Presumed sugar phosphate epimerase mutant strain, construction method thereof and application thereof in nucleoside production

Abstract

The invention provides an assumed sugar phosphate epimerase mutant strain as well as a construction method and application thereof in nucleoside production. The method comprises the following steps: carrying out point mutation on an iolH gene in wild type or nucleoside-producing bacillus amyloliquefaciens or bacillus subtilis by utilizing a gene engineering technology, so that an amino acid sequence for coding assumed sugar phosphate epimerase is changed, and / or the whole ORF frame is completely knocked out; and a new microorganism capable of efficiently producing nucleoside is successfully constructed.

Description

Technical Field

[0001] The invention relates to the fields of bioengineering and microbial fermentation, and in particular to a putative sugar-phosphate epimerase mutant strain and a construction method thereof, as well as an application thereof in nucleoside production. Background Art

[0002] Nucleoside is a glycoside formed by the condensation of D-ribose or DZ-deoxyribose with a pyrimidine base or a purine base. D-ribose condenses with adenine, guanine, hypoxanthine, cytosine, thymine or uracil to form the corresponding adenine ribonucleoside, guanine ribonucleoside, cytosine ribonucleoside, thymine ribonucleoside and uracil ribonucleoside, which are respectively abbreviated as adenosine (A), guanosine (G), inosine (I), cytidine (C), thymidine (T) and uridine (U).

[0003] Guanosine and inosine are widely used in the food and pharmaceutical industries. In the food field, guanosine and inosine are important precursors of disodium guanylate and disodium inosinate, respectively. The food flavor enhancer formed by the combination of disodium guanylate and disodium inosinate is 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 riboside, sodium guanosine triphosphate, etc., 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, optic atrophy, etc. Adenosine is an endogenous nucleoside that is distributed throughout human cells. It can directly enter the myocardium to generate adenosine through phosphorylation, participate in myocardial energy metabolism, and also participate in the dilation of coronary blood vessels and increase blood flow. Adenosine has important physiological effects on the cardiovascular system and many other systems and tissues of the body. In addition to being used as a specific drug for the treatment of heart disease, adenosine is also an important intermediate for the synthesis of adenosine triphosphate (ATP), adenine, adenylic acid, and adenosine arabinoside, and is widely used in the pharmaceutical industry and other industries.

[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. At present, the fermentation performance of nucleoside production strains is still poor, and the conversion rate of nucleosides is still low, which cannot meet the needs of large-scale industrial production. Therefore, it is still necessary to develop new metabolic engineering transformation targets and strains related to nucleoside production. Summary of the invention

[0005] The purpose of the present invention is to provide a putative sugar-phosphate epimerase mutant strain and a construction method thereof as well as an application thereof in nucleoside production.

[0006] To achieve the purpose of the present invention, in the first aspect, the present invention provides a modified microorganism, wherein the activity of its putative sugar-phosphate epimerase (encoded by the iolH gene) 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 the putative sugar-phosphate epimerase in NCBI is CAB16005.1 or CBI44907.1.

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

[0009] 1) reducing or losing the putative sugar phosphate epimerase by changing the amino acid sequence of the putative sugar phosphate epimerase;

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

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

[0012] Furthermore, mutagenesis, site-directed mutagenesis or homologous recombination are used to reduce the expression of the gene encoding the putative sugar-phosphate diastereomerase or knock out the gene encoding the putative sugar-phosphate diastereomerase.

[0013] In a specific embodiment of the present invention, the reduction or loss of the hypothetical sugar phosphate epimerase activity is achieved by A, B, C or D as follows:

[0014] A. Mutate the 105th amino acid of the putative sugar-phosphate epimerase from L to I;

[0015] B. Mutate the base encoding the 55th amino acid of the putative sugar-phosphate epimerase into a terminator;

[0016] C. The start codon of the gene encoding the putative sugar phosphate epimerase was mutated from atg to gtg;

[0017] D. Knockout of the ORF frame encoding the putative sugar phosphate epimerase.

[0018] The microorganism can be Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli, etc. For example, the starting strain is B. subtilis 168, B. subtilis A5 (see CN110257315B), Bs 833, Ba 836 (see CN112574934A).

[0019] In a second aspect, the present invention provides a method for constructing a nucleoside-producing strain, the method comprising: utilizing genetic engineering means to inactivate or weaken the coding gene of the putative sugar-phosphate diastereomerase in the microorganism.

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

[0021] a) reducing or losing the putative sugar phosphate epimerase by changing the amino acid sequence of the putative sugar phosphate epimerase;

[0022] b) reducing or losing the putative sugar phosphate epimerase by changing the nucleotide sequence encoding the putative sugar phosphate epimerase;

[0023] c) Loss by knocking out the coding sequence of the putative sugar phosphate 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 Mutate the 105th amino acid of the putative sugar-phosphate isomerase from L to I;

[0027] ② Mutate the base encoding the 55th amino acid of the putative sugar-phosphate isomerase into a terminator;

[0028] ③ The start codon of the gene encoding the putative sugar phosphate isomerase was mutated from atg to gtg;

[0029] ④ Knock out the ORF frame encoding the putative sugar phosphate isomerase.

[0030] In a third aspect, the present invention provides a putative sugar-phosphate epimerase mutant strain constructed according to the method.

[0031] In a fourth aspect, the present invention provides the use of the microorganism or the putative sugar-phosphate diastereomerase mutant strain 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 nucleoside derivatives of the present invention include but are not limited to:

[0033] ① Adenosine and adenosine-related derivatives, such as adenosine triphosphate (ATP), adenine, adenylic acid, adenosine arabinoside, etc.;

[0034] ②Inosine and its derivatives, such as inosinic acid, guanylic acid, adenylic acid, etc.;

[0035] ③Guanosine and guanosine-related derivatives, such as guanine, guanylic acid, riboflavin, diacetylguanylic acid, etc.

[0036] In a fifth aspect, the present invention provides a method for producing nucleosides, the method comprising the steps of:

[0037] i) culturing the microorganism or the putative sugar-phosphate epimerase mutant strain constructed according to the method to obtain a culture;

[0038] ii) collecting the produced nucleosides from the culture obtained in step i).

[0039] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0040] The present invention uses Bacillus amyloliquefaciens Bs 833 and Ba 836 (which have a certain guanosine or inosine production capacity) as starting strains to construct iolH gene weakened strains, and the weakening method is to perform point mutation on the iolH gene, and / or mutate its start codon ATG into GTG and / or completely knock out its entire ORF frame. The strains obtained by constructing have different degrees of improvement in nucleoside production capacity. At the same time, the present invention uses Bacillus subtilis B.subtilis A5 (which has a certain adenosine or inosine production capacity) as a starting strain to construct iolH mutation and / or mutate its start codon ATG into GTG and / or completely knock out its entire ORF frame. The engineered strains obtained by constructing have certain improvement in their ability to produce inosine or adenosine.

[0041] The above-mentioned transformation can be applied to Bacillus amyloliquefaciens and Bacillus subtilis, 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, guanosine, adenosine, etc. or corresponding nucleoside derivatives such as hypoxanthine, inosinic acid, guanine, guanylic acid, riboflavin, diacetylguanylic acid, etc. DETAILED DESCRIPTION

[0042] The present invention aims to provide a construction and application of a recombinant bacterium with high nucleoside production, and specifically relates to a mutant encoding a putative sugar-phosphate epimerase / isomerase IolH (with a reference sequence number of CAB16005.1 or CBI44907.1 on NCBI) and an application thereof.

[0043] Genetic engineering technology is used to create a new microorganism capable of efficiently producing purine nucleosides. The invention starts from the genome level of Bacillus subtilis or Bacillus amyloliquefaciens and improves the ability of strains to produce nucleosides by weakening the iolH gene.

[0044] The present invention adopts the following technical solution:

[0045] In the first aspect, the present invention provides a genetically engineered strain of Bacillus amyloliquefaciens or Bacillus subtilis, which weakens the activity of the IolH protein by weakening the hypothetical sugar-phosphate diastereomerase (encoded by the iolH gene), so that the microorganism can efficiently and quickly generate guanosine, adenine nucleotides or inosine, and successfully creates a new microorganism that can efficiently produce nucleosides.

[0046] In a second aspect, the present invention provides a method for constructing the genetically engineered strain of Bacillus amyloliquefaciens or Bacillus subtilis, comprising performing point mutation on the iolH gene in wild-type or nucleoside-producing Bacillus amyloliquefaciens or Bacillus subtilis to change the amino acid sequence encoding the putative sugar-phosphate epimerase, and / or mutating the start codon ATG to GTG and / or completely knocking out the entire ORF frame.

[0047] In a third aspect, the present invention provides use of the genetically engineered strain of Bacillus amyloliquefaciens or Bacillus subtilis in high-production of nucleosides.

[0048] 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.

[0049] The primer names and primer sequences involved in the following examples are shown in Table 1.

[0050] Table 1 Primer names and sequence information

[0051]

[0052]

[0053] Example 1: IolH in Bacillus subtilis L105I Construction of point mutation strains

[0054] The genome of the adenosine-producing strain B. subtilis A5 (strain B. subtilis A5, see CN110257315B) constructed in the laboratory was used as a template, and the left and right homologous arms were amplified using the primer pairs A5-IolHL105I-1f / 1r and A5-IolHL105I-2f / 2r, and then fused to obtain A5-IolHL L105I The full-length fragment (the sequence of the corresponding ORF frame is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6). The pKSU plasmid (pKSU plasmid was kindly donated by Professor Wang Shufang of Nankai University, see A markerless gene replacement method for B. amyloliquefaciens LL3 and its use ingenome reduction and improvement of poly-γ-glutamic acid production [J], Applied Microbiology and Biotechnology, 2014, 98 (21): 8963-8973. Zhang W, Gao W, Feng J, et al DOI: 10.1007 / s00253-014-5824-2) was double-digested with XbaI / PstI and recovered by gel. The linearized plasmid and A5-IolH were assembled using an assembly kit. L105I The fragments were assembled and transformed into TransT1 competent cells, and then the recombinant plasmid pKSU-A5-IolH was obtained by identification and screening. L105I . They were transformed into wild bacteria 168 and B. subtilis A5 strains, and A5-IolH was obtained by screening. L105I The strains were named B. subtilis A0134 and B. subtilis A0135.

[0055] The sequence of the ORF frame encoding the putative sugar-phosphate epimerase from Bacillus subtilis 168 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0056] Example 2: IolH in Bacillus subtilis Q55* Construction of point mutation strains

[0057] The genome of the adenosine-producing strain B. subtilis A5 (strain B. subtilis A5, see CN110257315B) constructed in the laboratory was used as a template, and the left and right homologous arms were amplified using the primer pairs A5-IolH55-1f / 1r and A5-IolH55-2f / 2r, and then fused to obtain A5-IolH Q55* The full-length fragment (the corresponding ORF frame sequence is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8). The recombinant plasmid pKSU-A5-IolH was obtained according to the construction method in Example 1. Q55* The mutants were transformed into wild bacteria 168 and B. subtilis A5, and the strains with the 55th amino acid codon of IolH mutated from CAA to TAA were screened and named B. subtilis A0136 and B. subtilis A0137, respectively.

[0058] Example 3: Construction of a strain with weakened initiation codon (ATG-GTG) of the iolH gene in Bacillus subtilis

[0059] The genome of the adenosine-producing strain B. subtilis A5 (strain B. subtilis A5, see CN110257315B) constructed in the laboratory was used as a template, and the left and right homologous arms were amplified using the primer pairs A5-IolH 1-1f / 1r and A5-IolH 1-2f / 2r, and then fused to obtain A5-IolH M1V The full-length fragment (the corresponding ORF frame sequence is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10). The recombinant plasmid pKSU-A5-IolH was obtained according to the construction method in Example 1. M1V The clones were transformed into wild bacteria 168 and B. subtilis A5, and strains with the IolH start codon ATG mutated to GTG were obtained. The strains were named B. subtilis A0138 and B. subtilis A0139, respectively.

[0060] Example 4: Construction of iolH knockout strain in Bacillus subtilis

[0061] The genome of the adenosine production strain B. subtilis A5 (strain B. subtilis A5, see CN110257315B) constructed in the laboratory was used as a template, and the left and right homology arms were amplified using the primer pairs A5-ΔIolH-1f / 1r and A5-ΔIolH-2f / 2r, and the full-length fragment of A5-ΔIolH was obtained by fusion. The recombinant plasmid pKSU-A5-ΔIolH was obtained according to the construction method in Example 1. It was transformed into wild bacteria 168 and B. subtilis A5 strains, and the A5-ΔIolH strains were screened and obtained. The strains were named B. subtilis A0140 and B. subtilis A0141, respectively.

[0062] Example 5: IolH in Bacillus amyloliquefaciens L105I Point mutation strain construction

[0063] Using the genome of DSM7 strain as template, the left and right homology arms were amplified using primer pairs IolHL105I-1f / 1r and IolHL105I-2f / 2r, and fused to obtain IolH L105I Full-length fragment (the sequence of the corresponding ORF box is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 12). The recombinant plasmid pKSU-IolH L105I was obtained according to the construction method in Example 1, and was transformed into the DSM7 model strain and two guanosine-producing strains B.s833 and Ba 836 constructed in the laboratory. The obtained strains were named B.a8471, B.s8472, and B.a8473.

[0064] The sequence of the ORF frame encoding the putative sugar-phosphate epimerase of Bacillus amyloliquefaciens DSM7 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

[0065] Example 6: IolH in Bacillus amyloliquefaciens Q55* Point mutation strain construction

[0066] Using the genome of DSM7 strain as template, the left and right homology arms were amplified using primer pairs IolH55-1f / 1r and IolH55-2f / 2r, and fused to obtain IolH Q55* The full-length fragment (the corresponding ORF frame sequence is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 14). The recombinant plasmid pKSU-IolH was obtained according to the construction method in Example 1. Q55*The guanosine was transformed into the DSM7 model strain and two guanosine-producing strains B.s833 and Ba 836 constructed in the laboratory. The strains with the CAA mutation of the 55th amino acid codon of IolH to TAA were screened and named B.a8474, B.s8475 and B.a8476 respectively.

[0067] Example 7: Construction of a strain with weakened initiation codon (ATG-GTG) of iolH gene in Bacillus amyloliquefaciens

[0068] Using the genome of DSM7 strain as template, the left and right homology arms were amplified using primer pairs IolH1-1f / 1r and IolH1-2f / 2r, and fused to obtain IolH M1V The full-length fragment (the corresponding ORF frame sequence is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16). The recombinant plasmid pKSU-IolH was obtained according to the construction method in Example 1. M1V The strains were transformed into the DSM7 model strain and two guanosine-producing strains B.s833 and B.a836 constructed in the laboratory. The strains in which the IolH start codon ATG mutated to GTG were screened and named B.a8477, B.s8478 and B.a8479 respectively.

[0069] Example 8: Construction of iolH knockout strain in Bacillus amyloliquefaciens

[0070] Using the genome of the DSM7 strain as a template, the left and right homology arms were amplified using the ΔIolH-1f / 1r and ΔIolH-2f / 2r primer pairs, and the full-length fragment of ΔIolH was obtained by fusion. The recombinant plasmid pKSU-ΔIolH was obtained according to the construction method in Example 1, and was transformed into the DSM7 model strain and two guanosine-producing strains B.s833 and B.a836 constructed in the laboratory. The obtained strains were named B.a8480, B.s8481, and B.a8482.

[0071] Example 9: Real-time quantitative fluorescence PCR verification of iolH expression levels in each engineered strain

[0072] All iolH-modified engineered bacteria and control strains B. subtilis A5 (control strain corresponding to Bacillus subtilis mutant strain) and DSM7, B.s833, B.a836 (control strains corresponding to Bacillus amyloliquefaciens mutant strain) were cultured in LB medium until the logarithmic growth phase, 1 mL of bacterial solution was treated with an appropriate amount of lysozyme, and total RNA was extracted for reverse transcription, and real-time quantitative PCR reaction was performed using cDNA as a template. Reaction conditions: 95℃ pre-denaturation for 10min; 95℃15s, 55℃1min, 40 cycles. After the reaction, the bacterial 16S rRNA was used as a reference, and the transcription level of the relevant gene was calculated according to the 2-ΔΔCT method. The results are shown in Table 2:

[0073] Table 2. Transcription level of strains

[0074] Strain number Transcription level (relative value) Reduction ratio% B. subtilis 168 1 - B. subtilis A0134 0.72 28％ B. subtilis A0136 0.29 71％ B. subtilis A0138 0.41 59％ B. subtilis A0140 0.06 99.4％ B. subtilis A5 1 - B. subtilis A0135 0.69 31％ B. subtilis A0137 0.25 75％ B. subtilis A0139 0.40 60％ B. subtilis A0141 0.04 99.6％ DSM7 1 - B.a8471 0.57 43％ B.a8474 0.26 73％ B.a8477 0.39 61％ B.a8480 0.07 99.3％ B.s833 1 - B.s8472 0.66 34％ B.s8475 0.31 69％ B.s8478 0.46 54％ B.s8481 0.07 99.3％ B.a836 1 - B.a8473 0.67 33％ B.a8476 0.33 67％ B.a8479 0.52 48％ B.a8482 0.05 99.5％

[0075] As can be seen from Table 2, the transcription levels of the modified bacteria were reduced to varying degrees compared with the starting strains, indicating that the above-mentioned modifications have achieved the effect of weakening the gene transcription level.

[0076] Example 10: Verification of nucleoside production performance of mutant strains

[0077] 1. Culture the bacteria stored in glycerol overnight at 37°C and isolate a single colony.

[0078] 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, sterilized at 121°C for 20 min), and culture at 110 rpm and 37°C for 7-8 h.

[0079] 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, sterilize at 121°C for 20 min) at a 10% v / v inoculum volume, shake at 130 rpm, and culture at 35°C for 70 h (Bs / a8471-8482 strain fermentation for 72 h; B. subtilis A0134-0141 strain fermentation for 48 h).

[0080] 4. Use liquid chromatography to detect the glycosides produced in the fermentation broth (Table 3).

[0081] Table 3 Evaluation results of mutant strains producing guanosine, inosine and adenosine by shake flask fermentation (mean of three replicates)

[0082] strains Guanosine yield (g / L) Inosine production (g / L) Adenosine production (g / L) <![CDATA[OD 562 ]]> B. subtilis 168 0 0 0 32.2 B. subtilis A0134 0.09 0.12 0.13 31.7 B. subtilis A0136 0.10 0.11 0.19 31.1 B. subtilis A0138 0.12 0.11 0.18 31.4 B. subtilis A0140 0.12 0.13 0.25 31.6 B. subtilis A5 0 1.11 8.73 24.9 B. subtilis A0135 0 0.97 9.26 25.3 B. subtilis A0137 0 1.71 8.97 25.6 B. subtilis A0139 0 2.12 9.95 24.3 B. subtilis A0141 0 2.75 10.09 24.8 DSM7 0 0 0 36.1 B.a8471 0.31 0.27 0.09 34.9 B.a8474 0.39 0.48 0.10 35.7 B.a8477 0.35 0.11 0.12 34.6 B.a8480 0.45 0.09 0.16 35.1 B.s833 1.28 0.22 0.81 25.8 B.s8472 1.49 1.23 1.17 25.6 B.s8475 1.65 1.32 1.21 25.7 B.s8478 2.35 1.19 1.08 26.3 B.s8481 2.68 1.44 0.97 26.4 B.a836 1.17 0.69 0.34 27.9 B.a8473 1.78 0.93 0.89 27.8 B.a8476 2.07 0.72 0.79 28.1 B.a8479 2.88 0.71 0.66 27.8 B.a8482 3.04 1.03 0.34 28.3

[0083] As can be seen from Table 3, when the L at position 105 of IolH was mutated to I, or the termination mutation at position 55 of IolH or the start codon was weakened or the entire ORF frame was knocked out and introduced into different starting strains, the corresponding nucleoside production was improved to varying degrees, indicating that the weakening of this site is effective in improving the nucleoside production capacity of the strain.

[0084] 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 activity of a putative sugar-phosphate epimerase compared to an unmodified microorganism, and the microorganism has an enhanced ability to produce nucleosides compared to an unmodified microorganism; The reference sequence number of the putative sugar-phosphate isomerase in NCBI is CAB16005.1 or CBI44907.

1.

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

4. The microorganism according to claim 1, characterized in that The hypothesized reduction or loss of sugar phosphate epimerase activity is achieved by A, B, C, or D as follows: A. Mutate the 105th amino acid of the putative sugar-phosphate epimerase from L to I; B. Mutate the base encoding the 55th amino acid of the putative sugar-phosphate epimerase into a terminator; C. The start codon of the gene encoding the putative sugar phosphate epimerase was mutated from atg to gtg; D. Knockout of the ORF frame encoding the putative sugar phosphate epimerase.

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 putative sugar-phosphate epimerase mutant strain, characterized in that: The method comprises: using genetic engineering means to inactivate or weaken the coding gene of the assumed sugar-phosphate diastereomerase in the microorganism; The weakening is achieved by selecting from the following a) to c), or any combination thereof: a) reducing or losing the putative sugar phosphate epimerase by changing the amino acid sequence of the putative sugar phosphate epimerase; b) reducing or losing the putative sugar phosphate epimerase by changing the nucleotide sequence encoding the putative sugar phosphate epimerase; c) lost by knocking out the coding sequence of the putative sugar phosphate epimerase; The reference sequence number of the putative sugar-phosphate isomerase in NCBI is CAB16005.1 or CBI44907.

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 Mutate the 105th amino acid of the putative sugar-phosphate isomerase from L to I; ② Mutate the base encoding the 55th amino acid of the putative sugar-phosphate isomerase into a terminator; ③ The start codon of the gene encoding the putative sugar phosphate isomerase was mutated from atg to gtg; ④ Knock out the ORF frame encoding the putative sugar phosphate isomerase; The microorganism is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli.

8. A putative sugar-phosphate epimerase mutant strain 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 putative sugar-phosphate epimerase mutant strain 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 adenosine, adenosine triphosphate, adenine, adenylic acid, adenosine arabinoside, inosine, inosinic acid, guanylic acid, adenylic acid, guanosine, 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 putative sugar-phosphate epimerase mutant strain according to claim 8 to obtain a culture; ii) collecting the produced nucleosides from the culture obtained in step i).

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