Construction method and application of recombinant bacteria for producing nucleoside and derivatives thereof

Abstract

The invention provides a construction method and application of recombinant bacteria for producing nucleoside and derivatives thereof. According to the invention, ribulose phosphate 3-epimerase (coded by rpe gene) of bacillus subtilis or bacillus amyloliquefaciens is modified, so that the activity of Rpe protein is weakened, guanine nucleoside or hypoxanthine nucleoside or adenosine can be rapidly generated by microorganisms at high rate, and a new microorganism capable of efficiently producing nucleoside is successfully created.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering and microbial fermentation, and in particular to a method for constructing a recombinant bacterium producing nucleosides and their derivatives and an application thereof. Background Art

[0002] Nucleoside is a general term for a class of glycosides. Nucleoside is a component of nucleic acid and nucleotide. Nucleoside is formed by the condensation of D-ribose or DZ-deoxyribose with pyrimidine base or purine base. Nucleoside is generally a colorless crystal, insoluble in common organic solvents, easily soluble in hot water, with a melting point of 160-240°C. Nucleosides generated from D-ribose are called ribonucleosides, which participate in the composition of RNA, and nucleosides generated from D-α-deoxyribose are called deoxyribonucleosides, which participate in the composition of DNA. D-ribose condenses with adenine, guanine, cytosine, thymine or uracil to generate the corresponding adenine ribonucleoside, guanine ribonucleoside, cytosine ribonucleoside, thymine ribonucleoside and uracil ribonucleoside, which are respectively referred to as adenosine (A), guanosine (G), cytidine (C), thymidine (T) and uridine (U).

[0003] Guanosine (guanosine) and inosine (inosine) play a wide range of roles in the food and pharmaceutical industries. In the food field, guanosine and inosine are important precursors of disodium guanylate and disodium inosinate, respectively. Disodium guanylate and disodium inosinate are used in combination as food flavor enhancers, and are widely used in condiments such as chicken essence and soy sauce. In the pharmaceutical field, guanosine and inosine can be used as pharmaceutical intermediates for a variety of antiviral drugs, such as acyclovir, triazole nucleoside, and sodium guanosine triphosphate, which all require guanosine as a synthetic raw material. Inosine is an important precursor of inosinic acid, and inosinic acid can be used as a precursor for the synthesis of adenosine (AMP) and guanylate (GMP), which is suitable for leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis, cirrhosis, etc. caused by various reasons. In addition, it can also treat central retinitis and optic atrophy.

[0004] At present, microbial fermentation is the main method for producing nucleosides, and the main microorganisms used include Bacillus subtilis, Bacillus amyloliquefaciens or Bacillus pumilus. In the process of selecting and transforming the growing strains, ultraviolet mutagenesis and diethyl sulfate mutagenesis breeding are used to selectively select high-yielding strains of nucleosides; or according to the metabolic pathways and regulatory mechanisms of nucleotides in bacteria, the genetic background and characteristics of the strains are deeply understood, and the strains are purposefully transformed through metabolic engineering methods to obtain production strains with excellent traits and high nucleoside production. However, the fermentation performance of nucleoside strains is still poor, and the conversion rate of nucleosides is still low, which cannot meet the needs of large-scale industrial production. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a recombinant bacterium producing nucleosides and their derivatives and its application.

[0006] To achieve the purpose of the present invention, in a first aspect, the present invention provides a modified microorganism, wherein the activity of ribulose phosphate 3-epimerase is reduced or lost compared to an unmodified microorganism, and the microorganism has an enhanced nucleoside production capacity compared to an unmodified microorganism.

[0007] In the present invention, the reference sequence number of ribulose-phosphate 3-epimerase from Bacillus amyloliquefaciens on NCBI is CBI42777.1, and the reference sequence number of ribulose-phosphate 3-epimerase from Bacillus subtilis on NCBI is CAB13452.1 (encoded by rpe gene).

[0008] Further, the activity reduction or loss of ribulose phosphate 3-epimerase in the microorganism is achieved by selecting from the following 1)-3), or an optional combination:

[0009] 1) reducing or losing the ribulose phosphate 3-epimerase by changing the amino acid sequence of the ribulose phosphate 3-epimerase;

[0010] 2) reducing or losing the ribulose phosphate 3-epimerase by changing the nucleotide sequence encoding the ribulose phosphate 3-epimerase;

[0011] 3) Loss by knocking out the coding sequence of the ribulose phosphate 3-epimerase.

[0012] Furthermore, the expression of the gene encoding the ribulose phosphate 3-epimerase is reduced or the gene encoding the ribulose phosphate 3-epimerase is knocked out by using mutagenesis, site-directed mutagenesis or homologous recombination.

[0013] In a specific embodiment of the present invention, the reduction or loss of ribulose phosphate 3-epimerase activity is achieved by the following A, B, C or D:

[0014] A, the 77th amino acid of ribulose phosphate 3-epimerase mutated from S to A;

[0015] B. The start codon of the gene encoding ribulose phosphate 3-epimerase mutates from atg to gtg or ttg;

[0016] C. Completely knocking out the entire ORF frame of the gene encoding ribulose phosphate 3-epimerase or mutating any amino acid in the ORF frame into a stop codon to cause inactivation (e.g., the start codon ATG is mutated to TAA, and the 171st amino acid undergoes a stop mutation, making the activity of the protein encoded by the gene lower than that of the wild type);

[0017] D. The codons of the gene encoding ribulose phosphate 3-epimerase were optimized, and the nucleotide mutation points of the optimized codons included G168A, T192C, A252G, and C264T.

[0018] The microorganism can be Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli, etc. For example, the starting strains are B. subtilis 168, B. subtilis A5 (the construction method of strain B. subtilis A5 is shown in CN110257315B), Ba 833 and Ba 836 (see CN112574934A).

[0019] In a second aspect, the present invention provides a method for constructing a recombinant bacterium that produces nucleosides and their derivatives, the method comprising: using genetic engineering means to inactivate or weaken the gene encoding ribulose phosphate 3-epimerase in a microorganism with nucleoside production ability.

[0020] The weakening is achieved by selecting from the following a) to c), or any combination thereof:

[0021] a) reducing or losing the ribulose phosphate 3-epimerase by changing the amino acid sequence of the ribulose phosphate 3-epimerase;

[0022] b) reducing or losing the ribulose phosphate 3-epimerase by changing the nucleotide sequence encoding the ribulose phosphate 3-epimerase;

[0023] c) loss by knocking out the coding sequence of the ribulose phosphate 3-epimerase.

[0024] The weakening method can be selected from at least one of mutagenesis, site-directed mutagenesis, homologous recombination, and the like.

[0025] Preferably, the weakening is achieved by ①, ②, ③ or ④ as follows:

[0026] ① The 77th amino acid of ribulose phosphate 3-epimerase mutated from S to A;

[0027] ② The start codon of the gene encoding ribulose phosphate 3-epimerase mutates from atg to gtg or ttg;

[0028] ③ Completely knock out the entire ORF frame of the gene encoding ribulose phosphate 3-epimerase or mutate any amino acid in the ORF frame into a stop codon to cause inactivation (such as the start codon ATG mutates to TAA, and the 171st amino acid undergoes a stop mutation, making the activity of the protein encoded by the gene lower than the wild type);

[0029] ④ The codons of the gene encoding ribulose phosphate 3-epimerase were optimized, and the nucleotide mutation points of the optimized codons included G168A, T192C, A252G, and C264T;

[0030] In a third aspect, the present invention provides a recombinant bacterium constructed according to the method.

[0031] In a fourth aspect, the present invention provides the use of the microorganism or the recombinant bacteria constructed according to the method in the fermentation production of nucleosides or nucleoside derivatives or in increasing the fermentation yield of nucleosides or nucleoside derivatives.

[0032] The nucleosides or nucleoside derivatives of the present invention include, but are not limited to, inosine, guanosine, hypoxanthine, inosinic acid, guanine, guanylic acid, riboflavin, diacetylguanylic acid, and the like.

[0033] In a fifth aspect, the present invention provides a method for producing nucleosides, the method comprising the steps of:

[0034] i) culturing the microorganism or the recombinant bacteria constructed according to the method to obtain a culture;

[0035] ii) collecting the produced nucleosides from the culture obtained in step i).

[0036] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0037] The present invention modifies the ribulose phosphate 3-epimerase (encoded by the rpe gene) of Bacillus subtilis or Bacillus amyloliquefaciens to weaken the activity of the Rpe protein, so that the microorganism can generate guanosine or inosine nucleoside at a high rate and quickly, and successfully creates a new microorganism that can efficiently produce nucleosides.

[0038] The above-mentioned transformation can be applied to Bacillus subtilis and Bacillus amyloliquefaciens, but is not limited to the above two strains. It can also be applied to host bacteria such as Bacillus pumilus and Escherichia coli to produce nucleosides such as inosine and guanosine or corresponding nucleoside derivatives such as hypoxanthine, inosinic acid, guanine, guanylic acid, riboflavin, diacetylguanylic acid, etc. DETAILED DESCRIPTION

[0039] The present invention aims to provide a method for producing purine nucleosides using microorganisms, and to construct new microorganisms capable of producing purine nucleosides with high efficiency.

[0040] The study found that by modifying the ribulose phosphate 3-epimerase (encoded by the rpe gene) of Bacillus subtilis or Bacillus amyloliquefaciens, the activity of the Rpe protein was weakened, allowing the microorganism to generate guanosine or inosine nucleoside at a high rate and quickly, and successfully creating a new microorganism that can efficiently produce nucleosides, thereby completing the present invention.

[0041] The present invention is the first to obtain corresponding mutants in Bacillus subtilis or Bacillus amyloliquefaciens by changing the amino acid sequence or appropriately optimizing the codons, so that the strain can efficiently produce nucleosides, and successfully create a microorganism that can efficiently produce nucleosides.

[0042] The present invention adopts the following technical solution:

[0043] Rpe catalyzes the reversible epimerization of D-ribulose 5-phosphate into D-xylulose 5-phosphate. The present invention provides a Bacillus amyloliquefaciens or Bacillus subtilis.

[0044] 1) The point mutation S77A in the rpe gene in the cell causes the activity of the protein encoded by the gene to be lower than that of the wild type; and / or

[0045] 2) A termination mutation occurs in 171, which makes the activity of the protein encoded by this gene lower than that of the wild type; and / or

[0046] 3) Inactivation, including complete knockout of the entire ORF frame or mutating any amino acid in the ORF frame into a stop codon, such as mutating the start codon ATG to TAA; and / or

[0047] 4) The codons of the rpe gene are optimized, and the nucleotide mutation points of the optimized codons include G168A, T192C, A252G, C264T or any combination thereof, so that the ability of the strain to produce nucleosides is enhanced compared with the unmodified strain.

[0048] The above mutation sites can be applied to Bacillus amyloliquefaciens, but are not limited to Bacillus amyloliquefaciens, such as Bacillus subtilis, Bacillus pumilus, Escherichia coli and other host bacteria, for producing nucleosides such as inosine and guanosine or corresponding nucleoside derivatives such as hypoxanthine, inosinic acid, guanine, guanylic acid, riboflavin, diacetylguanylic acid, etc.

[0049] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0050] The primer names and primer sequences involved in the following examples are shown in Table 1.

[0051] Table 1 Primer names and sequence information

[0052]

[0053]

[0054] Example 1 RPE in Bacillus amyloliquefaciens S77A Construction of point mutation strains

[0055] The genetic modification method used in the embodiment 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:

[0056] 1) Construction of specific pJOE8999-rpe S77A -N20 plasmid: Take 10 μl of each primer rpe-N2077-f / r, anneal at a constant speed from 94°C to 4°C in a PCR instrument, and obtain rpe S77A -N20 fragment. The pJOE8999 plasmid (pJOE8999 is a fully gene-synthesized plasmid, and the full sequence of the plasmid is shown in SEQ ID NO: 5) was digested with BsaI to obtain a linearized plasmid. S77A The -N20 fragment was ligated with T4 ligase at 22°C overnight and transformed into DH5α or BL21 chemical competent cells (competent cells were purchased from Quanshijin Biotechnology). The obtained single clone was identified by PCR and sequenced to construct pJOE8999-rpe S77A- N20 plasmid.

[0057] 2) pJOE8999-rpe S77A- Construction of N20-LR plasmid: Using the genome of DSM7 strain as a template, primers rpe77-1f / 1r and rpe77-2f / 2r were used to amplify the left and right homologous arms, and the full-length fragment rpeS77A-LR was obtained by fusion PCR. At the same time, the N20 plasmid obtained in 1) was digested with SalI / XbaI and collected by gel, and the above fragments were connected to the linearized vector using an assembly kit (purchased from Novozymes). Colonies were screened and verified by colony PCR and sequencing to obtain pJOE8999-rpe S77A-N20-LR plasmid.

[0058] 3) The above pJOE8999-rpe S77A -N20-LR plasmid was transformed into Bacillus amyloliquefaciens DSM7 (wild bacteria) / B.s833 / B.a836, respectively, and spread on LB plates containing 5 μg / mL kanamycin and 0.2% mannose for screening. The grown single colonies were verified by PCR and sequencing.

[0059] 4) The correctly identified strain was inoculated into a 37°C liquid culture medium and cultured overnight to eliminate the plasmid. The overnight culture liquid was diluted and spread on 10 -4 -10 -5 The strain was spread on a plate without resistance, and the single colonies grown were simultaneously spotted on plates with and without resistance. The colonies that grew on the plate without resistance but did not grow on the plate with resistance were screened as the target bacteria. The strain obtained after colony PCR and sequencing verification was named Ba 8414-8416. The rpe sequence on the genome of the modified strain is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0060] Strain B.s833 / B.a836, see CN112574934A.

[0061] Example 2 Optimization of rpe ORF frame codons in Bacillus amyloliquefaciens

[0062] According to the method in Example 1, the genome of DSM7 strain was used as a template, and the primers rpe77-1f / rpeT-1r and rpeT-2f / rpe77-2r were used to amplify the left and right homology arms, and the full-length fragment rpe was obtained by fusion PCR. T -N20-LR, and combine it with pJOE8999-rpe S77A- After the linearized plasmid N20 (after SalI / XbaI digestion) was connected, the recombinant plasmid pJOE8999-rpe was obtained. T -N20-LR, were transformed into DSM7 (wild bacteria) / B.s833 / B.a836 strains, and the strains obtained were named Ba 8417-8419. The corresponding nucleotide sequence is shown in SEQ ID NO: 3 (including nucleotide mutation sites G168A, T192C, A252G, C264T), and the amino acid sequence is shown in SEQ ID NO: 4.

[0063] Example 3 RPE in Bacillus amyloliquefaciens L171* Construction of strains

[0064] According to the method in Example 1, the pJOE8999-rpe171*-N20 plasmid was constructed using rpe-N201-f / r primers, and the Ba 8419 genome was used as a template, and primers rpe171-1f / 1r, rpe171-2f / 2r; rpe171-3f / 3r were used to amplify three fragments, which were then subjected to fusion PCR to obtain the full-length fragment rpe171*-LR, and the fragment was connected to the pJOE8999-rpe171*-N20 plasmid to construct the pJOE8999-rpe171*-N20-LR plasmid, which was electroporated into the B.s833 / B.a836 / Ba 8419 strain, and the screened strain was named Ba 8420-8422.

[0065] Example 4 Construction of a strain in which the rpe start codon is changed to a stop codon in Bacillus amyloliquefaciens

[0066] According to the method in Example 1, the DSM7 genome was used as a template, and primers rpe171-1f / rpe-star-1r and rpe-star-2f / rpe171-2r were used to amplify two fragments, which were then subjected to fusion PCR to obtain the full-length fragment rpe ATG-TAA -LR, and ligated the fragment with the pJOE8999-rpe171*-N20 linearized plasmid to construct pJOE8999-rpe ATG -TAA -N20-LR plasmid was electroporated into B.s833 / B.a836 / Ba 8419 strain, and the strain obtained by screening was named Ba 8423-8425.

[0067] Example 5 Construction of rpe knockout strain in Bacillus amyloliquefaciens

[0068] According to the method in Example 1, and using the DSM7 genome as a template, primers Drpe-1f / Drpe-1r and Drpe-2f / Drpe-2r were used to amplify two fragments, which were then subjected to fusion PCR to obtain the full-length fragment Drpe-LR, and the fragment was connected to the pJOE8999-rpe171*-N20 plasmid to construct the pJOE8999-Drpe-LR plasmid, which was electroporated into the B.s833 / B.a836 / Ba 8419 strain, and the strain obtained by screening was named Ba 8426-8428.

[0069] Example 6 Construction of rpe171* mutant strain in Bacillus subtilis

[0070] According to the method in Example 1, the N20 fragment was first amplified using primers Bsrpe171-N20-f / Bsrpe171-N20-r and the Bs-rpe171-N20 plasmid was constructed; and the genome of strain B. subtilis A5 was used as a template, and primers Bsrpe171-1f / Bsrpe71-1r and Bsrpe171-2f / Bsrpe171-2r were used to amplify two fragments, which were then subjected to fusion PCR to obtain the full-length fragment Bs-rpe171-LR, and the fragment was connected to the pJOE8999-Bs-rpe171-N20 linearized plasmid to construct the pJOE8999-Bs-rpe171-N20-LR plasmid, which was electrotransformed into the B. subtilis A5 strain, and the strain obtained by screening was named B. subtilis A0051.

[0071] Strain B. subtilis A5, see CN110257315B.

[0072] Example 7 Construction of a strain in which the rpe start codon ATG is mutated to TAA in Bacillus subtilis

[0073] According to the method in Example 6, the Bs-rpe171-N20 plasmid was used, the B. subtilis A0051 genome was used as a template, and the start codon mutant recombinant plasmid pJOE8999-Bs-star-N20-LR was constructed with Bs-star-1f / Bs-star-1r, Bs-star-2f / Bs-star-2r primers. It was then electroporated into the B. subtilis A5 strain, and the screened strain was named B. subtilis A0052.

[0074] Example 8 Construction of rpe knockout strain in Bacillus subtilis

[0075] According to the method in Example 6, the Bs-rpe171-N20 plasmid was used, the B. subtilis A0051 genome was used as a template, and the start codon mutant recombinant plasmid pJOE8999-Bs-Drpe-N20-LR was constructed using Bsrpe171-1f / Bs-Drpe-1r, Bs-Drpe-2f / Bsrpe171-2r primers. It was then electroporated into the B. subtilis A5 strain, and the strain obtained by screening was named B. subtilis A0053.

[0076] Example 9 Enzyme Activity Experiment Verification

[0077] The mixture contained 50 mM triethanolamine (pH 7.5), 5 mM MgCl 2, 0.2 mM NADH, 0.025 mM thiamine pyrophosphate (purchased from Shanghai Biotechnology Co., Ltd.), 1 U transketolase (tktA, EC number 2.2.1.1, reference Characterization and multi-step transketolase—transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube, Journal of Biotechnology, Amanatuzzakiah Abdul Halim et al. 2013), 20 U triosephosphate isomerase (tpiA, EC number 5.3.1.1, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), 2 U glyceraldehyde 3-phosphate dehydrogenase (gapA, EC number 1.2.1.12, purchased from Shanghai Yuanye Biotechnology Co., Ltd.), 2 mM 5-ribose phosphate (R5P, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and 1 mM A mixture of ribulose 5-phosphate (Ru5P, purchased from Shanghai Zhenzhun Biotechnology Co., Ltd.) was equilibrated at 30°C for 3 minutes. The cell-free extract was added first when the reagents were added, and the enzyme activity was determined by the consumption of NADH (Table 2).

[0078] Table 2 Enzyme activity determination results

[0079]

[0080]

[0081] Example 10 Verification of the glycoside production performance of the mutant strain

[0082] 1. Culture the bacteria stored in glycerol overnight at 37°C and isolate a single colony.

[0083] 2. Pick a single colony and inoculate it into 30 mL of seed culture medium (20 g / L glucose, 5 g / L yeast powder, 5 g / L corn steep liquor powder, 3 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.02 g / L ferrous sulfate, 0.01 g / L manganese sulfate, pH 7.0-7.2), and culture at 110 rpm and 37 °C for 7-8 h.

[0084] 3. Transfer the inoculum to 30 ml fermentation medium (120 g / L glucose, 3.5 g / L yeast powder, 3 g / L potassium dihydrogen phosphate, 25 g / L ammonium sulfate, 0.01 g / L manganese sulfate, 5 g / L magnesium sulfate, 10 g / L sodium glutamate, 15 g / L corn steep liquor powder, 25 g / L calcium carbonate, pH 7.0-7.2) at a 10% v / v inoculum volume, shake at 130 rpm, and culture at 35 °C for 70 h.

[0085] 4. Use liquid chromatography to detect the glycosides produced in the fermentation broth (Table 3).

[0086] Table 3 Evaluation results of guanosine and inosine production by shake flask fermentation of mutant strains (mean of three replicates)

[0087]

[0088]

[0089] As can be seen from Table 3, in Bacillus amyloliquefaciens, the rpe gene encoding protein was subjected to S77A point mutation by genetic engineering, the corresponding codons of G168A, T192C, A252G, and C264T were optimized, and the enzyme activity was reduced or completely inactivated by partial inactivation (stop mutation at position 171) or complete inactivation (knockout), which had different degrees of positive effects on the strain's production of guanosine or inosine (inosine); at the same time, in Bacillus subtilis, the 171 amino acid of the rpe gene encoding protein was inactivated, or the start codon was directly mutated to a stop codon, or the gene was directly knocked out, which had different degrees of improvement on the strain's ability to produce inosine and adenosine. This shows that weakening or inactivating rpe is beneficial to the nucleoside production ability of Bacillus amyloliquefaciens or Bacillus subtilis.

[0090] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A modified microorganism, characterized in that The microorganism has a reduced or lost ribulose phosphate 3-epimerase activity compared to an unmodified microorganism, and the microorganism has an enhanced nucleoside production capacity compared to an unmodified microorganism; Among them, the reference sequence number of ribulose phosphate 3-epimerase from Bacillus amyloliquefaciens on NCBI is CBI42777.1, and the reference sequence number of ribulose phosphate 3-epimerase from Bacillus subtilis on NCBI is CAB13452.

1.

2. The microorganism according to claim 1, characterized in that The activity reduction or loss of ribulose phosphate 3-epimerase in the microorganism is achieved by selecting from the following 1)-3), or an optional combination: 1) reducing or losing the ribulose phosphate 3-epimerase by changing the amino acid sequence of the ribulose phosphate 3-epimerase; 2) reducing or losing the ribulose phosphate 3-epimerase by changing the nucleotide sequence encoding the ribulose phosphate 3-epimerase; 3) Loss by knocking out the coding sequence of the ribulose phosphate 3-epimerase.

3. The microorganism according to claim 2, characterized in that The method of mutagenesis, site-directed mutagenesis or homologous recombination is used to reduce the expression of the gene encoding the ribulose phosphate 3-epimerase or knock out the gene encoding the ribulose phosphate 3-epimerase.

4. The microorganism according to claim 1, characterized in that The reduction or loss of ribulose phosphate 3-epimerase activity is achieved by A, B, C, or D: A, the 77th amino acid of ribulose phosphate 3-epimerase mutated from S to A; B. The start codon of the gene encoding ribulose phosphate 3-epimerase mutates from atg to gtg or ttg; C. Completely knocking out the entire ORF frame of the gene encoding ribulose phosphate 3-epimerase or mutating any amino acid in the ORF frame into a stop codon; D. The codons of the gene encoding ribulose phosphate 3-epimerase were optimized, and the nucleotide mutation points of the optimized codons included G168A, T192C, A252G, and C264T.

5. The microorganism according to any one of claims 1 to 4, characterized in that The microorganism is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli.

6. A method for constructing a recombinant bacterium producing nucleosides and their derivatives, characterized in that: The method comprises: using genetic engineering means to inactivate or weaken the coding gene of ribulose phosphate 3-epimerase in a microorganism having nucleoside production ability; The weakening is achieved by selecting from the following a) to c), or any combination thereof: a) reducing or losing the ribulose phosphate 3-epimerase by changing the amino acid sequence of the ribulose phosphate 3-epimerase; b) reducing or losing the ribulose phosphate 3-epimerase by changing the nucleotide sequence encoding the ribulose phosphate 3-epimerase; c) lost by knocking out the coding sequence of the ribulose phosphate 3-epimerase; Among them, the reference sequence number of ribulose phosphate 3-epimerase from Bacillus amyloliquefaciens on NCBI is CBI42777.1, and the reference sequence number of ribulose phosphate 3-epimerase from Bacillus subtilis on NCBI is CAB13452.

1.

7. The method according to claim 6, characterized in that The weakening method is selected from at least one of mutagenesis, site-directed mutagenesis, and homologous recombination; Preferably, the weakening is achieved by ①, ②, ③ or ④ as follows: ① The 77th amino acid of ribulose phosphate 3-epimerase mutated from S to A; ② The start codon of the gene encoding ribulose phosphate 3-epimerase mutates from atg to gtg or ttg; ③ Completely knock out the entire ORF frame of the gene encoding ribulose phosphate 3-epimerase or mutate any amino acid in the ORF frame into a stop codon; ④ The codons of the gene encoding ribulose phosphate 3-epimerase were optimized, and the nucleotide mutation points of the optimized codons included G168A, T192C, A252G, and C264T; The microorganism is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli.

8. The recombinant bacteria constructed according to the method of claim 6 or 7.

9. Use of the microorganism according to any one of claims 1 to 5 or the recombinant bacterium according to claim 8 in the fermentation production of nucleosides or nucleoside derivatives or in increasing the fermentation yield of nucleosides or nucleoside derivatives; The nucleosides or nucleoside derivatives include inosine, guanosine, hypoxanthine, inosinic acid, guanine, guanylic acid, riboflavin, and diacetylguanylic acid.

10. A method for producing nucleosides, characterized in that: The method comprises the following steps: i) culturing the microorganism according to any one of claims 1 to 5 or the recombinant bacterium according to claim 8 to obtain a culture; ii) collecting the produced nucleosides from the culture obtained in step i).

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