CN116926102A - 抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法 - Google Patents
抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法 Download PDFInfo
- Publication number
- CN116926102A CN116926102A CN202310889013.8A CN202310889013A CN116926102A CN 116926102 A CN116926102 A CN 116926102A CN 202310889013 A CN202310889013 A CN 202310889013A CN 116926102 A CN116926102 A CN 116926102A
- Authority
- CN
- China
- Prior art keywords
- glxr
- seq
- regulatory factor
- corynebacterium glutamicum
- factor 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
- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 20
- 238000013518 transcription Methods 0.000 title claims abstract description 19
- 230000035897 transcription Effects 0.000 title claims abstract description 19
- 101150075091 glxR gene Proteins 0.000 title claims abstract description 18
- 241000186226 Corynebacterium glutamicum Species 0.000 title claims abstract description 17
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 title claims description 7
- 238000000034 method Methods 0.000 claims abstract description 13
- 229960002749 aminolevulinic acid Drugs 0.000 claims abstract description 12
- 239000002773 nucleotide Substances 0.000 claims abstract description 12
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 12
- 239000012634 fragment Substances 0.000 claims description 23
- 239000013612 plasmid Substances 0.000 claims description 14
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 11
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 11
- 238000011144 upstream manufacturing Methods 0.000 claims description 11
- 238000010276 construction Methods 0.000 claims description 9
- 230000005764 inhibitory process Effects 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 2
- 239000002609 medium Substances 0.000 description 7
- 101100069458 Mus musculus Grhpr gene Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- DUJGMZAICVPCBJ-VDAHYXPESA-N 4-amino-1-[(1r,4r,5s)-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl]pyrimidin-2-one Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)C(CO)=C1 DUJGMZAICVPCBJ-VDAHYXPESA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 108091023040 Transcription factor Proteins 0.000 description 3
- 102000040945 Transcription factor Human genes 0.000 description 3
- 101150112623 hemA gene Proteins 0.000 description 3
- 238000012269 metabolic engineering Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical class CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 2
- 229930182821 L-proline Natural products 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 229960002429 proline Drugs 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 238000010446 CRISPR interference Methods 0.000 description 1
- 241001485655 Corynebacterium glutamicum ATCC 13032 Species 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 101100096533 Drosophila melanogaster SelD gene Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 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
- 239000004471 Glycine Substances 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 101150044508 key gene Proteins 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 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
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/265—Micrococcus
- C12R2001/28—Micrococcus glutamicus ; Corynebacterium glutamicum
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5‑ALA的方法，所述全局转录调控因子基因glxR的核苷酸序列如SEQ ID NO.1所示。实验证明，谷氨酸棒杆菌中全局转录调控因子基因glxR表达的抑制使得谷氨酸棒杆菌工程菌株中5‑氨基乙酰丙酸产量比CGS15‑h3提升约1.37倍。
Description
技术领域
本发明属于生物工程技术与应用领域，具体地涉及抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法。
背景技术
5-氨基乙酰丙酸(5-aminolevulinic acid，5-ALA)，是生物体内合成维生素B12、血红素、叶绿素等四吡咯类化合物的重要前体，广泛存在于微生物和动植物细胞中。由于其绿色无毒、易降解且无残留的特性，作为光动力学药物、生物可降解除草剂、植物生长调节剂以及饲料添加剂等在医药以及农业等领域得到广泛应用。目前5-ALA的大规模生产主要以化学法为主，但是合成过程中存在的高成本、高污染和低得率等问题大大限制了其大规模生产与应用。鉴于此，微生物合成法应运而生，并随着合成生物学，特别是代谢工程手段的兴起，5-ALA的生物法合成得到了进一步发展。但是，大多数生物合成方法产量低，并不能达到工业化生产的要求，难以实现大规模应用。
随着基因组技术的发展，转录因子(Transfer Factor，TF)作为改变复杂微生物代谢途径的调节因子，在转录水平的基因调控方面具有独特的优势，弥补了单个关键基因作用的不足，并降低了代谢工程操作中多个关键基因组成性致死表达的可能性。尤其是全局调节因子的修饰可以在转录水平上同时干扰数百个基因的表达。基于转录因子的生物传感器具有高灵敏度和特异性，在代谢工程中发挥着重要作用，显著提高了目标化合物的产量和宿主细胞耐受性。
但是，目前尚未有抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法的报道。
发明内容
本发明的目的是克服现有技术的不足，提供抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法。
本发明的技术方案概述如下：
抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法，所述全局转录调控因子基因glxR的核苷酸序列如SEQ ID NO.1所示。
优选地，上述方法，包括如下步骤：
1)pCRISPRi-low-GlxR质粒构建：
以pdCas9gRNA为模板，以l-GlxR-F1和cas-R1为上下游引物，用Phanta DNA聚合酶进行扩增，得到线性化载体片段1；
以pdCas9gRNA为模板，以ori-F和CRISPRi-R2为上下游引物，用Phanta DNA聚合酶进行扩增，得到线性化载体片段2；
将所述片段1和片段2连接起来，通过菌落PCR的方式进行验证得到pCRISPRi-low-GlxR质粒；
所述l-GlxR-F1的核苷酸序列如SEQ ID NO.8所示；
所述cas-R1的核苷酸序列如SEQ ID NO.3所示；
所述ori-F的核苷酸序列如SEQ ID NO.4所示；
所述CRISPRi-R2的核苷酸序列如SEQ ID NO.7所示；
2)5-氨基乙酰丙酸生产菌株构建及发酵：
将pCRISPRi-low-GlxR质粒通过电转导入谷氨酸棒杆菌CGS15-h3中，用菌落PCR的方式进行验证得到抑制全局转录调控因子基因glxR的表达菌株CGS15-l-GlxR；发酵，得到5-氨基乙酰丙酸。
本发明的优点：
实验证明，谷氨酸棒杆菌中全局转录调控因子基因glxR表达的抑制使得谷氨酸棒杆菌工程菌株中5-氨基乙酰丙酸产量比CGS15-h3提升约1.37倍。
附图说明
图1为pCRISPRi-high-GlxR质粒图谱。
图2为pCRISPRi-middle-GlxR质粒图谱。
图3为pCRISPRi-low-GlxR质粒图谱。
图4为菌株CGS15-h-GlxR，CGS15-m-GlxR和CGS15-l-GlxR在摇瓶条件下5-ALA产量图。
具体实施方式
下面结合具体实施例对本发明做进一步说明，下述实施例是为了使本领域的技术人员能够更好地理解本发明，但对本发明不作任何限制。
本发明所用到的底盘菌株谷氨酸棒杆菌Corynebacterium glutamicum ATCC13032来源为ATCC(The Global Bioresource Center，http://www.atcc.org/)，购买于2012年10月。
CGS15-h3的基因型为ATCC13032△ldhA△pqo::Psod*hemA△cat::Psod*hemA△pta△ackAPsod pyc Psod ppc,△aceA::Psod*hemA,Psod tal Psod tkt,Ptuf araE(del21bp),ackA-pta::Psod xylAB:C131T,NCgl2538:C311T
pdCas9gRNA的制备参见参考文献1。
5-氨基乙酰丙酸标准品从sigma公司(http://www.sigmaaldrich.com/sigmaaldrich)购买。
所用限制性内切酶、DNA连接酶等分子生物学试剂从Thermo公司购买(http://www.thermoscientificbio.com/fermentas)，所用其他生化试剂从生工生物工程(上海)股份有限公司购买(http://www.sangon.com/)。
E.coli DH5α感受态细胞、E.coli BL21感受态细胞通过常规CaCl2法制备；
LB液体培养基：酵母浸粉5g/L，胰蛋白胨10g/L，NaCl 10g/L，LB固体培养基中添加2％的琼脂粉。
CGIII培养基：酵母浸粉10g/L，胰蛋白胨10g/L，MOPS 21g/L，NaCl 2.5g/L，用5M的NaOH水溶液调节pH＝7。
BHIS液体培养基：脑心浸液肉汤粉74g/L。BHIS固体培养基中添加2％的琼脂粉。
抗生素浓度为：卡那霉素40μg/mL、15μg/mL，氯霉素10μg/mL。
5-ALA的检测方法：取250μL的5-氨基乙酰丙酸标准品溶液(终浓度为1、2、4、6、8mg/L)或稀释的发酵液或酶活力测定反应终止后的反应液，加入125μL pH＝4.6的乙酸钠缓冲液，然后加入62.5μL的乙酰丙酮，100℃金属浴温浴15min，冷却至室温后加入440μL现配的Modified Ehrlich's reagent试剂(0.2g对二甲氨基苯甲醛，1mL冰乙酸，1mL高氯酸，冰乙酸定容到10mL)混匀，室温反应20min后测反应液在554nm波长下的吸光值。利用测定5-氨基乙酰丙酸标准品得到的标准曲线计算发酵液或者酶活力测定反应终止后的反应液中的5-ALA含量。
实施例1
抑制谷氨酸棒杆菌(CGS15-h3)中全局转录调控因子基因glxR的表达生产5-氨基乙酰丙酸的方法，所述全局转录调控因子基因glxR的核苷酸序列如SEQ ID NO.1所示。
实施例2
pCRISPRi-GlxR抑制载体构建
(1)GlxR表达高抑制载体pCRISPRi-high-GlxR的构建
以pdCas9gRNA为模板，以GlxR-F1(SEQ ID NO.2)、cas-R1(SEQ ID NO.3)为上下游引物，用Phanta DNA聚合酶进行扩增，得到线性化载体片段3；
以pdCas9gRNA为模板，以ori-F(SEQ ID NO.4)、GlxR-R2(SEQ ID NO.5)为上下游引物，用Phanta DNA聚合酶进行扩增，得到线性化载体片段4；
用CPEC技术将片段3和片段4连接起来，测序验证正确后通过电转化的方式转入CGS15-h3中，并通过菌落PCR的方式进行验证，得到高抑制调控因子GlxR的工程菌株，最终的pCRISPRi-high-GlxR质粒图谱如图1所示。
(2)GlxR表达中抑制载体pCRISPRi-middle-GlxR的构建
以pdCas9gRNA为模板，以m-GlxR-F1(SEQ ID NO.6)、cas-R1(SEQ ID NO.3)为上下游引物，用Phanta DNA聚合酶进行扩增，得到线性化载体片段5；
以pdCas9gRNA为模板，以ori-F(SEQ ID NO.4)、CRISPRi-R2(SEQ ID NO.7)作为上下游引物，使用Phanta DNA聚合酶进行扩增，得到线性化载体片段2；
使用CPEC技术将片段5和片段2连接起来，测序验证正确后通过电转化的方式转入CGS15-h3中，并通过菌落PCR的方式进行验证，得到中抑制调控因子GlxR的工程菌株。最终的pCRISPRi-middle-GlxR质粒图谱如图2所示。
(3)GlxR表达低抑制载体pCRISPRi-low-GlxR的构建
以pdCas9gRNA为模板，以l-GlxR-F1(SEQ ID NO.8)、cas-R1(SEQ ID NO.3)为上下游引物，用Phanta DNA聚合酶进行扩增，得到线性化载体片段1；
以pdCas9gRNA为模板，以ori-F(SEQ ID NO.4)和CRISPRi-R2(SEQ ID NO.7)作为上下游引物，用Phanta DNA聚合酶进行扩增，得到线性化载体片段2。
使用CPEC技术将片段1和片段2连接起来，测序验证正确后通过电转化的方式转入CGS15-h3中，并通过菌落PCR的方式进行验证，得到低抑制调控因子GlxR的工程菌株。最终的pCRISPRi-low-GlxR质粒图谱如图3所示。
PCR扩增体系：模板2μL，上下游引物各2μL，dNTP 1μL，phanta Buffer 25μL，灭菌的双蒸水17μL，DNA聚合酶1μL，总体积50μL。
PCR反应条件为：95℃3min，30个循环(95℃20s,58℃20s,72℃1min)，72℃10min，4℃10min。采用胶回收试剂盒对PCR产物进行回收和纯化。
表1菌株构建所用引物序列
实施例2：5-ALA生产菌株构建及其摇瓶发酵
(1)5-ALA生产菌株构建
将测序结果正确的质粒pCRISPRi-high-GlxR，pCRISPRi-middle-GlxR和pCRISPRi-low-GlxR通过电转导入谷氨酸棒杆菌CGS15-h3中，均匀涂在氯霉素抗性(终浓度10μg/mL)的BHIS固体平板上。分别挑取单菌落以如下引物test-pX-1(SEQ ID NO.9)/test-pX-2(SEQ ID NO.10)进行PCR验证。测序无误的即为成功插入质粒的，能够不同程度抑制全局转录调控因子基因glxR的表达菌株的5-ALA生产菌株CGS15-h-GlxR、CGS15-m-GlxR、CGS15-l-GlxR。
(2)生产菌株的摇瓶发酵
对菌株CGS15-h-GlxR、CGS15-m-GlxR、CGS15-l-GlxR进行摇瓶发酵。
接种方式：首先将CGS15-h-GlxR、CGS15-m-GlxR、CGS15-l-GlxR在BHIS固体培养基上进行划线，置于30℃培养箱中培养约18h。挑取平板上的单菌落接种至5mL的BHIS液体培养基中，30℃，220rpm培养约12h，取1mL转接至CGIII液体培养基继续培养12h。以初始OD600约为0.5、转接葡萄糖的终浓度为10g/L的CGIII液体培养基，置于30℃，220rmp的恒温摇床中振荡培养4h至OD600约为5.0左右时，添加7.5g/L的前体物质甘氨酸。在培养期间测定其5-ALA产量(见图4)。培养72h后，菌株CGS15-h-GlxR的5-ALA产量比CGS15-h3提升约0.35倍，菌株CGS15-m-GlxR的5-ALA产量比CGS15-h3提升约0.5倍，菌株CGS15-l-GlxR的5-ALA产量为4.62g/L比CGS15-h3提升约1.37倍。
参考文献
1.Liu J.,Liu M.,Shi T.,Sun G.,Gao N.,Zhao X.,Guo X.,Ni X.,Yuan Q.,Feng J.,Liu Z.,Guo Y.,Chen J.,Wang Y.,Zheng P.,Sun J.CRISPR-assisted rationalflux-tuning and arrayed CRISPRi screening of an L-proline exporterfor L-proline hyperproduction.Nat Commun2022；13:891.
Claims (2)
1.抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法，其特征是所述全局转录调控因子基因glxR的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的方法，其特征是包括如下步骤：
1)pCRISPRi-low-GlxR质粒构建：
以pdCas9gRNA为模板，以l-GlxR-F1和cas-R1为上下游引物，用Phanta DNA聚合酶进行扩增，得到线性化载体片段1；
以pdCas9gRNA为模板，以ori-F和CRISPRi-R2为上下游引物，用Phanta DNA聚合酶进行扩增，得到线性化载体片段2；
将所述片段1和片段2连接起来，通过菌落PCR的方式进行验证得到pCRISPRi-low-GlxR质粒；
所述l-GlxR-F1的核苷酸序列如SEQ ID NO.8所示；
所述cas-R1的核苷酸序列如SEQ ID NO.3所示；
所述ori-F的核苷酸序列如SEQ ID NO.4所示；
所述CRISPRi-R2的核苷酸序列如SEQ ID NO.7所示；
2)5-氨基乙酰丙酸生产菌株构建及发酵：
将pCRISPRi-low-GlxR质粒通过电转导入谷氨酸棒杆菌CGS15-h3中，用菌落PCR的方式进行验证得到抑制全局转录调控因子基因glxR的表达菌株CGS15-l-GlxR；发酵，得到5-氨基乙酰丙酸。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310889013.8A CN116926102A (zh) | 2023-07-19 | 2023-07-19 | 抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310889013.8A CN116926102A (zh) | 2023-07-19 | 2023-07-19 | 抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116926102A true CN116926102A (zh) | 2023-10-24 |
Family
ID=88390476
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310889013.8A Pending CN116926102A (zh) | 2023-07-19 | 2023-07-19 | 抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116926102A (zh) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050196847A1 (en) * | 2002-05-16 | 2005-09-08 | Basf Ag | Use of the repressor glxr for the synthesis of lysine in corynebacterium glutamicum |
| CN1898259A (zh) * | 2003-12-18 | 2007-01-17 | 巴斯福股份公司 | 用于编码精细化学品的代谢途径的蛋白质的基因变体 |
| WO2012053794A2 (en) * | 2010-10-20 | 2012-04-26 | Cj Cheiljedang Corporation | Microorganism producing o-phosphoserine and method of producing l-cysteine or derivatives thereof from o-phosphoserine using the same |
| CN102906272A (zh) * | 2010-10-20 | 2013-01-30 | Cj第一制糖株式会社 | 生产o-磷酸丝氨酸的微生物和使用该微生物由o-磷酸丝氨酸生产l-半胱氨酸或其衍生物的方法 |
| CN104561077A (zh) * | 2014-12-22 | 2015-04-29 | 南京师范大学 | 重组工程介导的谷氨酸棒杆菌atcc 13032的基因敲除方法 |
| CN106434514A (zh) * | 2016-11-09 | 2017-02-22 | 天津大学 | 生产5‑氨基乙酰丙酸的谷氨酸棒杆菌工程菌株 |
| CN106636171A (zh) * | 2016-10-11 | 2017-05-10 | 天津大学 | 生产5‑氨基乙酰丙酸的谷氨酸棒杆菌工程菌株及构建 |
| CN113881611A (zh) * | 2021-02-02 | 2022-01-04 | 江南大学 | 一种提高谷氨酸棒杆菌合成l-谷氨酸产量的方法 |
| CN114426983A (zh) * | 2022-02-11 | 2022-05-03 | 天津大学 | 敲除谷氨酸棒杆菌中转录调控因子Ncgl0580生产5-氨基乙酰丙酸的方法 |
-
2023
- 2023-07-19 CN CN202310889013.8A patent/CN116926102A/zh active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050196847A1 (en) * | 2002-05-16 | 2005-09-08 | Basf Ag | Use of the repressor glxr for the synthesis of lysine in corynebacterium glutamicum |
| CN1898259A (zh) * | 2003-12-18 | 2007-01-17 | 巴斯福股份公司 | 用于编码精细化学品的代谢途径的蛋白质的基因变体 |
| WO2012053794A2 (en) * | 2010-10-20 | 2012-04-26 | Cj Cheiljedang Corporation | Microorganism producing o-phosphoserine and method of producing l-cysteine or derivatives thereof from o-phosphoserine using the same |
| CN102906272A (zh) * | 2010-10-20 | 2013-01-30 | Cj第一制糖株式会社 | 生产o-磷酸丝氨酸的微生物和使用该微生物由o-磷酸丝氨酸生产l-半胱氨酸或其衍生物的方法 |
| CN104561077A (zh) * | 2014-12-22 | 2015-04-29 | 南京师范大学 | 重组工程介导的谷氨酸棒杆菌atcc 13032的基因敲除方法 |
| CN106636171A (zh) * | 2016-10-11 | 2017-05-10 | 天津大学 | 生产5‑氨基乙酰丙酸的谷氨酸棒杆菌工程菌株及构建 |
| CN106434514A (zh) * | 2016-11-09 | 2017-02-22 | 天津大学 | 生产5‑氨基乙酰丙酸的谷氨酸棒杆菌工程菌株 |
| CN113881611A (zh) * | 2021-02-02 | 2022-01-04 | 江南大学 | 一种提高谷氨酸棒杆菌合成l-谷氨酸产量的方法 |
| CN114426983A (zh) * | 2022-02-11 | 2022-05-03 | 天津大学 | 敲除谷氨酸棒杆菌中转录调控因子Ncgl0580生产5-氨基乙酰丙酸的方法 |
Non-Patent Citations (2)
| Title |
|---|
| QI SHENG等: "Production of L-glutamate family amino acids in Corynebacterium glutamicum: Physiological mechanism, genetic modulation, and prospects", SYNTHETIC AND SYSTEMS BIOTECHNOLOGY, vol. 6, no. 4, 31 December 2021 (2021-12-31), pages 302 - 325, XP093044322, DOI: 10.1016/j.synbio.2021.09.005 * |
| QIUYU YANG等: "Microbial Synthesis of Heme b: Biosynthetic Pathways, Current Strategies, Detection, and Future Prospects", MOLECULES, vol. 28, no. 8, 21 April 2023 (2023-04-21), pages 1 - 22 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200347419A1 (en) | Recombinant bacterium capable of producing l-lysine, construction method thereof and production method of l-lysine | |
| US11512333B2 (en) | Method for producing tetrahydropyrimidine by fermenting recombinant Corynebacterium glutamicum | |
| JP2021500914A5 (zh) | ||
| CN103215291B (zh) | 用于生产l-2-氨基丁酸的载体、工程菌株及方法 | |
| CN109777763B (zh) | 一株用于l-茶氨酸生产的基因工程菌及其构建与应用 | |
| CN107502585A (zh) | 一株高效合成聚‑γ‑谷氨酸的地衣芽孢杆菌工程菌 | |
| CN111019878B (zh) | L-苏氨酸产量提高的重组大肠杆菌及其构建方法与应用 | |
| CN113774075B (zh) | 一株大肠杆菌基因工程菌及其发酵生产l-茶氨酸的方法 | |
| WO2022174597A1 (zh) | 一种用于l-肌氨酸生产的基因工程菌及构建方法与应用 | |
| CN109456987B (zh) | 高产l-亮氨酸的相关基因及工程菌构建方法与应用 | |
| CN103820485A (zh) | 一种减少l-丝氨酸生产菌株谷氨酸棒杆菌syps-062代谢副产物的方法 | |
| CN113564190A (zh) | 高产核黄素大肠杆菌工程菌株及构建方法 | |
| CN106635945B (zh) | 重组菌株及其制备方法和生产l-苏氨酸的方法 | |
| CN112980758A (zh) | 一种提高谷氨酸棒杆菌合成5-氨基乙酰丙酸产量的方法 | |
| CN108588108B (zh) | 一种高效代谢甘油的芽胞杆菌的制备方法和应用 | |
| CN111004761A (zh) | 一种l-酪氨酸基因工程菌及其生产l-酪氨酸的方法和应用 | |
| CN116926102A (zh) | 抑制谷氨酸棒杆菌中全局转录调控因子基因glxR的表达生产5-ALA的方法 | |
| CN107299074B (zh) | 甲酸脱氢酶工程菌株的构建方法及应用 | |
| CN113583930B (zh) | 一株不依赖抗生素并能高效生产γ-氨基丁酸的谷氨酸棒杆菌的构建 | |
| CN110862952B (zh) | 5-氨基乙酰丙酸生产菌株及其构建方法和应用 | |
| CN109929853B (zh) | 嗜热菌来源的热激蛋白基因的应用 | |
| CN113604413B (zh) | 一种重组菌株及制备方法与应用 | |
| CN118165907B (zh) | 一种γ-氨基丁酸生产菌株及其构建方法与应用 | |
| CN113957073B (zh) | 一种tkt基因启动子突变体及其在生产L-赖氨酸中的应用 | |
| WO2019154670A1 (en) | Corynebacterium spp. strains having increased rates of growth and/or sugar uptake |
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 |