Recombinant microorganism mutated for sucrose-6-phosphatase and its use in the production of nucleosides

Abstract

The present application relates to the technical field of microbial fermentation, in particular to sucrose-6-phosphate hydrolase mutant recombinant microorganism and its application in nucleoside production. The present application significantly improves the nucleoside production capacity and production efficiency of microorganism by weakening sucrose-6-phosphate hydrolase or its coding gene, and provides a new modification target and strategy for the construction of nucleoside production strain. The sucrose-6-phosphate hydrolase variant provided by the present application can effectively promote the accumulation of nucleoside in microorganism, and significantly improve the nucleoside production capacity of microorganism. The recombinant microorganism constructed by using the method for improving nucleoside production capacity and the sucrose-6-phosphate hydrolase variant provided by the present application can more efficiently accumulate nucleoside, and the nucleoside yield is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to recombinant microorganisms with sucrose-6-phosphate hydrolase mutations and their application in the production of nucleosides. Background Technology

[0002] Nucleosides are glycosides formed by the condensation of D-ribose or D2-deoxyribose with pyrimidine or purine bases. D-ribose condenses with adenine, guanine, hypoxanthine, cytosine, thymine, or uracil to form the corresponding adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, thymine ribonucleotide, and uracil ribonucleotide, which are abbreviated as adenosine (A), guanosine (G), inosine (I), cytidine (C), thymine (T), and uridine (U), respectively.

[0003] Guanosine and inosine have wide applications in the food and pharmaceutical industries. In the food sector, guanosine and inosine are important precursors of disodium guanylate and disodium inosinate, respectively, and the combination of disodium guanylate and disodium inosinate forms a food flavor enhancer widely used in condiments such as chicken essence and soy sauce. In the pharmaceutical field, guanosine and inosine can serve as pharmaceutical intermediates for various antiviral drugs, such as acyclic guanosine, triazole nucleoside, and sodium guanosine triphosphate, all of which require guanosine as a raw material for synthesis. Inosine is an important precursor of inosine monophosphate, which can serve as a precursor for the synthesis of adenosine monophosphate (AMP) and guanosine monophosphate (GMP), and is suitable for treating leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis, cirrhosis, etc., and can also be used to treat central retinitis and optic nerve atrophy. Adenosine is an endogenous nucleoside distributed throughout human cells. It can directly enter the myocardium, be phosphorylated to produce adenosine monophosphate, participate in myocardial energy metabolism, and also participate in the dilation of coronary arteries, increasing blood flow. Adenosine plays an important physiological role in the cardiovascular system and many other systems and tissues of the body. Besides being used as a specific drug for treating heart disease, adenosine is also an important intermediate in the synthesis of adenosine triphosphate (ATP), adenine, adenosine acid, and vidarabine, and is widely used in the pharmaceutical and other industries.

[0004] Currently, microbial fermentation is the main method for producing nucleosides, primarily using microorganisms such as Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus pumilus. However, the fermentation performance of current nucleoside-producing strains remains poor, and the conversion rate of nucleosides is still low, failing to meet the demands of large-scale industrial production. Therefore, it is still necessary to develop new metabolic engineering targets and strains related to nucleoside production. Summary of the Invention

[0005] This invention provides recombinant microorganisms with sucrose-6-phosphate hydrolase mutations and their application in nucleoside production.

[0006] In the process of developing microbial nucleoside production, this invention discovered that weakening the central metabolic pathway of sucrose-6-phosphate hydrolase or its encoding gene can significantly improve the nucleoside production capacity of microorganisms, enabling them to produce nucleosides more efficiently. Based on this discovery, sucrose-6-phosphate hydrolase mutants and recombinant microorganisms that can improve the nucleoside production capacity of microorganisms were developed.

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

[0008] This invention provides the application of weakening sucrose-6-phosphate hydrolase or its encoding gene in improving the ability of microorganisms to produce nucleosides or their derivatives.

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

[0010] Preferably, the improvement of the ability of microorganisms to produce nucleosides or their derivatives includes improving the yield and / or conversion rate of microorganisms to produce nucleosides or their derivatives.

[0011] This invention provides the application of a weakened form of sucrose-6-phosphate hydrolase or its encoding gene in the construction of microorganisms for the production of nucleosides or their derivatives.

[0012] The above applications include: constructing microorganisms for the production of nucleosides or their derivatives by weakening sucrose-6-phosphate hydrolase or its encoding gene.

[0013] In the above applications, the microorganisms are Bacillus, Corynebacterium, or Escherichia. Preferably, they are Bacillus or Escherichia.

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

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

[0016] The sequences of sucrose-6-phosphate hydrolases can be obtained by those skilled in the art from publicly available databases. The reference sequence numbers for one sucrose-6-phosphate hydrolase from Bacillus subtilis and Bacillus amyloliquefaciens are NP_391683.2 and KYC98802.1, respectively, in NCBI, and their coding gene is sacA.

[0017] In this invention, attenuated sucrose-6-phosphate hydrolase refers to a reduction or loss of sucrose-6-phosphate hydrolase activity. Attenuated sucrose-6-phosphate hydrolase encoding gene refers to a reduction or absence of expression (including transcription and / or translation) of the gene encoding sucrose-6-phosphate hydrolase.

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

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

[0020] (1) Replace, delete or insert one or more amino acids in the amino acid sequence of sucrose-6-phosphate hydrolase to reduce or eliminate the expression and / or enzyme activity of sucrose-6-phosphate hydrolase.

[0021] (2) Substitution, deletion or insertion of one or more bases in the nucleotide sequence of the gene encoding sucrose-6-phosphate hydrolase to reduce or eliminate the expression and / or activity of sucrose-6-phosphate hydrolase.

[0022] (3) Replace the transcriptional and / or translational regulatory elements of the gene encoding sucrose-6-phosphate hydrolase with elements of weaker activity to reduce or eliminate the expression of sucrose-6-phosphate hydrolase.

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

[0024] In some embodiments of the present invention, a single amino acid substitution is made to the amino acid sequence of sucrose-6-phosphate hydrolase to reduce the expression and / or activity of sucrose-6-phosphate hydrolase. Preferably, the expression and / or activity of sucrose-6-phosphate hydrolase is reduced by mutating amino acid position 448 to phenylalanine or isoleucine.

[0025] In some embodiments of the present invention, a nonsense mutation is made in the 245th amino acid of sucrose-6-phosphate hydrolase to reduce the expression and / or 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 activity of sucrose-6-phosphate hydrolase.

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

[0028] In this invention, the nucleosides preferably include purine nucleosides. Purine nucleosides include adenosine, guanosine, and inosine.

[0029] In this invention, the nucleoside derivatives include, but are not limited to, hypoxanthine, guanine, guanylic acid, riboflavin, diacetylguanylic acid, inosine, adenosine, adenosine triphosphate (ATP), adenine, vidarabine, etc.

[0030] The present invention provides a sucrose-6-phosphate hydrolase variant, which, compared with the wild-type Bacillus sucrose-6-phosphate hydrolase, contains a mutation at amino acid position 448, which is phenylalanine or isoleucine.

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

[0032] Alternatively, the sucrose-6-phosphate hydrolase variant contains a mutation in the first amino acid position, where methionine is changed to valine, compared to the wild-type Bacillus sucrose-6-phosphate hydrolase.

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

[0034] If the 448th amino acid of the wild-type Bacillus sucrose-6-phosphate hydrolase is phenylalanine, then 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 yield of nucleosides (especially purine nucleosides).

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

[0037] The present invention provides a nucleic acid molecule encoding the above-described sucrose-6-phosphate hydrolase variant.

[0038] Based on 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 as 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 biomaterials mentioned above include expression cassettes, vectors, or host cells.

[0042] The expression cassette is a recombinant nucleic acid molecule obtained by operatively linking the nucleic acid molecule with a transcriptional or translational regulatory element.

[0043] The vectors include, but are not limited to, plasmid vectors, viral vectors, and transposons.

[0044] The host cell includes microbial cells. Preferably, it is a Bacillus spp., a Corynebacterium spp., or an Escherichia spp.

[0045] The present invention provides recombinant microorganisms that are modified to weaken their sucrose-6-phosphate hydrolase or its encoding gene.

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

[0047] The recombinant microorganism exhibits increased nucleoside production compared to its originating strain. Preferably, the recombinant microorganism exhibits 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 originating strain.

[0048] Preferably, the microorganism is a Bacillus spp., a Corynebacterium spp., or an Escherichia spp.

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

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

[0051] Preferably, the recombinant microorganism is modified to express the above-described 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 a strain of Bacillus subtilis 168 or A5 (the construction method of B. subtilis A5 is described in patent CN110257315B, which has a certain adenosine and inosine production capacity) that is modified to weaken the sucrose-6-phosphate hydrolase or its encoding gene.

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

[0055] This invention significantly improves the nucleoside production capacity of wild-type Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus amyloliquefaciens and Bacillus subtilis with nucleoside production capacity by weakening sucrose-6-phosphate hydrolase or its encoding gene, enabling the strains to accumulate nucleosides more efficiently and rapidly.

[0056] The present invention provides a method for constructing the recombinant microorganisms described above, the method comprising: modifying the microorganisms to weaken their sucrose-6-phosphate hydrolase or its encoding gene.

[0057] This invention provides for any of the following applications 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 the construction of microorganisms for the production of nucleosides or their derivatives.

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

[0061] Preferably, the microorganism is a Bacillus spp., a Corynebacterium spp., or an Escherichia spp.

[0062] Among them, Bacillus bacteria include Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus pumilus; Corynebacterium bacteria include Corynebacterium glutamicum; and Escherichia bacteria 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 nucleosides or nucleoside derivatives from the culture.

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

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

[0066] The beneficial effects of this invention include at least the following: By weakening sucrose-6-phosphate hydrolase or its encoding gene, this invention significantly improves the nucleoside production capacity and efficiency of microorganisms, providing new modification targets and strategies for constructing nucleoside-producing strains. The sucrose-6-phosphate hydrolase variant provided by this invention can effectively promote the accumulation of nucleosides in microorganisms, significantly enhancing their nucleoside production capacity. The recombinant microorganisms constructed using the method for improving nucleoside production capacity provided by this invention and the sucrose-6-phosphate hydrolase variant can accumulate nucleosides more efficiently, resulting in a significantly increased nucleoside yield. Detailed Implementation

[0067] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this 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 embodiments of this invention.

[0070]

[0071]

[0072] Example 1: SacA in Bacillus subtilis L448F Construction of point mutant strains

[0073] Using the genome of adenosine-producing strain B. subtilis A5 (construction method of strain B. subtilis A5 can be found in CN110257315B) as a template, 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 corresponding ORF box sequence is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6) was digested with XbaI / PstI and then recovered by gel. The pKSU plasmid (the pKSU plasmid was kindly provided 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 then digested with XbaI / PstI and recovered by gel. The linearized plasmid and A5-sacA were then assembled using an assembly kit. L448F The fragments were assembled and transformed into TransT1 competent cells. Subsequent identification and screening yielded the recombinant plasmid pKSU-A5-sacA. L448F The plasmid pKSU-A5-sacA was used. L448F Transformed into Bacillus subtilis wild-type strain 168 and adenosine-producing strain A5 respectively, and screened for strains carrying A5-sacA. L448F The strains were named B. subtilis A0077 and B. subtilis A0078, respectively.

[0074] Example 2: SacA in Bacillus subtilis L448I Construction of point mutant strains

[0075] Using the genome of adenosine-producing strain B. subtilis A5 (construction method of strain B. subtilis A5 can be found in CN110257315B) as a template, the left and right homologous 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-sacA 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). The recombinant plasmid pKSU-A5-sacA was obtained according to the construction method in Example 1. L448I The strain was then transformed into Bacillus subtilis wild-type strain 168 and adenosine-producing strain A5, respectively, and A5-sacA-carrying strains were screened for. L448I The strains were named B. subtilis A0079 and B. subtilis A0080, respectively.

[0076] Example 3: SacA in Bacillus subtilis 245TAA Construction of point mutant strains

[0077] Using the genome of adenosine-producing strain B. subtilis A5 (construction method of strain B. subtilis A5 can be found in CN110257315B) as a template, 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). The recombinant plasmid pKSU-A5-sacA was obtained according to the construction method in Example 1. 245TAA The strain was then transformed into Bacillus subtilis wild-type strain 168 and adenosine-producing strain A5, respectively, and A5-sacA-carrying strains were screened for. 245TAA The strains were named B. subtilis A0081 and B. subtilis A0082, respectively.

[0078] Example 4: SacA in Bacillus subtilis M1* Construction of point mutant strains

[0079] Using the genome of adenosine-producing strain B. subtilis A5 (construction method of strain B. subtilis A5 can be found in CN110257315B) as a template, 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* The strain was then transformed into Bacillus subtilis wild-type strain 168 and adenosine-producing strain A5, respectively, and A5-sacA-carrying strains were screened for.M1* The strains were named B. subtilis A0083 and B. subtilis A0084, respectively.

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

[0081] Using the genome of adenosine-producing strain B. subtilis A5 (construction method of strain B. subtilis A5 is described in CN110257315B) as a template, the left and right homologous arms were amplified using primer pairs A5-ΔsacA-1f / 1r and A5-ΔsacA-2f / 2r, respectively, and then fused to obtain the full-length fragment of A5-ΔsacA. The recombinant plasmid pKSU-A5-ΔsacA was obtained according to the construction method in Example 1, and transformed into Bacillus subtilis wild-type strain 168 and adenosine-producing strain A5, respectively. Strains with the sacA gene knocked out were screened and named B. subtilis A0085 and B. subtilis A0086, respectively.

[0082] Example 6: sacA in Bacillus amyloliquefaciens F448I Construction of point mutant strains

[0083] Using the genome of Bacillus amyloliquefaciens DSM7 as a template, the left and right homologous arms were amplified using primer pairs sacAF448I-1f / 1r and sacAF448I-2f / 2r, respectively, and then 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 They were then transformed into the DSM7 model strain and two guanosine-producing strains, B.s833 and Ba 836, respectively, and the resulting strains were named B.a8459, B.s8460, and B.a8461, respectively.

[0084] Example 7: sacA in Bacillus amyloliquefaciens 245TAA Construction of point mutant strains

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

[0086] Example 8: sacA in Bacillus amyloliquefaciens M1* Construction of point mutant strains

[0087] Using the genome of Bacillus amyloliquefaciens DSM7 as a template, the left and right homologous arms were amplified using primer pairs sacA1-1f / 1r and sacA1-2f / 2r, respectively, and then fused to obtain sacA 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). The recombinant plasmid pKSU-sacA was obtained according to the construction method in Example 1. M1* They were then transformed into the DSM7 model strain and two guanosine-producing strains, B.s833 and Ba 836, respectively, and the resulting 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 primer pairs ΔsacA-1f / 1r and ΔsacA-2f / 2r, respectively, and then fused to obtain the full-length ΔsacA fragment. The recombinant plasmid pKSU-ΔsacA was obtained according to the construction method in Example 1, and transformed into the DSM7 model strain and two guanosine-producing strains B.s833 and Ba 836, respectively. The resulting strains were named B.a8468, B.s8469, and B.a8470, respectively.

[0090] Example 10: Real-time quantitative fluorescence PCR verification of sacA expression levels in various mutant strains

[0091] All sacA-modified mutant strains and control strains B. subtilis A5 (the control strain corresponding to the Bacillus subtilis mutant strain) and DSM7, B. s833, and B. a836 (the control strains corresponding to the Amyloliquefaciens mutant strain) were cultured in LB medium to the logarithmic growth phase. 1 mL of the bacterial culture was treated with an appropriate amount of lysozyme, and total RNA was extracted for reverse transcription. Real-time quantitative PCR was performed using cDNA as a template. The reaction conditions were as follows: 95℃ pre-denaturation for 10 min; 95℃ for 15 s, 55℃ for 1 min, 40 cycles. After the reaction, bacterial 16S rRNA was used as a reference, according to 2... -ΔΔCTThe transcriptional levels of relevant genes were calculated, and the percentage decrease in transcriptional levels compared to the corresponding starting strain was also calculated. The results are shown in Table 2.

[0092] Table 2. Transcription levels of the sacA gene in each strain

[0093]

[0094]

[0095] The above results indicate that the transcription level of the sacA gene in each mutant strain was reduced to varying degrees compared with its corresponding originating strain, suggesting that the above modifications all achieved the effect of weakening the transcription level of the sacA gene.

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

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

[0098] 1. Incubate the bacterial strain preserved in glycerol at 37°C overnight and cut out single colonies.

[0099] 2. Select single clones and inoculate them into 30 mL of 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℃ for 20 min), and incubate at 110 rpm and 37℃ for 7-8 h to obtain seed liquid.

[0100] 3. Transfer the seed culture obtained in step 2 to 30 mL of fermentation medium (g / L: glucose 120, yeast extract 3.5, potassium dihydrogen phosphate 3, ammonium sulfate 25, manganese sulfate 0.01, magnesium sulfate 5, monosodium glutamate 10, corn steep liquor powder 15, calcium carbonate 25, pH 7.0–7.2, sterilized at 121℃ for 20 min) at a shaking speed of 130 rpm and 35℃ for 70 h (Bs / a8459-8470 strain fermented for 72 h; B. subtilis A0077-0086 strain fermented for 48 h). Monitor cell growth (OD) during fermentation. 562 ).

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

[0102] Table 3. Results of guanosine, inosine, and adenosine production by the mutant strain during shake-flask fermentation (mean of three replicates).

[0103]

[0104]

[0105] The above results indicate that mutating the 448th amino acid of the sacA gene encoding sucrose-6-phosphate hydrolase to F / I, or performing a termination mutation on the 245th amino acid of the sacA gene encoding sucrose-6-phosphate hydrolase, or weakening the start codon of the sacA gene, or knocking out its entire ORF frame, all resulted in varying degrees of increased nucleoside production in the mutant strains. This demonstrates that weakening sucrose-6-phosphate hydrolase or its encoding gene has a significant effect on enhancing the nucleoside production capacity of the strains.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of attenuation of the gene encoding sucrose-6-phosphate hydrolase in enhancing the ability of microorganisms to produce nucleosides; among which, The weakening of the gene encoding sucrose-6-phosphate hydrolase is a decrease in the expression of the gene encoding sucrose-6-phosphate hydrolase. The microorganism is Bacillus amyloliquefaciens or Bacillus subtilis; The nucleoside is guanosine, inosine, or adenosine.

2. The application of attenuation of the gene encoding sucrose-6-phosphate hydrolase in the construction of microorganisms for nucleoside production; among which, The weakening of the gene encoding sucrose-6-phosphate hydrolase is a decrease in the expression of the gene encoding sucrose-6-phosphate hydrolase. The microorganism is Bacillus amyloliquefaciens or Bacillus subtilis; The nucleoside is guanosine, inosine, or adenosine.

3. The use of a sucrose-6-phosphate hydrolase variant or a nucleic acid molecule encoding said sucrose-6-phosphate hydrolase variant or biological material containing said nucleic acid molecule in the construction of microorganisms for the production of nucleosides; in, 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; The microorganism is Bacillus amyloliquefaciens or Bacillus subtilis, and the original sucrose-6-phosphate hydrolase of the microorganism is not expressed; The nucleoside is guanosine, inosine, or adenosine.

4. Application of recombinant microorganisms in nucleoside production; among which, The recombinant microorganism was modified to reduce the expression of its gene encoding sucrose-6-phosphate hydrolase. The microorganism is Bacillus amyloliquefaciens or Bacillus subtilis; The nucleoside is guanosine, inosine, or adenosine.

5. The application according to claim 4, characterized in that, The recombinant microorganism was modified to express a sucrose-6-phosphate hydrolase variant, and its original sucrose-6-phosphate hydrolase was not expressed. Alternatively, the recombinant microorganism is modified to inactivate its sucrose-6-phosphate hydrolase; The amino acid sequence of the original sucrose-6-phosphate hydrolase is shown in SEQ ID NO.2 or 4, and 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.

6. A method for producing nucleosides by fermentation, characterized in that, The method includes: culturing recombinant microorganisms to obtain a culture, and collecting nucleosides from the culture; The recombinant microorganism is modified to reduce the expression of its gene encoding sucrose-6-phosphate hydrolase. The microorganism is Bacillus amyloliquefaciens or Bacillus subtilis; The nucleoside is guanosine, inosine, or adenosine.

7. The method according to claim 6, characterized in that, The recombinant microorganism was modified to express a sucrose-6-phosphate hydrolase variant, and its original sucrose-6-phosphate hydrolase was not expressed. Alternatively, the recombinant microorganism is modified to inactivate its sucrose-6-phosphate hydrolase; The amino acid sequence of the original sucrose-6-phosphate hydrolase is shown in SEQ ID NO.2 or 4, and 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.

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