CN116790546A - Pyruvic acid kinase mutant and application thereof - Google Patents
Pyruvic acid kinase mutant and application thereof Download PDFInfo
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- CN116790546A CN116790546A CN202210255860.4A CN202210255860A CN116790546A CN 116790546 A CN116790546 A CN 116790546A CN 202210255860 A CN202210255860 A CN 202210255860A CN 116790546 A CN116790546 A CN 116790546A
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- Prior art keywords
- val
- gly
- ala
- ile
- glu
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- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 title abstract description 18
- 108091000080 Phosphotransferase Proteins 0.000 title abstract description 9
- 102000020233 phosphotransferase Human genes 0.000 title abstract description 9
- 229940107700 pyruvic acid Drugs 0.000 title abstract description 9
- 239000002777 nucleoside Substances 0.000 claims abstract description 40
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims abstract description 34
- 108020005115 Pyruvate Kinase Proteins 0.000 claims abstract description 30
- 102000013009 Pyruvate Kinase Human genes 0.000 claims abstract description 30
- 229930010555 Inosine Natural products 0.000 claims abstract description 19
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 claims abstract description 19
- 229960003786 inosine Drugs 0.000 claims abstract description 19
- 125000003835 nucleoside group Chemical group 0.000 claims abstract description 18
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims abstract description 17
- 229960005305 adenosine Drugs 0.000 claims abstract description 17
- 244000005700 microbiome Species 0.000 claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 claims abstract description 13
- 230000035772 mutation Effects 0.000 claims abstract description 11
- 150000001413 amino acids Chemical group 0.000 claims abstract description 9
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 4
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 24
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 claims description 18
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 17
- 238000000855 fermentation Methods 0.000 claims description 13
- 230000004151 fermentation Effects 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 10
- 150000007523 nucleic acids Chemical class 0.000 claims description 9
- 244000063299 Bacillus subtilis Species 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 8
- 102000039446 nucleic acids Human genes 0.000 claims description 8
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims description 7
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 claims description 7
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 claims description 7
- 229940029575 guanosine Drugs 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- 238000010276 construction Methods 0.000 claims description 6
- 241000194103 Bacillus pumilus Species 0.000 claims description 5
- 239000012620 biological material Substances 0.000 claims description 5
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- 108020004511 Recombinant DNA Proteins 0.000 claims description 2
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- 239000013603 viral vector Substances 0.000 claims description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 abstract description 9
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 abstract description 8
- 239000004473 Threonine Substances 0.000 abstract description 8
- 239000004472 Lysine Substances 0.000 abstract description 6
- 239000004475 Arginine Substances 0.000 abstract description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 abstract description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 abstract description 4
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- 239000002609 medium Substances 0.000 description 8
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- PXHABOCPJVTGEK-BQBZGAKWSA-N Glu-Gln-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O PXHABOCPJVTGEK-BQBZGAKWSA-N 0.000 description 6
- RFTVTKBHDXCEEX-WDSKDSINSA-N Glu-Ser-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RFTVTKBHDXCEEX-WDSKDSINSA-N 0.000 description 6
- ULZCYBYDTUMHNF-IUCAKERBSA-N Gly-Leu-Glu Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ULZCYBYDTUMHNF-IUCAKERBSA-N 0.000 description 6
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 6
- ISERLACIZUGCDX-ZKWXMUAHSA-N Val-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N ISERLACIZUGCDX-ZKWXMUAHSA-N 0.000 description 6
- AEFJNECXZCODJM-UWVGGRQHSA-N Val-Val-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](C(C)C)C(=O)NCC([O-])=O AEFJNECXZCODJM-UWVGGRQHSA-N 0.000 description 6
- PYMYPHUHKUWMLA-LMVFSUKVSA-N aldehydo-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 6
- 108010013835 arginine glutamate Proteins 0.000 description 6
- 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 6
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- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 6
- 108010089804 glycyl-threonine Proteins 0.000 description 6
- 108010010147 glycylglutamine Proteins 0.000 description 6
- 108010050848 glycylleucine Proteins 0.000 description 6
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000002342 ribonucleoside Substances 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- AUHDWARTFSKSAC-HEIFUQTGSA-N (2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-(6-oxo-1H-purin-9-yl)oxolane-2-carboxylic acid Chemical compound [C@]1([C@H](O)[C@H](O)[C@@H](CO)O1)(N1C=NC=2C(O)=NC=NC12)C(=O)O AUHDWARTFSKSAC-HEIFUQTGSA-N 0.000 description 3
- CWFMWBHMIMNZLN-NAKRPEOUSA-N (2s)-1-[(2s)-2-[[(2s,3s)-2-amino-3-methylpentanoyl]amino]propanoyl]pyrrolidine-2-carboxylic acid Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CWFMWBHMIMNZLN-NAKRPEOUSA-N 0.000 description 3
- NKJBKNVQHBZUIX-ACZMJKKPSA-N Ala-Gln-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NKJBKNVQHBZUIX-ACZMJKKPSA-N 0.000 description 3
- BGNLUHXLSAQYRQ-FXQIFTODSA-N Ala-Glu-Gln Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O BGNLUHXLSAQYRQ-FXQIFTODSA-N 0.000 description 3
- WKOBSJOZRJJVRZ-FXQIFTODSA-N Ala-Glu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WKOBSJOZRJJVRZ-FXQIFTODSA-N 0.000 description 3
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 3
- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 3
- NJWJSLCQEDMGNC-MBLNEYKQSA-N Ala-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](C)N)O NJWJSLCQEDMGNC-MBLNEYKQSA-N 0.000 description 3
- DVJSJDDYCYSMFR-ZKWXMUAHSA-N Ala-Ile-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O DVJSJDDYCYSMFR-ZKWXMUAHSA-N 0.000 description 3
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- QCTFKEJEIMPOLW-JURCDPSOSA-N Ala-Ile-Phe Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QCTFKEJEIMPOLW-JURCDPSOSA-N 0.000 description 3
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- SUHLZMHFRALVSY-YUMQZZPRSA-N Ala-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)NCC(O)=O SUHLZMHFRALVSY-YUMQZZPRSA-N 0.000 description 3
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- 238000012269 metabolic engineering Methods 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
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- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- DWRXFEITVBNRMK-JXOAFFINSA-N ribothymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 DWRXFEITVBNRMK-JXOAFFINSA-N 0.000 description 1
- 108010048734 sclerotin Proteins 0.000 description 1
- 235000013555 soy sauce Nutrition 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- XCSVCFZQFRBYFA-ZVQJTLEUSA-J tetrasodium;[[[(2r,3s,4r,5r)-5-(2-amino-6-oxo-3h-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-oxidophosphoryl]oxy-oxidophosphoryl] phosphate Chemical compound [Na+].[Na+].[Na+].[Na+].C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O XCSVCFZQFRBYFA-ZVQJTLEUSA-J 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- 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
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/0104—Pyruvate kinase (2.7.1.40)
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Abstract
The invention discloses a pyruvate kinase mutant and application thereof. After mutation of amino acid at position 101 of pyruvate kinase in bacillus microorganism from threonine (T) to lysine (K), arginine (R) or proline (P), adenosine yield is improved, and threonine mutation is used as lysineThe effect is best after the amino acid. Pyruvic acid kinase mutant strain pyk T101K Compared with the original strain A1, the adenosine yield is improved from 1.3g/L to 2.1 g/L; pyruvic acid kinase mutant strain pyk T101R Compared with the original strain A1, the inosine yield is improved from 0.6g/L to 1.1g/L, and the best is obtained. The invention provides an effective means for mass production of nucleosides and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of microbial engineering, and particularly relates to a pyruvate kinase mutant and application thereof.
Background
Nucleosides are a generic term for a class of glycosides. Nucleosides are constituents of nucleic acids and nucleotides. The nucleoside is formed by condensing D-ribose or D-Z-deoxyribose with pyrimidine base or purine base. The nucleoside is generally colorless crystals, insoluble in common organic solvents, soluble in hot water and having a melting point of 160-240 ℃. Nucleosides produced from D-ribose are called ribonucleosides, involved in RNA composition; nucleosides produced from D- α -deoxyribose are called deoxyribonucleosides and participate in DNA composition. D-ribose condenses with adenine, guanine, cytosine, thymine, or uracil to form the corresponding adenine ribonucleoside, guanine ribonucleoside, cytosine ribonucleoside, thymine ribonucleoside, and uracil ribonucleoside, which are abbreviated as adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U), respectively.
Guanosine and inosine have a wide range of roles in the food and pharmaceutical industries. In the food field, guanosine and inosine are important precursors of disodium guanylate and disodium inosinate, respectively, and the combination of disodium guanylate and disodium inosinate is used as a food flavor enhancer, and is widely applied to condiments such as chicken essence, soy sauce and the like. In the field of medicine, guanosine and inosine can be used as medicine intermediates of various antiviral medicines, such as acyclovir, ribavirin, guanosine triphosphate sodium and the like, and guanosine is required to be used as a synthetic raw material. Inosine is an important precursor of inosinic acid, and inosinic acid can be used as a precursor for synthesizing Adenylate (AMP) and Guanylate (GMP), and is suitable for treating leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis, liver cirrhosis and the like caused by various reasons, and can also treat central retinitis, optic atrophy and the like.
At present, microbial fermentation is a main method for producing nucleosides, and mainly used microorganisms include bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and the like. In the breeding and transformation process of the growing strain, the nucleoside high-yield strain is directionally bred by using ultraviolet mutagenesis and diethyl sulfate for mutation breeding; or based on the metabolic path and regulation mechanism of nucleotide in bacteria, the genetic background and the characteristics of the strain are deeply known, and the strain is purposefully modified by metabolic engineering means to obtain the production strain with excellent properties and high nucleoside yield. However, the fermentation performance of the current nucleoside strain is still poor, the conversion rate of the nucleoside is still low, and the requirement of large-scale industrial production cannot be met.
Disclosure of Invention
The invention aims to provide a pyruvate kinase mutant and application thereof.
Another object of the invention is to provide a nucleoside producing strain, and a method for constructing and using the same.
To achieve the object of the present invention, in a first aspect, the present invention provides a pyruvate kinase mutant comprising a mutation of amino acid 101 of pyruvate kinase from T to K, R or P.
In the present invention, the reference sequence number of pyruvate kinase on NCBI is NP-390796.1.
In a second aspect, the invention provides a nucleic acid molecule encoding the pyruvate kinase mutant.
In a third aspect, the invention provides biological materials comprising the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors or engineering bacteria.
In a fourth aspect, the invention provides any one of the following uses of the nucleic acid molecule or a biological material comprising the nucleic acid molecule:
(1) Used for fermentation production of nucleoside;
(2) For improving fermentation yield of nucleosides;
(3) Is used for constructing the genetic engineering bacteria for producing nucleosides.
In a fifth aspect, the present invention provides a method for constructing a nucleoside producing strain, wherein a mutation is introduced into the genome of a microorganism having nucleoside producing ability by genetic engineering means, such that the encoded pyruvate kinase comprises a T101K, T R or T101P mutation site.
Wherein the microorganism is a Bacillus species.
Preferably, the microorganism is Bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus) or the like, more preferably Bacillus subtilis A1, strain A1 can be seen in CN201910599510.8.
In a sixth aspect, the present invention provides a nucleoside producing strain constructed according to the method.
In a seventh aspect, the invention provides the use of said strain in the fermentative production of nucleosides or for increasing the yield of nucleoside fermentation.
In an eighth aspect, the present invention provides a method of producing nucleosides, the method comprising the steps of:
a) Culturing the nucleoside producing strain to obtain a culture of the strain;
b) Collecting the produced nucleosides from the culture obtained in step a).
The nucleoside includes adenosine, inosine, guanosine, etc. or corresponding nucleoside derivatives such as inosine, inosinic acid, guanine, guanylic acid, riboflavin, diacetylguanylic acid, etc.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
after the 101 st amino acid of pyruvate kinase in Bacillus microorganism is mutated from threonine (T) to lysine (K), arginine (R) or proline (P), the adenosine yield is improved, and the best effect is obtained especially after threonine (T) is mutated to lysine (K). Pyruvic acid kinase mutant strain pyk T101K Compared with the original strain A1, the adenosine yield is improved from 1.3g/L to 2.1 g/L; pyruvic acid kinase mutant strain pyk T101R Compared with the original strain A1, the inosine yield is improved from 0.6g/L to 1.1g/L, and the best is obtained. The invention provides an effective means for mass production of nucleosides and has wide application prospect.
Detailed Description
The present invention aims to provide a method for producing purine nucleosides by using a microorganism, and a novel microorganism capable of producing purine nucleosides with high efficiency.
It was found that pyruvate kinase (encoded by pyk gene) of bacillus subtilis or bacillus amyloliquefaciens was modified so that the microorganism can efficiently produce adenosine and inosine, and a novel microorganism capable of efficiently producing nucleosides was successfully created, thereby completing the present invention.
The invention adopts the following technical scheme:
the invention provides bacillus subtilis, wherein the 101 st amino acid of pyruvate kinase coded by pyk gene in cells is mutated from threonine (T) to other amino acids except threonine, preferably lysine (K), arginine (R) or proline (P), and pyruvate kinase mutants T101K, T101R, T101P are respectively obtained. The amino acid sequences of the pyruvate kinase mutant T101K, T101R, T P are shown as SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6, and the nucleic acid sequences of the pyruvate kinase mutant T101K, T101R, T P are shown as SEQ ID NO. 1, SEQ ID NO. 3and SEQ ID NO. 5.
pyk, which is a gene encoding pyruvate kinase, catalyzes the production of Pyruvate (PYR) from phosphoenolpyruvate (PEP), which is accompanied by the consumption of 1 molecule of ADP, while producing 1 molecule of ATP. According to the invention, the pyk gene is modified, so that mutation of pyruvate kinase is realized, the capability of producing nucleoside by the microorganism is enhanced compared with that of an unmodified strain, and the yields of adenosine and inosine are finally improved.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
The primers used in the following examples are shown in Table 1:
TABLE 1
Primer name | Primer sequence (5 '-3') |
pyk T101K -UP-1F | GGTTCTCCGAGTGCTGCTGAC |
pyk T101K -UP-1R | CATATGTCACTGAAATTTTATCTGTTGTTCCTACAACCTCGTCCA |
pyk T101K -DN-2F | TGGACGAGGTTGTAGGAACAACAGATAAAATTTCAGTGACATATG |
pyk T101K -DN-2R | ACAGGTTTGCCCAGCGCGTTG |
pyk T101R -UP-1R | CATATGTCACTGAAATTTTATCTCTTGTTCCTACAACCTCGTCCA |
pyk T101R -DN-2F | TGGACGAGGTTGTAGGAACAAGAGATAAAATTTCAGTGACATATG |
pyk T101P -UP-1R | CATATGTCACTGAAATTTTATCTGGTGTTCCTACAACCTCGTCCA |
pyk T101P -DN-2F | TGGACGAGGTTGTAGGAACACCAGATAAAATTTCAGTGACATATG |
EXAMPLE 1 construction of pyruvate kinase mutant strain pyk T101K
The genome of the strain B.subtilis A1 (hereinafter referred to as A1) was used as a template, and primer pyk was used T101K -UP-1F/pyk T101K -UP-1R and pyk T101K -DN-2F/pyk T101K DN-2R, 2 fragments were amplified using Phusion super Fidelity polymerase (New England BioLabs). By primer pyk T101K -UP-1F/pyk T101K The 2 fragments were fused by DN-2R to obtain recombinant fragments. The recombinant fragment was combined with the pKSU plasmid (pKSU plasmid is given by the teachings of university of south opening Wang Shufang, see A markerless gene replacement method for B. Amyloliquefaciens LL3and its use in genome reduction and improvement of poly-gamma-glutamic acid production [ J)]The recombinant plasmid pKSU-pyk is obtained after operations such as assembly and transformation of 8963-8973.Zhang W,Gao W,Feng J,et al DOI:10.1007/s00253-014-5824-2, etc. of Applied Microbiology and Biotechnology,2014,98 (21) T101K . Transforming into B.subtilis A1 strain, screening transformant with LB plate containing 2.5 mug/mL chloramphenicol at 30 ℃, inoculating obtained transformant into 5mL LB liquid medium, culturing at 42 ℃ at 200rpm for 12h and transferring to generation, diluting and coating to LB plate containing 5 mug/mL chloramphenicol to obtain primary recombinant; inoculating the primary recombinant into 5ml LB liquid medium, culturing at 42 deg.C and 200rpm for 12 hr, transferring to primary, diluting and coating LB plate containing 0.8 μm 5-FU for screening secondary recombinant, and screening to obtain pyk T101K Point mutant strain designated B.subtilis A1-pyk T101K Hereinafter abbreviated as pyk T101K 。
EXAMPLE 2 construction of pyruvate kinase mutant Strain pyk T101R
The strain B.subtilis A1 genome was used as a template, and primer pyk was used T101K -UP-1F/pyk T101R -UP-1R and pyk T101R -DN-2F/pyk T101K DN-2R, 2 fragments were amplified using Phusion super Fidelity polymerase (New England BioLabs). By primer pyk T101K -UP-1F/pyk T101K The 2 fragments were fused by DN-2R to obtain recombinant fragments. The recombinant plasmid pKSU-pyk is obtained after the recombinant fragment and the pKSU plasmid are assembled, transformed and the like T101R . Transformation into B.subtilis A1 Strain transformants were selected on LB plates containing 2.5. Mu.g/mL chloramphenicol at 30℃and the transformants obtainedInoculating the seed into 5mL LB liquid medium, culturing at 42 ℃ for 12 hours at 200rpm and transferring to the first generation, diluting and coating the seed onto LB plate containing 5 mug/mL chloramphenicol to obtain a first recombinant; inoculating the primary recombinant into 5ml LB liquid medium, culturing at 42 deg.C and 200rpm for 12 hr, transferring to primary, diluting and coating LB plate containing 0.8 μm 5-FU for screening secondary recombinant, and screening to obtain pyk T101R Point mutant strain designated B.subtilis A1-pyk T101R Hereinafter abbreviated as pyk T101R 。
EXAMPLE 3 construction of pyruvate kinase mutant Strain pyk T101P
The genome of strain B.subtilis A1 was used as a template, using primer pyk T101K -UP-1F/pyk T101P -UP-1R and pyk T101P -DN-2F/pyk T101K DN-2R, 2 fragments were amplified using Phusion super Fidelity polymerase (New England BioLabs). By primer pyk T101K -UP-1F/pyk T101K The 2 fragments were fused by DN-2R to obtain recombinant fragments. The recombinant plasmid pKSU-pyk is obtained after the recombinant fragment and the pKSU plasmid are assembled, transformed and the like T101P . Transforming into B.subtilis A1 strain, screening transformant with LB plate containing 2.5 mug/mL chloramphenicol at 30 ℃, inoculating obtained transformant into 5mL LB liquid medium, culturing at 42 ℃ at 200rpm for 12h and transferring to generation, diluting and coating to LB plate containing 5 mug/mL chloramphenicol to obtain primary recombinant; inoculating the primary recombinant into 5ml LB liquid medium, culturing at 42 deg.C and 200rpm for 12 hr, transferring to primary, diluting and coating LB plate containing 0.8 μm 5-FU for screening secondary recombinant, and screening to obtain pyk T101P Point mutant strain designated B.subtilis A1-pyk T101P Hereinafter abbreviated as pyk T101P 。
EXAMPLE 4 comparison of the production Capacity of engineering sclerotin
1. Culture medium:
(1) Seed culture formula (g/L): glucose 20, yeast powder 5, corn steep liquor dry powder 5, monopotassium phosphate 3, magnesium sulfate 0.5, ferrous sulfate 0.02, manganese sulfate 0.01 and pH 7.0-7.2. Sterilization conditions: sterilizing at 121deg.C for 20min.
(2) Fermentation medium formulation (g/L): glucose 60, yeast powder 3.5, monopotassium phosphate 3, ammonium sulfate 25, manganese sulfate 0.01, magnesium sulfate 5, monosodium glutamate 10, corn steep liquor dry powder 15, calcium carbonate 25 and pH 7.0-7.2. Sterilization conditions: sterilizing at 121deg.C for 20min.
2. Culture method
(1) Culturing the strain three-area line LB plate at 37 ℃ overnight;
(2) Inoculating the single colony into 30mL seed culture medium, and culturing at 36 ℃ for 7-8 h at 110 rpm;
(3) The culture medium was inoculated into 30mL of a fermentation medium at 10% of the inoculum size, and the culture was carried out at 36℃for 36 hours at 120rpm on a shaker.
3. Detection and results
The detection of nucleosides in the fermentation broth was performed using High Performance Liquid Chromatography (HPLC), the results are shown in table 2.
TABLE 2 engineering bacteria shake flask fermentation production ability evaluation results (three repeated mean values)
Strain | Adenosine yield (g/L) | Inosine yield (g/L) |
A1 | 1.3 | 0.6 |
pyk T101K | 2.1* | 1.0* |
pyk T101R | 1.9* | 1.1* |
pyk T101P | 1.6* | 0.8* |
Note that: * Indicating a significant difference (P < 0.01) compared to the starting strain.
From the above experimental results, it was found that pyruvate kinase mutant pyk T101K 、pyk T101R And pyk T101P Has positive effect on the improvement of the yields of the adenosine and the inosine, and the yield of the adenosine is improved from 1.3g/L to 2.1g/L and the yield of the inosine is improved from 0.6g/L to 1.1g/L.
The yield of adenosine is improved after the 101 st amino acid of pyruvate kinase is mutated from threonine (T) to lysine (K), arginine (R) or proline (P), and the effect is best especially when threonine (T) is mutated to lysine (K). Pyruvic acid kinase mutant strain pyk T101K Compared with the original strain A1, the adenosine yield is improved from 1.3g/L to 2.1 g/L; pyruvic acid kinase mutant strain pyk T101R Compared with the original strain A1, the inosine yield is improved from 0.6g/L to 1.1g/L, and the best is obtained.
The mutant and the mutant strain of the pyruvate kinase provided by the invention have remarkable promotion effect on the increase of the yield of target products of adenosine and inosine. The pyruvic acid kinase mutant and the recombinant microorganism thereof provide reference for the construction of production strains for producing adenosine and inosine and derivatives taking the mutant and the inosine as precursors.
The order of the steps of the construction of the strain of the present invention is not limited, and it is within the scope of the present invention for a person skilled in the art to achieve the object of the present invention according to the present disclosure.
The strain code of the invention is pyk T101K 、pyk T101R And pyk T101P Etc. are for convenience of description and should not be construed as limiting the invention. The bacillus subtilis pyruvate kinase mutation pyk constructed by the method T101K 、pyk T101R And pyk T101P Including, but not limited to, adenosine, inosine.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
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gcgatcttcg acggaacaga tgcgatcatg ctttctggtg aaactgctgc cggaagttac 960
ccggttgaag cagttcaaac aatgcataac atcgcgtccc gttctgaaga agcattaaat 1020
tataaagaaa ttctctcaaa acgcagagac caagtgggca tgacaattac agacgcaatt 1080
ggacaatctg tcgcacatac ggcgattaac ctgaatgctg ctgcgatcgt aacgccgaca 1140
gaaagcggcc atacagcacg tatgattgca aaataccgtc cgcaggctcc gattgttgcg 1200
gttactgtaa atgactctat ttccagaaag cttgccctcg tatctggcgt attcgcggaa 1260
agcggccaaa atgcgagctc aacagatgag atgcttgagg atgctgtcca aaaatcattg 1320
aacagcggaa ttgtaaaaca cggcgatctt atcgttatta cagctggcac tgtcggtgag 1380
tccggcacta cgaacttaat gaaggttcat actgtcggcg atatcatcgc taaaggccaa 1440
ggcattggac gcaaatcagc ttacggtccg gttgtcgttg cacaaaatgc aaaagaagct 1500
gagcaaaaaa tgactgacgg tgcggtactt gttaccaaaa gcactgaccg tgatatgatt 1560
gcatcccttg aaaaagcgtc tgctcttatt acagaagaag gcggtttgac tagccatgct 1620
gcggtagtcg gattaagcct tggcatcccg gttatcgtgg gtctggaaaa tgcgacatct 1680
attttgacag atggccagga tattacagtt gacgcttcca gaggcgcagt ctatcaaggc 1740
cgtgcgagcg ttctttaa 1758
<210> 2
<211> 585
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Arg Lys Thr Lys Ile Val Cys Thr Ile Gly Pro Ala Ser Glu Ser
1 5 10 15
Ile Glu Met Leu Thr Lys Leu Met Glu Ser Gly Met Asn Val Ala Arg
20 25 30
Leu Asn Phe Ser His Gly Asp Phe Glu Glu His Gly Ala Arg Ile Lys
35 40 45
Asn Ile Arg Glu Ala Ser Lys Lys Leu Gly Lys Asn Val Gly Ile Leu
50 55 60
Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr His Thr Met Glu Asn Gly
65 70 75 80
Gly Ile Glu Leu Glu Thr Gly Lys Glu Leu Ile Ile Ser Met Asp Glu
85 90 95
Val Val Gly Thr Lys Asp Lys Ile Ser Val Thr Tyr Glu Gly Leu Val
100 105 110
His Asp Val Glu Gln Gly Ser Thr Ile Leu Leu Asp Asp Gly Leu Ile
115 120 125
Gly Leu Glu Val Leu Asp Val Asp Ala Ala Lys Arg Glu Ile Lys Thr
130 135 140
Lys Val Leu Asn Asn Gly Thr Leu Lys Asn Lys Lys Gly Val Asn Val
145 150 155 160
Pro Gly Val Ser Val Asn Leu Pro Gly Ile Thr Glu Lys Asp Ala Arg
165 170 175
Asp Ile Val Phe Gly Ile Glu Gln Gly Val Asp Phe Ile Ala Pro Ser
180 185 190
Phe Ile Arg Arg Ser Thr Asp Val Leu Glu Ile Arg Glu Leu Leu Glu
195 200 205
Glu His Asn Ala Gln Asp Ile Gln Ile Ile Pro Lys Ile Glu Asn Gln
210 215 220
Glu Gly Val Asp Asn Ile Asp Ala Ile Leu Glu Val Ser Asp Gly Leu
225 230 235 240
Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Ala Glu Glu Val
245 250 255
Pro Leu Val Gln Lys Glu Leu Ile Lys Lys Cys Asn Ala Leu Gly Lys
260 265 270
Pro Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Gln Arg Asn Pro
275 280 285
Arg Pro Thr Arg Ala Glu Ala Ser Asp Val Ala Asn Ala Ile Phe Asp
290 295 300
Gly Thr Asp Ala Ile Met Leu Ser Gly Glu Thr Ala Ala Gly Ser Tyr
305 310 315 320
Pro Val Glu Ala Val Gln Thr Met His Asn Ile Ala Ser Arg Ser Glu
325 330 335
Glu Ala Leu Asn Tyr Lys Glu Ile Leu Ser Lys Arg Arg Asp Gln Val
340 345 350
Gly Met Thr Ile Thr Asp Ala Ile Gly Gln Ser Val Ala His Thr Ala
355 360 365
Ile Asn Leu Asn Ala Ala Ala Ile Val Thr Pro Thr Glu Ser Gly His
370 375 380
Thr Ala Arg Met Ile Ala Lys Tyr Arg Pro Gln Ala Pro Ile Val Ala
385 390 395 400
Val Thr Val Asn Asp Ser Ile Ser Arg Lys Leu Ala Leu Val Ser Gly
405 410 415
Val Phe Ala Glu Ser Gly Gln Asn Ala Ser Ser Thr Asp Glu Met Leu
420 425 430
Glu Asp Ala Val Gln Lys Ser Leu Asn Ser Gly Ile Val Lys His Gly
435 440 445
Asp Leu Ile Val Ile Thr Ala Gly Thr Val Gly Glu Ser Gly Thr Thr
450 455 460
Asn Leu Met Lys Val His Thr Val Gly Asp Ile Ile Ala Lys Gly Gln
465 470 475 480
Gly Ile Gly Arg Lys Ser Ala Tyr Gly Pro Val Val Val Ala Gln Asn
485 490 495
Ala Lys Glu Ala Glu Gln Lys Met Thr Asp Gly Ala Val Leu Val Thr
500 505 510
Lys Ser Thr Asp Arg Asp Met Ile Ala Ser Leu Glu Lys Ala Ser Ala
515 520 525
Leu Ile Thr Glu Glu Gly Gly Leu Thr Ser His Ala Ala Val Val Gly
530 535 540
Leu Ser Leu Gly Ile Pro Val Ile Val Gly Leu Glu Asn Ala Thr Ser
545 550 555 560
Ile Leu Thr Asp Gly Gln Asp Ile Thr Val Asp Ala Ser Arg Gly Ala
565 570 575
Val Tyr Gln Gly Arg Ala Ser Val Leu
580 585
<210> 3
<211> 1758
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atgagaaaaa ctaaaattgt ttgtaccatc ggtccggcaa gtgaaagtat tgaaatgctt 60
acgaaattaa tggagtcagg aatgaacgtg gctcgattaa acttttctca cggagatttt 120
gaggagcacg gtgcaagaat taaaaatatc cgcgaagcaa gtaaaaaact tggcaagaac 180
gttggaattc tgcttgatac aaaaggtcct gaaatccgca cacatacaat ggaaaacggc 240
ggtattgagc ttgaaacagg caaagagctc attatttcaa tggacgaggt tgtaggaaca 300
agagataaaa tttcagtgac atatgaaggt ttagtccatg acgttgaaca aggttcaacg 360
attctgttag atgacggcct tatcggtctt gaggtacttg atgtagatgc cgctaaacgc 420
gaaatcaaaa caaaagtatt aaacaacgga acactcaaaa ataaaaaagg tgttaacgta 480
ccgggcgtaa gtgtcaatct tccggggatt actgaaaagg atgcgcgaga catcgttttc 540
ggtattgagc aaggagtaga cttcatcgca ccatctttca ttcgacgttc tacggatgtg 600
ctcgaaatcc gtgagcttct tgaagagcac aacgctcagg atattcaaat catccctaaa 660
atcgaaaacc aagagggcgt tgacaacatc gatgcgattc tcgaagtgtc tgacggctta 720
atggttgcac gcggagactt aggtgtggaa attccagctg aagaagtgcc gcttgtgcaa 780
aaagaactga tcaaaaaatg caacgcgctg ggcaaacctg ttattacagc gacacaaatg 840
cttgacagca tgcagcgcaa cccgcgtccg actcgtgcgg aagcaagtga cgttgcaaac 900
gcgatcttcg acggaacaga tgcgatcatg ctttctggtg aaactgctgc cggaagttac 960
ccggttgaag cagttcaaac aatgcataac atcgcgtccc gttctgaaga agcattaaat 1020
tataaagaaa ttctctcaaa acgcagagac caagtgggca tgacaattac agacgcaatt 1080
ggacaatctg tcgcacatac ggcgattaac ctgaatgctg ctgcgatcgt aacgccgaca 1140
gaaagcggcc atacagcacg tatgattgca aaataccgtc cgcaggctcc gattgttgcg 1200
gttactgtaa atgactctat ttccagaaag cttgccctcg tatctggcgt attcgcggaa 1260
agcggccaaa atgcgagctc aacagatgag atgcttgagg atgctgtcca aaaatcattg 1320
aacagcggaa ttgtaaaaca cggcgatctt atcgttatta cagctggcac tgtcggtgag 1380
tccggcacta cgaacttaat gaaggttcat actgtcggcg atatcatcgc taaaggccaa 1440
ggcattggac gcaaatcagc ttacggtccg gttgtcgttg cacaaaatgc aaaagaagct 1500
gagcaaaaaa tgactgacgg tgcggtactt gttaccaaaa gcactgaccg tgatatgatt 1560
gcatcccttg aaaaagcgtc tgctcttatt acagaagaag gcggtttgac tagccatgct 1620
gcggtagtcg gattaagcct tggcatcccg gttatcgtgg gtctggaaaa tgcgacatct 1680
attttgacag atggccagga tattacagtt gacgcttcca gaggcgcagt ctatcaaggc 1740
cgtgcgagcg ttctttaa 1758
<210> 4
<211> 585
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Met Arg Lys Thr Lys Ile Val Cys Thr Ile Gly Pro Ala Ser Glu Ser
1 5 10 15
Ile Glu Met Leu Thr Lys Leu Met Glu Ser Gly Met Asn Val Ala Arg
20 25 30
Leu Asn Phe Ser His Gly Asp Phe Glu Glu His Gly Ala Arg Ile Lys
35 40 45
Asn Ile Arg Glu Ala Ser Lys Lys Leu Gly Lys Asn Val Gly Ile Leu
50 55 60
Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr His Thr Met Glu Asn Gly
65 70 75 80
Gly Ile Glu Leu Glu Thr Gly Lys Glu Leu Ile Ile Ser Met Asp Glu
85 90 95
Val Val Gly Thr Arg Asp Lys Ile Ser Val Thr Tyr Glu Gly Leu Val
100 105 110
His Asp Val Glu Gln Gly Ser Thr Ile Leu Leu Asp Asp Gly Leu Ile
115 120 125
Gly Leu Glu Val Leu Asp Val Asp Ala Ala Lys Arg Glu Ile Lys Thr
130 135 140
Lys Val Leu Asn Asn Gly Thr Leu Lys Asn Lys Lys Gly Val Asn Val
145 150 155 160
Pro Gly Val Ser Val Asn Leu Pro Gly Ile Thr Glu Lys Asp Ala Arg
165 170 175
Asp Ile Val Phe Gly Ile Glu Gln Gly Val Asp Phe Ile Ala Pro Ser
180 185 190
Phe Ile Arg Arg Ser Thr Asp Val Leu Glu Ile Arg Glu Leu Leu Glu
195 200 205
Glu His Asn Ala Gln Asp Ile Gln Ile Ile Pro Lys Ile Glu Asn Gln
210 215 220
Glu Gly Val Asp Asn Ile Asp Ala Ile Leu Glu Val Ser Asp Gly Leu
225 230 235 240
Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Ala Glu Glu Val
245 250 255
Pro Leu Val Gln Lys Glu Leu Ile Lys Lys Cys Asn Ala Leu Gly Lys
260 265 270
Pro Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Gln Arg Asn Pro
275 280 285
Arg Pro Thr Arg Ala Glu Ala Ser Asp Val Ala Asn Ala Ile Phe Asp
290 295 300
Gly Thr Asp Ala Ile Met Leu Ser Gly Glu Thr Ala Ala Gly Ser Tyr
305 310 315 320
Pro Val Glu Ala Val Gln Thr Met His Asn Ile Ala Ser Arg Ser Glu
325 330 335
Glu Ala Leu Asn Tyr Lys Glu Ile Leu Ser Lys Arg Arg Asp Gln Val
340 345 350
Gly Met Thr Ile Thr Asp Ala Ile Gly Gln Ser Val Ala His Thr Ala
355 360 365
Ile Asn Leu Asn Ala Ala Ala Ile Val Thr Pro Thr Glu Ser Gly His
370 375 380
Thr Ala Arg Met Ile Ala Lys Tyr Arg Pro Gln Ala Pro Ile Val Ala
385 390 395 400
Val Thr Val Asn Asp Ser Ile Ser Arg Lys Leu Ala Leu Val Ser Gly
405 410 415
Val Phe Ala Glu Ser Gly Gln Asn Ala Ser Ser Thr Asp Glu Met Leu
420 425 430
Glu Asp Ala Val Gln Lys Ser Leu Asn Ser Gly Ile Val Lys His Gly
435 440 445
Asp Leu Ile Val Ile Thr Ala Gly Thr Val Gly Glu Ser Gly Thr Thr
450 455 460
Asn Leu Met Lys Val His Thr Val Gly Asp Ile Ile Ala Lys Gly Gln
465 470 475 480
Gly Ile Gly Arg Lys Ser Ala Tyr Gly Pro Val Val Val Ala Gln Asn
485 490 495
Ala Lys Glu Ala Glu Gln Lys Met Thr Asp Gly Ala Val Leu Val Thr
500 505 510
Lys Ser Thr Asp Arg Asp Met Ile Ala Ser Leu Glu Lys Ala Ser Ala
515 520 525
Leu Ile Thr Glu Glu Gly Gly Leu Thr Ser His Ala Ala Val Val Gly
530 535 540
Leu Ser Leu Gly Ile Pro Val Ile Val Gly Leu Glu Asn Ala Thr Ser
545 550 555 560
Ile Leu Thr Asp Gly Gln Asp Ile Thr Val Asp Ala Ser Arg Gly Ala
565 570 575
Val Tyr Gln Gly Arg Ala Ser Val Leu
580 585
<210> 5
<211> 1758
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
atgagaaaaa ctaaaattgt ttgtaccatc ggtccggcaa gtgaaagtat tgaaatgctt 60
acgaaattaa tggagtcagg aatgaacgtg gctcgattaa acttttctca cggagatttt 120
gaggagcacg gtgcaagaat taaaaatatc cgcgaagcaa gtaaaaaact tggcaagaac 180
gttggaattc tgcttgatac aaaaggtcct gaaatccgca cacatacaat ggaaaacggc 240
ggtattgagc ttgaaacagg caaagagctc attatttcaa tggacgaggt tgtaggaaca 300
ccagataaaa tttcagtgac atatgaaggt ttagtccatg acgttgaaca aggttcaacg 360
attctgttag atgacggcct tatcggtctt gaggtacttg atgtagatgc cgctaaacgc 420
gaaatcaaaa caaaagtatt aaacaacgga acactcaaaa ataaaaaagg tgttaacgta 480
ccgggcgtaa gtgtcaatct tccggggatt actgaaaagg atgcgcgaga catcgttttc 540
ggtattgagc aaggagtaga cttcatcgca ccatctttca ttcgacgttc tacggatgtg 600
ctcgaaatcc gtgagcttct tgaagagcac aacgctcagg atattcaaat catccctaaa 660
atcgaaaacc aagagggcgt tgacaacatc gatgcgattc tcgaagtgtc tgacggctta 720
atggttgcac gcggagactt aggtgtggaa attccagctg aagaagtgcc gcttgtgcaa 780
aaagaactga tcaaaaaatg caacgcgctg ggcaaacctg ttattacagc gacacaaatg 840
cttgacagca tgcagcgcaa cccgcgtccg actcgtgcgg aagcaagtga cgttgcaaac 900
gcgatcttcg acggaacaga tgcgatcatg ctttctggtg aaactgctgc cggaagttac 960
ccggttgaag cagttcaaac aatgcataac atcgcgtccc gttctgaaga agcattaaat 1020
tataaagaaa ttctctcaaa acgcagagac caagtgggca tgacaattac agacgcaatt 1080
ggacaatctg tcgcacatac ggcgattaac ctgaatgctg ctgcgatcgt aacgccgaca 1140
gaaagcggcc atacagcacg tatgattgca aaataccgtc cgcaggctcc gattgttgcg 1200
gttactgtaa atgactctat ttccagaaag cttgccctcg tatctggcgt attcgcggaa 1260
agcggccaaa atgcgagctc aacagatgag atgcttgagg atgctgtcca aaaatcattg 1320
aacagcggaa ttgtaaaaca cggcgatctt atcgttatta cagctggcac tgtcggtgag 1380
tccggcacta cgaacttaat gaaggttcat actgtcggcg atatcatcgc taaaggccaa 1440
ggcattggac gcaaatcagc ttacggtccg gttgtcgttg cacaaaatgc aaaagaagct 1500
gagcaaaaaa tgactgacgg tgcggtactt gttaccaaaa gcactgaccg tgatatgatt 1560
gcatcccttg aaaaagcgtc tgctcttatt acagaagaag gcggtttgac tagccatgct 1620
gcggtagtcg gattaagcct tggcatcccg gttatcgtgg gtctggaaaa tgcgacatct 1680
attttgacag atggccagga tattacagtt gacgcttcca gaggcgcagt ctatcaaggc 1740
cgtgcgagcg ttctttaa 1758
<210> 6
<211> 585
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Met Arg Lys Thr Lys Ile Val Cys Thr Ile Gly Pro Ala Ser Glu Ser
1 5 10 15
Ile Glu Met Leu Thr Lys Leu Met Glu Ser Gly Met Asn Val Ala Arg
20 25 30
Leu Asn Phe Ser His Gly Asp Phe Glu Glu His Gly Ala Arg Ile Lys
35 40 45
Asn Ile Arg Glu Ala Ser Lys Lys Leu Gly Lys Asn Val Gly Ile Leu
50 55 60
Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr His Thr Met Glu Asn Gly
65 70 75 80
Gly Ile Glu Leu Glu Thr Gly Lys Glu Leu Ile Ile Ser Met Asp Glu
85 90 95
Val Val Gly Thr Pro Asp Lys Ile Ser Val Thr Tyr Glu Gly Leu Val
100 105 110
His Asp Val Glu Gln Gly Ser Thr Ile Leu Leu Asp Asp Gly Leu Ile
115 120 125
Gly Leu Glu Val Leu Asp Val Asp Ala Ala Lys Arg Glu Ile Lys Thr
130 135 140
Lys Val Leu Asn Asn Gly Thr Leu Lys Asn Lys Lys Gly Val Asn Val
145 150 155 160
Pro Gly Val Ser Val Asn Leu Pro Gly Ile Thr Glu Lys Asp Ala Arg
165 170 175
Asp Ile Val Phe Gly Ile Glu Gln Gly Val Asp Phe Ile Ala Pro Ser
180 185 190
Phe Ile Arg Arg Ser Thr Asp Val Leu Glu Ile Arg Glu Leu Leu Glu
195 200 205
Glu His Asn Ala Gln Asp Ile Gln Ile Ile Pro Lys Ile Glu Asn Gln
210 215 220
Glu Gly Val Asp Asn Ile Asp Ala Ile Leu Glu Val Ser Asp Gly Leu
225 230 235 240
Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Ala Glu Glu Val
245 250 255
Pro Leu Val Gln Lys Glu Leu Ile Lys Lys Cys Asn Ala Leu Gly Lys
260 265 270
Pro Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Gln Arg Asn Pro
275 280 285
Arg Pro Thr Arg Ala Glu Ala Ser Asp Val Ala Asn Ala Ile Phe Asp
290 295 300
Gly Thr Asp Ala Ile Met Leu Ser Gly Glu Thr Ala Ala Gly Ser Tyr
305 310 315 320
Pro Val Glu Ala Val Gln Thr Met His Asn Ile Ala Ser Arg Ser Glu
325 330 335
Glu Ala Leu Asn Tyr Lys Glu Ile Leu Ser Lys Arg Arg Asp Gln Val
340 345 350
Gly Met Thr Ile Thr Asp Ala Ile Gly Gln Ser Val Ala His Thr Ala
355 360 365
Ile Asn Leu Asn Ala Ala Ala Ile Val Thr Pro Thr Glu Ser Gly His
370 375 380
Thr Ala Arg Met Ile Ala Lys Tyr Arg Pro Gln Ala Pro Ile Val Ala
385 390 395 400
Val Thr Val Asn Asp Ser Ile Ser Arg Lys Leu Ala Leu Val Ser Gly
405 410 415
Val Phe Ala Glu Ser Gly Gln Asn Ala Ser Ser Thr Asp Glu Met Leu
420 425 430
Glu Asp Ala Val Gln Lys Ser Leu Asn Ser Gly Ile Val Lys His Gly
435 440 445
Asp Leu Ile Val Ile Thr Ala Gly Thr Val Gly Glu Ser Gly Thr Thr
450 455 460
Asn Leu Met Lys Val His Thr Val Gly Asp Ile Ile Ala Lys Gly Gln
465 470 475 480
Gly Ile Gly Arg Lys Ser Ala Tyr Gly Pro Val Val Val Ala Gln Asn
485 490 495
Ala Lys Glu Ala Glu Gln Lys Met Thr Asp Gly Ala Val Leu Val Thr
500 505 510
Lys Ser Thr Asp Arg Asp Met Ile Ala Ser Leu Glu Lys Ala Ser Ala
515 520 525
Leu Ile Thr Glu Glu Gly Gly Leu Thr Ser His Ala Ala Val Val Gly
530 535 540
Leu Ser Leu Gly Ile Pro Val Ile Val Gly Leu Glu Asn Ala Thr Ser
545 550 555 560
Ile Leu Thr Asp Gly Gln Asp Ile Thr Val Asp Ala Ser Arg Gly Ala
565 570 575
Val Tyr Gln Gly Arg Ala Ser Val Leu
580 585
Claims (10)
1. A pyruvate kinase mutant, characterized in that the mutant comprises a mutation of amino acid 101 of pyruvate kinase from T to K, R or P;
wherein, the reference sequence number of the pyruvate kinase on NCBI is NP_390796.1.
2. A nucleic acid molecule encoding the pyruvate kinase mutant of claim 1.
3. A biological material comprising the nucleic acid molecule of claim 2, said biological material being a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineering bacterium.
4. The nucleic acid molecule of claim 2 or any of the following applications of the biological material of claim 3:
(1) Used for fermentation production of nucleoside;
(2) For improving fermentation yield of nucleosides;
(3) Is used for constructing the genetic engineering bacteria for producing nucleosides.
5. The construction method of the nucleoside producing strain is characterized in that a mutation is introduced into a microorganism genome with nucleoside producing capacity by utilizing a genetic engineering means, so that the encoded pyruvate kinase contains a T101K, T R or T101P mutation site; wherein, the reference sequence number of the pyruvate kinase on NCBI is NP_390796.1;
wherein the microorganism is a Bacillus species.
6. The method of claim 5, wherein the microorganism is Bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus).
7. A nucleoside producing strain constructed according to the method of claim 5 or 6.
8. Use of the strain of claim 7 for nucleoside fermentation production or for increasing the yield of nucleoside fermentation.
9. A method of producing nucleosides, said method comprising the steps of:
a) Culturing the strain of claim 7 to obtain a culture of the strain;
b) Collecting the produced nucleosides from the culture obtained in step a).
10. The method of claim 9, wherein the nucleoside comprises adenosine, inosine, guanosine, and their corresponding nucleoside derivatives.
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