CN111996208A - 一种利用重组枯草芽孢杆菌生产烟酰胺单核苷酸的方法 - Google Patents
一种利用重组枯草芽孢杆菌生产烟酰胺单核苷酸的方法 Download PDFInfo
- Publication number
- CN111996208A CN111996208A CN202010944821.6A CN202010944821A CN111996208A CN 111996208 A CN111996208 A CN 111996208A CN 202010944821 A CN202010944821 A CN 202010944821A CN 111996208 A CN111996208 A CN 111996208A
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- nicotinamide
- culture medium
- promoter
- gene
- 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
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 47
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 47
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 15
- DAYLJWODMCOQEW-TURQNECASA-O NMN(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(O)=O)O2)O)=C1 DAYLJWODMCOQEW-TURQNECASA-O 0.000 title abstract 5
- 239000001963 growth medium Substances 0.000 claims abstract description 20
- 230000014509 gene expression Effects 0.000 claims abstract description 19
- 108010064862 Nicotinamide phosphoribosyltransferase Proteins 0.000 claims abstract description 18
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
- 102000015532 Nicotinamide phosphoribosyltransferase Human genes 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 6
- 210000000349 chromosome Anatomy 0.000 claims abstract description 5
- DAYLJWODMCOQEW-TURQNECASA-N NMN zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)([O-])=O)O2)O)=C1 DAYLJWODMCOQEW-TURQNECASA-N 0.000 claims description 29
- 239000013612 plasmid Substances 0.000 claims description 28
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 18
- 238000009630 liquid culture Methods 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 12
- 230000010354 integration Effects 0.000 claims description 9
- 210000004027 cell Anatomy 0.000 claims description 8
- 241000276408 Bacillus subtilis subsp. subtilis str. 168 Species 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 108010043652 Transketolase Proteins 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 241000894007 species Species 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 101100001650 Geobacillus stearothermophilus amyM gene Proteins 0.000 claims description 5
- 229930193140 Neomycin Natural products 0.000 claims description 5
- 229960004927 neomycin Drugs 0.000 claims description 5
- 230000001737 promoting effect Effects 0.000 claims description 5
- 241000194108 Bacillus licheniformis Species 0.000 claims description 4
- 150000001447 alkali salts Chemical class 0.000 claims description 4
- 238000010353 genetic engineering Methods 0.000 claims description 4
- 108010061330 glucan 1,4-alpha-maltohydrolase Proteins 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 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 claims description 3
- 241000589496 Meiothermus ruber Species 0.000 claims description 3
- 241000589596 Thermus Species 0.000 claims description 3
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims description 3
- 241000606834 [Haemophilus] ducreyi Species 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 abstract description 10
- 229960003966 nicotinamide Drugs 0.000 abstract description 5
- 235000005152 nicotinamide Nutrition 0.000 abstract description 5
- 239000011570 nicotinamide Substances 0.000 abstract description 5
- 239000000758 substrate Substances 0.000 abstract description 5
- 241000193830 Bacillus <bacterium> Species 0.000 abstract description 3
- 238000000034 method Methods 0.000 description 11
- 239000012634 fragment Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000010276 construction Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 4
- 102000003960 Ligases Human genes 0.000 description 4
- 108090000364 Ligases Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 230000032683 aging Effects 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- KWOLFJPFCHCOCG-UHFFFAOYSA-N Acetophenone Chemical compound CC(=O)C1=CC=CC=C1 KWOLFJPFCHCOCG-UHFFFAOYSA-N 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 2
- 230000003712 anti-aging effect Effects 0.000 description 2
- 238000003287 bathing Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 229940079919 digestives enzyme preparation Drugs 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229950006238 nadide Drugs 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 241000498991 Bacillus licheniformis DSM 13 = ATCC 14580 Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000331993 Erythraeus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000009084 cardiovascular function Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 208000035474 group of disease Diseases 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 208000001076 sarcopenia Diseases 0.000 description 1
- 230000001235 sensitizing effect Effects 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 229960000268 spectinomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 108010045348 trehalose synthase Proteins 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1077—Pentosyltransferases (2.4.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
- C12Y204/02012—Nicotinamide phosphoribosyltransferase (2.4.2.12), i.e. visfatin
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明公开了一种利用重组枯草芽孢杆菌生产烟酰胺单核苷酸的方法。具体是将烟酰胺磷酸核糖转移酶表达元件整合到优化改造后的枯草芽孢杆菌染色体中，以该菌作为菌种，使用优化后的培养基，加入烟酰胺为底物，发酵生产烟酰胺单核苷酸。本发明的优点是：本发明利用食品安全的表达系统生产烟酰胺单核苷酸，整合型表达的重组菌株所含的外源基因能够稳定传代和表达，使用优化后的重组菌株和培养基，烟酰胺单核苷酸产量高于其他文献报道的水平。
Description
技术领域
本发明属于生物技术领域，具体是一种重组枯草芽孢杆菌生产烟酰胺单核苷酸的方法。
背景技术
心血管疾病是现代社会对人类健康和生命威胁最大的一类疾病。同时，常见的老年疾病和症状，如阿尔茨海默病、骨质疏松、肌肉减少以及肝脏的功能减退都与毛细血管的减少有关。研究发现，烟酰胺腺嘌呤二核苷酸(nad+，也称辅酶I)是一个关键的物质，在动物和人类中的正常年龄增长中，nad+在细胞中的生物可利用度及相关代谢程度是逐步下降的，进而造成生理衰老。
多项研究开始探索通过提升体内nad+的活力来延缓衰老。一个nad+的前体物质，烟酰胺单核苷酸(NMN)表现出非常好的前景。在动物模型试验中，口服补充烟酰胺单核苷酸(NMN),能够改善动物的多种生理功能，包括改善心血管功能。还有研究发现，中年小鼠口服烟酰胺单核苷酸作为抗衰老补充剂，能够延长寿命长达29％。除了在保健抗衰老方面的应用，研究也发现，NMN在一些具体疾病的治疗中也有良好表现。在这个老龄化日益严重的社会背景下，烟酰胺单核苷酸有难以估量的市场容量。
目前，烟酰胺单核苷酸的来源主要为化学合成。化学合成的方法操作较为困难，耗时长、成本高、收率低，难以实现工厂化大规模生产。近来，有研究开发使用微生物发酵法和体外酶法转化来进行生产。体外酶法转化需要收集纯化所需的酶制剂，且需要使用酶转化反应的各种底物，底物成本较高；微生物发酵法只需使用烟酰胺作为反应底物，成本低，但目前所报道的大肠杆菌发酵法和和酵母发酵法获得的产品得率较低，只有不到16mg/L发酵液，达不到商业化生产要求。枯草芽孢杆菌(Bacillus subtilis)有长期制备发酵食品的历史，是非致病的，不产内毒素和致热致敏蛋白质，是一种食品级的安全微生物，已被广泛应用于酶制剂等生物制品的生产。本发明通过酶基因克隆及改造、代谢通路优化等手段改造枯草芽孢杆菌作为细胞工厂，高效发酵生产烟酰胺单核苷酸。
发明内容
本发明的目的是提供一种利用重组枯草芽孢杆菌生产烟酰胺单核苷酸的方法。利用整合质粒将烟酰胺磷酸核糖转移酶基因表达元件整合到枯草芽孢杆菌染色体基因组中，构建整合型重组枯草芽孢杆菌，利用该菌在液体培养基中进行发酵，转化烟酰胺得到烟酰胺单核苷酸。
本发明解决上述技术问题的技术方案如下：
1.构建一种能够以整合型的方式表达烟酰胺磷酸核糖转移酶的重组枯草芽孢杆菌菌株，具体方法为：
将烟酰胺磷酸核糖转移酶表达元件克隆到枯草芽孢杆菌整合质粒上，得到的重组质粒转化宿主枯草芽孢杆菌，挑选外源基因通过双交换整合到枯草芽孢杆菌染色体中的重组枯草芽孢杆菌；
上述烟酰胺磷酸核糖转移酶表达元件包含能够在枯草芽孢杆菌中高效启动基因表达的启动子和烟酰胺磷酸核糖转移酶基因；
上述的能够在枯草芽孢杆菌中高效启动基因表达的启动子为来源于枯草芽孢杆菌(Bacillus subtilis)重叠启动子P43，或来源于地衣芽孢杆菌(Baclicuslincheniformis)麦芽糖淀粉酶基因amyM的启动子；
上述烟酰胺磷酸核糖转移酶基因来源于红色亚栖热菌(Meiothermus ruber)或杜克雷嗜血杆菌(Haemophilus ducreyi)；
枯草芽孢杆菌整合质粒为整合质粒pMLK83及其衍生质粒；
宿主枯草芽孢杆菌是枯草芽孢杆菌168或转酮酶基因缺失的枯草芽孢杆菌168衍生菌株。
2.利用上述一种能够以整合型的方式表达表达烟酰胺磷酸核糖转移酶的重组枯草芽孢杆菌生产烟酰胺单核苷酸的方法，工艺步骤如下：
1)一级种的制备：从含新霉素20ug/ml的LB平板上接上述枯草芽孢杆菌基因工程菌株单菌落在4ml液体培养基中，于37℃、220rpm培养过夜，所得的菌种为一级种；
2)二级种的制备：将一级种接种于800ml液体培养基，于37℃，220rpm培养至OD600为0.6左右，时间为4～5小时；
3)三级种的制备：将二级种接种到80L液体发酵罐中，在37℃条件下，以柠檬酸、NaOH控pH 7.0左右，通风搅拌，溶氧控制在20～30％，培养至OD600为0.6左右，时间为5～6小时；
4)生产罐发酵：将三级种接种到3T发酵罐中，加入0.1％-1％烟酰胺的液体培养基，在36～38℃条件下，通风搅拌，溶氧控制在20～30％，以柠檬酸、NaOH控pH 6～8,培养24～48小时,破胞后分离纯化可得烟酰胺单核苷酸产品；
上述液体培养基为基础盐培养基，具体成分为：每升培养基包含7g磷酸氢二钾，2g磷酸二氢钾，0.5g柠檬酸钠，0.1g硫酸镁，1g硫酸铵，10g葡萄糖。
本发明首次将烟酰胺磷酸核糖转移酶表达元件整合到优化改造后的枯草芽孢杆菌染色体中，以该菌作为菌种，使用优化后的培养基，加入烟酰胺为底物，发酵生产烟酰胺单核苷酸。
本发明的优点是：本发明利用食品安全的表达系统生产烟酰胺单核苷酸，整合型表达的菌株所含的外源基因能够稳定传代和表达，表达的烟酰胺单核苷酸超过200ug/ml，远高于其他文献报道的水平。
具体实施方式
下面结合实施例对本发明作进一步描述。但需要说明的是，实施例并不构成对本发明要求保护范围的限制。
实施例1
本实例将由来源于枯草芽孢杆菌(Bacillus subtilis)重叠启动子P43启动子和来源于杜克雷嗜血杆菌(Haemophilus ducreyi)的烟酰胺磷酸核糖转移酶基因构成的烟酰胺磷酸核糖转移酶表达元件克隆到整合质粒pMLK83。
1.构建重组质粒pMLK83-P43
根据Genbank中注释的启动子P43序列，设计上游引物为5’attgctggacgcttatggac3’和下游引物为5’cgggatccattcctctcttacctataat 3’。PCR反应体系100ul：DNA模板(枯草芽孢杆菌1A751总DNA)1ul(约20ng)，5×PrimeSTAR Buffer 20ul，10pmol/ul dNTP 2ul，10pmol/ul正反向引物各为2ul，2.5U/ul PrimeSTAR HS DNA聚合酶1ul，添加ddH2O至100ul。PCR反应程序：94℃5min；94℃30s，60℃30s，72℃1min，30个循环；72℃10min；4℃保存。PCR片段和质粒pMLK83用限制性内切酶BamH I、Hind III分别进行双酶切后用T4连接酶进行连接，转化到大肠杆菌DH5ɑ中，经筛选鉴定获得重组质粒pMLK83-P43。
2.构建重组质粒pMLK83-P43-hdNadV
人工合成如下DNA片断：
将合成的DNA片段和质粒pMLK83-P43用限制性内切酶BamH I和Sac II进行双酶切后用T4连接酶进行连接，转化到大肠杆菌DH5ɑ感受态细胞中，经筛选鉴定获得重组质粒pMLK83-p43-hdNadV。
实施例2
本实例将由来源于来源于地衣芽孢杆菌(Baclicus lincheniformis)麦芽糖淀粉酶基因amyM的启动子和来源于红色亚栖热菌(Meiothermus ruber)的烟酰胺磷酸核糖转移酶基因的海藻糖合成酶表达元件克隆到整合质粒pMLK83。
1.构建重组质粒pMLK83-amyM
根据Genbank中注释的启动子amyM序列，设计上游引物为5’cccaagcttctgtacacttgcgtcctcca 3’和下游引物为5’cgggatcctctcctcccctttcaatgtg3’。PCR反应体系100ul：DNA模板(Bacillus licheniformis ATCC 14580总DNA)1ul(约20ng)，5×PrimeSTAR Buffer 20ul，10pmol/ul dNTP 2ul，10pmol/ul正反向引物各为2ul，2.5U/ul PrimeSTAR HS DNA聚合酶1ul，添加ddH2O至100ul。PCR反应程序：94℃5min；94℃30s，60℃30s，72℃30s，30个循环；72℃10min；4℃保存。PCR片段和质粒pMLK83用限制性内切酶BamH I、Hind III分别进行双酶切后用T4连接酶进行连接，转化到大肠杆菌DH5ɑ中，经筛选鉴定获得重组质粒pMLK83-amyM。
2.构建重组质粒pMLK83-amyM-mrNadV
人工合成如下DNA片断：
将合成的DNA片段和质粒pMLK83-amyM用限制性内切酶BamH I和Sac II进行双酶切后用T4连接酶进行连接，转化到大肠杆菌DH5ɑ感受态细胞中，经筛选鉴定获得重组质粒pMLK83-amyM-mrNadV.
实施例3
转酮酶基因缺失的枯草芽孢杆菌168衍生菌株的构建
人工合成如下DNA片断：
合成的片段克隆在Puc57质粒上，命名为Puc57-TST。
用EcoR I/Pst I酶切Puc57-TST质粒，胶纯化1.7kb片段，用常规转化方法转化枯草芽孢杆菌1A751，筛选抗壮观霉素(60ug/ml)菌落。用PCR方法筛选同源双交换的转化子即得到转酮酶基因缺失的枯草芽孢杆菌168衍生菌株。筛选同源双交换转化子的PCR方法如下：
合成如下引物：DT1:5’gacccagaaggcaatgatgt 3’；DT2:5’cttcaagccccgtgtatttg3’；DTC:5’caccatcaccgcaaatactg 3’.将转化子在LB培养基中过夜培养后，提取总DNA，然后分别用引物对DT1/DT2和DT1/DTC进行PCR.DT1/DT2引物对的PCR结果作为菌落DNA提取成功的阳性对照。如果用DT1/DT2引物对可以扩增出2kb左右的片段，而用DT1/DTC引物对不能扩增出709bp的产物，则此转化子为转酮酶基因缺失的同源双交换的转化子。转酮酶基因缺失的枯草芽孢杆菌1A751命名为1A751(tkt)。
实施例4
重组枯草芽孢杆菌的构建及烟酰胺单核苷酸的生成
用常规转化方法，将pMLK83-amyM-mrNadV转化枯草芽孢杆菌1A751，取菌液涂布新霉素(20ug/ml)LB平板，筛选淀粉酶缺失转化子即得枯草芽孢杆菌基因工程菌株1A751[amyM-mrNadV]。
用同样方法可得1A751(tkt)[amyM-mrNadV]，1A751[P43-hdNadV]和1A751(tkt)[P43-hdNadV]。
将基因工程菌株分别接入基础盐培养基(含10ug/ml新霉素和0.5％烟酰胺)，培养30小时后，测定烟酰胺单核苷酸的含量。测得的烟酰胺单核苷酸最高表达量见表1。
表1
| 菌种 | 烟酰胺单核苷酸含量(ug/ml) |
| 1A751[amyM-mrNadV] | 152 |
| 1A751(tkt)[amyM-mrNadV] | 185 |
| 1A751[P43-hdNadV] | 159 |
| 1A751(tkt)[P43-hdNadV] | 203 |
烟酰胺单核苷酸的含量测定方法如下：
将培养液稀释20倍后超声破胞，离心取上清液。将200ul上清液与100ul 20％苯乙酮(溶解于DMSO溶液中)及100ul 2M氢氧化钾溶液混匀后，冰浴2min,然后加入450ul 88％甲酸溶液，混合后在37℃温浴10min。反应产物用荧光分光光度计检测。用10ug/ml烟酰胺单核苷酸纯品作为检测对照。
实施例5
烟酰胺单核苷酸的发酵生产的方法，操作步骤如下：
1)一级种的制备：从含新霉素20ug/ml的LB平板上接上述枯草芽孢杆菌基因工程菌株单菌落在4ml液体培养基37℃、220rpm培养过夜，所得的菌种为一级种；
2)二级种的制备：一级种接种于800ml液体培养基，于37℃，220rpm培养至OD600为0.6左右(约4～5小时)；
3)三级种的制备：将二级种接种到80L液体发酵罐中，37℃，以柠檬酸、NaOH控pH7.0左右，通风搅拌，溶氧控制在20～30％，培养至OD600为0.6左右(约5～6小时)；
4)生产罐发酵：将三级种接种到3T发酵罐中，加入0.3％烟酰胺的液体培养基，36～38℃，通风搅拌，溶氧控制在20～30％，以柠檬酸、NaOH控pH 6～8,培养约40小时,破胞后分离纯化可得烟酰胺单核苷酸产品。
液体培养基为基础盐培养基，具体成分为：每升培养基包含7g磷酸氢二钾，2g磷酸二氢钾，0.5g柠檬酸钠，0.1g硫酸镁，1g硫酸铵，10g葡萄糖。
序列表
<110> 南宁邦尔克生物技术有限责任公司
南宁市拜欧生物工程有限责任公司
<120> 一种利用重组枯草芽孢杆菌生产烟酰胺单核苷酸的方法
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
attgctggac gcttatggac 20
<210> 2
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cgggatccat tcctctctta cctataat 28
<210> 3
<211> 1500
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggatccatgg ataatctgct taactattca agcagagcat ctgcgattcc gtcactgctg 60
tgcgattttt acaaaacatc tcatcgcatc atgtatccgg aatgttctca aatcatctac 120
tcaacattta caccgagatc aaatgaacaa gcaccgtatc ttacacaggt tgtgagcttt 180
ggctttcagg cgtttatcat caaatacctg atccattact ttaatgataa tttcttttca 240
agagataaac atgatgtcgt tacagaatac tcagctttta tcgaaaaaac actgcaactg 300
gaagatacag gcgaacatat cgccaaactt catgaattag gatatctgcc gattagaatc 360
aaagcaattc cggaaggaaa aacagtggcg attaaagtgc cggtcatgac aatcgaaaac 420
acacattctg atttcttttg gctgacaaac taccttgaaa cactgatcaa cgtctcactt 480
tggcagccga tgacatctgc atcaattgct tttgcctatc gcacagcatt aatcaaattt 540
gcgaatgaaa catgcgataa ccaagaacat gtcccgtttc agagccatga tttttctatg 600
agaggcatgt cttcactgga atcagctgaa acaagcggcg ccggacattt aacaagcttt 660
ctgggaacag atacaattcc ggctttatct tttgttgaag cctattatgg cagctcttca 720
ctgattggaa caagcatccc ggcaagcgaa cattctgtga tgagctctca tggcgtcgat 780
gaattatcta catttcgcta tctgatggcg aaatttccgc ataatatgct ttcaattgtc 840
agcgatacaa cagatttttg gcataacatc acagttaacc tgccgctttt aaaacaagaa 900
attatcgcac gcccggaaaa tgcgagactt gtgattcgcc cggattcagg caatttcttt 960
gctattatct gcggagatcc gacagccgat acagaacatg aaagaaaagg acttatcgaa 1020
tgtctgtggg atatctttgg cggaacagtt aaccaaaaag gctacaaagt gatcaacccg 1080
catattggag cgatctatgg cgatggagtt acatacgaaa aaatgtttaa aatcctggaa 1140
ggccttcagg ctaaaggatt tgcctcaagc aatatcgtct ttggcgttgg agctcaaaca 1200
tatcagagaa acacacgcga tacactgggc tttgcactta aagcgacatc aatcacaatc 1260
aacggagaag aaaaagccat ctttaaaaac ccgaaaacag atgatggctt taaaaaatca 1320
cagaaaggaa gagttaaagt gcttagccgc gatacatatg ttgatggctt aacaagcgct 1380
gatgattttt ctgatgatct gcttgaactg ctgtttgaag atggaaaact tctgagacaa 1440
acagattttg atgaaatcag acagaatctg cttgtgagcc gcacaacact gtaaccgcgg 1500
<210> 4
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cccaagcttc tgtacacttg cgtcctcca 29
<210> 5
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgggatcctc tcctcccctt tcaatgtg 28
<210> 6
<211> 1404
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggatccatga agactctcaa tccgcataat ctgattctga atacagattc atataaagcg 60
tcacatttcg cccagtttcc gaaaggcatg acatatgcgt catggtatat tgaatcaaga 120
ggcggcgatt caaatttcgt ccgtttcttc gggttacaag cgtttctgat tgaatatctg 180
tcaaagggtg tgtcactggc ggatgttgaa gaagcgcaag aagtattcct tgcccatggc 240
ctgccgtttc cgacagaagg ctggagatat attgcgcaag atctgggcgg cagactgccg 300
gttagaatta gagcggttcc ggaaggcaaa gttgttccgg ttcataatcc gctggttatt 360
attgaatcaa cagatccgaa agttccgtgg ctgccgggct ggctggaaac agcgctgctg 420
agagcggttt ggtatccgac aacagtttgc acagtttcat ggggcataag gaatacaatt 480
aaagaatatc tggagaagac ggccgatgat ccggaagcgg aactgccgtt taaactgcat 540
gatttcggag ccagaggcgt ttcatcactg gaatcagcgg gcctgggcgg catggcgcat 600
ctggttaatt tcatgggtac agatacagtt acagcgctga tttatgcgag aaattattat 660
ggcgcggaaa tggcgggcta ttcaattccg gcgatgcaac attcaacagt tacatcattt 720
ggcagaacag gcgaagcgca agcgtataga caaatgctgg aaacatttgc gaaaccgggc 780
gcgctgatgg cgatggttat tgattcatat aatagagaac atgcggttgg ccaaattatt 840
ggcgaagaac tgagagaact gattcaacaa tcaggcgcga cagttgttat tagaccggat 900
tcaggcgatc cgccgtttgt tgttctgaga acagttcaaa cactggaagc gaaatttggc 960
gcgacactga atagaaaggg atacaaagtt ctgaatggcg ttagagttat tcaaggcgat 1020
ggcgttaatg cggattcaat tagaaaggta ctctttctgc tggaacaatg gggctactca 1080
gcgtcaaatg ttgcgtttgg catgggcggc gcgctgctgc aacatccgca tagagataca 1140
cagaagttcg cacagaagct tcacctggtt acagttaatg gcgaaacata tggcgttggc 1200
aaatcaccgg ttgatgatcc gggcaaactg tcaaagaagg gtcggttaga tgttattcaa 1260
gatgaaagag gcattagaac agttgaactg ccgctggaag cggcgcaacc gcatccgcaa 1320
tcaattctgc aaacagtctt cgagaacggt tcaattacaa gaagatatac atgggaagaa 1380
gttagaaata atgcgtaacc gcgg 1404
<210> 7
<211> 1773
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gaattccgac ccagaaggca atgatgttac accagaaaag ctaaaaagag aacaaagaaa 60
taacaaactt cactaatata agaggaatac ggcaatatcg tattcctctt ttgcatatac 120
tataatcaaa actcttatgt gacaaaattc aacaagtttc tctcaaattt aagctgaaac 180
agttgagaaa aagtcgtcag acatttatga tgtaagggta caacacataa ggaaggggat 240
ttttatggat acaattgaaa agaaatcagt tgctaccatt cgcacatgct ataataacta 300
taactaataa cgtaacgtga ctggcaagag atatttttaa aacaatgaat aggtttacac 360
ttactttagt tttatggaaa tgaaagatca tatcatatat aatctagaat aaaattaact 420
aaaataatta ttatctagat aaaaaattta gaagccaatg aaatctataa ataaactaaa 480
ttaagtttat ttaattaaca actatggata taaaataggt actaatcaaa atagtgagga 540
ggatatattt gaatacatac gaacaaatta ataaagtgaa aaaaatactt cggaaacatt 600
taaaaaataa ccttattggt acttacatgt ttggatcagg agttgagagt ggactaaaac 660
caaatagtga tcttgacttt ttagtcgtcg tatctgaacc attgacagat caaagtaaag 720
aaatacttat acaaaaaatt agacctattt caaaaaaaat aggagataaa agcaacttac 780
gatatattga attaacaatt attattcagc aagaaatggt accgtggaat catcctccca 840
aacaagaatt tatttatgga gaatggttac aagagcttta tgaacaagga tacattcctc 900
agaaggaatt aaattcagat ttaaccataa tgctttacca agcaaaacga aaaaataaaa 960
gaatatacgg aaattatgac ttagaggaat tactacctga tattccattt tctgatgtga 1020
gaagagccat tatggattcg tcagaggaat taatagataa ttatcaggat gatgaaacca 1080
actctatatt aactttatgc cgtatgattt taactatgga cacgggtaaa atcataccaa 1140
aagatattgc gggaaatgca gtggctgaat cttctccatt agaacatagg gagagaattt 1200
tgttagcagt tcgtagttat cttggagaga atattgaatg gactaatgaa aatgtaaatt 1260
taactataaa ctatttaaat aacagattaa aaaaattata aaaaaattga aaaaatggtg 1320
gaaacacttt tttcaatttt tttgttttat tatttaatat ttgggaaata ttcattctaa 1380
ttggtaatca gattttagaa aacaataaac ccttgcatat ttctttgatc cgtccagcag 1440
acggcaatga gacagcagca gcatggaagc ttgcagtgca aagcactgac cacccaacag 1500
cgctagtgct tacacgtcaa aaccttccta ccatcgatca aacatctgaa gaagcattgg 1560
caggagtaga aaaaggtgca tatgtcgttt ctaaatctaa aaacgaaaca cctgacgctc 1620
ttctcatcgc ttccggatca gaggtaggtc ttgcaattga agcgcaggct gaattggcaa 1680
aagaaaatat cgatgtttct gttgtcagca tgccttcaat ggaccgtttt gagaaacaat 1740
ctgatgaata caaaaacgaa gtccttcctg cag 1773
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gacccagaag gcaatgatgt 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cttcaagccc cgtgtatttg 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caccatcacc gcaaatactg 20
Claims (2)
1.一种能够以整合型的方式表达烟酰胺磷酸核糖转移酶的重组枯草芽孢杆菌菌株，其特征在于，其获得方法为：
将烟酰胺磷酸核糖转移酶表达元件克隆到枯草芽孢杆菌整合质粒上，得到的重组质粒转化宿主枯草芽孢杆菌，挑选外源基因通过双交换整合到枯草芽孢杆菌染色体中的重组枯草芽孢杆菌；
所述的烟酰胺磷酸核糖转移酶表达元件包含能够在枯草芽孢杆菌中高效启动基因表达的启动子和烟酰胺磷酸核糖转移酶基因；
所述的能够在枯草芽孢杆菌中高效启动基因表达的启动子为来源于枯草芽孢杆菌(Bacillus subtilis)重叠启动子P43，或来源于地衣芽孢杆菌(Baclicuslincheniformis)麦芽糖淀粉酶基因amyM的启动子；
所述的能够在枯草芽孢杆菌中高效启动基因表达的启动子为来源于枯草芽孢杆菌(Bacillus subtilis)重叠启动子P43，或来源于地衣芽孢杆菌(Baclicuslincheniformis)麦芽糖淀粉酶基因amyM的启动子；
所述的烟酰胺磷酸核糖转移酶基因来源于红色亚栖热菌(Meiothermus ruber)或杜克雷嗜血杆菌(Haemophilus ducreyi)；
所述的枯草芽孢杆菌整合质粒为整合质粒pMLK83；
所述的宿主枯草芽孢杆菌是枯草芽孢杆菌168或转酮酶基因缺失的枯草芽孢杆菌168衍生菌株。
2.一种利用权利要求1所述一种能够以整合型的方式表达烟酰胺磷酸核糖转移酶的重组枯草芽孢杆菌生产烟酰胺单核苷酸的方法，其特征在于，工艺步骤如下：
1)一级种的制备：从含新霉素20ug/ml的LB平板上接上述枯草芽孢杆菌基因工程菌株单菌落在4ml液体培养基中，于37℃、220rpm培养过夜，所得的菌种为一级种；
2)二级种的制备：将一级种接种于800ml液体培养基，于37℃，220rpm培养至OD600为0.6左右，时间为4～5小时；
3)三级种的制备：将二级种接种到80L液体发酵罐中，在37℃条件下，以柠檬酸、NaOH控pH 7.0左右，通风搅拌，溶氧控制在20～30％，培养至OD600为0.6左右，时间为5～6小时；
4)生产罐发酵：将三级种接种到3T发酵罐中，加入0.1％-1％烟酰胺的液体培养基，在36～38℃条件下，通风搅拌，溶氧控制在20～30％，以柠檬酸、NaOH控pH 6～8,培养24-48小时,破胞后分离纯化可得烟酰胺单核苷酸产品；
所述液体培养基为基础盐培养基，具体成分为：每升培养基包含7g磷酸氢二钾，2g磷酸二氢钾，0.5g柠檬酸钠，0.1g硫酸镁，1g硫酸铵，10g葡萄糖。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2020104492250 | 2020-05-25 | ||
| CN202010449225 | 2020-05-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111996208A true CN111996208A (zh) | 2020-11-27 |
Family
ID=73468537
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010944821.6A Pending CN111996208A (zh) | 2020-05-25 | 2020-09-10 | 一种利用重组枯草芽孢杆菌生产烟酰胺单核苷酸的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111996208A (zh) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852678A (zh) * | 2021-03-08 | 2021-05-28 | 泓博元生命科技（深圳）有限公司 | 一株产烟酰胺单核苷酸的成都肠杆菌及其应用 |
| CN113278558A (zh) * | 2021-05-26 | 2021-08-20 | 南宁市拜欧生物工程有限责任公司 | 一种高产脲酶的枯草芽孢杆菌及其应用方法 |
| CN113736714A (zh) * | 2021-10-20 | 2021-12-03 | 福建君生益本色生物科技有限公司 | 嗜酸乳杆菌及其应用 |
| CN113832204A (zh) * | 2021-09-22 | 2021-12-24 | 杭州吾尾科技有限公司 | Nmn的制备方法及含nmn的犬猫抗衰老保健品配方 |
| CN114164190A (zh) * | 2021-10-12 | 2022-03-11 | 南宁邦尔克生物技术有限责任公司 | 一种生产烟酰胺单核苷酸的融合酶及其应用 |
| CN115287278A (zh) * | 2022-06-17 | 2022-11-04 | 武汉真福医药股份有限公司 | 一种枯草芽孢杆菌纤溶酶及其制备方法和抗氧化溶血栓组合物 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215300A (zh) * | 2013-05-13 | 2013-07-24 | 南宁中诺生物工程有限责任公司 | 以整合型重组枯草芽孢杆菌生产海藻糖合成酶及制造海藻糖的方法 |
| CN111051520A (zh) * | 2017-09-29 | 2020-04-21 | 三菱化学株式会社 | 烟酰胺单核苷酸的制造方法及该方法所使用的转化体 |
-
2020
- 2020-09-10 CN CN202010944821.6A patent/CN111996208A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215300A (zh) * | 2013-05-13 | 2013-07-24 | 南宁中诺生物工程有限责任公司 | 以整合型重组枯草芽孢杆菌生产海藻糖合成酶及制造海藻糖的方法 |
| CN111051520A (zh) * | 2017-09-29 | 2020-04-21 | 三菱化学株式会社 | 烟酰胺单核苷酸的制造方法及该方法所使用的转化体 |
Non-Patent Citations (2)
| Title |
|---|
| MARINESCU GC等: "β-nicotinamide mononucleotide (NMN) production in Escherichia coli" * |
| 廖湘萍等: "产D-核糖菌株的选育研究" * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852678A (zh) * | 2021-03-08 | 2021-05-28 | 泓博元生命科技（深圳）有限公司 | 一株产烟酰胺单核苷酸的成都肠杆菌及其应用 |
| CN113278558A (zh) * | 2021-05-26 | 2021-08-20 | 南宁市拜欧生物工程有限责任公司 | 一种高产脲酶的枯草芽孢杆菌及其应用方法 |
| CN113832204A (zh) * | 2021-09-22 | 2021-12-24 | 杭州吾尾科技有限公司 | Nmn的制备方法及含nmn的犬猫抗衰老保健品配方 |
| CN114164190A (zh) * | 2021-10-12 | 2022-03-11 | 南宁邦尔克生物技术有限责任公司 | 一种生产烟酰胺单核苷酸的融合酶及其应用 |
| CN114164190B (zh) * | 2021-10-12 | 2023-11-21 | 南宁邦尔克生物技术有限责任公司 | 一种生产烟酰胺单核苷酸的融合酶及其应用 |
| CN113736714A (zh) * | 2021-10-20 | 2021-12-03 | 福建君生益本色生物科技有限公司 | 嗜酸乳杆菌及其应用 |
| CN115287278A (zh) * | 2022-06-17 | 2022-11-04 | 武汉真福医药股份有限公司 | 一种枯草芽孢杆菌纤溶酶及其制备方法和抗氧化溶血栓组合物 |
| CN115287278B (zh) * | 2022-06-17 | 2023-11-10 | 武汉真福医药股份有限公司 | 一种枯草芽孢杆菌纤溶酶及其制备方法和抗氧化溶血栓组合物 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111996208A (zh) | 一种利用重组枯草芽孢杆菌生产烟酰胺单核苷酸的方法 | |
| US9469858B2 (en) | Sporulation-deficient thermophilic microorganisms for the production of ethanol | |
| WO2022095591A1 (zh) | 复合酶及其在制备麦角硫因中的应用 | |
| CN108949852B (zh) | 一种利用全细胞催化制备木糖醇的方法 | |
| CN106755015B (zh) | 一种新型普鲁兰酶基因和高产菌株的获得方法及产酶工艺 | |
| CN112662637B (zh) | 一种甲酸脱氢酶突变体及其制备方法和应用 | |
| JP5496356B2 (ja) | アラビノース代謝経路が導入されたキシリトール生産菌株及びそれを用いたキシリトール生産方法 | |
| CN105420265B (zh) | 一种过量表达atp酶的基因工程醋酸菌及其构建方法和应用 | |
| WO2021036788A1 (zh) | 一种2-酮基-l-古龙酸生产菌株及其构建方法 | |
| CN112852911A (zh) | 一种熊去氧胆酸的制备方法 | |
| CN114164190B (zh) | 一种生产烟酰胺单核苷酸的融合酶及其应用 | |
| CN113957065B (zh) | 一种高转化率的蔗糖异构酶及其应用 | |
| CN114717250B (zh) | 一种基于辅因子代谢工程策略改造蛹虫草菌提高虫草素产量的方法及应用 | |
| KR101219685B1 (ko) | 고정화 효소 알파-엘-아라비노퓨라노시데이즈와 엔도-1,5-알파-엘-아라비난에이즈 함유 생물반응기에서 엘-아라비노스의 제조 방법 | |
| CN111286509B (zh) | 一种烯还原酶突变体及其编码基因和应用 | |
| CN107955805B (zh) | 一种稳定性提高的nadh氧化酶及其在乙偶姻生产中的应用 | |
| WO2022217694A1 (zh) | 制备β-烟酰胺单核苷酸的方法、酶组合物及其应用 | |
| CN118291353A (zh) | 一株产普鲁兰酶菌株及其构建方法与应用 | |
| CN117535217A (zh) | 一种重组枯草芽孢杆菌工程菌株及其在生物制备熊去氧胆酸中应用 | |
| CN116218813A (zh) | 一种烟酰胺核糖激酶突变体的应用 | |
| Sokolenko et al. | A Novel Saccharomyces cerevisiae G strain Possessing Inuliase Activity and Good Baking Characteristics | |
| CN117904067A (zh) | 一种用于生产西他列汀中间体的转氨酶突变体 | |
| WO2024130778A1 (zh) | 一种热稳定性提高的(2r,3r)-丁二醇脱氢酶突变体及其应用 | |
| JP2015062357A (ja) | 酵母 | |
| EP4388115A1 (en) | Genetically modified yeast and fermentation processes for the production of lactate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20201127 |