AimA mutant as well as construction method and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to an AimA mutant as well as a construction method and application thereof. It is found for the first time that the activity of glutamic acid / serine transporter is weakened, and the nucleoside production capacity of microorganisms can be improved. Based on this, the present invention provides a modified microorganism in which the glutamic acid / serine transporter activity is reduced or lost as compared to an unmodified microorganism, and the nucleoside production capacity of the modified microorganism is enhanced as compared to the unmodified microorganism, and more specifically, the modified microorganism has a reduced or lost glutamic acid / serine transporter activity as compared to the unmodified microorganism. The modified bacillus subtilis or bacillus amyloliquefaciens provided by the invention can generate guanine nucleoside or hypoxanthine nucleoside with high efficiency and high speed.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to an AimA mutant, a construction method thereof, and an application thereof. Background Art

[0002] Nucleosides are a general term for a class of glycosides. Nucleosides are components of nucleic acids and nucleotides. Nucleosides are formed by the condensation of D-ribose or D-α-deoxyribose with pyrimidine bases or purine bases. Nucleosides are generally colorless crystals, insoluble in common organic solvents, soluble in hot water, and have a melting point of 160-240°C. Nucleosides formed from D-ribose are called ribonucleosides and participate in the composition of RNA. Nucleosides formed from D-α-deoxyribose are called deoxyribonucleosides and participate in the composition of DNA. D-ribose condenses with adenine, guanine, cytosine, thymine, or uracil to form the corresponding adenosine ribonucleoside, guanosine ribonucleoside, cytidine ribonucleoside, thymidine ribonucleoside, and uridine ribonucleoside, which are abbreviated as adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U), respectively.

[0003] Guanosine and inosine have a wide range of functions in the food and pharmaceutical industries. In the food field, guanosine and inosine are important precursors of disodium guanylate and disodium inosinate, respectively. The combination of disodium guanylate and disodium inosinate is used as a food flavor enhancer and is widely used in seasonings 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. For example, acyclovir, ribavirin, sodium guanosine triphosphate, etc. all require guanosine as a synthesis raw material. Inosine is an important precursor of inosinic acid, and inosinic acid can be used as a precursor for the synthesis of adenylic acid (AMP) and guanylic acid (GMP). It is applicable to leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis, cirrhosis, etc. caused by various reasons. In addition, it can also treat central retinitis, optic nerve atrophy, etc.

[0004] Adenosine, that is, adenosine ribonucleoside, has the chemical name of 6-amino-9-β-D-ribofuranosyl-9H-purine. It is the product after the dephosphorylation of adenine nucleotide and belongs to an important nucleotide derivative. Adenosine is an endogenous nucleoside that pervades human cells. It can directly enter the myocardium and be phosphorylated to form adenylic acid, participating in myocardial energy metabolism. At the same time, it also participates in dilating coronary blood 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, adenylic acid, and vidarabine, and is widely used in industries such as medicine.

[0005] At present, microbial fermentation is the main method for producing nucleosides. The main microorganisms used include Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus pumilus, etc. During the breeding and modification of growth strains, high-yield nucleoside strains are directionally selected through the use of ultraviolet mutagenesis and diethyl sulfate mutagenesis breeding; or based on 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 modified through metabolic engineering means to obtain production strains with excellent traits and high nucleoside yields.

[0006] However, the fermentation performance of current nucleoside strains is still poor, and the conversion rate of nucleosides is still low, unable to meet the requirements of large-scale industrial production. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for producing purine nucleosides using microorganisms. To achieve the purpose of the present invention, the present invention provides new microorganisms capable of efficiently producing purine nucleosides, and uses the new microorganisms to produce purine nucleosides.

[0008] In the first aspect, the present invention provides a modified microorganism. Compared with the unmodified microorganism, the activity of its glutamate / serine transporter is reduced or lost, and the microorganism has enhanced nucleoside production ability compared with the unmodified microorganism;

[0009] Among them, the amino acid sequence of the glutamate / serine transporter from Bacillus amyloliquefaciens is shown in SEQ ID NO.2; the amino acid sequence of the glutamate / serine transporter from Bacillus subtilis is shown in SEQ ID NO.4.

[0010] The coding of AimA protein in Bacillus amyloliquefaciens on NCBI is CBI41329.1; the coding of AimA protein in Bacillus subtilis on NCBI is CAB12006.2.

[0011] The reduction or loss of the activity of the glutamate / serine transporter in the modified microorganism provided by the present invention is achieved by selecting from the following 1)-3), or an optional combination:

[0012] 1) Changing the amino acid sequence of the glutamate / serine transporter;

[0013] 2) Changing the nucleotide sequence encoding the glutamate / serine transporter;

[0014] 3) Knocking out the coding sequence of the glutamate / serine transporter.

[0015] The modified microorganism provided by the present invention uses mutagenesis, site-directed mutagenesis or homologous recombination to reduce the expression of the coding gene of the glutamate / serine transporter or knock out the coding gene of the glutamate / serine transporter.

[0016] In the modified microorganism provided by the present invention, the reduction or loss of glutamate / serine transporter activity is achieved by the following A, B or C:

[0017] A. The 506th amino acid of the glutamate / serine transporter undergoes a nonsense mutation from W;

[0018] B. The 93rd amino acid of the glutamate / serine transporter undergoes a nonsense mutation from W;

[0019] C. The start codon ATG of the glutamate / serine transporter is weakened to GTG or TTG.

[0020] The microorganism of the present invention is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli.

[0021] The present invention constructs the above-mentioned modified microorganisms using Bacillus subtilis and Bacillus amyloliquefaciens (wild-type strains, genetically engineered production strains) with different genetic backgrounds as starting strains. After verification, after replacing the genes of the glutamate / serine transporters of these starting strains with the coding genes of glutamate / serine transporter mutants, the nucleoside production of the strains is significantly increased. Thus, it can be seen that the improvement effect of the glutamate / serine transporter mutant on the nucleoside production ability of the strain does not depend on other genetic modifications contained in the above strains, and these genetic modifications are only to enable the strain to have a certain nucleoside production ability. Therefore, the improvement effect of the glutamate / serine transporter mutant on the nucleoside production ability of microorganisms is universal for starting strains that can synthesize and accumulate nucleosides.

[0022] Thus, those skilled in the art can understand that introducing the glutamate / serine transporter mutant into other nucleoside-producing strains can effectively promote the nucleoside production.

[0023] In a second aspect, the present invention provides a method for constructing a nucleoside-producing engineering bacterium, and the construction method includes: using genetic engineering means to inactivate or weaken the coding gene of the glutamate / serine transporter in a microorganism having nucleoside production ability;

[0024] The weakening is achieved by the following a)-c), or an optional combination:

[0025] a) By changing the amino acid sequence of the glutamate / serine transporter;

[0026] b) By changing the nucleotide sequence encoding the glutamate / serine transporter;

[0027] c) By knocking out the coding sequence of the glutamate / serine transporter;

[0028] Wherein, the coding sequence of the glutamate / serine transporter is as shown in SEQ ID NO.1 or SEQ ID NO.3.

[0029] In the method for constructing the nucleoside-producing engineering bacteria provided by the present invention, the weakening method is selected from at least one of mutagenesis, site-directed mutagenesis, and homologous recombination;

[0030] Preferably, the weakening is achieved by the following (1), (2), or (3):

[0031] (1) The 506th amino acid of the glutamate / serine transporter undergoes a nonsense mutation from W;

[0032] (2) The 93rd amino acid of the glutamate / serine transporter undergoes a nonsense mutation from W;

[0033] (3) The start codon ATG of the glutamate / serine transporter is weakened to GTG or TTG;

[0034] The microorganism is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, or Escherichia coli.

[0035] In the third aspect, according to the understanding of those skilled in the art, the present invention provides an engineering bacterium constructed by using the method for constructing the nucleoside-producing engineering bacterium described above.

[0036] In the fourth aspect, the present invention provides the use of the above microorganism or the above engineering bacterium in the fermentation production of nucleosides or nucleoside derivatives or in increasing the fermentation yield of nucleosides or nucleoside derivatives;

[0037] The nucleosides or nucleoside derivatives include adenosine, adenosine triphosphate, adenine, adenylic acid, vidarabine, inosine, inosinic acid, guanylic acid, adenylic acid, guanosine, guanine, guanylic acid, riboflavin, and diacetyl guanylic acid.

[0038] In the fifth aspect, the present invention provides a method for producing nucleosides, and the method includes the following steps:

[0039] i) Cultivate the above-mentioned microorganism or the above-mentioned engineered bacterium to obtain a culture;

[0040] ii) Collect the produced nucleoside from the culture obtained in step i).

[0041] In the sixth aspect, there is currently no patent or literature reporting that weakening or inactivating AimA expression can improve the ability of microorganisms to produce nucleosides. The present invention also provides the application of the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.4; or an amino acid sequence having 80-90% homology with the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.4 and also having the function of glutamate / serine transporter in regulating the ability of microorganisms to produce nucleosides.

[0042] Through in-depth research, the present inventors found that modifying the glutamate / serine transporter (encoded by the aimA gene) of Bacillus subtilis or Bacillus amyloliquefaciens, weakening the activity of the AimA protein, enabling microorganisms to efficiently and rapidly produce guanosine or inosine, and successfully creating a new microorganism capable of efficiently producing nucleosides, thus completing the present invention.

[0043] The beneficial effects of the present invention are as follows:

[0044] The present invention discovers for the first time that weakening the activity of the glutamate / serine transporter can enhance the ability of microorganisms to produce nucleosides. In Bacillus amyloliquefaciens or Bacillus subtilis, point mutations, start codon weakening or interruption, and knockout of the AimA protein are used to obtain corresponding mutants for efficient production of nucleosides, and a microorganism capable of efficiently producing nucleosides is successfully created. Detailed implementation manners

[0045] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without making creative efforts based on the embodiments in the present invention belong to the scope of protection of the present invention.

[0046] The primer names and sequence information involved in the present invention are shown in Table 1.

[0047] Table 1 Primer names and sequence information

[0048]

[0049]

[0050] Example 1 AimA mutant strain construction in Bacillus amyloliquefaciens W506* Construction of mutant strains

[0051] This example provides a method for constructing the AimAW506* mutant strain in Bacillus amyloliquefaciens as follows: Using the B.a836 (see CN112574934B) genome as a template, with the primer pairs Ba-aimA-506-1f / Ba-aimA-506-1r (SEQ ID NO.15-16) and Ba-aimA-506-2f / Ba-aimA-506-2r (SEQ ID NO.17-18), the upstream and downstream homologous arms of aimA were amplified by pfu high-fidelity DNA polymerase. The obtained fragments were gel-extracted and fused, and the AimA full-length fragment was amplified and then gel-extracted. W506* The full-length fragment was gel-extracted.

[0052] The pKSU plasmid (the pKSU plasmid was kindly provided by Professor Wang Shufang of Nankai University, see Amarkerless gene replacement method for B.amyloliquefaciens LL3 and its use in genome reductionand i mprovement of poly-γ-glutamic acid production[J], Applied Microbiologyand Biotechn ology,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 to obtain a linearized plasmid, which was then gel-extracted.

[0053] The assembled kit was used to assemble the digested linearized plasmid and the AimA fragment, and the mixture was transformed into TransT1 competent cells. Subsequently, identification and screening were carried out to obtain the recombinant plasmid pKSU-AimA. W506* The recombinant plasmid pKSU-AimA was obtained. W506* The recombinant plasmid pKSU-Ai mA W506*Transfer it into the laboratory-constructed B.a 836 (see CN112574934B) and the wild-type strain DSM7. Screen the transformants on LB plates containing 2.5 μg / mL chloramphenicol at 30 °C. Transfer the obtained transformants into 5 mL of LB liquid medium, culture them at 42 °C and 200 rpm for 12 h and then subculture once. Dilute and spread them on LB plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. Transfer the primary recombinants into 5 mL of LB liquid medium, culture them at 42 °C and 200 rpm for 12 h and then subculture once. Dilute and spread them on LB plates containing 0.8 μM 5-FU to screen for secondary recombinants. Screen the strains with a nonsense mutation at the 506th position, and name them B.a 8509 and B.a 8510 respectively.

[0054] The gene sequence encoding the glutamate / serine transporter in Bacillus amyloliquefaciens is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0055] Example 2 AimA in Bacillus amyloliquefaciens W93* Construction of mutant strains

[0056] This example provides AimA in Bacillus amyloliquefaciens W93* The method for constructing mutant strains is as follows: Use primer pairs Ba-aimA-1f / Ba-aimA-93-1r and Ba-aimA-93-2f / Ba-aimA-2r respectively, and use the genome of B.a 836 strain as a template to amplify and obtain the left homologous arm AimA W93* -L, AimA W93* -R fragments and fuse them. Obtain the AimA W93* full-length fragment.

[0057] Obtain the plasmid pKSU-AimA according to the plasmid construction method in Example 1 W93* plasmid, transfer it into B.a 836 and the wild-type strain DSM7, and name the strains with a nonsense mutation at the 93rd tryptophan as B.a 8515 and B.a 8516.

[0058] Example 3 AimA in Bacillus amyloliquefaciens A1G Construction of mutant strains

[0059] This example provides AimA in Bacillus amyloliquefaciens A1G The method for constructing mutant strains is as follows: Use primer pairs Ba-aimA-1f / Ba-aimA-AG-1r and Ba-aimA-AG-2f / Ba-aimA-2r respectively, and use the genome of B.a 836 strain as a template to amplify and obtain the left homologous arm Ba-aimA A1G -L, Ba-aimAA1G -R fragment and fuse. Obtain Ba-aimA A1G The full-length fragment was constructed to obtain pKSU-Ba-AimA according to the plasmid construction method in Example 1 A1G Plasmid, which was transformed into B.a836 and wild-type strain DSM7 strains constructed in the laboratory, and the screened mutant strains were named B.a 8511 and B.a8512 respectively.

[0060] Example 4 AimA in Bacillus amyloliquefaciens A1T Construction of mutant strains

[0061] This example provides the construction method of AimA mutant strains in Bacillus amyloliquefaciens A1T as follows: Using primer pairs Ba-aimA-1f / Ba-aimA-AT-1r and Ba-aimA-AT-2f / Ba-aimA-2r respectively, with the genome of B.a 836 strain as the template, amplify to obtain the left homologous arm Ba-aimA A1T -L, Ba-aimA A1T -R fragment and fuse to obtain Ba-aimA A1T full-length fragment.

[0062] Construct pKSU-Ba-aimA according to the plasmid construction method in Example 1 A1T Plasmid, which was transformed into B.a 836 and wild-type strain DSM7 strains constructed in the laboratory, and the screened mutant strains were named B.a 8513 and B.a 8514 respectively.

[0063] Example 5 Construction of AimA knockout strains in Bacillus amyloliquefaciens

[0064] This example provides the construction method of AimA knockout strains in Bacillus amyloliquefaciens, as follows: Using primer pairs Ba-aimA-1f / Ba-DaimA-1r and Ba-DaimA-2f / Ba-aimA-2r respectively, with the genome of B.a 836 strain as the template, amplify to obtain the left homologous arm Ba-DaimA-L and Ba-DaimA-R fragments and fuse to obtain the Ba-DaimA full-length fragment.

[0065] Construct pKSU-Ba-DaimA plasmid according to the plasmid construction method in Example 1, transform it into B.a836 and wild-type DSM7 strains, and screen the obtained aimA knockout strains named B.a 8517 and B.a 8518.

[0066] Example 6 AimA in Bacillus subtilis A1G Construction of strains

[0067] This example provides a method for constructing the Bacillus subtilis AimA A1G strain, as follows:

[0068] Using primer pairs A5-aimA-1f / A5-aimA-AG-1r and A5-aimA-AG-2f / A5-aimA-2r respectively, with the genome of strain 168 as the template, the left homologous arms A5-aimA A1G -L and A5-aimA A1G -R fragments were amplified and fused to obtain the A5-AimA A1G full-length fragment. According to the plasmid construction method in Example 1, the plasmid pKSU-A5-AimA A1G was obtained and transformed into B. subtilis A5 strain and wild-type B. subtilis 168 strain. Strains with the start codon mutated from ATG to GTG were screened and named B. subtilis A0054 and B. subtilis A0055.

[0069] The gene sequence encoding the glutamate / serine transporter in Bacillus subtilis is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0070] Example 7 Construction of AimA A1T in Bacillus subtilis

[0071] This example provides a method for constructing the Bacillus subtilis AimA A1T strain, as follows: Using primer pairs A5-aimA-1f / A5-aimA-AT-1r and A5-aimA-AT-2f / A5-aimA-2r respectively, with the genome of B. subtilis A5 strain as the template, the left homologous arms A5-aimA A1T -L and A5-aimA A1T -R fragments were amplified and fused to obtain the A5-AimA A1T full-length fragment.

[0072] According to the plasmid construction method in Example 1, the plasmid pKSU-A5-AimA A1T was obtained and transformed into B. subtilis A5 strain and wild-type B. subtilis 168 strain. Strains with the start codon mutated from ATG to TTG were screened and named B. subtilis A0056 and B. subtilis A0106.

[0073] Example 8 Construction of AimA W93* in Bacillus subtilis

[0074] This example provides AimA in Bacillus subtilis W93* The construction method of the strain is as follows: primer pairs A5-aimA-1f / A5-aimA-AT-1r and A5-aimA-AT-2f / A5-aimA-2r are used respectively, and the genome of B. subtilis A5 strain is used as a template to amplify the left homologous arm A5-aimA A1T -L, A5-aimA A1T -R fragments and fused to obtain A5-AimA A1T Full-length clip.

[0075] According to the plasmid construction method in Example 1, pKSU-A5-AimA was obtained. A1T The plasmid was transformed into B. subtilis A5 strain and wild-type B. subtilis168 strain, and strains with nonsense mutation at tryptophan position 93 were screened and named B. subtilis A0107 and B. subtilis A0108.

[0076] Example 9 Construction of AimA knockout strain in Bacillus subtilis

[0077] This example provides a method for constructing an AimA knockout strain in Bacillus subtilis, as follows: using primer pairs A5-aimA-1f / A5-DaimA-1r, A5-DaimA-2f / A5-aimA-2r, respectively, the B. subtilis A5 strain genome is used as a template, the left homologous arm A5-DaimA-L and A5-DaimA-R fragments are amplified and fused to obtain the A5-DAimA full-length fragment.

[0078] The pKSU-A5-DAimA plasmid was obtained according to the plasmid construction method in Example 1, and transformed into B. subtilisA5 strain and wild-type B. subtilis168 strain, and AimA knockout strains were obtained by screening and named B. subtilisA0109 and B. subtilis A0110.

[0079] Example 10 Verification of the glycoside production performance of the engineered strain

[0080] This example provides verification of the glycoside production performance of the engineered strain, and the steps are as follows:

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

[0082] 2. Inoculate a single colony into 30 mL of seed medium and culture at 37 °C with a rotation speed of 110 rpm for 7 - 8 h. The formula of the seed medium is as follows (in g / L): glucose 20, yeast powder 5, corn steep liquor dry powder 5, potassium dihydrogen phosphate 3, magnesium sulfate 0.5, ferrous sulfate 0.02, manganese sulfate 0.01, pH 7.0 - 7.2.

[0083] 3. Transfer the inoculum at an inoculation amount of 10% (v / v) to 30 ml of fermentation medium, and culture in a shaker at a rotation speed of 130 rpm at 35 °C for 72 h or 48 h (the engineered Bacillus amyloliquefaciens is cultured for 72 h; the engineered Bacillus subtilis is cultured for 48 h). The formula of the fermentation medium is as follows (in g / L): glucose 120, yeast powder 3.5, potassium dihydrogen phosphate 3, ammonium sulfate 25, manganese sulfate 0.01, magnesium sulfate 5, sodium glutamate 10, corn steep liquor dry powder 15, calcium carbonate 25, pH 7.0 - 7.2.

[0084] 4. Use a liquid chromatograph to detect the production of nucleosides in the fermentation broth, and the detection results are shown in Table 2.

[0085] Table 2 Evaluation results of guanosine and inosine production by mutant strains in shake - flask fermentation (average value of three replicates)

[0086]

[0087]

[0088] Judging from the results in Table 2, nonsense mutations at tryptophan at position 93 or 506 of AimA in Bacillus subtilis or Bacillus amyloliquefaciens, or weakening the start codon ATG to GTG or TTG, or directly inactivating the gene can all improve the ability of nucleoside production, and also have a certain promoting effect on the growth of microorganisms.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A modified microorganism, characterized in that, The microorganism has reduced or lost glutamate / serine transporter activity compared to the unmodified microorganism, and the microorganism has enhanced nucleoside production ability compared to the unmodified microorganism; Among them, the amino acid sequence of the glutamate / serine transporter from Bacillus amyloliquefaciens is shown in SEQ ID NO.2; the amino acid sequence of the glutamate / serine transporter from Bacillus subtilis is shown in SEQ ID NO.

4.

2. The microorganism according to claim 1, characterized in that, The reduction or loss of glutamate / serine transporter activity in the microorganism is achieved by selecting from the following 1)-3), or an optional combination: 1) Reducing or losing by changing the amino acid sequence of the glutamate / serine transporter; 2) Reducing or losing by changing the nucleotide sequence encoding the glutamate / serine transporter; 3) Losing by knocking out the coding sequence of the glutamate / serine transporter.

3. The microorganism according to claim 2, characterized in that, Use mutagenesis, site-directed mutagenesis or homologous recombination methods to reduce the expression of the coding gene of the glutamate / serine transporter or knock out the coding gene of the glutamate / serine transporter.

4. The microorganism according to claim 1, wherein The reduction or loss of glutamate / serine transporter activity is achieved by the following A, B or C: A. A nonsense mutation occurs at the 506th amino acid W of the glutamate / serine transporter; B. The 93rd amino acid of the glutamate / serine transporter changes from W to a nonsense mutation; C. The start codon ATG of the glutamate / serine transporter is weakened to GTG or TTG.

5. Method for constructing nucleoside-producing engineering bacteria, characterized in that, The construction method includes: using genetic engineering means to inactivate or weaken the coding gene of the glutamate / serine transporter in a microorganism with nucleoside production ability; The weakening is achieved by selecting from the following a)-c), or an optional combination: a) Changing the amino acid sequence of the glutamate / serine transporter; b) Changing the nucleotide sequence encoding the glutamate / serine transporter; c) Knocking out the coding sequence of the glutamate / serine transporter; Among them, the coding sequence of the glutamate / serine transporter is shown in SEQ ID NO.1 or SEQ ID NO.

3.

6. The construction method according to claim 5, wherein The weakening method is selected from at least one of mutagenesis, site-directed mutagenesis, and homologous recombination; Preferably, the weakening is achieved by the following (1), (2) or (3): (1) The 506th amino acid of the glutamate / serine transporter changes from W to a nonsense mutation; (2) The 93rd amino acid of the glutamate / serine transporter changes from W to a nonsense mutation; (3) The start codon ATG of the glutamate / serine transporter is weakened to GTG or TTG; The microorganism is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli.

7. An engineered bacterium constructed by the construction method according to claim 5 or 6.

8. Use of the microorganism according to any one of claims 1-4 or the engineered bacterium according to claim 7 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, vidarabine, inosine, inosinic acid, guanylic acid, adenylic acid, guanosine, guanine, guanylic acid, riboflavin, diacetyl guanylic acid.

9. A method for producing a nucleoside, characterized in that, The method comprises the following steps: i) Culturing the microorganism according to any one of claims 1-4 or the engineered bacterium according to claim 7 to obtain a culture; ii) Collecting the produced nucleosides from the culture obtained in step i).

10. Use of a glutamate / serine transporter in regulating the nucleoside-producing ability of a microorganism, wherein the amino acid sequence of the glutamate / serine transporter is as shown in SEQ ID NO.2 or SEQ ID NO.4; or an amino acid sequence having 80-90% homology with the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.4 and also having the function of a glutamate / serine transporter.

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