Construction method and application of nucleoside-producing engineering bacteria

Abstract

The invention provides a construction method and application of nucleoside-producing engineering bacteria. By weakening or inactivating N-acetyl-gamma-glutamyl-phosphate reductase (encoded by an argC gene) in a central metabolic pathway, synthesis of a by-product arginine is inhibited, carbon source shunting is effectively blocked, nucleoside production is maximized, the actual conversion rate is increased, and efficient and rapid nucleoside production of microorganisms is promoted.

Description

Technical Field

[0001] The invention relates to the fields of bioengineering and microbial fermentation, and in particular to a construction method of nucleoside-producing engineering bacteria and its application. 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] Adenosine is adenine nucleoside, and its chemical name is 6-amino-9-β-D-ribofuranosyl-9-hydrogen purine. It is the product of adenine nucleotide dephosphorylation and is an important nucleotide derivative. Adenosine is an endogenous nucleoside that is found throughout human cells. It can directly enter the myocardium and generate adenosine acid through phosphorylation, and participate in myocardial energy metabolism. It also participates in dilating coronary vessels and increasing blood flow. Adenosine has 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 treating the heart, adenosine is also an important intermediate for the synthesis of adenosine triphosphate (ATP), adenine, adenosine acid, and adenosine arabinoside, and is widely used in the pharmaceutical and other industries.

[0005] 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

[0006] The purpose of the present invention is to provide a method for constructing a nucleoside-producing engineering bacterium and its application.

[0007] To achieve the purpose of the present invention, in a first aspect, the present invention provides a modified microorganism, wherein the activity of N-acetyl-γ-glutamyl-phosphate reductase is reduced or lost compared to an unmodified microorganism, and the microorganism has an enhanced nucleoside production capacity compared to an unmodified microorganism.

[0008] In the present invention, the reference sequence number of the N-acetyl-γ-glutamyl-phosphate reductase from Bacillus subtilis on NCBI is NP_389001.2 (encoded by the argC gene). The reference sequence number of the N-acetyl-γ-glutamyl-phosphate reductase from Bacillus amyloliquefaciens on NCBI is WP_013351802.1.

[0009] Further, the activity reduction or loss of N-acetyl-γ-glutamyl-phosphate reductase in the microorganism is achieved by being selected from the following 1)-3), or an optional combination:

[0010] 1) reducing or losing the N-acetyl-γ-glutamyl-phosphate reductase by changing the amino acid sequence of the N-acetyl-γ-glutamyl-phosphate reductase;

[0011] 2) reducing or losing the N-acetyl-γ-glutamyl-phosphate reductase by changing the nucleotide sequence encoding the N-acetyl-γ-glutamyl-phosphate reductase;

[0012] 3) Loss by knocking out the coding sequence of the N-acetyl-γ-glutamyl-phosphate reductase.

[0013] Furthermore, the expression of the gene encoding the N-acetyl-γ-glutamyl-phosphate reductase is reduced or the gene encoding the N-acetyl-γ-glutamyl-phosphate reductase is knocked out by using mutagenesis, site-directed mutagenesis or homologous recombination.

[0014] In a specific embodiment of the present invention, the reduction or loss of N-acetyl-γ-glutamyl-phosphate reductase activity is achieved by the following A, B or C:

[0015] A, amino acid 93 of N-acetyl-γ-glutamyl-phosphate reductase mutated from T to A, K or P (Bacillus subtilis);

[0016] B, amino acid 93 of N-acetyl-γ-glutamyl-phosphate reductase mutated from P to A or K (Bacillus amyloliquefaciens);

[0017] C. The 109th amino acid of N-acetyl-γ-glutamyl-phosphate reductase mutates from Y to C or S.

[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 nucleoside-producing engineered bacterium, the method comprising: inactivating or weakening the coding gene of N-acetyl-γ-glutamyl-phosphate reductase in a microorganism having the ability to produce nucleosides by means of genetic engineering.

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

[0021] a) reducing or losing the N-acetyl-γ-glutamyl-phosphate reductase by changing the amino acid sequence of the N-acetyl-γ-glutamyl-phosphate reductase;

[0022] b) reducing or losing the N-acetyl-γ-glutamyl-phosphate reductase by changing the nucleotide sequence encoding the N-acetyl-γ-glutamyl-phosphate reductase;

[0023] c) Loss by knocking out the coding sequence of the N-acetyl-γ-glutamyl-phosphate reductase.

[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 or ③ as follows:

[0026] ① The 93rd amino acid of N-acetyl-γ-glutamyl-phosphate reductase mutates from T to A, K or P (Bacillus subtilis);

[0027] ② The 93rd amino acid of N-acetyl-γ-glutamyl-phosphate reductase mutates from P to A or K (Bacillus amyloliquefaciens);

[0028] ③The 109th amino acid of N-acetyl-γ-glutamyl-phosphate reductase mutates from Y to C or S.

[0029] In a third aspect, the present invention provides an engineered bacterium constructed according to the method.

[0030] In a fourth aspect, the present invention provides the use of the microorganism or the engineered 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.

[0031] The nucleoside derivatives of the present invention include but are not limited to:

[0032] (1) Adenosine and adenosine-related derivatives, such as adenosine triphosphate (ATP), adenine, adenylic acid, adenosine arabinoside, etc.;

[0033] (2) Inosine and its derivatives, such as inosinic acid, guanylic acid, adenylic acid, etc.

[0034] (3) Guanosine and guanosine-related derivatives, such as guanine, guanylic acid, riboflavin, diacetylguanylic acid, etc.

[0035] In a fifth aspect, the present invention provides a method for producing nucleosides, the method comprising the steps of:

[0036] i) culturing the microorganism or the engineered bacteria constructed according to the method to obtain a culture;

[0037] ii) collecting the produced nucleosides from the culture obtained in step i).

[0038] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0039] The invention provides a Bacillus subtilis or Bacillus amyloliquefaciens, which inhibits the synthesis of byproduct arginine by weakening or inactivating N-acetyl-γ-glutamyl-phosphate reductase (encoded by argC gene) in the central metabolic pathway, and promotes microorganisms to efficiently and quickly generate nucleosides.

[0040] 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, guanosine, adenosine, etc. or corresponding nucleoside derivatives such as hypoxanthine, inosinic acid, guanine, guanylic acid, riboflavin, diacetylguanylic acid, etc. DETAILED DESCRIPTION

[0041] The present invention aims to provide a novel microorganism capable of efficiently producing purine nucleosides by means of genetic engineering. Starting from the genome level of Bacillus subtilis or Bacillus amyloliquefaciens, the present invention uses metabolic engineering technology to perform point mutation on the argC gene in the central metabolic pathway to improve the nucleoside production ability of the strain.

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

[0043] The invention provides a genetically engineered strain of Bacillus subtilis or Bacillus amyloliquefaciens. The engineered strain inhibits the synthesis of byproduct arginine by weakening or inactivating N-acetyl-γ-glutamyl-phosphate reductase (encoded by argC gene) in the central metabolic pathway, promotes microorganisms to efficiently and quickly generate guanosine, adenine nucleotides or inosine, and successfully creates a new microorganism capable of efficiently producing nucleosides.

[0044] ArgC is weakened by gene modification, and the weakening methods include amino acid mutation, gene inactivation, etc., but are not limited to the above two weakening methods.

[0045] The present invention also provides a method for constructing the genetically engineered strain of the above-mentioned Bacillus subtilis or Bacillus amyloliquefaciens, which comprises performing point mutation on the argC gene in the wild-type Bacillus subtilis or Bacillus amyloliquefaciens with nucleoside production ability, thereby changing the amino acid sequence encoding N-acetyl-γ-glutamyl-phosphate reductase and / or inactivating it.

[0046] Starting from Bacillus subtilis A5 (which has a certain ability to produce adenosine or inosine), mutant weakened or inactivated strains of argC were constructed, and the engineered strains obtained had a certain improvement in their ability to produce inosine or adenosine (Table 3).

[0047] The argC gene weakened strains were constructed from Bacillus amyloliquefaciens B.s833 and B.a836 (which have certain guanosine or inosine production capabilities), respectively, by point mutation of the argC gene, or / and inactivation of the gene. The nucleoside production capabilities of the constructed strains were improved to varying degrees (Table 3).

[0048] 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, guanosine, adenosine, etc. or corresponding nucleoside derivatives such as hypoxanthine, inosinic acid, guanine, guanylic acid, riboflavin, diacetylguanylic acid, etc.

[0049] The present invention further provides the use of the genetically engineered strain of Bacillus subtilis or Bacillus amyloliquefaciens in high-yield nucleosides.

[0050] 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.

[0051] The molecular biology reagents used in the present invention, such as DNA polymerase, DNA purification kit, restriction endonuclease, DNA ligase, etc., were purchased from Beijing Quanshijin Company (https: / / www.transgen.com / ), and other biochemical reagents were purchased from Sangon Biotechnology (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0052] The primer names and primer sequences involved in the following examples are shown in Table 1.

[0053] Table 1 Primer names and sequence information

[0054]

[0055]

[0056] Example 1 argC in Bacillus subtilis T93A Construction of point mutation strains

[0057] 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 upstream and downstream homologous arms of the argC gene were amplified using the primer pairs argC-1f / argC-T93A-1r and argC-T93A-2f / argC-2r and pfu high-fidelity DNA polymerase. The obtained fragments were gel-recovered and fused, and argC was amplified. T93AThe full-length fragment was gel-recovered (the nucleotide sequence of the corresponding ORF frame is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2). 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 gel-recovered. The linearized plasmid and argCT93A fragment after enzyme digestion were assembled using an assembly kit and transformed into TransT1 competent medium, and the recombinant plasmid pKSU-argCT93A was obtained by identification and screening at a later stage. The strain was transformed into B. subtilis A5, and transformants were screened at 30°C on LB plates containing 2.5 μg / mL chloramphenicol. The obtained transformants were inoculated into 5 ml LB liquid culture medium, cultured at 42°C 200 rpm for 12 h and passed for one generation, and diluted and spread on LB plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. The primary recombinants were inoculated into 5 ml LB liquid culture medium, cultured at 42°C 200 rpm for 12 h and passed for one generation, and diluted and spread on LB plates containing 0.8 μM 5-FU to screen secondary recombinants. The argCT93A point mutation strain was obtained and named B. subtilis A0046.

[0058] Example 2 argC in Bacillus subtilis T93K Construction of point mutation strains

[0059] Using the B.subtilis A5 genome as a template, using argC-1f / argC-T93K-1r, argC-T93K-2f / argC-2r primer pairs, pfu high-fidelity DNA polymerase amplification to obtain the upstream and downstream homologous arms of the argCT93K gene. The obtained fragments were gel-recovered and fused, and the argCT93K full-length fragment was amplified and gel-recovered (the nucleotide sequence of the corresponding ORF box is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4). The plasmid pKSU-argCT93K was constructed according to the construction method in Example 1, and transformed into the B.subtilis A5 strain, and the obtained strain was named B.subtilisA0047.

[0060] Example 3 argC in Bacillus subtilis T93P Construction of point mutation strains

[0061] Using the B.subtilis A5 genome as a template, using argC-1f / argC-T93P-1r, argC-T93P-2f / argC-2r primer pairs, pfu high-fidelity DNA polymerase amplification to obtain the upstream and downstream homologous arms of the argCT93P gene. The obtained fragments were gel-recovered and fused, and the argCT93P full-length fragment was amplified and gel-recovered (the nucleotide sequence of the corresponding ORF box is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6). The plasmid pKSU-argCT93P was constructed according to the construction method in Example 1, and transformed into the B.subtilis A5 strain, and the obtained strain was named B.subtilisA0048.

[0062] Example 4 argC in Bacillus subtilis Y109C Construction of point mutation strains

[0063] Using the B.subtilis A5 genome as a template, using argC-1f / argC-Y109C-1r, argC-Y109C-2f / argC-2r primer pairs, pfu high-fidelity DNA polymerase amplification to obtain the upstream and downstream homologous arms of the argCY109C gene. The obtained fragments were gel-recovered and fused, and the argCY109C full-length fragment was amplified and gel-recovered (the nucleotide sequence of the corresponding ORF frame is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8). The plasmid pKSU-argCY109C was constructed according to the construction method in Example 1, and transformed into the B.subtilis A5 strain. The obtained strain was named B.subtilisA0049.

[0064] Example 5 Construction of argC inactivated strain in Bacillus subtilis

[0065] Using the B.subtilis A5 genome as a template, using argC-1f / △argC-1r, △argC-2f / argC-2r primer pairs, pfu high-fidelity DNA polymerase amplification to obtain the upstream and downstream homologous arms of the △argC gene. The obtained fragments were gel-recovered and fused, and the full-length △argC fragment was amplified and gel-recovered (the nucleotide sequence of the corresponding ORF frame is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10). The plasmid pKSU-△argC was constructed according to the construction method in Example 1, and it was transformed into the B.subtilis A5 strain. The obtained strain was named B.subtilis A0050.

[0066] Example 6 argC in Bacillus amyloliquefaciens P93A Construction of point mutation strains

[0067] To determine whether the above point mutation has the same effect in Bacillus amyloliquefaciens, the mutation is also introduced therein for verification, using the DSM7 strain genome as a template, using DSM7-argC-1f / DSM7-argCP93A-1r, DSM7-argCP93A-2f / DSM7-argC-2r primer pairs, and pfu high-fidelity DNA polymerase amplification to obtain upstream and downstream homologous arms. The obtained fragments are gel-recovered and fused, and the argCP93A full-length fragment is amplified and gel-recovered (the nucleotide 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 plasmid pKSU-DSM7-argCP93A was constructed according to the construction method in Example 1, and was transformed into wild bacteria ATCC13952 and two guanosine-producing bacteria B.s833 and B.a836 constructed in the laboratory, and the strains obtained by screening were named Ba 8524, Bs 8406, and B.a8407, respectively.

[0068] Example 7 argC in Bacillus amyloliquefaciens Y109C Construction of point mutation strains

[0069] Using the DSM7 strain genome as a template, using DSM7-argC-1f / DSM7-argCY109C-1r, DSM7-argCY109C-2f / DSM7-argC-2r primer pairs, amplified to obtain the full-length fragment (the nucleotide sequence of the corresponding ORF box is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 14), and the plasmid pKSU-argCY109C was constructed according to the construction method in Example 1, and transformed into wild bacteria ATCC13952 and two guanosine-producing bacteria B.s833 and B.a836 constructed in the laboratory. The obtained strains were named Ba 8525, B.s8408, and B.a8409.

[0070] Example 8 argC in Bacillus amyloliquefaciens Y109S Construction of point mutation strains

[0071] Using the DSM7 strain genome as a template, using the DSM7-argC-1f / DSM7-argCY109S-1r and DSM7-argCY109S-2f / DSM7-argC-2r primer pairs, the full-length fragment was amplified (the nucleotide sequence of the corresponding ORF box is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16). The plasmid pKSU-argCY109S was constructed according to the construction method in Example 1, and transformed into the wild bacteria ATCC13952 and two guanosine-producing bacteria B.s833 and B.a836 constructed in the laboratory. The obtained strains were named Ba 8526, B.s8410, and B.a8411.

[0072] Example 9 Construction of argC inactivated strain in Bacillus amyloliquefaciens

[0073] Using the DSM7 strain genome as a template, using DSM7-argC-1f / DSM7-△argC-1r, DSM7-△argC-2f / DSM7-argC-2r primer pairs, amplified to obtain the full-length fragment (the nucleotide sequence of the corresponding ORF box is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18), and the plasmid pKSU-△argC was constructed according to the construction method in Example 1, and transformed into wild bacteria ATCC13952 and two guanosine-producing bacteria B.s833 and B.a836 constructed in the laboratory. The obtained strains were named Ba 8527, B.s8412, and B.a8413.

[0074] Example 10 Real-time quantitative fluorescence PCR verification of argC expression levels in each engineered strain

[0075] All argC mutant engineered bacteria and control strains B.subtilis A5 (control strain corresponding to Bacillus subtilis mutant strain) and 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:

[0076] Table 2. Transcription level of strains

[0077]

[0078]

[0079] As can be seen from the table, the transcription levels of the modified bacteria are reduced to varying degrees compared with the starting strains, indicating that the above-mentioned point mutations have achieved the effect of weakening the gene transcription level.

[0080] Example 11 Verification of the nucleoside production performance of mutant strains

[0081] 1. Culture the bacteria stored in glycerol overnight at 37°C and isolate a single colony.

[0082] 2. Pick a single colony and inoculate it into 30 mL 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 37°C, 110 rpm shaking for 7-8 h.

[0083] 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 35.5°C (Bs / a8406-8413 strain fermentation for 72 h; B. subtilis A0046-0050 strain fermentation for 48 h).

[0084] 4. Use liquid chromatography to detect the glycosides produced in the fermentation broth. The results are shown in Table 3.

[0085] Table 3 Evaluation results of mutant strains producing guanosine, inosine and adenosine by shake flask fermentation (mean of three replicates)

[0086]

[0087]

[0088] As can be seen from the table above, the mutated amino acid T / P at position 93 of argC to A / K / P, or the amino acid Y at position 109 to C / S, as well as the direct inactivation of the gene, and the introduction of them into different starting strains, all increased the corresponding nucleoside production to varying degrees. Combined with the conversion rate level data, it shows that the weakening of this site is effective in improving the nucleoside production capacity of the strain.

[0089] 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 Compared with an unmodified microorganism, the activity of N-acetyl-γ-glutamyl-phosphate reductase in the microorganism is reduced or lost, and the microorganism has an enhanced nucleoside production capacity compared with an unmodified microorganism; Among them, the reference sequence number of N-acetyl-γ-glutamyl-phosphate reductase from Bacillus subtilis on NCBI is NP_389001.2, and the reference sequence number of N-acetyl-γ-glutamyl-phosphate reductase from Bacillus amyloliquefaciens on NCBI is WP_013351802.

1.

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

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 N-acetyl-γ-glutamyl-phosphate reductase or knock out the gene encoding the N-acetyl-γ-glutamyl-phosphate reductase.

4. The microorganism according to claim 1, characterized in that The reduction or loss of N-acetyl-γ-glutamyl-phosphate reductase activity is achieved by A, B, or C as follows: A, amino acid 93 of N-acetyl-γ-glutamyl-phosphate reductase mutated from T to A, K or P; B, amino acid 93 of N-acetyl-γ-glutamyl-phosphate reductase mutated from P to A or K; C. The 109th amino acid of N-acetyl-γ-glutamyl-phosphate reductase mutates from Y to C or S.

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 nucleoside-producing engineering bacterium, characterized in that: The method comprises: using genetic engineering means to inactivate or weaken the coding gene of N-acetyl-γ-glutamyl-phosphate reductase in a microorganism having the ability to produce nucleosides; The weakening is achieved by selecting from the following a) to c), or any combination thereof: a) reducing or losing the N-acetyl-γ-glutamyl-phosphate reductase by changing the amino acid sequence of the N-acetyl-γ-glutamyl-phosphate reductase; b) reducing or losing the N-acetyl-γ-glutamyl-phosphate reductase by changing the nucleotide sequence encoding the N-acetyl-γ-glutamyl-phosphate reductase; c) lost by knocking out the coding sequence of the N-acetyl-γ-glutamyl-phosphate reductase; The reference sequence number of the N-acetyl-γ-glutamyl-phosphate reductase on NCBI is NP_389001.

2.

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 or ③ as follows: ① The 93rd amino acid of N-acetyl-γ-glutamyl-phosphate reductase mutates from T to A, K or P; ② The 93rd amino acid of N-acetyl-γ-glutamyl-phosphate reductase mutates from P to A or K; ③ The 109th amino acid of N-acetyl-γ-glutamyl-phosphate reductase mutates from Y to C or S; The microorganism is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli.

8. An engineered bacterium 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 engineered bacteria 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 engineered bacteria according to claim 8 to obtain a culture; ii) collecting the produced nucleosides from the culture obtained in step i).

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