Recombinant microorganism with mutated sucrose-6-phosphohydrolase and application of recombinant microorganism in nucleoside production

Abstract

The invention relates to the technical field of microbial fermentation, in particular to a recombinant microorganism with mutated sucrose-6-phosphohydrolase and application of the recombinant microorganism in nucleoside production. By weakening the sucrose-6-phosphohydrolase or the coding gene thereof, the nucleoside production capacity and production efficiency of microorganisms are remarkably improved, and a new modification target and strategy are provided for construction of nucleoside production strains. The sucrose-6-phosphohydrolase variant provided by the invention can effectively promote microorganisms to accumulate nucleoside and remarkably improve the nucleoside production capacity of the microorganisms. According to the method for improving the nucleoside production capacity and the recombinant microorganism constructed by the sucrose-6-phosphohydrolase variant provided by the invention, nucleoside can be more efficiently accumulated, and the nucleoside yield is remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, in particular to a recombinant microorganism with a sucrose-6-phosphate hydrolase mutation and an application thereof in producing nucleosides. 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 invention provides a recombinant microorganism with a sucrose-6-phosphate hydrolase mutation and an application of the microorganism in producing nucleosides.

[0006] The present invention has found in the process of developing microbial nucleoside production that a sucrose-6-phosphate hydrolase or a gene encoding the sucrose-6-phosphate hydrolase that weakens the central metabolic pathway can significantly improve the nucleoside production capacity of the microorganism, so that the microorganism can produce nucleosides more efficiently. Based on this discovery, a sucrose-6-phosphate hydrolase mutant and a recombinant microorganism that can improve the nucleoside production capacity of the microorganism have been developed.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] The invention provides application of sucrose-6-phosphate hydrolase or weakened coding gene thereof in improving the ability of microorganisms to produce nucleosides or derivatives thereof.

[0009] The above application includes: improving the ability of microorganisms to produce nucleosides or their derivatives by weakening sucrose-6-phosphate hydrolase or its encoding gene.

[0010] Preferably, said improving the ability of microorganisms to produce nucleosides or derivatives thereof comprises improving the yield and / or conversion rate of nucleosides or derivatives thereof produced by microorganisms.

[0011] The invention provides application of sucrose-6-phosphate hydrolase or weakened encoding gene thereof in constructing microorganisms for producing nucleosides or derivatives thereof.

[0012] The above application includes: constructing a microorganism for producing nucleosides or their derivatives by weakening sucrose-6-phosphate hydrolase or its encoding gene.

[0013] In the above application, the microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Corynebacterium or a bacterium of the genus Escherichia, preferably a bacterium of the genus Bacillus or a bacterium of the genus Escherichia.

[0014] Among them, the bacteria of the genus Bacillus include Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus pumilus; the bacteria of the genus Corynebacterium include Corynebacterium glutamicum; and the bacteria of the genus Escherichia include Escherichia coli.

[0015] In some embodiments of the present invention, the microorganism is Bacillus subtilis or Bacillus amyloliquefaciens.

[0016] The sequence of sucrose-6-phosphate hydrolase can be obtained by those skilled in the art through public databases. The reference sequence numbers of sucrose-6-phosphate hydrolase of Bacillus subtilis and Bacillus amyloliquefaciens in NCBI are NP_391683.2 and KYC98802.1, respectively, and the encoding gene is sacA.

[0017] In the present invention, weakening sucrose-6-phosphate hydrolase means that the activity of sucrose-6-phosphate hydrolase is reduced or lost. Weakening the coding gene of sucrose-6-phosphate hydrolase means that the expression (including transcription and / or translation) of the coding gene of sucrose-6-phosphate hydrolase is reduced or not expressed.

[0018] Weakening of sucrose-6-phosphate hydrolase or its encoding gene can be achieved by conventional genetic engineering means.

[0019] Specifically, weakening sucrose-6-phosphate hydrolase or its encoding gene can be achieved by any one or more of the following methods:

[0020] (1) replacing, deleting or inserting one or more amino acids in the amino acid sequence of sucrose-6-phosphate hydrolase so that the expression and / or enzyme activity of sucrose-6-phosphate hydrolase is reduced or lost;

[0021] (2) replacing, deleting or inserting one or more bases in the nucleotide sequence of the gene encoding sucrose-6-phosphate hydrolase so as to reduce or lose the expression and / or enzyme activity of sucrose-6-phosphate hydrolase;

[0022] (3) Replacing the transcriptional and / or translational regulatory elements of the gene encoding sucrose-6-phosphate hydrolase with elements with weaker activity so that the expression of sucrose-6-phosphate hydrolase is reduced or lost.

[0023] The transcription and translation regulatory elements mentioned above include promoters, ribosome binding sites, etc.

[0024] In some embodiments of the invention, an amino acid is substituted to the amino acid sequence of sucrose-6-phosphate hydrolase to reduce the expression and / or enzymatic activity of sucrose-6-phosphate hydrolase. Preferably, the expression and / or enzymatic activity of sucrose-6-phosphate hydrolase is reduced by mutating the 448th amino acid to phenylalanine or isoleucine.

[0025] In some embodiments of the present invention, a nonsense mutation is performed on the amino acid at position 245 of sucrose-6-phosphate hydrolase to reduce the expression and / or enzyme activity of sucrose-6-phosphate hydrolase.

[0026] In some embodiments of the present invention, the start codon of the gene encoding sucrose-6-phosphate hydrolase is mutated from ATG to GTG to reduce the expression and / or enzyme activity of sucrose-6-phosphate hydrolase.

[0027] In some embodiments of the present invention, the entire ORF frame of the gene encoding sucrose-6-phosphate hydrolase is knocked out to lose the expression and / or enzyme activity of sucrose-6-phosphate hydrolase.

[0028] In the present invention, the nucleoside preferably includes purine nucleosides, including adenine nucleoside (adenosine), guanosine nucleoside (guanosine), and inosine nucleoside (inosine).

[0029] In the present invention, the nucleoside derivatives include but are not limited to hypoxanthine, guanine, guanylic acid, riboflavin, diacetylguanylic acid, inosinic acid, adenylic acid, adenosine triphosphate (ATP), adenine, adenosine arabinoside and the like.

[0030] The present invention provides a sucrose-6-phosphate hydrolase variant, wherein the sucrose-6-phosphate hydrolase variant comprises a mutation in which the amino acid at position 448 is mutated to phenylalanine or isoleucine compared with a wild-type sucrose-6-phosphate hydrolase of Bacillus;

[0031] Alternatively, the sucrose-6-phosphate hydrolase variant has a nonsense mutation at amino acid position 245 compared to the Bacillus wild-type sucrose-6-phosphate hydrolase;

[0032] Alternatively, the sucrose-6-phosphate hydrolase variant comprises a mutation in which the amino acid at position 1 is mutated from methionine to valine compared to the wild-type sucrose-6-phosphate hydrolase of Bacillus.

[0033] Preferably, the amino acid sequence of the wild-type sucrose-6-phosphate hydrolase of Bacillus is shown as SEQ ID NO. 2 or 4, and the encoding gene sequence thereof is shown as SEQ ID NO. 1 or 3, respectively.

[0034] If the amino acid at position 448 of the wild-type Bacillus sucrose-6-phosphate hydrolase is phenylalanine, it is mutated to isoleucine to obtain the sucrose-6-phosphate hydrolase variant.

[0035] The sucrose-6-phosphate hydrolase variants described above can significantly improve the nucleoside production capacity of microorganisms (especially Bacillus bacteria) and significantly promote the increase in the production of nucleosides (especially purine nucleosides).

[0036] Preferably, the amino acid sequence of the sucrose-6-phosphate hydrolase variant is shown in SEQ ID NO. 6, 8, 10, 12, 14, 16 or 18.

[0037] The present invention provides nucleic acid molecules encoding the above-mentioned sucrose-6-phosphate hydrolase variants.

[0038] According to the amino acid sequence and codon rules of the sucrose-6-phosphate hydrolase variant, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the sucrose-6-phosphate hydrolase variant.

[0039] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.5, 7, 9, 11, 13, 15 or 17.

[0040] The present invention provides biological materials comprising the nucleic acid molecule or expressing the sucrose-6-phosphate hydrolase variant.

[0041] The biological materials mentioned above include expression cassettes, vectors or host cells.

[0042] Wherein, the expression cassette is a recombinant nucleic acid molecule obtained by operably linking the nucleic acid molecule with a transcription or translation regulatory element.

[0043] The vector includes but is not limited to a plasmid vector, a viral vector, and a transposon.

[0044] The host cell includes microbial cells, preferably Bacillus bacteria, Corynebacterium bacteria, and Escherichia bacteria.

[0045] The present invention provides a recombinant microorganism which is modified so that its sucrose-6-phosphate hydrolase or a gene encoding it is weakened.

[0046] The recombinant microorganism is a nucleoside-producing recombinant microorganism.

[0047] The recombinant microorganism has an increased nucleoside production compared to its starting strain. Preferably, the recombinant microorganism has an increased nucleoside production by at least 10%, at least 20%, at least 30%, at least 50%, at least 80% or at least 100% compared to its starting strain.

[0048] Preferably, the microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Corynebacterium or a bacterium of the genus Escherichia.

[0049] Among them, the bacteria of the genus Bacillus include Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus pumilus; the bacteria of the genus Corynebacterium include Corynebacterium glutamicum; and the bacteria of the genus Escherichia include Escherichia coli.

[0050] In some embodiments of the present invention, the recombinant microorganism is a recombinant Bacillus amyloliquefaciens or a recombinant Bacillus subtilis.

[0051] Preferably, the recombinant microorganism is modified to express the above-mentioned sucrose-6-phosphate hydrolase variant, and its original sucrose-6-phosphate hydrolase is not expressed.

[0052] Alternatively, the recombinant microorganism is modified to inactivate its sucrose-6-phosphate hydrolase.

[0053] In some embodiments of the present invention, the recombinant microorganism is based on Bacillus subtilis 168 or A5 (the construction method of B. subtilis A5 refers to patent CN110257315B, which has a certain adenosine and inosine production capacity) as the starting strain, which is modified so that the sucrose-6-phosphate hydrolase or its encoding gene is weakened.

[0054] In some embodiments of the present invention, the recombinant microorganism is based on Bacillus amyloliquefaciens DSM7, B.s833 or Ba 836 (the construction method of B.s833 and Ba 836 refers to patent application CN112574934A, which has a certain guanosine or inosine production capacity) as the starting strain, which is modified so that the sucrose-6-phosphate hydrolase or its encoding gene is weakened.

[0055] The present invention significantly improves the nucleoside production capacity of the strain by weakening sucrose-6-phosphate hydrolase or its encoding gene in wild-type Bacillus amyloliquefaciens and Bacillus subtilis and in Bacillus amyloliquefaciens and Bacillus subtilis with nucleoside production capacity, so that the strain can accumulate nucleosides more efficiently and quickly.

[0056] The present invention provides a method for constructing the above-mentioned recombinant microorganism, which comprises: modifying the microorganism so that its sucrose-6-phosphate hydrolase or its encoding gene is weakened.

[0057] The present invention provides any one of the following uses of the sucrose-6-phosphate hydrolase variant, the nucleic acid molecule, the biological material, or the recombinant microorganism:

[0058] (1) Application in the production of nucleosides or their derivatives;

[0059] (2) Application in constructing microorganisms for producing nucleosides or their derivatives.

[0060] The present invention also provides use of the sucrose-6-phosphate hydrolase variant, the nucleic acid molecule or the biological material in improving the nucleoside production of microorganisms.

[0061] Preferably, the microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Corynebacterium or a bacterium of the genus Escherichia.

[0062] Among them, the bacteria of the genus Bacillus include Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus pumilus; the bacteria of the genus Corynebacterium include Corynebacterium glutamicum; and the bacteria of the genus Escherichia include Escherichia coli.

[0063] The present invention provides a method for producing nucleosides or nucleoside derivatives by fermentation, the method comprising: culturing the recombinant microorganism to obtain a culture, and collecting the nucleosides or nucleoside derivatives from the culture.

[0064] In some embodiments of the present invention, the method comprises: inoculating the recombinant microorganism into a seed culture medium for seed culture to obtain a seed solution, inoculating the seed solution into a fermentation culture medium for culture to obtain a fermentation solution, and separating and extracting nucleosides or their derivatives from the fermentation solution.

[0065] The present invention provides a method for increasing the yield of nucleosides or their derivatives. The method comprises: modifying the production strain of nucleosides or their derivatives to weaken the sucrose-6-phosphate hydrolase or its coding gene.

[0066] The beneficial effects of the present invention include at least: the present invention significantly improves the nucleoside production capacity and production efficiency of microorganisms by weakening sucrose-6-phosphate hydrolase or its encoding gene, and provides a new transformation target and strategy for the construction of nucleoside production strains. The sucrose-6-phosphate hydrolase variant provided by the present invention can effectively promote the accumulation of nucleosides by microorganisms and significantly improve the nucleoside production capacity of microorganisms. The recombinant microorganism constructed by the method for improving nucleoside production capacity provided by the present invention and the sucrose-6-phosphate hydrolase variant can accumulate nucleosides more efficiently, and its nucleoside yield is significantly improved. DETAILED DESCRIPTION

[0067] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0068] The primer names and primer sequences involved in the following examples are shown in Table 1.

[0069] Table 1 Primer names and sequence information used in the examples of the present invention

[0070]

[0071]

[0072] Example 1: sacA in Bacillus subtilis L448F Construction of point mutation strains

[0073] The genome of adenosine-producing strain B. subtilis A5 (the construction method of strain B. subtilis A5 is shown in CN110257315B) was used as a template, and the left and right homologous arms were amplified using primer pairs A5-sacAL448F-1f / 1r and A5-sacAL448F-2f / 2r, respectively, and then fused to obtain A5-sacA L448F 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 in genome 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-sacA were assembled using the assembly kit. L448F The fragments were assembled and transformed into TransT1 competent cells, and then the recombinant plasmid pKSU-A5-sacA was obtained by identification and screening. L448F . Plasmid pKSU-A5-sacA L448F The strains were transformed into wild Bacillus subtilis 168 and adenosine-producing strain A5, and the strains carrying A5-sacA were screened. L448F The strains were named B. subtilis A0077 and B. subtilis A0078 respectively.

[0074] Example 2: sacA in Bacillus subtilis L448I Construction of point mutation strains

[0075] The genome of adenosine-producing strain B. subtilis A5 (the construction method of strain B. subtilis A5 is shown in CN110257315B) was used as a template, and the left and right homology arms were amplified using primer pairs A5-sacAL448F-1f / A5-sacAL448I-1r and A5-sacAL448I-2f / A5-sacAL448F-2r, respectively, and then fused to obtain A5-sacAL448F-2r. L448IThe 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) was obtained according to the construction method in Example 1. L448I , and transformed them into wild strain 168 of Bacillus subtilis and adenosine-producing strain A5, respectively, to obtain strains carrying A5-sacA L448I The strains were named B. subtilis A0079 and B. subtilis A0080 respectively.

[0076] Example 3: sacA in Bacillus subtilis 245TAA Construction of point mutation strains

[0077] The genome of adenosine-producing strain B. subtilis A5 (the construction method of strain B. subtilis A5 is shown in CN110257315B) was used as a template, and the left and right homologous arms were amplified using primer pairs A5-sacA245-1f / 1r and A5-sacA245-2f / 2r, respectively, and then fused to obtain A5-sacA 245TAA 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) was obtained according to the construction method in Example 1. 245TAA , and transformed them into wild strain 168 of Bacillus subtilis and adenosine-producing strain A5, respectively, to obtain strains carrying A5-sacA 245TAA strains, and the strains were named B. subtilis A0081 and B. subtilis A0082, respectively.

[0078] Example 4: sacA in Bacillus subtilis M1* Construction of point mutation strains

[0079] The genome of adenosine-producing strain B. subtilis A5 (the construction method of strain B. subtilis A5 is shown in CN110257315B) was used as a template, and the left and right homologous arms were amplified using primer pairs A5-sacA1-1f / 1r and A5-sacA1-2f / 2r, respectively, and then fused to obtain A5-sacA M1* The full-length fragment (the corresponding ORF frame sequence is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12). The recombinant plasmid pKSU-A5-sacA was obtained according to the construction method in Example 1. M1* , and transformed them into wild strain 168 of Bacillus subtilis and adenosine-producing strain A5, respectively, to obtain strains carrying A5-sacAM1* The strains were named B. subtilis A0083 and B. subtilis A0084 respectively.

[0080] Example 5: Construction of a sacA knockout strain in Bacillus subtilis

[0081] The genome of adenosine-producing strain B. subtilis A5 (the construction method of strain B. subtilis A5 is shown in CN110257315B) was used as a template, and the left and right homologous arms were amplified using primer pairs A5-ΔsacA-1f / 1r and A5-ΔsacA-2f / 2r, respectively, and the full-length fragment of A5-ΔsacA was obtained by fusion. The recombinant plasmid pKSU-A5-ΔsacA was obtained according to the construction method in Example 1, and was transformed into wild strain 168 of Bacillus subtilis and adenosine-producing strain A5, respectively, and strains with knockout of sacA gene were obtained by screening, and the strains were named B. subtilis A0085 and B. subtilis A0086, respectively.

[0082] Example 6: sacA in Bacillus amyloliquefaciens F448I Point mutation strain construction

[0083] Using the genome of Bacillus amyloliquefaciens DSM7 as a template, the left and right homology arms were amplified using the primer pairs sacAF448I-1f / 1r and sacAF448I-2f / 2r, respectively, and fused to obtain sacA F448I 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-sacA was obtained according to the construction method in Example 1. F448I and transformed them into the DSM7 model strain and two guanosine-producing strains B.s833 and Ba 836, respectively. The obtained strains were named B.a8459, B.s8460, and B.a8461, respectively.

[0084] Example 7: sacA in Bacillus amyloliquefaciens 245TAA Point mutation strain construction

[0085] Using the genome of Bacillus amyloliquefaciens DSM7 as a template, the left and right homology arms were amplified using the primer pairs sacA245-1f / 1r and sacA245-2f / 2r, respectively, and sacA was fused to obtain 245TAA 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) was obtained according to the construction method in Example 1. 245TAAand transformed them into the DSM7 model strain and two guanosine-producing strains B.s833 and Ba 836, respectively. The obtained strains were named B.a8462, B.s8463, and B.a8464, respectively.

[0086] Example 8: sacA in Bacillus amyloliquefaciens M1* Point mutation strain construction

[0087] Using the genome of Bacillus amyloliquefaciens DSM7 as a template, the left and right homology arms were amplified using the primer pairs sacA1-1f / 1r and sacA1-2f / 2r, respectively, and sacA was fused to obtain M1* The full-length fragment (the corresponding ORF frame sequence is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.18) was obtained according to the construction method in Example 1. M1* and transformed them into the DSM7 model strain and two guanosine-producing strains B.s833 and Ba 836, respectively. The obtained strains were named B.a8465, B.s8466, and B.a8467, respectively.

[0088] Example 9: Construction of sacA knockout strain in Bacillus amyloliquefaciens

[0089] Using the genome of Bacillus amyloliquefaciens DSM7 as a template, the left and right homologous arms were amplified using the ΔsacA-1f / 1r and ΔsacA-2f / 2r primer pairs, respectively, and the ΔsacA full-length fragment was obtained by fusion. The recombinant plasmid pKSU-ΔsacA 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, respectively. The obtained strains were named B.a8468, B.s8469, and B.a8470, respectively.

[0090] Example 10: Real-time quantitative fluorescence PCR verification of the expression level of sacA in each mutant strain

[0091] All sacA-modified mutant strains and control strains B. subtilis A5 (control strain corresponding to Bacillus subtilis mutant strains) and DSM7, B.s833, B.a836 (control strains corresponding to Bacillus amyloliquefaciens mutant strains) were cultured in LB medium until the logarithmic growth phase. 1 mL of bacterial solution was treated with an appropriate amount of lysozyme to extract total RNA for reverse transcription, and real-time quantitative PCR was performed using cDNA as a template. The reaction conditions were as follows: 95°C pre-denaturation for 10 min; 95°C, 15 s, 55°C, 1 min, 40 cycles. After the reaction, bacterial 16S rRNA was used as a reference, according to 2 -ΔΔCTThe transcription level of the relevant gene was calculated by the method, and the reduction ratio of the transcription level compared with the starting strain corresponding to the strain was calculated. The results are shown in Table 2.

[0092] Table 2 Transcription levels of sacA genes in various strains

[0093]

[0094]

[0095] The above results show that the transcription level of the sacA gene in each mutant strain is reduced to varying degrees compared with its corresponding starting strain, indicating that the above-mentioned transformations have achieved the effect of weakening the transcription level of the sacA gene.

[0096] Example 11: Verification of nucleoside production performance of mutant strains

[0097] The nucleoside production performance of the mutant strains constructed in Examples 1-9 was verified by fermentation as follows:

[0098] 1. Culture the bacteria stored in glycerol at 37°C overnight and streak out a single colony.

[0099] 2. Pick a single clone and inoculate it into 30 mL seed culture medium (g / L: glucose 20, yeast powder 5, corn steep liquor powder 5, potassium dihydrogen phosphate 3, magnesium sulfate 0.5, ferrous sulfate 0.02, manganese sulfate 0.01, pH 7.0-7.2, sterilized at 121°C for 20 min), culture at 110 rpm and 37°C for 7-8 h to obtain seed solution.

[0100] 3. Transfer the seed solution obtained in step 2 to 30 mL fermentation medium (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 powder 15, calcium carbonate 25, pH 7.0-7.2, sterilized at 121°C for 20 min) at a 10% v / v inoculation volume, and culture at 35°C for 70 h (Bs / a8459-8470 strain fermentation 72 h; B. subtilis A0077-0086 strain fermentation 48 h). Monitor the bacterial growth during the fermentation process (OD 562 ).

[0101] 4. The nucleoside content in the fermentation broth was detected using a liquid chromatograph. The results are shown in Table 3.

[0102] Table 3 Results of shake flask fermentation of mutant strains to produce guanosine, inosine and adenosine (average of three replicates)

[0103]

[0104]

[0105] The above results show that the nucleoside production of the mutant strains obtained by mutating the 448th amino acid of the sucrose-6-phosphate hydrolase encoded by the sacA gene in the strain to F / I, or performing a termination mutation on the 245th amino acid of the sacA gene encoded by the sacose-6-phosphate hydrolase, or weakening the start codon of the sacA gene, or knocking out the entire ORF frame are all improved to varying degrees; this indicates that weakening of sucrose-6-phosphate hydrolase or its encoding gene has a significant effect on improving the nucleoside production capacity of the strain.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of sucrose-6-phosphate hydrolase or weakened encoding gene thereof in improving the ability of microorganisms to produce nucleosides or their derivatives.

2. Use of sucrose-6-phosphate hydrolase or a weakened version of its encoding gene in constructing a microorganism for producing nucleosides or their derivatives.

3. The use according to claim 1 or 2, characterized in that: The microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Corynebacterium or a bacterium of the genus Escherichia.

4. A sucrose-6-phosphate hydrolase variant, characterized in that The sucrose-6-phosphate hydrolase variant contains a mutation in which the amino acid at position 448 is mutated to phenylalanine or isoleucine compared to the wild-type sucrose-6-phosphate hydrolase of Bacillus; Alternatively, the sucrose-6-phosphate hydrolase variant has a nonsense mutation at amino acid position 245 compared to the Bacillus wild-type sucrose-6-phosphate hydrolase; Alternatively, the sucrose-6-phosphate hydrolase variant comprises a mutation in which the amino acid at position 1 is mutated from methionine to valine compared to the wild-type sucrose-6-phosphate hydrolase of Bacillus sp.; Preferably, the amino acid sequence of the sucrose-6-phosphate hydrolase variant is shown in SEQ ID NO. 6, 8, 10, 12, 14, 16 or 18.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the sucrose-6-phosphate hydrolase variant of claim 4.

6. Biomaterial, characterized in that The biological material comprises the nucleic acid molecule of claim 5 or expresses the sucrose-6-phosphate hydrolase variant of claim 4.

7. A recombinant microorganism, characterized in that The recombinant microorganism is modified so that its sucrose-6-phosphate hydrolase or its encoding gene is weakened; Preferably, the microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Corynebacterium or a bacterium of the genus Escherichia.

8. The recombinant microorganism according to claim 7, characterized in that The recombinant microorganism is modified to express the sucrose-6-phosphate hydrolase variant of claim 4, and its original sucrose-6-phosphate hydrolase is not expressed; Alternatively, the recombinant microorganism is modified to inactivate its sucrose-6-phosphate hydrolase.

9. Any of the following uses of the sucrose-6-phosphate hydrolase variant according to claim 4, the nucleic acid molecule according to claim 5, the biological material according to claim 6, or the recombinant microorganism according to claim 7 or 8: (1) Application in the production of nucleosides or their derivatives; (2) Application in constructing microorganisms for producing nucleosides or their derivatives; Preferably, the microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Corynebacterium or a bacterium of the genus Escherichia.

10. A method for producing nucleosides or nucleoside derivatives by fermentation, characterized in that: The method comprises: culturing the recombinant microorganism according to claim 7 or 8 to obtain a culture, and collecting nucleosides or nucleoside derivatives from the culture.

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