CN118166052A - Method for improving guanosine yield, engineering bacteria thereof and application thereof - Google Patents
Method for improving guanosine yield, engineering bacteria thereof and application thereof Download PDFInfo
- Publication number
- CN118166052A CN118166052A CN202410321729.2A CN202410321729A CN118166052A CN 118166052 A CN118166052 A CN 118166052A CN 202410321729 A CN202410321729 A CN 202410321729A CN 118166052 A CN118166052 A CN 118166052A
- Authority
- CN
- China
- Prior art keywords
- guanosine
- strain
- glck
- recombinant plasmid
- yield
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 title claims abstract description 84
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 title claims abstract description 42
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 title claims abstract description 42
- 229940029575 guanosine Drugs 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 22
- 241000894006 Bacteria Species 0.000 title abstract description 6
- 101150083237 glcK gene Proteins 0.000 claims abstract description 32
- 239000013612 plasmid Substances 0.000 claims abstract description 31
- 108010021582 Glucokinase Proteins 0.000 claims abstract description 30
- 238000000855 fermentation Methods 0.000 claims abstract description 28
- 230000004151 fermentation Effects 0.000 claims abstract description 28
- 239000012634 fragment Substances 0.000 claims abstract description 20
- 238000004519 manufacturing process Methods 0.000 claims description 14
- 244000063299 Bacillus subtilis Species 0.000 claims description 10
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 7
- 230000002018 overexpression Effects 0.000 claims description 5
- 239000013598 vector Substances 0.000 claims description 3
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 2
- 241000555281 Brevibacillus Species 0.000 claims description 2
- 241000186216 Corynebacterium Species 0.000 claims description 2
- 241000179039 Paenibacillus Species 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 239000013613 expression plasmid Substances 0.000 claims description 2
- 238000010367 cloning Methods 0.000 abstract description 5
- 239000002253 acid Substances 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 102000004190 Enzymes Human genes 0.000 abstract description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 2
- 230000006860 carbon metabolism Effects 0.000 abstract description 2
- 230000006798 recombination Effects 0.000 abstract description 2
- 238000005215 recombination Methods 0.000 abstract description 2
- 239000002609 medium Substances 0.000 description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000002585 base Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 235000010633 broth Nutrition 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229960004150 aciclovir Drugs 0.000 description 3
- MKUXAQIIEYXACX-UHFFFAOYSA-N aciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCO)C=N2 MKUXAQIIEYXACX-UHFFFAOYSA-N 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 229960000329 ribavirin Drugs 0.000 description 2
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 150000004683 dihydrates Chemical class 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical class OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 1
- 239000004198 disodium guanylate Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- IYWCBYFJFZCCGV-UHFFFAOYSA-N formamide;hydrate Chemical compound O.NC=O IYWCBYFJFZCCGV-UHFFFAOYSA-N 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 1
- 239000004226 guanylic acid Substances 0.000 description 1
- 235000013928 guanylic acid Nutrition 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229940127554 medical product Drugs 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- MRWXACSTFXYYMV-FDDDBJFASA-N nebularine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC=C2N=C1 MRWXACSTFXYYMV-FDDDBJFASA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002212 purine nucleoside Substances 0.000 description 1
- OENLEHTYJXMVBG-UHFFFAOYSA-N pyridine;hydrate Chemical compound [OH-].C1=CC=[NH+]C=C1 OENLEHTYJXMVBG-UHFFFAOYSA-N 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the field of genetic engineering, and particularly relates to a method for improving guanosine yield, engineering bacteria thereof and application thereof. The method for improving the guanosine yield comprises the steps of carrying out seamless cloning recombination on a glucokinase gene glcK fragment to a pBG-BamHI enzyme fragment to form a recombinant plasmid, then converting the recombinant plasmid into a strain SHE166E to obtain a recombinant strain, producing guanosine by shake flask fermentation, and obviously improving the guanosine fermentation yield of the recombinant strain which overexpresses the glucokinase gene glcK in a final fermentation product, thereby providing data support for improving the guanosine yield and the acid yield by optimizing the carbon metabolism of the strain.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a method for improving guanosine yield, engineering bacteria thereof and application thereof.
Background
Guanosine is one of purine nucleoside substances, and has a molecular formula of C 10H13N5O5, a relative molecular mass of 283.24, white crystalline powder, needle-like crystals of dihydrate, water loss at 110 ℃, a melting point of 240 ℃ (decomposition), a specific optical rotation of-60 ℃, and can be dissolved in dilute mineral acid, hot acetic acid and dilute alkali (1 g/33 mL), boiling water, and slightly dissolved in cold water (1 g/1320 mL), and is insoluble in alcohol, ether, chloroform and benzene.
The guanosine has very wide application, is an important intermediate of food and medical products, can be used for synthesizing food flavoring agents, namely, 5' -disodium guanylate, flavor disodium nucleotide, nucleoside antiviral drugs such as ribavirin, acyclovir and the like, and is also a main raw material for preparing drugs such as acyclovir (Acyclovir), ribavirin (ATC), guanosine Triphosphate (GTP) and the like.
Industrial guanosine production is mainly chemical synthesis, RNA hydrolysis and microbial fermentation. The guanylic acid is produced by chemical synthesis, and the nucleoside is usually subjected to phosphorylation reaction by phosphoric acid or active derivative of pyrophosphoric acid, so that the chemical synthesis is less and less dominant along with strict environmental protection requirements. The RNA hydrolysis method is to make RNA react in strong ammonia water at high temperature and high pressure of 175-180 ℃, or boil in pyridine water solution for several days, or heat lamp method in formamide water solution to obtain nucleotide, but the method has few adoption in modern industrial production because of the problems of complex process, low yield, high cost, environmental pollution of raw material source base and the like. The production of guanosine by a microbial fermentation method enters an industrial production scale and becomes a main development direction of guanosine production in the future; microbial fermentation studies of guanosine have now been carried out industrially, but the yield and conversion rate have yet to be improved. Based on the background, we plan to develop a genetically engineered bacillus subtilis with high guanosine yield, in the process, we find that glcK has great advantages in SHE166E genetically engineered strains, and is beneficial to improving the guanosine yield produced by fermenting the genetically engineered strains.
Disclosure of Invention
The method for producing the guanosine comprises the steps of carrying out seamless cloning recombination on a glucokinase gene glcK fragment to a pBG-BamHI restriction enzyme plasmid fragment to obtain a recombinant plasmid, then converting the recombinant plasmid into a strain SHE166E to obtain a recombinant strain, producing the guanosine through shake flask fermentation, and obviously improving the guanosine yield of the strain which overexpresses the glucokinase gene glcK in a final fermentation product.
In one aspect, the invention provides a method for increasing guanosine fermentation yield comprising: the glucokinase gene glcK is overexpressed in the basal strain.
The nucleotide fragment of the glucokinase gene glcK comprises SEQ ID NO.1 or a functional fragment thereof.
Specifically, the basic strain comprises bacillus, paenibacillus, brevibacillus and corynebacterium;
Preferably, the said base bacterial species is bacillus subtilis (Bacillus subtilis);
Preferably, the basic strain is bacillus subtilis SHE166E, and the preservation number is CGMCC No.29449.
Specifically, the overexpression of the glucokinase gene glcK is realized by constructing a recombinant plasmid or is directly integrated into a genome.
Preferably, the vector skeleton of the recombinant plasmid is pBG102,102 plasmid.
Preferably, the insertion site of the glucokinase gene glcK in pBG102 plasmid is BamHI.
SEQ ID NO.1：
TTCCCATATAAAGGAGGAAGGATCatggacgagatatggtttgcgggcattgacctgggaggaacgacgattaaactcgcttttattaatcaatatggcgaaattcagcataagtgggaagttccgacagataaaaccggcgacacgattactgtcacaattgcaaaaacaatcgacagcaagctggatgagctgcaaaaaccgaagcacatcatcaaatacatcggaatgggtgcaccaggccctgtagatatggcggcaggagtggtttatgaaacagtaaatctagggtggaaaaattatgctttgaaaaaccatctggagacagaaaccggcatcccagctgttatagaaaatgacgcgaatattgctgcgctcggggaaatgtggaagggagcgggtgatggcgcaaaagacgtcattctcgtgacgcttggcacaggagttggcggcggcatcattgcaaatggtgaaattgtacatggtataaatggcgccggcggagaaatcggccatatttgcagcatccctgaaggcggagcgccctgcaactgcggcaaaacgggctgtatcgaaacaattgcgtcagcaaccggaattgtaagaattgcaaaagaaaaaatagcaaatgctaaaaagacgacacgtttaaaagcaaccgaacaattgtcagcgcgagatgtgtttgaagcggcgggtgaaaatgatgaaattgcccttgaggtggttgattatgtagccaagcatcttggtttggtgctcggaaatttggcaagctcgcttaatccatccaaaatcgttcttggcggcggcgtatcgagagccggagaactgctgagatcaaaagtcgagaaaacattccgcaaatgcgcgtttccgcgggcagcccaagctgctgatatttcaatcgcagcacttggaaatgatgccggcgttatcggaggcgcttggatcgctaaaaatgaatggctgaaacatcaaaattgttaaGATCCTCTAGAGTCGACgtccc.
Specifically, the nucleotide sequence of the primer for amplifying the glucokinase gene glcK fragment comprises SEQ ID NO.2 and SEQ ID NO.3.
Specifically, amplification of the glcK fragment of the glucokinase gene was performed by PCR technique.
Specifically, the sequence length of the glucokinase gene glcK fragment is 1012bp.
SEQ ID NO.2：TTCCCATATAAAGGAGGAAGGATCatggacgagatatggtttgcgg。
SEQ ID NO.3：gggacGTCGACTCTAGAGGATCttaacaattttgatgtttcagccattc。
In yet another aspect, the present invention also provides a recombinant plasmid comprising the expression of the aforementioned glucokinase gene glcK.
Specifically, the backbone plasmid of the recombinant plasmid is a bacillus subtilis expression plasmid vector.
Specifically, the backbone plasmid is selected from pHT01, pHT254, pHT43, pMA5, pWB980 or pBG102.
Preferably, the backbone plasmid is pBG102,102.
Specifically, the nucleotide sequence of the recombinant plasmid comprises SEQ ID NO.4.
Specifically, the construction method of the recombinant plasmid comprises the following steps: and (3) recombining the glucokinase gene glcK onto pBG-BamHI enzyme fragments in a seamless cloning mode to obtain recombinant plasmids.
SEQ ID NO.4：gtaaaacgacggccagtgaattcgagctcaggccttaactcacattaattgcgttgcgctc actgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttgacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcatcaaaatcgtctccctccgtttgaatatttgattgatcgtaaccagatgaagcactctttccactatccctacagtgttatggcttgaacaatcacgaaacaataattggtacgtacgatctttcagccgactcaaacatcaaatcttacaaatgtagtctttgaaagtattacatatgtaagatttaaatgcaaccgttttttcggaaggaaatgatgacctcgtttccaccggaattagcttggtaccaaaggaggtaaggatcactagaaaattttttaaaaaatctcttgacattggaagggagatatgttattataagaattgcggAATTGTGAGCGGATAACAATTCCCATATAAAGGAGGAAGGATCatggacgagatatggtttgcgggcattgacctgggaggaacgacgattaaactcgcttttattaatcaatatggcgaaattcagcataagtgggaagttccgacagataaaaccggcgacacgattactgtcacaattgcaaaaacaatcgacagcaagctggatgagctgcaaaaaccgaagcacatcatcaaatacatcggaatgggtgcaccaggccctgtagatatggcggcaggagtggtttatgaaacagtaaatctagggtggaaaaattatgctttgaaaaaccatctggagacagaaaccggcatcccagctgttatagaaaatgacgcgaatattgctgcgctcggggaaatgtggaagggagcgggtgatggcgcaaaagacgtcattctcgtgacgcttggcacaggagttggcggcggcatcattgcaaatggtgaaattgtacatggtataaatggcgccggcggagaaatcggccatatttgcagcatccctgaaggcggagcgccctgcaactgcggcaaaacgggctgtatcgaaacaattgcgtcagcaaccggaattgtaagaattgcaaaagaaaaaatagcaaatgctaaaaagacgacacgtttaaaagcaaccgaacaattgtcagcgcgagatgtgtttgaagcggcgggtgaaaatgatgaaattgcccttgaggtggttgattatgtagccaagcatcttggtttggtgctcggaaatttggcaagctcgcttaatccatccaaaatcgttcttggcggcggcgtatcgagagccggagaactgctgagatcaaaagtcgagaaaacattccgcaaatgcgcgtttccgcgggcagcccaagctgctgatatttcaatcgcagcacttggaaatgatgccggcgttatcggaggcgcttggatcgctaaaaatgaatggctgaaacatcaaaattgttaaGATCCTCTAGAGTCGACgtccccggggcagcccgcctaatgagcgggcttttttcacgtcacgcgtccatggagatctttgtctgcaactgaaaagtttataccttacctggaacaaatggttgaaacatacgaggctaatatcggcttattaggaatagtccctgtactaataaaatcaggtggatcagttgatcagtatattttggacgaagctcggaaagaatttggagatgacttgcttaattccacaattaaattaagggaaagaataaagcgatttgatgttcaaggaatcacggaagaagatactcatgataaagaagctctaaaactattcaataaccttacaatggaattgatcgaaagggtggaaggttaatggtacgaaaattaggggatctacctagaaagccacaaggcgataggtcaagcttaaagaacccttacatggatcttacagattctgaaagtaaagaaacaacagaggttaaacaaacagaaccaaaaagaaaaaaagcattgttgaaaacaatgaaagttgatgtttcaatccataataagattaaatcgctgcacgaaattctggcagcatccgaagggaattcatattacttagaggatactattgagagagctattgataagatggttgagacattacctgagagccaaaaaactttttatgaatatgaattaaaaaaaagaaccaacaaaggctgagacagactccaaacgagtctgtttttttaaaaaaaatattaggagcattgaatatatattagagaattaagaaagacatgggaataaaaatattttaaatccagtaaaaatatgataagattatttcagaatatgaagaactctgtttgtttttgatgaaaaaacaaacaaaaaaaatccacctaacggaatctcaatttaactaacagcggccaaactgagaagttaaatttgagaaggggaaaaggcggatttatacttgtatttaactatctccattttaacattttattaaaccccatacaagtgaaaatcctcttttacactgttcctttaggtgatcgcggagggacattatgagtgaagtaaacctaaaaggaaatacagatgaattagtgtattatcgacagcaaaccactggaaataaaatcgccaggaagagaatcaaaaaagggaaagaagaagtttattatgttgctgaaacggaagagaagatatggacagaagagcaaataaaaaacttttctttagacaaatttggtacgcatataccttacatagaaggtcattatacaatcttaaataattacttctttgatttttggggctattttttaggtgctgaaggaattgcgctctatgctcacctaactcgttatgcatacggcagcaaagacttttgctttcctagtctacaaacaatcgctaaaaaaatggacaagactcctgttacagttagaggctacttgaaactgcttgaaaggtacggttttatttggaaggtaaacgtccgtaataaaaccaaggataacacagaggaatccccgatttttaagattagacgtaaggttcctttgctttcagaagaacttttaaatggaaaccctaatattgaaattccagatgacgaggaagcacatgtaaagaaggctttaaaaaaggaaaaagagggtcttccaaaggttttgaaaaaagagcacgatgaatttgttaaaaaaatgatggatgagtcagaaacaattaatattccagaggccttacaatatgacacaatgtatgaagatatactcagtaaaggagaaattcgaaaagaaatcaaaaaacaaatacctaatcctacaacatcttttgagagtatatcaatgacaactgaagaggaaaaagtcgacagtactttaaaaagcgaaatgcaaaatcgtgtctctaagccttcttttgatacctggtttaaaaacactaagatcaaaattgaaaataaaaattgtttattacttgtaccgagtgaatttgcatttgaatggattaagaaaagatatttagaaacaattaaaacagtccttgaagaagctggatatgttttcgaaaaaatcgaactaagaaaagtgcaataaactgctgaagtatttcagcagttttttttatttagaaatagtgaaaaaaatataatcagggaggtatcaatatttaatgagtactgatttaaatttatttagactggaattaataattaacacgtagactaattaaaatttaatgagggataaagaggatacaaaaatattaatttcaatccctattaaattttaacaagggggggattaaaatttaattagaggtttatccacaagaaaagaccctaataaaatttttactagggttataacactgattaatttcttaatgggggagggattaaaatttaatgacaaagaaaacaatcttttaagaaaagcttttaaaagataataataaaaagagctttgcgattaagcaaaactctttactttttcattgacattatcaaattcatcgatttcaaattgttgttgtatcataaagttaattctgttttgcacaaccttttcaggaatataaaacacatctgaggcttgttttataaactcagggtcgctaaagtcaatgtaacgtagcatatgatatggtatagcttccacccaagttagcctttctgcttcttctgaatgtttttcatatacttccatgggtatctctaaatgattttcctcatgtagcaaggtatgagcaaaaagtttatggaattgatagttcctctctttttcttcaacttttttatctaaaacaaacactttaacatctgagtcaatgtaagcataagatgtttttccagtcataatttcaatcccaaatcttttagacagaaattctggacgtaaatcttttggtgaaagaatttttttatgtagcaatatatccgatacagcaccttctaaaagcgttggtgaatagggcattttacctatctcctctcattttgtggaataaaaatagtcatattcgtccatctacctatcctattatcgaacagttgaactttttaatcaaggatcagtcctttttttcattattcttaaactgtgctcttaactttaacaactcgatttgtttttccagatctcgagggtaactagcctcgccgatcccgcaagaggcccggcagtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcatgcttaagttattggtatgactggttttaagcgcaaaaaaagttgctttttcgtacctattaatgtatcgttttagaaaaccgactgtaaaaagtacagtcggcattatctcatattataaaagccagtcattaggcctatctgacaattcctgaatagagttcataaacaatcctgcatgataaccatcacaaacagaatgatgtacctgtaaagatagcggtaaatatattgaattacctttattaatgaattttcctgctgtaataatgggtagaaggtaattactattattattgatatttaagttaaacccagtaaatgaagtccatggaataatagaaagagaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaaccattatatttctctacatcagaaaggtataaatcataaaactctttgaagtcattctttacaggagtccaaataccagagaatgttttagatacaccatcaaaaattgtataaagtggctctaacttatcccaataacctaactctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatcacccttgtcactaagaaaataaatgcagggtaaaatttatatccttcttgttttatgtttcggtataaaacactaatatcaatttctgtggttatactaaaagtcgtttgttggttcaaataatgattaaatatctcttttctcttccaattgtctaaatcaattttattaaagttcatttgatatgcctcctaaatttttatctaaagtgaatttaggaggcttacttgtctgctttcttcattagaatcaatccttttttaaaagtcaatattactgtaacataaatatatattttaaaaatatcccactttatccaattttcgtttgttgaactaatgggtgctttagttgaagaataaagaccacattaaaaaatgtggtcttttgtgtttttttaaaggatttgagcgtagcgaaaaatccttttctttcttatcttgataataagggtaactattgccgatcgtccattccgacagcatcgccagtcactatggcgtgctgctagcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgtt.
In yet another aspect, the invention also provides a genetically engineered strain comprising the recombinant plasmid.
Specifically, the recombinant plasmid was transformed into the base strain SHE 166E.
Specifically, the genetically engineered strain in the present invention refers to a genetically engineered microorganism strain.
In yet another aspect, the invention also provides the use of the overexpression of the aforementioned glucokinase gene glcK for increasing the yield of guanosine fermentation.
In yet another aspect, the invention also claims the use of the aforementioned recombinant plasmid and/or the aforementioned genetically engineered bacterium in the production of guanosine.
In yet another aspect, the invention also provides a method for producing guanosine.
Specifically, guanosine is produced by a shake flask fermentation method.
The shaking flask fermentation: i.e. this is a laboratory scale fermentation process, typically used for small scale production or testing. In this method, the bottles containing microorganisms and nutrients are shaken under controlled conditions to promote growth and metabolism.
In the present invention, guanosine is produced by a shake flask fermentation method in which a genetically engineered microorganism such as bacteria or yeast is used to efficiently produce a target compound.
Specifically, the shake flask fermentation comprises the following steps:
(1) Activation of the aforementioned genetically engineered strain;
(2) Fermenting;
(3) Extracting guanosine.
Specifically, the step (1) includes: inoculating the genetic engineering strain to an LB culture medium containing antibiotics for culture, and transferring the cultured seed liquid to a liquid culture medium containing antibiotics for continuous culture;
The step (2) comprises the following steps: transferring the activated seeds into a fermentation medium, fermenting and culturing, adding IPTG, and continuing fermenting and culturing;
The step (3) comprises the following steps: taking fermentation liquor, adding NaOH, diluting, centrifuging, filtering supernatant by using an aqueous phase membrane, and detecting by HPLC.
The invention has the beneficial effects that:
After the glucose kinase gene glcK is overexpressed in the base strain bacillus subtilis SHE166E, the obtained recombinant strain has improved guanosine fermentation yield compared with the base strain SHE 166E. In the strain SHE166E, the glcK fragment of the glucokinase gene was overexpressed, and guanosine yield was increased by more than 20%.
Preservation certificate
Preserving the strain: SHE166E;
Classification naming: bacillus subtilis Bacillus subtilis;
Preservation number: CGMCC No.29449;
preservation time: 2023, 12, 28;
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection).
Drawings
FIG. 1 is a peak control plot of HPLC.
FIG. 2 shows the results of guanosine production after shake flask fermentation of the strain.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
In the following examples:
basic example 1 method for producing guanosine by shake flask fermentation
1. Reagent:
(1) LB medium: each liter of the medium contained 5g of yeast powder, 10g of sodium chloride, 10g of peptone, and was fixed to a volume of 1L with deionized water (J.Sam Brookfield. Huang Peitang. TM., molecular cloning guide 2002,1595).
Sterilizing the above solution with high pressure steam at 121deg.C for 20-30min.
(2) Fermentation medium (per liter): 50g of glucose, 5g of corn steep liquor dry powder, 5g of yeast extract, 10g of monosodium glutamate, 5g of ammonium sulfate, 1g of monopotassium phosphate, 0.4g of magnesium sulfate heptahydrate and 20g of calcium carbonate, and after being dissolved by deionized water, regulating the pH value to 7.0, and fixing the volume to 1L by deionized water.
Sterilizing the above solution with high pressure steam at 121deg.C for 20-30min. Simultaneously preparing empty shake flasks, weighing 0.4g of calcium carbonate per flask, and fermenting at 121 ℃ for 20-30min to obtain final concentration of calcium carbonate.
2. Instrument: constant temperature shaking table, constant temperature incubator.
3. And (3) shaking and fermenting:
(1) The recombinant strain was inoculated into 3mL of LB medium containing the antibiotic, and cultured at 37℃with shaking table 250 rpm.
(2) After 16h of cultivation, 500. Mu.L of the seed solution was transferred to 1.5mL of LB liquid medium containing antibiotics, and cultured at 37℃for 4h with shaking table 250 rpm.
(3) 2ML of the secondary seeds were all transferred to a shake flask containing 18mL of fermentation medium, and incubated at 250rpm in a shaker at 37℃for 4h.
(4) The final concentration of IPTG was 0.1mM, and fermentation was continued at 37℃for about 40 hours.
(5) The OD 600 of the broth was determined after dilution with 0.1mL of broth and 2.9mL of 30mM dilute hydrochloric acid.
(6) After the rest of the fermentation broth was diluted with 0.2M NaOH at a multiple, it was centrifuged at 12000rpm for 1min, and the supernatant was filtered with a 0.22 μm filter membrane and then subjected to HPLC detection.
4. Determination of adenosine in fermentation broths by HPLC
(1) The HPLC parameters were as follows:
Chromatographic column: XBridge C18.6 x 150mm 5um
Mobile phase: a is methanol and B is 10mM ammonium acetate (pH 4.0)
Column temperature: 30 DEG C
Initial flow rate: 1.0mL/min
Detection wavelength: 260nm of
| Time (min) | Phase A (% v/v) | Phase B (% v/v) | Flow(mL/min) |
| 0.01 | 2 | 98 | 1.0 |
| 3.50 | 15 | 85 | 1.0 |
| 4.00 | 22 | 98 | 1.0 |
| 8.00 | 2 | 98 | 1.0 |
(2) HPLC profile is shown in fig. 1, T (guanine) =3.1 min, T (guanosine) =5.9 min.
EXAMPLE 1 construction of glcK overexpression plasmid
(1) The Bacilllus subtilis genome (NCBI Reference Sequence: NC_ 000964.3) is used as a template, and a primer pair YHB1521 and YHB1522 is used for PCR to obtain 1012bp glucokinase gene glcK fragments, and the nucleotide sequence of the fragments comprises SEQ ID NO.1 or functional fragments thereof.
TABLE 1 sequences of primers used in the above (1)
(2) The expression vector was pBG as described in patent CN113755412B, and the glcK fragment of the glucokinase gene obtained above was recombined onto pBG-BamHI restriction enzyme fragment by a seamless cloning method (GBclonart seamless cloning kit, su zhou shenzhou gene limited) to obtain a recombinant plasmid pHB477 (the nucleic acid sequence is shown in SEQ ID No. 4).
EXAMPLE 2 overexpression of the glucokinase Gene glcK
(1) The recombinant plasmids pHB477 and pBG102,102 constructed in example 1 were transformed into SHE166E (preservation number: CGMCC No. 29449) and GA01 (guanosine industrial production strain), respectively, according to the transformation method described in patent CN113755412B, to obtain recombinant strains.
(2) Referring to the shake flask fermentation method in basic example 1, the recombinant strain obtained in the above (1) was subjected to shake flask fermentation to verify the guanosine yield, and the result is shown in fig. 2, in which the glcK fragment of the glucokinase gene was overexpressed in the strain SHE166E, and the guanosine yield was increased by 20% or more; in the strain GA01, the glucose kinase gene glcK is overexpressed, and the guanosine yield is improved by nearly one time, namely, the glucose kinase gene glcK has obvious advantages in the guanosine production strain, and data support is provided for improving the guanosine yield and the acid production rate by optimizing the carbon metabolism of the strain subsequently.
Claims (10)
1. A method for increasing guanosine fermentation yield comprising: the glucose kinase gene glcK is overexpressed in the basic strain, and the nucleotide sequence of the glucose kinase gene glcK comprises SEQ ID NO.1 or a functional fragment thereof.
2. The method of claim 1, wherein the base strain comprises bacillus, paenibacillus, brevibacillus, or corynebacterium.
3. The method of claim 2, wherein the base strain is bacillus subtilis (Bacillus subtilis) with a preservation number of CGMCC No.29449.
4. The method according to claim 1, wherein the overexpression of the glucokinase gene glcK is achieved by constructing a recombinant plasmid or directly integrating into the genome.
5. The recombinant plasmid for expressing the glucokinase gene glcK is characterized in that the backbone plasmid is a bacillus subtilis expression plasmid vector; the backbone plasmid is selected from pHT01, pHT254, pHT43, pMA5, pWB980 or pBG102.
6. The recombinant plasmid according to claim 5, wherein the nucleotide sequence of the glucokinase gene glcK comprises SEQ ID No.1 or a functional fragment thereof; the nucleotide sequence of the recombinant plasmid comprises SEQ ID NO.4.
7. A genetically engineered strain comprising the recombinant plasmid of claim 5.
8. Use of the overexpressed glucokinase gene glcK for increasing the production of guanosine, comprising overexpressing the glucokinase gene glcK in a base strain, said glucokinase gene glcK having a nucleotide sequence comprising SEQ ID No.1 or a functional fragment thereof.
9. Use of the recombinant plasmid of claim 5 and/or the genetically engineered strain of claim 7 for increasing guanosine production.
10. A method for producing guanosine, comprising the steps of:
(1) Activation of the genetically engineered strain of claim 7;
(2) Fermenting;
(3) Extracting guanosine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410321729.2A CN118166052A (en) | 2024-03-20 | 2024-03-20 | Method for improving guanosine yield, engineering bacteria thereof and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410321729.2A CN118166052A (en) | 2024-03-20 | 2024-03-20 | Method for improving guanosine yield, engineering bacteria thereof and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118166052A true CN118166052A (en) | 2024-06-11 |
Family
ID=91346704
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410321729.2A Pending CN118166052A (en) | 2024-03-20 | 2024-03-20 | Method for improving guanosine yield, engineering bacteria thereof and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118166052A (en) |
-
2024
- 2024-03-20 CN CN202410321729.2A patent/CN118166052A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100491524C (en) | Process and materials for production of glucosamine | |
| CN112342176A (en) | Genetic engineering bacterium for producing 2' -fucosyllactose and application thereof | |
| CN114134093B (en) | Recombinant microorganism producing cytosine and method for producing cytosine | |
| CN108753669B (en) | Adenine production strain and construction method and application thereof | |
| CN107916283B (en) | A kind of production technology of niacinamide | |
| CN103409485A (en) | Method for improving adenosine fermentation output through feeding organic nitrogen source | |
| CN105154381A (en) | Novel mutant microorganism producing succinic acid simultaneously using sucrose and glycerol, and method for preparing succinic acid using same | |
| CN116463273A (en) | Method for enhancing accumulation of 5' -cytidine acid and application thereof | |
| CN104561195A (en) | Preparation method of uridine diphosphate glucose | |
| CN108018252A (en) | A kind of preparation method of intermediate 2 '-deoxyguanosine | |
| CN110551781A (en) | Method for preparing 5' -guanylic acid by enzyme method | |
| KR101371954B1 (en) | Novel process | |
| CN114480461B (en) | Recombinant microorganism for producing beta-nicotinamide mononucleotide and construction method and application thereof | |
| CN106834176B (en) | Nucleoside phosphorylase, coding gene, high-yield strain thereof and application | |
| CN118166052A (en) | Method for improving guanosine yield, engineering bacteria thereof and application thereof | |
| CN115851855A (en) | Method and composition for enzymatic synthesis of purine nucleosides | |
| CN113025550B (en) | High yield of vitamin B2Bacillus subtilis engineering strain, construction and application thereof | |
| Kang et al. | Preparative synthesis of dTDP‐l‐rhamnose through combined enzymatic pathways | |
| JP4066121B2 (en) | Process for producing guanosine and its intermediate | |
| CN114107143A (en) | Method for producing 5' -cytidylic acid | |
| Taran et al. | Synthesis of 2-chloro-2′-deoxyadenosine by microbiological transglycosylation using a recombinant Escherichia coli strain | |
| CN104894022B (en) | The gene and application of a kind of organic solvent-resistant galactosidase Producing Strain and the galactosidase | |
| CN116925993B (en) | Genetically engineered strains and methods for enzyme-catalyzed production of cytidine acids | |
| CN116948928B (en) | Seed culture medium and fermentation production method of 2' -fucosyllactose without antibiotics and IPTG inducer | |
| CN117106680B (en) | Recombinant microorganism and method for producing cytosine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |