The present disclosure relates to the field of microbial engineering, and in particular to a method for producing inosine guanosine by using bacterial fermentation. BACKGROUND

[0004] Nucleosides are a general term for a class of glycosides. Nucleosides are the constituent elements of nucleic acids and nucleotides. Nucleosides are formed by condensation of D-ribose or D-Z-deoxyribose with pyrimidine bases or purine bases. Nucleosides are generally colorless crystals, insoluble in ordinary organic solvents, easily soluble in hot water, and have a melting point of 160-240°C. Nucleosides formed from D-ribose are called ribonucleosides, which participate in the composition of RNA. Nucleosides formed from D-a-deoxyribose are called deoxyribonucleosides, which participate in the composition of DNA. Condensation of D-ribose with adenine, guanine, cytosine, thymine, or uracil generates the corresponding adenine ribonucleoside, guanine ribonucleoside, cytosine ribonucleoside, thymine ribonucleoside, and uracil ribonucleoside, which are simply referred to as adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U), respectively.

[0005] Guanine riboside (guanosine) and hypoxanthine riboside (inosine) have a wide range of applications in the food and pharmaceutical industries. In the food industry, guanosine and inosine are important precursors of guanylate disodium and inosinate disodium, respectively, and the combination of guanylate disodium and inosinate disodium is used as a food enhancer and widely used in condiments such as chicken essence and soy sauce. In the pharmaceutical industry, guanosine and inosine can be used as pharmaceutical intermediates for a variety of antiviral drugs, such as acyclovir, ribavirin, and guanosine triphosphate sodium, all of which require guanosine as a synthetic raw material. Inosine is an important precursor of inosinic acid, which can be used as a precursor for the synthesis of adenosine acid (AMP) and guanosine acid (GMP), and is suitable for treating leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis, cirrhosis, and other conditions, in addition to treating central retinitis and optic atrophy.

[0006] At present, microbial fermentation is the main method for producing nucleosides, and the main microorganisms used include Bacillus subtilis, Bacillus amyloliquefaciens or Bacillus pumilus, etc. In the process of breeding and modification of growing strains, high-yield nucleoside strains are bred by using ultraviolet mutagenesis, diethyl sulfate mutagenesis, and directional breeding; or according to the metabolic pathway and regulation mechanism of nucleotides in bacteria, the genetic background and strain characteristics of the strain are deeply understood, and the strain is modified by metabolic engineering means, so as to obtain a production strain with excellent properties and high yield of nucleosides. However, the fermentation performance of the nucleoside strain is still poor, and the conversion rate of nucleosides is still low, which cannot meet the needs of large-scale industrial production.

[0007] Studies have shown that adding P43 strong promoter in front of purF gene, the expression level of purF gene and its downstream purM, purN, purH and purD genes is enhanced, the adenosine yield is increased by 17.5%, and the glycoside conversion rate is increased by 26.1%. However, at the 5L level, the growth is affected to a certain extent (Liu Yue et al., Modification of Purine Biosynthetic Pathway in Bacillus subtilis Enhances Adenosine Accumulation, Acta Microbiologica Sinica, 2014, 54(6): 641-647). Asahara et al. inactivated purA, guaB, punA and deoD in Bacillus subtilis 168, disrupted the purR gene encoding the repressor protein of the purine operon and the 5'-untranslated region, and finally enhanced the promoter of the purine operon. The inosine yield of the engineering strain reached 6g / L (Asahara T, et al., Accumulation of Gene Targeted Bacillus subtilis Mutations that Enhance Fermentative Inosine Production. Applied Microbiology and Biotechnology, 2010, 87(6): 2195 2207). Xie Xixian et al. used E. coli MG1655 as the starting strain, integrated pbuE at the yjiT pseudogene site, purFK316Q at the yeeP pseudogene site, purEKBCSQLFK316QMNHD at the yghE pseudogene site, replaced purAP242N with the original purA, and knocked out purine nucleoside phosphorylase genes deoD, ppnP, and nucleoside hydrolase genes rihA, rihB, rihC, etc. The inosine yield reached 20.16g / L after 48h fermentation in a 5L tank (Zhu Yankai et al., Metabolic engineering of Escherichia coli for inosine production, Food and Fermentation Industries, 2022).Liao, Y., Ye, Y., Wang, B. et al. Optimization of the purine operon and energy generation in Bacillus amyloliquefaciens for guanosine production. Biotechnol Lett 2017 (39): 1675-1682. https: / / doi.org / 10.1007 / s10529-017-2412-4.

[0008] The present application mainly strengthens the purM gene, increases the yield and conversion rate of guanosine or inosine, and the strains obtained by the construction herein grow better after amplification. SUMMARY

[0009] The present disclosure provides a mutant promoter comprising a sequence as set forth in SEQ ID NO: 28 or a sequence having at least 70%, 80%, 90%, 95%, 99% or more identity to SEQ ID NO: 28.

[0010] The present disclosure provides a chimeric promoter sequence, wherein it comprises, in the 5’ to 3’ direction, a mutant promoter as previously described and a P43 promoter.

[0011] In some embodiments, the P43 promoter comprises a sequence as set forth in SEQ ID NO: 25 or 26 or a variant thereof having a promoter function, preferably the variant comprises a sequence having at least 70%, 80%, 90%, 95%, 99% or more identity to SEQ ID NO: 25 or 26.

[0012] In some embodiments, the chimeric promoter comprises a sequence as set forth in SEQ ID NO: 30 or a sequence having at least 70%, 80%, 90%, 95%, 99% or more identity to SEQ ID NO: 30.

[0013] The present disclosure provides a microorganism comprising a mutant promoter as previously described and / or a chimeric promoter as previously described.

[0014] In another aspect, the present disclosure provides a modified bacteria for producing inosine guanosine, wherein compared with the bacteria before modification, a modification of an expression control sequence of a gene encoding phosphoribosylformylglycinamidine cyclase (PurM) is comprised, wherein the modification of the expression control sequence comprises a) substitution of G to C at the 1st position before ATG, substitution of A to T at the 4th position before ATG, substitution of G to C at the 7th position before ATG, substitution of A to T at the 10th position before ATG of the purM gene ORF frame; b) insertion of a P43 promoter before ATG in the ORF region of the purM gene.

[0015] In some embodiments, the bacteria is a bacteria for producing inosine or guanosine.

[0016] In some embodiments, the bacteria for producing inosine is a Bacillus bacteria, preferably Bacillus amyloliquefaciens.

[0017] In some embodiments, the bacteria for producing guanosine is a Bacillus bacteria, preferably Bacillus amyloliquefaciens.

[0018] In some embodiments, the P43 promoter is derived from Bacillus subtilis, preferably the P43 promoter comprises a sequence mutation compared with the wild-type P43 promoter. The P43 promoter comprises a sequence as set forth in SEQ ID NO: 25 or 26, or a variant thereof having a promoter function, preferably the variant comprises a sequence having at least 70%, 80%, 90%, 95%, 99% or more identity to SEQ ID NO: 25 or 26.

[0019] In some embodiments, it comprises a nucleotide sequence as set forth in SEQ ID NO: 24, or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 99% or more identity to SEQ ID NO: 24.

[0020] In another aspect, use of the modified bacteria of the present disclosure in improving inosine production is provided.

[0021] In yet another aspect, use of the modified bacteria of the present disclosure in improving guanosine production is provided.

[0022] In another aspect, a method for producing inosine is provided, comprising culturing the modified bacteria of the present disclosure in a culture medium.

[0023] In yet another aspect, a method for producing guanosine is provided, comprising culturing the modified bacteria of the present disclosure in a culture medium.

[0024] In one embodiment, the culture medium comprises glucose.

[0025] In one embodiment, the method further comprises isolating inosine or guanosine.

[0026] In another aspect, a bioreactor comprising the modified bacteria described in the present disclosure. Advantages

[0027] The genetically engineered bacteria provided by the present disclosure have high inosine and guanosine conversion rates. DETAILED DESCRIPTION

[0028] The following description of the present disclosure is merely intended to illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various different equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments will be included herein. All references cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0029] In order to better understand the present disclosure by those skilled in the art, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, not all.

[0030] Table 1. Instruments used in the present application

[0031] Table 2. Reagents used in the present application

[0032] Table 3. Primer sequence information

[0033] The gene sequence information is as follows in Table 4:

[0034] Table 4. Sequence information

[0035] Example 1: Experimental method

[0036] 1.1. Reagent preparation method

[0037] Growth medium: composed of 2xLB medium and 2x sorbitol solution, when used, the two reagents should be mixed in equal proportions before use, and the preparation methods of the two reagents are as follows:

[0038] (1) 2xLB medium: Tryptone 20 g / L, Sodium chloride 20 g / L, Yeast extract 10 g / L, Distilled water to volume, according to 50 ml / bottle, 250 ml conical flask, 121℃ sterilization for 20 minutes, ready for use.

[0039] (2) 2x Sorbitol: Sorbitol 91.1 g / L, Distilled water to volume, according to 50 ml / bottle, 150 ml conical flask, 121℃ sterilization for 20 minutes, ready for use.

[0040] (3) Washing medium: Sorbitol 91.1 g / L, Mannitol 91.1 g / L, Glycerol 126 g / L, Add ultrapure water to 1000 ml, 121℃ sterilization for 20 minutes, ready for use.

[0041] (4) Resuscitation medium: composed of 2xLB medium and 2x (Sorbitol + Mannitol), 2xLB medium is configured according to 1), 2x (Sorbitol + Mannitol) is a mixed solution of Sorbitol 91.1 g / L and Mannitol 69.2 g / L.

[0042] (5) LB medium: Tryptone 10 g / L, Sodium chloride 10 g / L, Yeast extract 5 g / L, Distilled water to 1 L, 121℃ sterilization for 20 minutes, ready for use. Inosine strains need to add guanine during culture, the final concentration is 50 mg / L, when configuring the medium, directly weigh the corresponding concentration into the medium and sterilize together, below, unless otherwise specified, all inosine strains add guanine with a final concentration of 50 mg / L during culture.

[0043] 1.2. Genetic modification method

[0044] The genetic modification method is carried out according to the reference (Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System, Applied and Environmental Microbiology, Josef Altenbuchner et al. 2016), and the specific steps are as follows:

[0045] 1.2.1. pJOE8999-N20 plasmid construction

[0046] 1) Extract pJOE8999 plasmid (HonorGene, product code: HG-VCH1431), according to the enzyme digestion system, use BsaI enzyme to digest the plasmid, the specific system is shown in Table 5, 37℃, reaction for 1 hour, then add 1 μL FastAP and react for 1 hour, recover the product and reserve it.

[0047] Table 5. Enzyme digestion system

[0048] 2) Take 10 μL of each of N20-f and N20-r primers with a concentration of 10 μM in a PCR tube; perform amplification according to the procedure of 98°C for 2 minutes; at a speed of 0.1°C / s, from 98°C to 4°C; after amplification, dilute 10 times, and name it gene-N20, and linearized pJOE8999 plasmid obtained in 1) is connected at 22°C overnight using T4 DNA ligase, and the connection system is as shown in Table 6.

[0049] Table 6. Connection system

[0050] 3) The above 10 μL of connection product is electroporated into EC135 (Pm.Bam) strain (for specific construction method of strain, see document AMimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria. Plos Genetics. 2012, 8(9): e1002987.), and cultured at 30°C for 2 days to grow single colonies, and PCR verification is performed using 8999-F1 and 8999-R1, and the correct transformant is identified for sequencing verification. The correctly sequenced plasmid is used for the next step experiment.

[0051] 1.2.2. pJOE8999-N20-LR plasmid construction

[0052] 1) The primers corresponding to the scheme are used to amplify the upstream and downstream homologous arms, and full-length fragments are obtained by fusion PCR.

[0053] 2) The pJOE8999-N20 plasmid constructed in 1.2.1 is incubated in a 37°C incubator for 1 hour using SalI and XbaI restriction endonucleases, and the enzyme digestion system is as shown in Table 7. The above obtained linearized vector is connected according to the ClonExpress II One Step Cloning Kit instruction manual with the full-length fragments in 1), and is transformed into EC135 (Pm.Bam) cells, and cultured at 30°C for 48 hours, and the single colonies grown are identified by PCR using 8999-F1 and 8999-R1 and sequenced.

[0054] Table 7. Enzyme digestion system

[0055] 1.2.3. Strain construction

[0056] The above-mentioned sequencing correct pJOE8999-N20-LR plasmid (about 1 μg) is mixed with the competent cells of the strain to be modified and placed in a 1 mm electroporation cup, and an electric shock is performed at a voltage of 1.8 kv. The cells after the electric shock are transferred to a 15 ml centrifuge tube containing 5 ml of recovery liquid, and placed in a 30°C, 180-200 rpm shaker for 2.5 hours of recovery. After centrifugation, the supernatant is discarded, and the bacterial pellet is resuspended and plated on LB plates containing 5 mg / L kanamycin resistance (when the strain is modified for inosine, the kanamycin concentration should be 15 mg / L) and 0.2% (w / w) mannose, and cultured in a 30°C incubator for about 48 hours until single colonies are formed. PCR identification is performed, and the correct transformants are sequenced for verification.

[0057] 1.2.4. Plasmid elimination

[0058] The above-mentioned sequencing correct strain is picked as a single colony in LB liquid medium and cultured at 37°C for 24 hours. It is diluted 10 5 times and plated on antibiotic-free LB plates and cultured at 37°C until single colonies are formed. Single colonies are selected and spotted on antibiotic-free LB and LB+Kan5 resistance plates. Single colonies that grow on the antibiotic-free plates but not on the resistance plates are selected for PCR verification (primers outside the homologous arms to avoid false positives) and sequencing verification. The correct strain is the successfully modified strain.

[0059] Example 2: Construction of 4 mutation + P43 promoter purM enhanced engineering strain

[0060] 2.1 Plasmid construction

[0061] SMBI0321 strain construction process: ATCC 13952 (this strain has been disclosed in US3111459A, and the genomic information has been analyzed in “Whole genome sequencing and sequence analysis of inosine-producing Bacillus subtilis ATCC 13952”) is used as the starting strain. The adeC gene is inactivated (470 is inserted between 471 and 13 bp to inactivate the gene), the guaC (E8*) and yeaC (Q71*) genes are inactivated, the gmk gene is mutated to V105E, the ylzA gene is mutated to E81G, the ATG of the purR gene is deleted by 23 bp, and the first 25 and 34 A are mutated to T. The strain IG1 is constructed. On the basis of this strain, the nupC (Q231*) gene is inactivated, the purL gene is mutated to S444F, and the purF gene is mutated to L312P. The strain SMBI0321 is obtained. The inosine production level of SMBI0321 is 27.4 g / L, and the conversion rate is 31.4%, which is a high-yield inosine-producing strain.

[0062] The pJOE8999-P43-purM-N20 plasmid was constructed by using the primers purM-N20-f1 / purM-N20-r1 according to the modification method described in Example 1, using the SMBI0321 strain genome as a template, using Pfu enzyme, primers purM-1f / purM-1r, purM-2f / purM-2r, and purM-3f / purM-3r to amplify the left homologous arm, the P43 promoter, and the right homologous arm fragment, respectively, to obtain the full-length fragment P43-purM-LR by fusion PCR, and the linearized pJOE8999-P43-purM-N20 plasmid was connected with the P43-purM-LR fragment according to the method in Example 1, and finally the pJOE8999-P43-purM-N20-LR plasmid was constructed and obtained, and the plasmid was named pBGR029.

[0063] 2.2 SMBI0321→SMBI9124 strain construction (SMBI9124 inosine bacteria)

[0064] The pJOE8999-P43-purM-N20-LR (pBGR029) plasmid obtained in 2.1 was transformed into the SMBI0321 strain, spread on LB plates containing 5 μg / mL kanamycin and 0.2% (w / w) mannose for screening, and the single colonies grown were subjected to PCR verification and sequencing verification. The correct strain identified above was deplasmidized, and PCR verification was performed. The correct strain was subjected to sequencing verification and named SMBI9124.

[0065] 2.3 SMBG001→SMBG97b strain construction (SMBG97b guanosine bacteria)

[0066] SMBG001 strain construction: The strain IG1 obtained in 2.1 above was used as the starting strain, the E at position 481 of the guaB gene was mutated to K, and the V at position 234 of the guaA gene was mutated to I, and the strain was named SMBG001. The guanosine production level of the SMBG001 strain was 22.6 g / L, and the conversion rate was 20.4%, which was a guanosine high-yield strain.

[0067] The plasmid pJOE8999-P43-purM-N20-LR (pBGR029) constructed in 2.1 was transformed into the SMBG001 strain, spread on LB plates containing 5 μg / mL kanamycin and 0.2% mannose for screening, and the single colonies grown were subjected to PCR verification and sequencing verification. The correct strain identified above was deplasmidized, and PCR verification was performed. The correct strain was subjected to sequencing verification and the strain obtained by screening was named SMBG97b.

[0068] Example 3: Construction of P43 promoter purM enhanced engineering strain

[0069] 3.1 Plasmid construction

[0070] The plasmid construction method is the same as that in Example 2, wherein the primers used for constructing the N20 plasmid are purM-N20-f2 / purM-N20-r2, the left and right homologous arm amplifications use the plasmid pBGR029 obtained in Example 2 as a template, and purM-1f / purM-2r2 and purM-3f / purM-3r are used for amplification, respectively, to obtain the left and right homologous arms, and fusion PCR is performed, and the obtained plasmid is named pBGR048.

[0071] 3.2 Construction of SMBI0321→SMBI9126 strain (SMBI9126 myo-inosine bacteria)

[0072] The pBGR048 plasmid obtained in 3.1 is transformed into the SMBI0321 strain, spread on an LB plate containing 5 μg / mL kanamycin and 0.2% mannose for screening, and the single colonies grown are subjected to PCR verification and sequencing verification. The strain identified correctly above is disintegrated, and PCR verification is performed, and the strain verified correctly is subjected to sequencing verification, and the obtained strain is named SMBI9126.

[0073] 3.3 Construction of SMBG001→SMBG010 strain (SMBG010 guano sine bacteria)

[0074] The pBGR048 plasmid obtained in 3.1 is transformed into the SMBG001 strain, spread on an LB plate containing 5 μg / mL kanamycin and 0.2% mannose for screening, and the single colonies grown are subjected to PCR verification and sequencing verification. The strain identified correctly above is disintegrated, and PCR verification is performed, and the strain verified correctly is subjected to sequencing verification, and the constructed strain is named SMBG011.

[0075] Example 4: Construction of ΔPpurM+P43 promoter strain

[0076] 4.1 Plasmid construction

[0077] The plasmid construction method is exactly the same as in Example 2, wherein the primers used for constructing the N20 plasmid are purM-N20-F / purM-N20-R, the left and right homologous arm amplifications are obtained using purM-up-F / purM-up-Rm and purM-down-Fm / purM-down-R, respectively, with the genome of the SMBG001 strain as the template, the P43 promoter fragment is obtained by amplifying the primers MP43-Fm / MP43-Rm with the plasmid containing the P43 promoter as the template, the fusion PCR of the above left and right homologous arms and the P43 promoter fragment is performed, and the plasmid obtained by the plasmid construction method is named pBGRpurM.

[0078] 4.2 Construction of SMBI0321→SMBI9213 strain (SMBI9211 inosine bacteria)

[0079] The pBGRpurM plasmid is transformed into the SMBI0321 strain, spread on an LB plate containing 5 μg / mL kanamycin and 0.2% mannose for screening, and the single colonies grown are subjected to PCR verification and sequencing verification. The correct strain is identified by plasmid removal and PCR verification, and the correct strain is subjected to sequencing verification, and the strain obtained is named SMBI9213.

[0080] 4.3 Construction of SMBG001→SMBG011 strain (SMBG011 guanosine bacteria)

[0081] The pBGRpurM plasmid is transformed into the SMBG001 strain, spread on an LB plate containing 5 μg / mL kanamycin and 0.2% mannose for screening, and the single colonies grown are subjected to PCR verification and sequencing verification. The correct strain is identified by plasmid removal and PCR verification, and the correct strain is subjected to sequencing verification and named SMBG010.

[0082] Example 5: Shake flask verification of the strain

[0083] The recombinant bacteria constructed above are subjected to fermentation to verify the production performance of inosine and guanosine, and the method for verifying the yield of inosine and guanosine is as follows:

[0084] 1. The bacteria stored in glycerol are incubated at 37°C overnight, and a single colony is streaked.

[0085] 2. The single colony is inoculated into 30 mL of seed culture medium, and incubated at 110 rpm and 37°C for 7-8 hours. The formula of the seed culture medium is as follows in g / L: glucose 20, yeast powder 5, corn syrup dry powder 5, potassium dihydrogen phosphate 3, magnesium sulfate 0.5, ferrous sulfate 0.02, manganese sulfate 0.01, and pH 7.0-7.2.

[0086] 3. Transfer to 30 ml fermentation medium at 10% (v / v) inoculation amount, 130 rpm shaking speed, 35 °C for 72 hours. The fermentation medium is formulated as follows in g / L: glucose 120, yeast powder 3.5, potassium dihydrogen phosphate 3, ammonium sulfate 25, manganese sulfate 0.01, magnesium sulfate heptahydrate 5, sodium glutamate 10, corn syrup dry powder 15, calcium carbonate 25, pH 7.0-7.2.

[0087] 4. Use liquid chromatograph to detect the inosine produced in the fermentation broth, and the detection results are shown in the table.

[0088] 5.1 Inosine bacterial shake flask fermentation

[0089] Table 8. Inosine modified bacterial shake flask fermentation results

[0090] The performance of new bacteria SMBI 9124, SMBI 9126, and SMBI 9213 is improved compared with SMBI 0321, with inosine production increased by 1.2 g / L, 1 g / L, and 0.4 g / L, respectively, and conversion rate increased by 0.9%, 0.4%, and 0.5%. Based on the inosine production, conversion rate, and sugar consumption of the three strains, 9124 is planned to be verified in 5L. Conclusion: The conversion rate of 9124 is increased by 3.5% compared with the control 0321 at the 5L level. At the 50L level, the conversion rate of SMBI 9124 is increased by 4.3%, which is the dominant strain.

[0091] Table 9. Inosine modified bacterial 5L fermentation content detection

[0092] Table 10. Inosine bacterial 50L fermentation content detection

[0093] 5.2 Guanosine bacterial shake flask fermentation

[0094] Table 11. Guanosine bacterial content detection table

[0095] The new bacteria SMBG 97b, SMBG 010, and SMBG 011 are all improved compared with the control strain SMBG 001, with inosine production increased by 0.8 g / L, 0.7 g / L, and 0.6 g / L, respectively, and conversion rate increased by 1.4%, 0.8%, and 0.3%, respectively, with little difference in sugar consumption and OD. The inosine production of new bacteria SMBG 97b is increased by 2.12 g / L compared with the control, and the conversion rate is increased by 1.53%. The conversion rate of SMBG 97b is increased by about 0.9-1.4% compared with the control strain.

[0096] Table 12. Guanosine bacterial 5L fermentation content detection

[0097] Table 13. Guanosine content detection of 50L fermentation of G. candidum

[0098] incorporated by reference

[0099] The entire contents of each patent and scientific document referred to herein are incorporated by reference for all purposes.

[0100] equivalents

[0101] The present disclosure can be embodied in other specific ways without departing from the spirit or essential characteristics thereof. Therefore, the above embodiments are to be regarded as illustrative in all respects and not restrictive, the scope of the disclosure being indicated by the appended claims rather than by the description and all changes which come within the meaning and range of equivalency of the claims are to be embraced therein.

Claims

1. A mutant promoter comprising a sequence as set forth in SEQ ID NO: 28 or a sequence having at least 70%, 80%, 90%, 95%, 99% or more identity to SEQ ID NO:

28.

2. The mutant promoter of claim 1, comprising a sequence as set forth in SEQ ID NO:

28.

3. A chimeric promoter sequence, wherein, 3. A chimeric promoter comprising a mutant promoter as set forth in claim 1 or 2 and a P43 promoter in 5’ to 3’ direction.

4. The chimeric promoter of claim 3, wherein the P43 promoter comprises a sequence as set forth in SEQ ID NO: 25 or 26 or a variant thereof having a promoter function.

5. The chimeric promoter of claim 3 or 4, wherein the chimeric promoter comprises a sequence as set forth in SEQ ID NO:

30.

6. The chimeric promoter of claim 3 or 4, wherein the chimeric promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99% or more identity to SEQ ID NO:

30.

7. A microorganism comprising a mutant promoter as set forth in claim 1 or 2 and / or a chimeric promoter as set forth in any one of claims 3 to 6.

8. A modified bacterium produced, wherein compared to the bacterium before modification, a modification of an expression control sequence of a gene encoding phosphoribosylformylglycinamidine cyclase (PurM) is comprised, wherein the modification of the expression control sequence comprises a) substitution of G to C at position 1 before ATG, substitution of A to T at position 4 before ATG, substitution of G to C at position 7 before ATG, substitution of A to T at position 10 before ATG of the purM gene ORF frame; b) insertion of a P43 promoter in the ORF region before ATG of the purM gene.

9. The modified bacterium of claim 8, wherein the bacterium is a bacterium producing inosine or guanosine.

10. The modified bacterium of claim 9, wherein the bacterium producing inosine is a Bacillus bacterium, preferably B. amyloliquefaciens or B. subtilis.

11. The modified bacterium of claim 9, wherein the bacterium producing guanosine is a Bacillus bacterium, preferably B. amyloliquefaciens or B. subtilis.

12. The modified bacterium of claim 8, wherein the P43 promoter is derived from B. subtilis.

13. The modified bacterium of claim 8, comprising a nucleotide sequence as set forth in SEQ ID NO:

24.

14. The modified bacterium of claim 8, comprising a nucleotide sequence having at least 70%, 80%, 90%, 95%, 99% or more identity to SEQ ID NO:

24.

15. Use of the modified bacterium of any one of claims 8 to 14 for increasing inosine production.

16. Use of the modified bacterium of any one of claims 8 to 14 for increasing guanosine production.

17. A method of producing inosine, comprising culturing the modified bacterium of any one of claims 8 to 14 in a culture medium.

18. A method of producing guanosine comprising culturing the modified bacteria of any one of claims 8 to 14 in a culture medium.

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