CN103898150B - Produce ALANINE and the bacterial strain of tolerance tap water and construction process - Google Patents
Produce ALANINE and the bacterial strain of tolerance tap water and construction process Download PDFInfo
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- CN103898150B CN103898150B CN201410140656.3A CN201410140656A CN103898150B CN 103898150 B CN103898150 B CN 103898150B CN 201410140656 A CN201410140656 A CN 201410140656A CN 103898150 B CN103898150 B CN 103898150B
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- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 title claims abstract description 66
- 235000004279 alanine Nutrition 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 42
- 239000008399 tap water Substances 0.000 title claims abstract description 40
- 235000020679 tap water Nutrition 0.000 title claims abstract description 40
- 230000001580 bacterial effect Effects 0.000 title abstract description 23
- 238000010276 construction Methods 0.000 title abstract description 5
- 229960003767 alanine Drugs 0.000 claims abstract description 65
- 241000894006 Bacteria Species 0.000 claims abstract description 57
- 101150093586 clpA gene Proteins 0.000 claims abstract description 41
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 38
- 239000002773 nucleotide Substances 0.000 claims description 16
- 238000013467 fragmentation Methods 0.000 claims description 14
- 238000006062 fragmentation reaction Methods 0.000 claims description 14
- 235000018102 proteins Nutrition 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 230000006801 homologous recombination Effects 0.000 claims description 8
- 238000002744 homologous recombination Methods 0.000 claims description 8
- 150000001413 amino acids Chemical group 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- 230000000968 intestinal effect Effects 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 108010031025 Alanine Dehydrogenase Proteins 0.000 claims description 4
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 3
- 229960000310 isoleucine Drugs 0.000 claims description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 3
- 108010092060 Acetate kinase Proteins 0.000 claims description 2
- 108010041525 Alanine racemase Proteins 0.000 claims description 2
- 108010021809 Alcohol dehydrogenase Proteins 0.000 claims description 2
- 101710088194 Dehydrogenase Proteins 0.000 claims description 2
- 241000193385 Geobacillus stearothermophilus Species 0.000 claims description 2
- 108090000854 Oxidoreductases Proteins 0.000 claims description 2
- 230000002759 chromosomal effect Effects 0.000 claims description 2
- 210000000349 chromosome Anatomy 0.000 claims description 2
- 108010008221 formate C-acetyltransferase Proteins 0.000 claims description 2
- 238000010353 genetic engineering Methods 0.000 claims description 2
- 230000010354 integration Effects 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 claims description 2
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 abstract description 3
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 239000002609 medium Substances 0.000 description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 25
- 239000012153 distilled water Substances 0.000 description 17
- 238000000855 fermentation Methods 0.000 description 15
- 230000004151 fermentation Effects 0.000 description 15
- 238000004519 manufacturing process Methods 0.000 description 15
- 239000013612 plasmid Substances 0.000 description 9
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- 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 5
- 239000008103 glucose Substances 0.000 description 5
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- XBDQKXXYIPTUBI-UHFFFAOYSA-N Propionic acid Chemical compound CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 3
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- MONMFXREYOKQTI-UHFFFAOYSA-N 2-bromopropanoic acid Chemical compound CC(Br)C(O)=O MONMFXREYOKQTI-UHFFFAOYSA-N 0.000 description 2
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 2
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- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
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- 229910001628 calcium chloride Inorganic materials 0.000 description 2
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- LELOWRISYMNNSU-UHFFFAOYSA-N hydrogen cyanide Chemical compound N#C LELOWRISYMNNSU-UHFFFAOYSA-N 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 2
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- 239000003921 oil Substances 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
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- 101150082527 ALAD gene Proteins 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 241000191070 Escherichia coli ATCC 8739 Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
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- 238000012408 PCR amplification Methods 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
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- 230000036506 anxiety Effects 0.000 description 1
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- 230000003115 biocidal effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
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- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 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 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 239000010779 crude oil Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
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- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
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- 239000000796 flavoring agent Substances 0.000 description 1
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- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000021096 natural sweeteners Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 229940049547 paraxin Drugs 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- 235000012976 tarts Nutrition 0.000 description 1
- 238000005891 transamination reaction Methods 0.000 description 1
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- 239000011726 vitamin B6 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
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Classifications
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- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- 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)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- 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
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Molecular Biology (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
The invention discloses and a kind ofly produce ALANINE and the tolerance bacterial strain of tap water and construction process.The invention provides a kind of method building recombinant bacterium, be the clpA gene set out in bacterium that suddenlys change, obtain recombinant bacterium; The described sudden change clpA gene set out in bacterium is that the described clpA gene nucleotide series set out in bacterium is sported G from the T of 5 ' end the 1895th.Experiment of the present invention proves, the present invention is by suddenling change the clpA gene in colibacillus engineering XZ-A26, the recombinant bacterium XZ-A45 obtained, ALANINE output can not only be improved, but also L-alanine with high yield in the fermention medium that can configure at tap water, adopting tap water to configure can be cost-saving.
Description
Technical field
The present invention relates to biological technical field, particularly relate to and produce ALANINE and the bacterial strain of tolerance tap water and construction process.
Background technology
ALANINE, as human body non-essential amino acid, is formed to pyruvic acid by the transamination of glycine in vivo.ALANINE is a kind of white crystals or crystalline powder, is with pleasantly sweet, soluble in water, has been widely used at food and medicine industry field tool.In field of food industry, ALANINE can improve Nutritive value of food, can significantly improve protein utilization in food and beverage after adding ALANINE.ALANINE can improve the sense of taste of artificial synthesis edulcorant, makes it as natural sweeteners.In addition, ALANINE can also improve organic acid tart flavour, makes it closer to natural taste.At field of medicaments, ALANINE is often used as amino acids nutritional drugs, and ALANINE is also the important source material manufacturing vitamin B6, synthetic pantothenic acid calcium and other organic compound simultaneously.
At present, the production method of ALANINE mainly contains chemical synthesis and biological synthesis process.Wherein chemical synthesis mainly contains propanoate ammoniation process, α-bromo-propionic acid chlorination process and cyanalcohol method.These methods all need petroleum base starting material, and as propionic acid, α-bromo-propionic acid, acetaldehyde and prussic acid etc., therefore cost locks into crude oil price.Along with the lifting of oil price, cost can be more and more higher.In addition, these methods are all through that complicated chemical catalysis completes, and pollute heavy, separation and Extraction cost is high, is not suitable for the needs of Sustainable development.
It take mainly L-Aspartic acid as raw material at present that biological process produces ALANINE, carries out decarboxylic reaction and produce ALANINE under the catalysis of aspartic acid-beta-decarboxylase.The method is the production technology that current domestic ALANINE manufacturer mainly uses.But because the production of the starting material aspartic acid in the method is raw material with cis-butenedioic anhydride, therefore the production cost of ALANINE still depends on oil price.Along with the scarcity of petroleum resources and the lifting of price, there is huge hidden danger by causing the supply of aspartic acid in the anxiety of cis-butenedioic anhydride resource and price increase, thus can have influence on production and the cost of ALANINE.
Along with the development of synthetic biology and metabolic engineering, the research of Production by Microorganism Fermentation ALANINE is used more and more to come into one's own in recent years.Microbe fermentation method can realize with carbohydrates such as glucose as raw material production ALANINE.Glucose belongs to reproducible biomass resource, can be obtained by the ligocellulose degradation being extensively present in occurring in nature.Therefore, use it as starting material, the production cost of ALANINE can be made to remain on stable level, there are long-range economic advantages.At present, many strains have been had can to produce the report of the bacterial strain of ALANINE.Smith etc. construct strain E.coliALS929 (pTrc99A-alaD) bacterial strain, the conversion of pyruvate generated in born of the same parents can be ALANINE by the alanine dehydrogenase AlaD wherein expressed in plasmid, this bacterial strain, after 48 hours fermentation, can produce the ALANINE of 88g/L.Lee etc. construct strain E.coliALA887 (pTrc99A-alaD) bacterial strain, can produce the ALANINE of 32g/L during fermentative production in 27 hours.In these bacterial strains, alanine dehydrogenase is the committed step of producing ALANINE, and in the bacterial strain of report, the gene of this albumen of encoding generally is expressed by high copy number plasmid.Carrying out in strain cultures fermenting process, needing to add the genetic stability that antibiosis usually maintains plasmid.These factors will cause fermentation process technique complicated and improve ALANINE production cost.Therefore, along with the development of synthetic biology and metabolic engineering, the ALANINE building inheritance stability produce bacterial strain and the method that undergoes microbial fermentation to produce ALANINE will be following development trend.
What current strain fermentation produced ALANINE use is all the substratum configured by distilled water, and distilled water improves production cost in the industrial production.The substratum that exploitation can directly use tap water to configure carry out fermentative production ALANINE bacterial strain and for the production of, the production cost of ALANINE will be reduced greatly.But relative to distilled water, there is a large amount of ions in tap water and concentration is higher.In order to obtain the bacterial strain that the substratum that can directly use tap water to configure carries out fermenting, need to improve bacterial strain to the tolerance of high ion concentration.
Summary of the invention
An object of the present invention is to provide and produce ALANINE and the construction process of the bacterial strain of tolerance tap water.
The method of structure recombinant bacterium (XZ-A45) provided by the invention, comprises the steps: the encoding gene clpA protein coding gene set out on bacterium karyomit(e) being replaced with clpA* albumen, the recombinant bacterium obtained;
The aminoacid sequence of described clpA* albumen is that the 632nd of described clpA protein amino acid sequence the Isoleucine I is sported Serine S.
In aforesaid method, described clpA* albumen is by the albumen of sequence in sequence table 2 from 5 ' end 1-2272 position nucleotide coding.
In aforesaid method, described clpA* protein coding gene is be the gene that T sports G and obtains by the base of described clpA protein coding gene nucleotide sequence the 1895th.
In aforesaid method, the nucleotides sequence of described clpA* protein coding gene to be classified as in sequence table sequence 2 from 5 ' end 1-2272 position Nucleotide.
In aforesaid method, the described clpA* protein coding gene that replaced with by the clpA protein coding gene set out on bacterium karyomit(e) is DNA fragmentation II homologous recombination containing described clpA* protein coding gene set out in bacterium to described.
In aforesaid method, the nucleotides sequence of described DNA fragmentation II is classified as sequence 2 in sequence table.
In aforesaid method, the described bacterium that sets out be by by the L-alanine dehydrogenase gene integration on Geobacillus stearothermophilus karyomit(e) at the chromosomal serum lactic dehydrogenase place of intestinal bacteria ATCC8739, knock out the pyruvate formate-lyase gene of gained escherichia coli chromosome, alcohol dehydrogenase gene, Acetokinase gene, fumaric reductase gene and alanine racemase gene more successively, then in fermentor tank, continuous passage is cultivated and the genetic engineering bacterium that obtains;
The described bacterium that sets out is specially intestinal bacteria XZ-A26CGMCCNo.4036.
Above-mentioned homologous recombination is carried out especially by two steps:
1) DNA fragmentation I is imported with in the colibacillus engineering strain XZ-A26 of pKD46, carry out first time homologous recombination, obtain middle bacterium XZ-A44;
2) DNA fragmentation II is imported in described middle bacterium XZ-A44, carry out second time homologous recombination, obtain recombinant bacterium XZ-A45.
The recombinant bacterium prepared by above-mentioned method is also the scope of protection of the invention.
Above-mentioned recombinant bacterium is also the scope of protection of the invention in generation and/or the application improved in ALANINE.
In above-mentioned application, described generation and/or to improve ALANINE be to be fermented in the fermention medium that solvent is prepared generation at tap water by recombinant bacterium according to claim 7.
Another order of the present invention is to provide a kind of method producing ALANINE.
Method provided by the invention, comprises the steps: to ferment in the fermention medium prepared as solvent at tap water above-mentioned recombinant bacterium, collects tunning, namely obtains ALANINE.
Tap water is the fermention medium II in embodiment as the fermention medium that solvent is prepared.
Above-mentioned tap water, for taking from Shanhai Pass branch office of pioneering Running-water Company, has following feature: hardness: 2-3mmol/L, pH=6.5-7.5, specific conductivity: 400-600 μ s/cm, muriate 100-250mg/L, vitriol 100-250mg/L.
Experiment of the present invention proves, the present invention is by suddenling change the clpA gene in colibacillus engineering XZ-A26, the recombinant bacterium XZ-A45 obtained, ALANINE output can not only be improved, but also L-alanine with high yield in the fermention medium that can configure at tap water, adopting tap water to configure can be cost-saving.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Colibacillus engineering XZ-A26CGMCCNo.4036, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 26th, 2010 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCCNO.4036, and Classification And Nomenclature is colon bacillus Escherichiacoli.This bacterial strain can in minimal medium fermentative production ALANINE.
Shanhai Pass branch office of pioneering Running-water Company (hardness: 2-3mmol/L, pH=6.5-7.5, specific conductivity: 400-600 μ s/cm, muriate 100-250mg/L, vitriol 100-250mg/L) taken from by tap water in following embodiment.
Distilled water (hardness 0, pH=7.0-8.0, specific conductivity: 5 μ s/cm) in following embodiment
Embodiment 1, produce ALANINE and the bacterial strain of resistance to tap water with the Screening of Media of tap water configuration
1, the substratum of distilled water and tap water configuration is contrasted on the impact of colibacillus engineering XZ-A26 fermentative production ALANINE
Colibacillus engineering XZ-A26CGMCCNo.4036(specific features is in table 1), in the minimal medium of distilled water configuration, ALANINE can be produced by glucose fermentation.But, owing to using distilled water cost too high in industrial fermentation, therefore wish directly to use tap water to configure substratum.
Therefore, compared for the substratum of distilled water and tap water configuration to the impact of colibacillus engineering XZ-A26 fermentative production ALANINE.
The recombination bacillus coli of ALANINE produced by table 1
Concrete steps are as follows:
Seed culture medium: by following solute dissolves in solvent distilled water, obtain seed culture medium, the concentration provided is final concentration:
Glucose 120g/L, ammonium chloride 5g/L, NaH
2pO
45g/L, Na
2hPO
45g/L, MgSO
47H
2o1g/L, CaCl
22H
2o0.1g/L, small-scale inorganic salt 5ml/L, medium pH 6.5.
Small-scale inorganic salt consists of: FeCl
36H
2o1.5mg, CoCl
26H
2o0.1mg, CuCl
22H
2o0.1mg, ZnCl
20.1mg, Na
2moO
42H
2o0.1mg, MnCl
24H
2o0.2mg, distilled water is settled to 1L, filtration sterilization.
Fermention medium I consists of: same to seed culture medium.
Fermention medium II consists of: same to seed culture medium, just substitutes distilled water as solvent with tap water.
In 250ml triangular flask, seed culture medium is 150ml, 121 DEG C of sterilizing 15min.Access XZ-A26 after cooling, at 30 DEG C, shaking speed is 50rpm, cultivates 18h, inoculates for fermention medium.
3L fermentation cylinder for fermentation culture volume is 2.4L, 121 DEG C of sterilizing 15min.Inoculum size is 0.1%(V/V).Leavening temperature is 30 DEG C, and mixing speed is 100rpm, fermentation 48h.Neutralizing agent is ammoniacal liquor, and the pH of fermentor tank is controlled 6.5.
Analytical procedure: use Agilent (Agilent-1200) high performance liquid chromatography to measure the component in fermented liquid.Glucose in fermented liquid and organic acid concentration adopt the AminexHPX-87H organic acid analysis column of Bole (Biorad) company.Quantitative and the chiral determination of ALANINE adopts the aglucon crossover chiral isomer liquid chromatography separation column (ChiralpakMA (+)) of Daicel (Daciel) company.
The results are shown in Table 2, fermentation 48 hours in the fermention medium I that strain X Z-A26 configures at distilled water, ALANINE output reaches 115g/L.Fermentation 48 hours in the fermention medium II of tap water configuration, ALANINE output reaches 80g/L, and output reduces 30%.
Table 2 recombination bacillus coli fermentative production ALANINE
ause the fermentor tank of 3L, fermention medium is 2.4L.The neutralizing agent used is ammoniacal liquor, and the pH of fermentor tank is controlled 6.5.
bin the fermention medium II configured at tap water with XZ-A26 bacterial strain, ALANINE output is defined as 100%.
2, adaptive evolution is adopted to improve the ability of engineering bacteria tolerance tap water
Adopt adaptive evolution technology, continuous passage engineering bacteria XZ-A26 in the substratum of tap water configuration, to improve the ability of its tolerance tap water.
The fermention medium that evolution metabolism uses is identical with the composition of fermention medium II described in above-mentioned 1.Evolution metabolic process uses the fermentor tank of 500ml, and the volume of fermention medium II is 250ml, and 121 DEG C of sterilizing 15min, access XZ-A26 after cooling, and inoculum size is 0.1%(V/V).Leavening temperature is 30 DEG C, and mixing speed is 100rpm.Use ammoniacal liquor for neutralizing agent in fermenting process, the pH of fermentor tank is controlled 6.5.Continuous passage culturing engineering bacterium in fermentor tank, is transferred to the bacterium liquid in fermentor tank in a new fermentor tank according to the ratio of 1:1000 for every 24 hours.Through the switching of 820 generations, final acquisition strain X Z-A41(table 1).
Use with the method described in above-mentioned 1, ferment the engineering bacteria XZ-A41 obtained in the fermention medium II configured with tap water, and after 48 hours, ALANINE output reaches 114g/L, with fermentation yield substantially the same (table 2) in the substratum configured at distilled water.
The above results shows, engineering bacteria XZ-A41 not only can L-alanine with high yield, and tolerance tap water.Therefore, whether caused by transgenation to study its tolerance, its genome is checked order.
3, the gene order-checking of engineering bacteria XZ-A41
(1) prepared by fermentation culture and genome
The mono-clonal of picking engineering strain XZ-A41 is inoculated in the LB liquid nutrient medium of 4ml, is 37 DEG C in culture temperature, and rotating speed is shake overnight incubation under the condition of 250rpm, set three parallel.By cultured three parallel in cell mixing and collecting cell, use
genomicDNAPurificationKit (promega) extracting bacterial genomes DNA.The detection of DNA concentration is quantitatively completed by QubitFluorometer and agarose gel electrophoresis.
(2) genome is resurveyed sequence
Genome sequence of resurveying is completed by Shenzhen Huada Genetic Technology Co., Ltd.Adopt whole-genome shotgun sequencing, build Paired-End fragment library and check order, the overall order-checking degree of depth is more than 100 times, and expected data amount is 500Mbp.The technological method that order-checking adopts and route are: DNA sample preparation--above machine checks order--data processing-analysis of biological information.The reference sequences of sequential analysis is the genome sequence (http://www.ncbi.nlm.nih.gov/nuccore/NC_010468.1) of E.coliATCC8739.
Result: analysis is carried out to the heavy sequencing result of engineering bacteria XZ-A41 genome and finds, clpA gene (coding ATP dependent form molecular chaperone protein ClpA, GenBankNo.ADT74495.1) T that nucleotide sequence is the 1895th sports G, the I of the 632nd, the amino acid of its correspondence is mutated into S, be clpA* by this unnamed gene, the albumen of coding is clpA*.
In addition, find that the C of lon gene (coding ATP dependent form proteolytic enzyme La, GenBankNo.AFH10177.1) nucleotide sequence the 1310th sports A, the A of the aminoacid sequence the 437th of its correspondence is mutated into D.Be lon* by this unnamed gene, the albumen of coding is lon*.
Therefore, think that resistance to water-based from the beginning may cause due to clpA gene or lon transgenation, next step suddenlys change to the clpA gene of the bacterium that sets out, thus obtains the bacterial strain of resistance to tap water.
Embodiment 2, clpA transgenation obtain produces ALANINE and resistance to tap water strain X Z-A45
In order to verify the impact of clpA transgenation (T1895G) on engineering strain tolerance tap water ability, clpA* being introduced XZ-A26 by the method for two step homologous recombination and replaces original clpA gene, obtaining XZ-A45(table 1).Concrete steps are as follows:
The first step, with pXZ-CS plasmid (Tanetal., ApplEnvironMicrobiol.2013,79:4838-4844; The public can obtain from Anhui permanent biotechnology art limited-liability company of China; ) DNA is template, uses primer XZ-clpA*cat-up/XZ-clpA*sacB-down to amplify the DNA fragmentation I(sequence 1 of 2719bp).
Amplification system is: NewEnglandBiolabsPhusion5X damping fluid 10 μ l, dNTP (often kind of each 10mM of dNTP) 1 μ l, DNA profiling 20ng, each 2 μ l, PhusionHigh-FidelityDNA polysaccharases (2.5U/ μ l) of primer (10 μMs) 0.5 μ l, distilled water 33.5 μ l, cumulative volume is 50 μ l.
Amplification condition is 98 DEG C of denaturations 2 minutes (1 circulation); 98 DEG C of sex change, 10 seconds, 56 DEG C annealing 10 seconds, 72 DEG C extend 30 seconds (30 circulations); 72 DEG C extend 5 minutes (1 circulation).
DNA fragmentation I comprises clpA upstream region of gene homology arm 50 bases (sequence 1 is from 5 ' end 1-50 position Nucleotide), cat-sacB gene DNA fragment (sequence 1 is from 5 ' end 51-2669 position Nucleotide) and clpA downstream of gene homology arm 50 bases (sequence 1 is from 5 ' end 2670-2719 position Nucleotide).
DNA fragmentation I is used for first time homologous recombination, first pKD46 plasmid (derive from Hefei hundred and step Bioisystech Co., Ltd) is converted into colibacillus engineering strain XZ-A26 by calcium chloride transformation, obtain the colibacillus engineering strain XZ-A26 with pKD46, then DNA fragmentation I electricity is gone to the colibacillus engineering strain XZ-A26 with pKD46.
Electricity turns condition: the Electroporation-competent cells first preparing the colibacillus engineering strain XZ-A26 with pKD46 plasmid; 50 μ l competent cells are placed on ice, add 50ngDNA fragment I, place 2 minutes on ice, be transferred to the Bio-Rad electric shock cup of 0.2cm.Use MicroPulser(Bio-Rad company) electroporation apparatus, shock parameters is voltage 2.5kv.Rapid in 1mlLB media transfer extremely electric shock cup after electric shock, be transferred in test tube after blowing and beating 5 times, 75 turns, hatch 2 hours for 30 DEG C.Getting 200 μ l bacterium liquid is coated on the LB flat board containing paraxin (final concentration is 17ug/ml), after 37 DEG C of incubated overnight, select 5 single bacterium colonies and carry out PCR checking, use primer XZ-clpA*-up/XZ-clpA*-down to verify, correct bacterium colony amplified production is the fragment of 3419bp.Select a correct single bacterium colony, by its called after XZ-A44.
Second step, the genomic dna of the engineering strain XZ-A41 obtained with embodiment 1 is template, primer XZ-clpA*-up/XZ-clpA*-down is used to carry out pcr amplification, obtain 2272bp its nucleotides sequence of DNA fragmentation II(be classified as sequence 2 in sequence table), DNA fragmentation II is used for second time homologous recombination.First pKD46 plasmid is converted into XZ-A44 by calcium chloride transformation, obtains the colibacillus engineering strain XZ-A44 with pKD46, then DNA fragmentation II electricity is converted into the XZ-A44 with pKD46 plasmid.
DNA fragmentation II comprises clpA* gene, the nucleotides sequence of clpA* gene is classified as sequence 2 from 5 ' end 1-2272 position, clpA* gene is that the 1895th T of clpA gene sports G, and the albumen of clpA* genes encoding is that the 632nd of the albumen of clpA genes encoding the Isoleucine I is sported Serine S.
Electricity turns condition: the Electroporation-competent cells first preparing the colibacillus engineering strain XZ-A44 with pKD46 plasmid; 50 μ l competent cells are placed on ice, add 50ngDNA fragment II, place 2 minutes on ice, be transferred to the Bio-Rad electric shock cup of 0.2cm.Use MicroPulser(Bio-Rad company) electroporation apparatus, shock parameters is voltage 2.5kv.Rapid in 1mlLB media transfer extremely electric shock cup after electric shock, be transferred in test tube after blowing and beating 5 times, 75 turns, hatch 4 hours for 30 DEG C.Being transferred to by bacterium liquid does not have the LB liquid nutrient medium of sodium-chlor (filling 50ml substratum in 250ml flask) containing 10% sucrose, cultivate after 24 hours containing 6% sucrose do not have on the LB solid medium of sodium-chlor streak culture.Through PCR checking, the primer is XZ-clpA*-up/XZ-clpA*-down, and correct bacterium colony amplified production is the fragment of 2272bp.Select a correct single bacterium colony, by its called after XZ-A45.
Above-mentioned the primer sequence is in table 3.
Use the method described in same embodiment 1, ferment the engineering bacteria XZ-A45 obtained in the fermention medium II configured with tap water, and after 48 hours, ALANINE output reaches 102g/L, and relative starting strain XZ-A26 improves 28%.
The primer used in table 3 the present invention
The substratum that embodiment 3, contrast distilled water and tap water configure is on the impact of colibacillus engineering XZ-A45 fermentative production ALANINE
Use method described in same embodiment 1, ferment XZ-A45 bacterial strain respectively in the fermention medium with distilled water and tap water configuration.
Found that, after the fermentation of 48h, the ALANINE of 114g/L can be produced in the fermention medium I that XZ-A45 bacterial strain configures at distilled water, and the ALANINE of 102g/L when fermenting in the medium ii using tap water configuration, can be produced.When XZ-A45 bacterial strain to be compared with XZ-A26 and is fermented in the medium ii using tap water configuration, ALANINE output increased 27.5%.
Claims (9)
1. build a method for recombinant bacterium, comprise the steps: the encoding gene clpA protein coding gene set out on bacterium karyomit(e) being replaced with clpA* albumen, the recombinant bacterium obtained;
The aminoacid sequence of described clpA* albumen is that the 632nd of described clpA protein amino acid sequence the Isoleucine I is sported Serine S;
The nucleotides sequence of described clpA* protein coding gene to be classified as in sequence table sequence 2 from 5 ' end 1-2272 position Nucleotide;
The described bacterium that sets out is intestinal bacteria.
2. method according to claim 1, is characterized in that:
Described clpA* protein coding gene is be the gene that T sports G and obtains by the base of described clpA protein coding gene nucleotide sequence the 1895th.
3. method according to claim 1, is characterized in that:
The described clpA* protein coding gene that replaced with by the clpA protein coding gene set out on bacterium karyomit(e) is DNA fragmentation II homologous recombination containing described clpA* protein coding gene set out in bacterium to described.
4. method according to claim 3, is characterized in that:
The nucleotides sequence of described DNA fragmentation II is classified as sequence 2 in sequence table.
5. the method according to claim 3 or 4, it is characterized in that: described in set out bacterium be by by the L-alanine dehydrogenase gene integration on Geobacillus stearothermophilus karyomit(e) at the chromosomal serum lactic dehydrogenase place of intestinal bacteria ATCC8739, knock out the pyruvate formate-lyase gene of gained escherichia coli chromosome, alcohol dehydrogenase gene, Acetokinase gene, fumaric reductase gene and alanine racemase gene more successively, then in fermentor tank, continuous passage is cultivated and the genetic engineering bacterium that obtains;
Described intestinal bacteria are intestinal bacteria XZ-A26CGMCCNo.4036.
6. the recombinant bacterium prepared by described method arbitrary in claim 1-5.
7. recombinant bacterium according to claim 6 is in the application producing ALANINE and/or improve in ALANINE output.
8. application according to claim 7, is characterized in that: described generation ALANINE and/or to improve ALANINE output be to be fermented in the fermention medium that solvent is prepared generation at tap water by recombinant bacterium according to claim 6.
9. produce a method for ALANINE, it is characterized in that: ferment recombinant bacterium according to claim 6 in the fermention medium that tap water is prepared as solvent, collect tunning, namely obtain ALANINE.
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