Xanthine nucleoside production strain as well as construction method and application thereof
The xanthine nucleoside production strain XL8 constructed through genetic modification solves the problem of low production efficiency of xanthine nucleoside fermentation method in the existing technology, and achieves efficient and simple xanthine nucleoside fermentation production, with good industrial application prospects.
Patent Information
- Application Number
- CN202510101075.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-22
- Publication Date
- 2025-05-09
AI Technical Summary
The prior art is difficult to efficiently produce xanthine nucleosides through fermentation, mainly because of its long synthetic route and feedback inhibition, and the natural extraction method and chemical synthesis method have problems such as low yield and high equipment requirements.
A xanthine nucleoside production strain XL8 was constructed through genetic modification methods. The specific steps include introducing the purine operon gene pur in E. coli W3110, knocking out the purine nucleotide repressor protein gene purR, introducing the anti-feedback variants purFBazK326Q and prsEcoD128A, knocking out the nucleoside phosphate enzyme gene ppnP and deoD, integrating the subpartic acid aminolytic enzyme gene aspA, and knocking out the guanosine monophosphate synthase gene guaA to remove the feedback inhibition and degradation pathways and promote the synthesis of xanthine nucleosides.
A genetically engineered strain XL8, which does not contain plasmids and synthesizes xanthine nucleosides from de novo, has the advantages of clear genetic background and sustainable transformation. It can produce xanthine nucleosides simply and efficiently through fermentation methods, which is suitable for industrial applications.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of genetic engineering, in particular to a xanthine nucleoside production strain and a construction method and application thereof. Background Art
[0002] Xanthine nucleoside, also known as 9-furanosyl-3,9-dihydro-1H-purine-2,6-dione, is a nucleoside composed of xanthine and a sugar group, which can generate xanthine and nucleotides through hydrolysis reaction. As an inhibitor of non-equilibrium cell kinetic division mode, xanthine nucleoside can not only realize the transformation of the division mode of bone marrow mesenchymal stem cells from a non-equilibrium state to an equilibrium state, but also does not affect its liver differentiation characteristics, which helps to improve the in vitro proliferation efficiency of bone marrow mesenchymal stem cells and has a wide range of applications. Xanthine nucleoside is the substrate of the first step reaction in the caffeine synthesis pathway, so during the fermentation culture process, a certain concentration of xanthine nucleoside is added to the fermentation, and the recombinant bacterial cells are permeabilized by pressurization to promote the absorption of the substrate xanthine nucleoside by the recombinant bacteria, thereby increasing the fermentation yield of caffeine.
[0003] At present, there are two methods for synthesizing xanthine nucleosides: natural extraction and chemical synthesis. The natural extraction method mainly extracts and purifies xanthine nucleosides from organisms. Its advantage is that the source is wide, but it also has problems such as low yield and high cost. The chemical synthesis method is to artificially synthesize xanthine nucleosides, including synthesizing xanthine and sugar compounds, and then connecting them together. Its advantage is that the raw materials are easy to obtain, but the preparation process is complicated, the equipment requirements are high, and the yield is low.
[0004] Microbial fermentation has now become the mainstream method for large-scale production of most natural small molecule products. Although the biosynthetic route of purine nucleosides in cells has long been explored, due to the long synthesis route, strong feedback inhibition of the synthesis pathway, and strict regulation in many aspects of the cell, no one has used fermentation to produce xanthine nucleosides. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a xanthine nucleoside producing strain.
[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned xanthine nucleoside-producing strain.
[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned xanthine nucleoside producing strain.
[0008] In order to solve the above technical problems, the technical solution of the present invention is:
[0009] A xanthine nucleoside production strain, strain XL8, is obtained by genetic modification using wild-type Escherichia coli (E. coli) W3110 as a starting strain: firstly, the purine operon gene pur (EKBCSQLFMNHD) in Bacillus amyloliquefaciens is introduced into the yghX site of the genome of wild-type Escherichia coli (E. coli) W3110, and then the purine operon gene pur (EKBCSQLFMNHD) in Bacillus amyloliquefaciens is introduced into the yghX site of the genome of wild-type Escherichia coli (E. coli) W3110. trc The promoter was activated; then the purine nucleotide repressor protein gene purR was knocked out in the genome, and the anti-feedback variant purF of PRPP transamidase was introduced into the rph and ilvG sites of the genome respectively. Baz K326Q and the feedback-resistant variant of PRPP synthase, prs Eco D128A , both by P trc The promoter was started; then the nucleoside phosphorylase gene ppnP and the nucleoside phosphorylase gene deoD were knocked out in the genome to weaken the degradation of xanthine nucleoside, and the aspartate ammonia lyase gene aspA was integrated at the mbhA site on the genome to strengthen the supply of the strain's precursor aspartate, and then the guanosine monophosphate (GMP) synthetase gene guaA was knocked out to block the strain's branch metabolism to guanosine monophosphate (GMP), so that it would flow more to the synthesis pathway of xanthine nucleoside, and finally XL8 was obtained.
[0010] Preferably, the above-mentioned xanthine riboside producing strain has an integrated heterologous purine operon gene pur (EKBCSQLFMNHD) from Bacillus amyloliquefaciens, and the NCBI-GeneID of the purine operon gene pur (EKBCSQLFMNHD) is: purE: 75094376; purK: 75094377; purB: 75094378; purC: 75094379; purS: 75094380; purQ: 75094381; purL: 75094382; purF: 75094383; purM: 75094384; purN: 75094385; purH: 75094386; purD: 75094387.
[0011] Preferably, in the above-mentioned xanthine nucleoside producing strain, the NCBI-GeneID of the purine nucleotide repressor protein gene purR is: 945226; the anti-feedback variant purF Baz K326Q NCBI-GeneID: 75094383; the anti-feedback variant prs Eco D128ANCBI-GeneID: 545772; NCBI-GeneID: 945048 of the nucleoside phosphorylase gene ppnP; NCBI-GeneID: 945654 of the nucleoside phosphorylase gene deoD; NCBI-Gene ID: 948658 of the aspartate ammonia lyase gene aspA; NCBI-Gene ID: 947334 of the guanosine monophosphate (GMP) synthetase gene guaA.
[0012] Preferably, the above-mentioned xanthine riboside producing strain, the P trc The nucleotide sequence of the promoter is shown in SEQ ID NO.95.
[0013] The above-mentioned method for constructing a xanthine nucleoside production strain uses CRISPR / Cas9-mediated gene editing technology to gradually transform wild-type Escherichia coli (E. coli) W3110. The specific steps are as follows:
[0014] (1) The heterologous purine operon gene pur (EKBCSQLFMNHD) was introduced into the yghX locus of the E. col i W3110 genome by P trc The promoter is activated, enhancing the carbon metabolic flow of the purine metabolic pathway;
[0015] (2) Knock out the purine nucleotide repressor protein gene purR and introduce the anti-feedback variant purF of the PRPP amidotransferase of Bacillus amyloliquefaciens into the ilvG and rph sites of the genome, respectively. Baz K326Q and the feedback-resistant variant of PRPP synthase from Escherichia coli, prs Eco D128A , both by P trc The promoter is activated, relieving the feedback inhibition of the final product and key intermediates;
[0016] (3) Knock out the ppnP gene and deoD gene in the genome to weaken the degradation of xanthine nucleotides;
[0017] (4) Overexpression of the aspartate ammonia lyase gene aspA further strengthens the xanthine nucleoside biosynthesis pathway;
[0018] (5) By knocking out the guaA gene, the strain's branch metabolism to guanosine monophosphate (GMP) was blocked, allowing more of the metabolism to flow to the xanthine nucleoside synthesis pathway.
[0019] The application of the xanthosine nucleoside producing strain in producing xanthosine nucleoside.
[0020] Preferably, the xanthosine nucleoside producing strain is used to produce xanthosine nucleoside by a fermentation method.
[0021] Preferably, the xanthoside producing strain is used to contact the xanthoside producing strain with a fermentation medium for fermentation culture to prepare xanthoside.
[0022] Preferably, in the application of the above-mentioned xanthine nucleoside-producing strain, the fermentation culture includes shake flask fermentation or fermentation tank fermentation.
[0023] Preferably, in the application of the above-mentioned xanthine nucleoside production strain, the inoculation amount during shake flask fermentation is 10%-15%, the temperature is maintained at 37°C±0.2, and the shaking culture is carried out at 220r / min. During the fermentation process, 25% ammonia water is added to maintain the pH at 7.0; 60% glucose solution is added to supplement the carbon source required by the bacteria.
[0024] Preferably, in the application of the above-mentioned xanthine nucleoside production strain, the specific steps of the fermentation tank fermentation are as follows:
[0025] (1) Activation of bacterial strains: transfer the bacterial strains from the glycerol tube to the slant culture medium for activation and culture for 11-13 hours; transfer the bacterial strains from the slant culture medium to the eggplant flask culture medium for further activation and expansion culture for 10-12 hours, culture temperature: 37°C;
[0026] (2) Seed culture: During the culture process, the pH was maintained at 7.0±0.1, the temperature was maintained at 37±0.2°C, and the dissolved oxygen was maintained at 25%-35%. When the OD reached 10, the culture was transferred to a fermenter for fermentation.
[0027] (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at an inoculum rate of 20% for fermentation culture. The fermentation pH was maintained at 7.0±0.1, the temperature was maintained at 37±0.2°C, and the dissolved oxygen was maintained at 25%-30%. During the culture process, 80% glucose solution was added to maintain the carbon source required by the fermentation process, and 25% ammonia water was added to adjust the pH to maintain at 7.0±0.1.
[0028] Preferably, the application of the above-mentioned xanthine riboside production strain uses a slant culture medium and an eggplant flask culture medium containing: 10 g / L beef extract, 5 g / L glucose, 5 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast powder, 100 mg / L xanthine, 25 g / L agar powder, the pH is adjusted to 7.0-7.2, and sterilized at 121°C for 20 min.
[0029] Preferably, the application of the above-mentioned xanthine riboside production strain adopts a seed culture medium comprising: 30 g / L glucose, 5 g / L yeast extract powder, 1 g / L peptone, 1.25 g / L citric acid, 1.5 g / L MgSO4·7H2O, 3.0 g / L KH2PO4, and vitamin B1 (V B1 )1mg / L, biotin(VH )1mg / L, guanine 200mg / L, and the rest is water.
[0030] Preferably, the fermentation medium used in the application of the above-mentioned xanthine riboside production strain is: glucose 20g / L, yeast extract 3g / L, peptone 1g / L, citric acid 2g / L, MgSO4·7H2O 2g / L, KH2PO4 6g / L, FeSO4·7H2O 20mg / L, MnSO410mg / L, vitamin B1 (V B1 )2mg / L, biotin (V H )2mg / L, guanine 200mg / L, and the rest is water.
[0031] The above culture media can be prepared using standard methods.
[0032] Beneficial effects:
[0033] The xanthine nucleoside production strain is constructed by analyzing the purine metabolism-related pathways of wild-type Escherichia coli (E. coli) and Bacillus amyloliquefaciens (Bac illus amyloliquefaciens), introducing the purine operon of Bacillus amyloliquefaciens into the chassis microorganism wild-type Escherichia coli (E. coli) W3110, knocking out the xanthine nucleoside metabolic replenishment pathway and the key branch pathway, releasing the key feedback inhibition, knocking out the xanthine nucleoside decomposition pathway and other related molecular modifications, thereby successfully constructing a genetically engineered bacterium XL8 that does not contain a plasmid and synthesizes xanthine nucleoside from scratch. The strain has the advantages of clear genetic background and sustainable modification. The fermentation production of xanthine nucleoside by the strain is simple to operate, and xanthine nucleoside can be directly synthesized with a cheap carbon source, and has a good industrial application prospect. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is a diagram of xanthine nucleotide metabolism.
[0035] Figure 2 This is a comparison chart of the shake flask fermentation yield of xanthine nucleoside production strain XL8.
[0036] Figure 3 This is the fermentation yield diagram of xanthine riboside production strain XL8 in the fermenter. DETAILED DESCRIPTION
[0037] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.
[0038] The starting strain used in the following examples is wild-type Escherichia coli (E. coli) W3110, the strain information is shown in Table 1, and the corresponding promoter, terminator and gene are shown in the sequence table. The primers used in the construction process of the involved strains are shown in Table 2.
[0039] Table 1 Strain construction information
[0040] Strain number genotype XL0 E.coli W3110 XL1 XL0,yghX::pur(EKBCSQLFMNHD) XL2 XL1,ΔpurR XL3 <![CDATA[XL2,Δrph::Ptrc-purF Baz K326Q ]]> XL4 <![CDATA[XL3,ilvG::Ptrc-prs Eco D128A ]]> XL5 XL4,ΔppnP XL6 XL5,ΔdeoD XL7 XL6,mbhA::Ptrc-aspA XL8 XL7,ΔguaA
[0041] Table 2 Primers involved in strain construction
[0042]
[0043]
[0044]
[0045]
[0046]
[0047] Example 1
[0048] like Figure 1 As shown, this example is intended to illustrate the specific construction steps of strain XL8. In particular, if there is a method for operating a gene of the same type in the example, it will only be provided once and annotated without further elaboration.
[0049] 1.1 Integration of the purine nucleoside operon pur (EKBCSQLFMNHD) gene:
[0050] Since the purine nucleoside operon pur (EKBCSQLFMNHD) gene in the genome of Bacillus amyloliquefaciens strain is 12797 bp in length, we integrated it into the [4] The five segments were integrated into the yghX site of the genome in sequence. The five segments are pur1, pur2, pur3, pur4, and pur5.
[0051] 1.1.1 Integration of gene pur1:
[0052] Using the genome of wild-type Escherichia coli E.coliW3110 (strain XL0) as a template, yghX-UP-S, yghX-UP-A and pur1-DN-S, pur1-DN-A as primers, upstream and downstream homology arms were obtained by PCR amplification; using the genome of Bacillus amyloliquefaciens as a template, pur1-UP-1, pur1-DN-1 and pur1-UP-2, pur1-DN-2 as primers, target gene fragments pur1-1 and pur1-2 were obtained by PCR amplification; then, using pur1-1 and pur1-2 fragments as templates and pur1-UP-1 and pur1-UP-1 as primers, target gene fragment pur1 was amplified by PCR; then, using the upstream and downstream homology arms and the target gene fragment as templates and yghX-UP-S and pur1-DN-A as primers, overlapping fragments were obtained by overlapping PCR amplification. Since P was included in the primer design, trc The promoter is attached to the 5' end of the primer, so the overlapping fragment has a P trc The overlapping fragment is composed of the upstream homology arm-P trc The primers yghX-pGRB-UP and yghX-pGRB-DN were used to anneal the gRNA fragment, and the gRNA fragment was connected to the pGRB vector to obtain yghX-pGRB; wild-type Escherichia coli (E. coli) W3110 electroporation competent cells were prepared, the overlapping fragments and yghX-pGRB were electroporated into the competent cells, and positive transformants were screened to obtain the strain XL1-pur1.
[0053] 1.1.2 Integration of gene pur2:
[0054] The same operation method as in 1.1 is used, except that P is not used in primer design. trc The promoter was connected to the 5' end of the primers, and the primers used were pur2-UP-S, pur2-UP-A, pur2-UP-1, pur2-DN-1, pur2-UP-2, pur2-DN-2, pur2-DN-S, pur2-DN-A, 4-pGRB-UP, and 4-pGRB-DN. The competent cell was XL1-pur1, and the strain XL1-pur2 was obtained.
[0055] 1.1.3 Integration of gene pur3:
[0056] The same operation method as in 1.2 is used, except that the primers used are pur3-UP-S, pur3-UP-A, pur3-UP, pur3-DN, pur3-DN-S, pur3-DN-A, 1-pGRB-UP, 1-pGRB-DN. The competent cell is XL1-pur2, and the strain XL1-pur3 is obtained.
[0057] 1.1.4 Integration of gene pur4:
[0058] The same operation method as in 1.2 was used, and the primers used were pur4-UP-S, pur4-UP-A, pur4-UP, pur4-DN, pur4-DN-S, pur4-DN-A, 4-pGRB-UP, and 4-pGRB-DN. The competent cell was XL1-pur3, and the strain XL1-pur4 was obtained.
[0059] 1.1.5 Integration of gene pur5:
[0060] The same operation method as in 1.2 is used, except that P trc terminator (the P trc The nucleotide sequence of the terminator is shown in SEQ ID NO.96) was connected to the 3' end of the primer, and the primers used were pur5-UP-S, pur5-UP-A, pur5-UP-1, pur5-DN-1, pur5-UP-2, pur5-DN-2, pur5-DN-S, pur5-DN-A, 1-pGRB-UP, and 1-pGRB-DN. The competent cell was XL1-pur4, and the strain XL1 was obtained.
[0061] 1.2 Knockout of purR:
[0062] The genome of wild-type Escherichia coli (E. coli) W3110 was used as a template, except that purR-UP-S, purR-UP-A and purR-DN-S, purR-DN-A were used to obtain the upstream and downstream homology arms by PCR amplification, respectively. Then, the upstream and downstream homology arms were used as templates and purR-UP-S and purR-DN-A were used as primers to obtain overlapping fragments by overlapping PCR amplification, that is, the overlapping fragments consisted of "upstream homology arm-downstream homology arm"; purR-pGRB-UP and purR-pGRB-DN were used as primers to anneal the gRNA fragments, and the gRNA fragments were connected to the pGRB vector to obtain purR-pGRB; XL1 electroporation competent cells were prepared, the overlapping fragments and purR-pGRB were electroporated into the competent cells together, and the positive transformants were screened to obtain the strain XL2.
[0063] 1.3 Overexpression of heterologous purF Baz K326Q Gene:
[0064] The genome of wild-type Escherichia coli (E. coli) W3110 was used as a template, and rph-UP-S, rph-UP-A and rph-DN-S, rph-DN-A were used as primers to obtain the upstream and downstream homology arms by PCR amplification; the genome of Bacillus amyloliquefaciens was used as a template, and purF-UP-1, purF-DN-1, purF-UP-2, and purF-DN-2 were used as primers to obtain purF-1 and purF-2 fragments by PCR amplification; the purF-1 and purF-2 fragments were used as templates, and purF-UP-1 and purF-DN-2 were used as primers to obtain the purF gene fragment by PCR amplification; finally, the upstream homology arm, the downstream homology arm, and the target gene fragment were used as templates, and rph-UP-S and rph-DN-A were used as primers to obtain overlapping fragments by overlapping PCR amplification. Since P was included in the primer design, trc The promoter and terminator are attached to the 5' and 3' ends of the primers, respectively, so the overlapping fragments have P trc Promoter and P trc terminator, that is, the overlapping fragment is composed of the "upstream homology arm-P trc Promoter-target gene-P trc terminator-downstream homology arm"; using rph-pGRB-UP and rph-pGRB-DN as primers, annealing to obtain gRNA fragments, and connecting them with pGRB vector to obtain rph-pGRB; preparing XL2 electroporation competent cells, electroporating the overlapping fragments and rph-pGRB into competent cells, and screening to obtain positive transformants to obtain strain XL3.
[0065] 1.4 Overexpression of pRS Eco D128A Gene:
[0066] The three methods have the same operation method, except that the target gene template is the genome of wild-type Escherichia coli (E. coli) W3110, the primers are ilvG-UP-S, ilvG-UP-A; ilvG-DN-S, ilvG-DN-A, prs-UP-1, prs-DN-1, prs-UP-2, prs-DN-2, ilvG-pGRB-UP, ilvG-pGRB-DN. The competent cell is XL3, and the strain XL4 is obtained.
[0067] 1.5 Knockout of ppnP gene:
[0068] The two methods have the same operation method, except that the primers are ppnP-UP-S, ppnP-UP-A, ppnP-DN-S, ppnP-DN-A, ppnP-pGRB-UP, ppnP-pGRB-DN, the competent cell used is XL4, and the strain XL5 is obtained.
[0069] 1.6 Knockout of deoD gene:
[0070] The two methods have the same operation method, except that the primers are deoD-UP-S, deoD-UP-A, deoD-DN-S, deoD-DN-A, deoD-pGRB-UP, deoD-pGRB-DN, the competent cell used is XL5, and the strain XL6 is obtained.
[0071] 1.7 Integration of aspA gene:
[0072] The three methods have the same operation method, except that the target gene template is the genome of wild-type Escherichia coli (E. coli) W3110, the primers are mbhA-UP-S, mbhA-UP-A, mbhA-DN-S, mbhA-DN-A, aspA-UP, aspA-DN, mbhA-pGRB-UP, mbhA-pGRB-DN. The competent cell is XL6, and the strain XL7 is obtained.
[0073] 1.8 Knockout of guaA gene:
[0074] The two methods have the same operation method, except that the primers are guaA-UP-S, guaA-UP-A, guaA-DN-S, guaA-DN-A, guaA-pGRB-UP, guaA-pGRB-DN, the competent cell used is XL7, and the final strain XL8 is obtained.
[0075] Example 2
[0076] The strains XL0 and XL8 in Example 1 were respectively used for 24 h fermentation in 500 mL Erlenmeyer flasks, and the specific steps were as follows:
[0077] 2.1 Activation of bacteria: Transfer the bacteria from the glycerol tube to the LB test tube for activation and culture for 10-12 hours at 37°C and 220 rpm for about 10-12 hours;
[0078] 2.2 Seed culture: Inoculate the bacteria on the slant medium in step 2.1 into 30 mL seed culture medium under sterile conditions and culture at 37°C and 220 rpm for about 10-12 hours;
[0079] 2.3 Fermentation culture: Under sterile conditions, use a sterile pipette to inoculate the seed culture solution in step 2.2 into the fermentation medium at a 10-15% inoculation rate, so that the total volume of the fermentation medium is 30mL, 37°C, 220rpm. During the fermentation process, the pH value and glucose content are determined by the color change of the phenol red indicator. The pH value is maintained at 7.0-7.2 by adding ammonia water in small amounts and multiple times, and the strain growth is maintained by adding 60% (m / v) glucose. The fermentation cycle is 24h.
[0080] In the above shake flask fermentation culture process, the LB test tube culture medium used is: sodium chloride 10g / L, peptone 10g / L, yeast powder 5g / L, xanthine 100mg / L 121℃, 20min sterilization; after sterilization, the test tube culture medium is prepared. The seed culture medium used is: glucose 30g / L, yeast extract powder 2g / L, peptone 1g / L, citric acid 1.25g / L, glutamic acid 1g / L, ammonium sulfate 3g / L, MgSO4·7H2O0.4g / L, KH2PO41.5g / L, vitamin B1 (V B1 )0.5mg / L, guanine 100mg / L, and the rest is water. The fermentation medium used is: glucose 30g / L, yeast extract 4g / L, peptone 1g / L, citric acid 1g / L, ammonium sulfate 3g / L, glutamic acid 1g / L, MgSO4·7H2O 1g / L, KH2PO43.5g / L, FeSO4·7H2O 20mg / L, MnSO410mg / L, vitamin B1 (V B1 )0.5mg / L, guanine 100mg / L, and the rest is water.
[0081] like Figure 2 As shown, after 24 h fermentation in a 500 mL Erlenmeyer flask, the OD of strain XL0 was 600 17.5, no xanthine nucleoside was produced in the fermentation broth; strain XL80 D 600 17.3, the concentration of xanthine nucleotides in the fermentation broth can reach 3.6 g / L.
[0082] Example 3
[0083] The strain XL8 in Example 1 was used for fermentation culture in a 5L fermentation tank, and the specific steps were as follows:
[0084] 3.1 Activation of strains: transfer the strains from the glycerol tube to the slant culture medium for activation and culture for 11-13 hours; transfer the strains from the slant culture medium to the eggplant flask culture medium for further activation and expansion culture for 10-12 hours, culture temperature: 37°C;
[0085] 3.2 Seed culture: During the culture process, the pH is maintained at 7.0±0.1, the temperature is maintained at 37±0.2℃, and the dissolved oxygen is maintained at 25%-35%. When the OD reaches 10, the culture is transferred to a fermenter for fermentation culture;
[0086] 3.3 Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 20% for fermentation culture. The fermentation pH is maintained at 7.0±0.1, the temperature is maintained at 37±0.2°C, and the dissolved oxygen is maintained at 25%-30%. During the culture process, 80% glucose solution is added to maintain the carbon source required by the fermentation process. During this period, 25% ammonia water is added to adjust the pH to maintain at 7.0±0.1 to prepare xanthine nucleoside.
[0087] In the fermentation tank culture process, the slant medium and eggplant flask medium used are: beef extract 10g / L, glucose 5g / L, sodium chloride 5g / L, peptone 10g / L, yeast powder 5g / L, xanthine 100mg / L, agar powder 25g / L, pH adjusted to 7.0-7.2, 121℃, 20min sterilization; after sterilization, test tube slant medium and eggplant flask medium are prepared respectively. The seed culture medium used is: glucose 30g / L, yeast extract powder 5g / L, peptone 1g / L, citric acid 1.25g / L, MgSO4·7H2O1.5g / L, KH2PO4 3.0g / L, vitamin B1 (V B1 )1mg / L, biotin(V H )1mg / L, guanine 200mg / L, and the rest is water. The fermentation medium used is: glucose 20g / L, yeast extract 3g / L, peptone 1g / L, citric acid 2g / L, MgSO4·7H2O 2g / L, KH2PO46g / L, FeSO4·7H2O 20mg / L, MnSO410mg / L, vitamin B1 (V B1 )2mg / L, biotin (V H )2mg / L, guanine 200mg / L, and the rest is water.
[0088] like Figure 3 As shown, xanthocyanine riboside was produced by fermentation in a 5L fermenter. After 26h, the OD of the strain was 600 60.8 The xanthine nucleoside production reached 10.25g / L, laying the foundation for the subsequent fermentation production of xanthine nucleoside.
[0089] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.
Claims
1. A xanthosine nucleoside producing strain, characterized in that: The wild-type Escherichia coli E.co / iW3110 was used as the starting strain by genetic modification: first, the purine operon gene pur (EKBCSQLFMNHD) in Bacillus amyloliquefaciens was introduced into the yghX site of the genome of wild-type Escherichia coli E.co / iW3110, and then P trc The promoter was activated; then the purine nucleotide repressor gene purR was knocked out in the genome, and the anti-feedback variant purF of PRPP transamidase was introduced into the rph and i / vG sites of the genome respectively. Baz K326Q and the feedback-resistant variant of PRPP synthase, prs Eco D128A , both by P trc Promoter is started; then the nucleoside phosphorylase gene ppnP and the nucleoside phosphorylase gene deoD are knocked out in the genome, and the aspartate ammonia lyase gene aspA is integrated at the mbhA site in the genome, and then the guanosine monophosphate synthetase gene guaA is knocked out.
2. The xanthosine nucleoside producing strain according to claim 1, characterized in that: The NCBI-GeneID of the purine operon gene pur (EKBCSOLFMNHD) is: purE: 75094376; purK: 75094377; purB: 75094378; purC: 75094379; purS: 75094380; purQ: 75094381; purL: 75094382; purF: 75094383; purM: 75094384; purN: 75094385; purH: 75094386; purD: 75094387; the NCBI-GeneID of the purine nucleotide repressor protein gene purR is 945226; the anti-feedback variant purF Baz K326Q NCBI-GeneID: 75094383; the anti-feedback variant prs Eco D128A NCBI-GeneID: 545772; NCBI-GeneID: 945048 of the nucleoside phosphorylase gene ppnP; NCBI-GeneID: 945654 of the nucleoside phosphorylase gene deoD; NCBI-Gene ID: 948658 of the aspartate ammonia lyase gene aspA; NCBl-Gene ID: 947334 of the guanosine monophosphate synthase gene guaA.
3. The xanthosine nucleoside producing strain according to claim 1, characterized in that: The P trc The nucleotide sequence of the promoter is shown in SEQ ID NO.
95.
4. The method for constructing a xanthosine nucleoside producing strain according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: (1) The heterologous purine operon gene pur (EKBCSQLFMNHD) was introduced into the yghX locus of the E. co / i W3110 genome by P trc Promoter start; (2) Knock out the purine nucleotide repressor protein gene purR and introduce the anti-feedback variant purF of the PRPP amidotransferase of Bacillus amyloliquefaciens into the i / vG and rph sites of the genome, respectively. Baz K326Q and the feedback-resistant variant prS of PRPP synthase from Escherichia coli Eco D128A , both by P trc Promoter start; (3) Knock out the ppnP gene and deoD gene in the genome; (4) overexpression of the aspartate ammonia lyase gene aspA; (5) Knock out the guanosine monophosphate synthase gene guaA.
5. Use of the xanthosine nucleoside producing strain according to any one of claims 1 to 3 in producing xanthosine nucleoside.
6. The use according to claim 5, characterized in that: Xanthine ribosides are produced by fermentation.
7. The use according to claim 6, characterized in that: The fermentation culture includes shake flask fermentation or fermenter fermentation.
8. The use according to claim 7, characterized in that: The inoculation amount during the shake flask fermentation is 10%-15%, the temperature is maintained at 37°C±0.2, and the shaking culture is carried out at 220r / min. During the fermentation process, ammonia water is added to maintain the pH at 7.0; and glucose solution is added to supplement the carbon source required by the bacteria.
9. The use according to claim 7, characterized in that: The specific steps of the fermentation tank fermentation are as follows: (1) Activation of bacterial strains: transfer the bacterial strains from the glycerol tube to the slant culture medium for activation and culture for 11-13 hours; transfer the bacterial strains from the slant culture medium to the eggplant flask culture medium for further activation and expansion culture for 10-12 hours, culture temperature: 37°C; (2) Seed culture: During the culture process, the pH was maintained at 7.0±0.1, the temperature was maintained at 37±0.2°C, and the dissolved oxygen was maintained at 25%-35%. When the OD reached 10, the culture was transferred to a fermenter for fermentation. (3) Fermentation culture: The seed liquid is inoculated into the fermentation medium at an inoculum rate of 20% for fermentation culture. The fermentation pH is maintained at 7.0±0.1, the temperature is maintained at 37±0.2°C, and the dissolved oxygen is maintained at 25%-30%. During the culture process, glucose solution is added to maintain the carbon source required by the fermentation process, and ammonia water is added to adjust the pH to maintain at 7.0±0.
1.
10. The use according to claim 9, characterized in that: The slant culture medium and eggplant flask culture medium used are: beef extract 10g / L, glucose 5g / L, sodium chloride 5g / L, peptone 10g / L, yeast powder 5g / L, xanthine 100mg / L, agar powder 25g / L, pH adjusted to 7.0-7.2; the seed culture medium used is: glucose 30g / L, yeast extract powder 5g / L, peptone 1g / L, citric acid 1.25g / L, MgS04·7H2O1.5g / L, KH2PO4 3.0g / L, vitamin B11mg / L, biotin 1mg / L, guanine 200mg / L, and the rest is water; the fermentation medium used is: glucose 20g / L, yeast extract powder 3g / L, peptone 1g / L, citric acid 2g / L, MgSO4·7H2O 2g / L, KH2PO4 6g / L, FeSO4·7H2O 20mg / L, MnSO4 10mg / L, vitamin B12mg / L, biotin 2mg / L, guanine 200mg / L, and the rest is water.