CN103642766B - Albumen, DNA molecular, convert host and this conversion host for the method producing L valine containing this DNA - Google Patents
Albumen, DNA molecular, convert host and this conversion host for the method producing L valine containing this DNA Download PDFInfo
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- CN103642766B CN103642766B CN201310275401.3A CN201310275401A CN103642766B CN 103642766 B CN103642766 B CN 103642766B CN 201310275401 A CN201310275401 A CN 201310275401A CN 103642766 B CN103642766 B CN 103642766B
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- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 title claims abstract description 59
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 239000004474 valine Substances 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000006243 chemical reaction Methods 0.000 title abstract description 20
- 241000894006 Bacteria Species 0.000 claims abstract description 28
- 108010028658 Leucine Dehydrogenase Proteins 0.000 claims abstract description 20
- 239000002773 nucleotide Substances 0.000 claims abstract description 18
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 18
- 150000001413 amino acids Chemical group 0.000 claims description 26
- 238000000855 fermentation Methods 0.000 claims description 23
- 230000004151 fermentation Effects 0.000 claims description 23
- 239000001963 growth medium Substances 0.000 claims description 10
- 238000011218 seed culture Methods 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 5
- 241000572303 Corynebacterium pekinense Species 0.000 claims description 4
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 3
- 241000186216 Corynebacterium Species 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 abstract description 14
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical group C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 abstract description 8
- 230000001580 bacterial effect Effects 0.000 abstract description 6
- 230000003834 intracellular effect Effects 0.000 abstract description 6
- 241000193830 Bacillus <bacterium> Species 0.000 abstract description 5
- 101100342797 Geobacillus stearothermophilus ldh gene Proteins 0.000 abstract description 5
- 238000010353 genetic engineering Methods 0.000 abstract description 4
- 229960004295 valine Drugs 0.000 description 49
- 230000006870 function Effects 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- 229940024606 amino acid Drugs 0.000 description 11
- 235000001014 amino acid Nutrition 0.000 description 10
- 239000007788 liquid Substances 0.000 description 9
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 5
- 108090000340 Transaminases Proteins 0.000 description 5
- 102000003929 Transaminases Human genes 0.000 description 5
- 229960003136 leucine Drugs 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 150000005693 branched-chain amino acids Chemical class 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
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- 238000004519 manufacturing process Methods 0.000 description 4
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- 244000005700 microbiome Species 0.000 description 4
- NMDWGEGFJUBKLB-YFKPBYRVSA-N (2S)-2-hydroxy-2-methyl-3-oxobutanoic acid Chemical compound CC(=O)[C@](C)(O)C(O)=O NMDWGEGFJUBKLB-YFKPBYRVSA-N 0.000 description 3
- QHKABHOOEWYVLI-UHFFFAOYSA-N 3-methyl-2-oxobutanoic acid Chemical compound CC(C)C(=O)C(O)=O QHKABHOOEWYVLI-UHFFFAOYSA-N 0.000 description 3
- 241000194107 Bacillus megaterium Species 0.000 description 3
- 101710088194 Dehydrogenase Proteins 0.000 description 3
- 108700016168 Dihydroxy-acid dehydratases Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
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- 108010088278 Branched-chain-amino-acid transaminase Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
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- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
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- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- CTMXBOCTJPQVDZ-UHFFFAOYSA-N 2,2-dihydroxy-3-methylbutanoic acid Chemical compound CC(C)C(O)(O)C(O)=O CTMXBOCTJPQVDZ-UHFFFAOYSA-N 0.000 description 1
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- 125000003580 L-valyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(C([H])([H])[H])(C([H])([H])[H])[H] 0.000 description 1
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- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
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- 125000003118 aryl group Chemical group 0.000 description 1
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- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
-
- 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
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
- C12Y104/01009—Leucine dehydrogenase (1.4.1.9)
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- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to biological technical field, the DNA molecular particularly to albumen, encoding this albumen, conversion host this conversion host containing this DNA molecular are for the method producing L valine.By by the encoding gene leuDH(nucleotide sequence of leucine dehydrogenase that cofactor is NADH as shown in SEQ ID No.2) convert in introduction rod bacillus, obtain the genetic engineering bacterium that intracellular redox equilibrium changes, cultivating this bacterial strain to can be used in producing L valine, pole significantly improves the yield of L valine.
Description
Technical field
The present invention relates to biological technical field, the DNA molecular that particularly to albumen, encodes this albumen, containing this DNA molecular
Convert host this conversion host for the method that produces Valine.
Background technology
Valine (L-valine), chemical name is L-valine, and molecular formula is C5H11NO2, average molecular
Quality is 117.15.White crystallization or crystalline powder, odorless, bitter in the mouth, dissolubility in water: 25 DEG C is 88.5g/L, 50 DEG C
For 96.2g/L, insoluble in cold ethanol, ether, acetone.Isoelectric point, IP is 5.96, fusing point 315 DEG C.Valine (L-Val) is human body
One of eight kinds of essential amino acids, are again one of three kinds of branched-chain amino acid (including valine, leucine, isoleucine), Yin Qite
Different 26S Proteasome Structure and Function, has the status of particular importance in human life's metabolism.Can be widely applied to medical industry, food
Industry and feed industry etc..In medical industry, amino acid transfusion, the main component of comprehensive amino acid preparation can be made, liver can be treated
Nonfunction, central nervous system function are disorderly.Neurological disorder, stasi, weight loss, anemia etc. can be caused as lacked.
In food industry, can be used as food additive, nutritional supplement liquid and flavoring agent etc..Metric system cake adds valine (1g/kg),
Product has Semen Sesami fragrant, also can improve local flavor for bread.Valine also is used as amino acid energy beverage and sports drink,
There is formation muscle, strengthen liver function, alleviate the effect such as muscle fatigue.Mammary gland tissue lactation in feed industry, to animal
There is important facilitation.Use it in chicken feed, the chickling immunocompetence to newcastle disease virus can be improved.And L-
Valine is a kind of limiting amino acid in animal feed, so Valine can improve animal diets as feed additive
The deficiency of middle amino acid content.
The production method of Valine has three kinds: extraction method, chemical synthesis, microbe fermentation method.Extraction method and chemistry
Synthetic method is owing to raw material sources are restricted, production cost is high, pollute environment, it is difficult to realize industrialized production.Microbe fermentation method
Produce Valine and have that cost of material is low, reaction condition is gentle, easily realize the advantages such as large-scale production, be to produce L-at present
The topmost method of valine.By the selection-breeding of bacterial strain, with the feedback suppression released in Metabolism regulation with check, reach excess long-pending
The purpose of tired Valine, is the most commonly used means of microbe fermentation method commercial Application.
Bar bacterium is the representative microorganism for producing l-amino acid, particularly Corynebacterium glutamicum
(Corynebacterium glutamicum), Beijing corynebacterium (Corynebacterium pekinense) and yellow quarter butt
Bacterium (Breviabacterium flavum).But, the yield that existing strain fermentation produces Valine is relatively low, it is impossible to meet
The market demand.
In order to improve the Valine production capacity of microorganism, can be by the method such as classic mutagenesis and metabolic engineering to life
Produce bacterial strain constantly to transform.Classic mutagenesis breeding, refers to by specific starting strain carries out physics, chemistry or the two conjunction
And ground mutagenic treatment, then select auxotroph and/or Amino acid analogue resistant mutant strain, to release Metabolism regulation
In feedback suppression or check effect, thus reach certain amino acid whose purpose of excess accumulation.Metabolic engineering breeding, it is simply that right
Metabolism network system uses genetic engineering techniques cellular metabolism system to improve efficiency of pcr product or improvement on the basis of analyzing
Cell performance.Mainly include following means: to the transformation of original metabolic pathway, the structure of novel metabolic pathways, the generation of group scale
Thank to the identification of critical path or target spot.Build amino acid preparation strain by the metabolic engineering of rationality, be increasingly becoming aminoacid
The main policies of breeding.
Summary of the invention
In view of this, the present invention provides a kind of albumen, the DNA molecular encoding this albumen, conversion place containing this DNA molecular
Main frame this conversion host is for the method producing Valine.By by the coding of leucine dehydrogenase that cofactor is NADH
Gene leuDH converts in introduction rod bacillus, it is thus achieved that the genetic engineering bacterium that intracellular redox equilibrium changes, cultivating this bacterial strain can
For producing Valine, pole significantly improves the yield of Valine.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides leucine dehydrogenase or its merit of a kind of aminoacid sequence having as shown in SEQ ID No.1
Can equivalent.
The metabolic pathway of synthesizing of valine, is from the beginning of dimolecular acetone acid, through acetolactic acid, dihydroxyisovalerate
With three intermediate products such as ketoisovalerate, ultimately form valine.And these mesostates pass through acetohydroxy acid respectively
The catalysis of synzyme, Acetohydroxy acid isomeroreductase, dihydroxyacid dehydratase and transaminase is active and produces, as shown in Figure 1.
Acetohydroxy acid synthetase (AHAS) be first shared enzyme on Valine route of synthesis be also key enzyme, catalysis
2 molecule pyruvate decarboxylation synthesize 1 molecule acetolactic acid, the i.e. precursor of valine.Any by three kinds of branched-chain amino acid of AHAS
A kind of feedback suppression.
Dihydroxy acid reduction isomerase (AHAIR) is second enzyme on Valine route of synthesis, is a tetramer enzyme, by
Size is the subunit of the same race composition of 53kDa, and catalysis acetolactic acid generates 2,3-dihydroxyisovalerate.Reaction includes the isomery of alkyl
And reduction；This reaction is occurred to also need to Mg2+(activator) and NADPH (hydrogen donor).
Dihydroxyacid dehydratase (DHAD) is the 3rd enzyme on Valine route of synthesis, two be made up of two subunits
Aggressiveness enzyme, can be catalyzed 2, and 3-dihydroxyisovalerate generates 2-ketoisovalerate.
Transaminase (TA) B is last enzyme in valine building-up process.Transaminase B, Transaminase C and aromatic aminotransferase
(gene is respectively ilvE, avtA and tyrB) all has catalysis activity in the synthesis of branched-chain amino acid, but main by Transaminase B
It is catalyzed the final step reaction of three kinds of branched-chain amino acid synthesis.
Leucine dehydrogenase (Leucine Dehydrogenase, LDH, EC1.4.1.9) is a kind of NAD+The oxygen of dependent form
Changing reductase, it is reversibly catalyzed L-Leu and generates corresponding keto acid and the like with the reaction of some branched-chain l-amino acids,
As follows:
The leucine dehydrogenase deriving from bacillus megaterium (Bacillus megaterium) ATCC14945 has good
Good heat stability, chemical stability and operational stability.
NADH and NADPH is intracellular important coenzyme, involved in sugar, fat, the overwhelming majority of protein three class substance metabolism
Redox reaction, NAD/NADH and NADP/NADPH is the redox couple of its correspondence respectively.Huge spore bar will be derived from
The leucine dehydrogenase encoding gene leuDH of bacterium imports in bar bacterium or Flavobacterium, and its coded product is with NADH as coenzyme.Should
The expression of enzyme can supplement the function of the original transaminase with NADPH as coenzyme with NADH as coenzyme, thus reduces NADPH
Consumption, reach to regulate the effect of the balance of intracellular NADH and NADPH, the result converting and expressing is to make intracellular oxidoreduction put down
Weighing apparatus is changed the most accordingly.
As preferably, what the present invention provided has the leucine dehydrogenase of the aminoacid sequence as shown in SEQ ID No.1
Function equivalent by replacing, lack or add one or more amino in the aminoacid sequence shown in SEQ ID No.1
Acid residue obtains.
Present invention also offers leucine dehydrogenase or its function with the aminoacid sequence as shown in SEQ ID No.1
Equivalent generates the application of valine for being catalyzed 2-ketoisovalerate.
Present invention also offers coding have the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or its
The DNA molecular of function equivalent.
In some embodiments of the invention, the coding that the present invention provides has the aminoacid as shown in SEQ ID No.1
The DNA molecular nucleotide sequence of the leucine dehydrogenase of sequence or its function equivalent is as shown in SEQ ID No.2.
Present invention also offers coding have the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or its
The DNA molecular of function equivalent, its nucleotide sequence is more than 95% with the homology of the nucleotide sequence shown in SEQ ID No.2
Above.
Present invention also offers coding have the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or its
The carrier of the DNA molecular of function equivalent；Coding has the leucine dehydrogenase of the aminoacid sequence as shown in SEQ ID No.1
Or the DNA molecular of its function equivalent can be for nucleotide sequence DNA molecular as shown in SEQ ID No.2 or nucleotide sequence
The DNA molecular that homology with the nucleotide sequence shown in SEQ ID No.2 is more than more than 95%.
In some embodiments of the invention, coding has the leucine of the aminoacid sequence as shown in SEQ ID No.1
The carrier of the DNA molecular of dehydrogenase or its function equivalent is pMX119.
Present invention also offers coding have the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or its
The conversion host of the DNA molecular of function equivalent；Coding has the leucine of the aminoacid sequence as shown in SEQ ID No.1 and takes off
The DNA molecular of hydrogen enzyme or its function equivalent can be for nucleotide sequence DNA molecular as shown in SEQ ID No.2 or nucleotide
The homology of sequence and the nucleotide sequence shown in the SEQ ID No.2 DNA molecular more than more than 95%.
In some embodiments of the invention, coding has the leucine of the aminoacid sequence as shown in SEQ ID No.1
The DNA molecular of dehydrogenase or its function equivalent can be for nucleotide sequence DNA molecular as shown in SEQ ID No.2 or nucleoside
Acid sequence is more than the DNA molecular of more than 95% by electroporated method with the homology of the nucleotide sequence shown in SEQ ID No.2
Import.
In some embodiments of the invention, coding has the leucine of the aminoacid sequence as shown in SEQ ID No.1
The DNA molecular of dehydrogenase or its function equivalent can be for nucleotide sequence DNA molecular as shown in SEQ ID No.2 or nucleoside
The homology of acid sequence and the nucleotide sequence shown in the SEQ ID No.2 DNA molecular more than more than 95% is expressed and is strengthened.
As preferably, expressing the mode strengthened is the bright ammonia that coding has the aminoacid sequence as shown in SEQ ID No.1
The DNA molecular of acidohydrogenase or its function equivalent can be for nucleotide sequence DNA molecular as shown in SEQ ID No.2 or core
Nucleotide sequence is more than the DNA molecular of more than 95% as strong promoter with the homology of the nucleotide sequence shown in SEQ ID No.2
Control under or increase its copy number.
In some embodiments of the invention, converting host is bar bacterium or brevibacterium.
In other embodiments of the present invention, converting host is bar bacterium, and its deposit number is that its deposit number is
CGMCC No.7041。
Present invention also offers the above-mentioned conversion host purposes for fermenting and producing Valine.
The method that present invention also offers above-mentioned conversion host's fermenting and producing Valine, it is characterised in that take described turning
After change host expands in seed culture medium, it is placed in fermentation medium and cultivates 50～120h in 28 DEG C～37 DEG C, to obtain final product.
The present invention provides a kind of albumen, the DNA molecular encoding this albumen, conversion this turn of host containing this DNA molecular
Change host for the method producing Valine.By by the encoding gene of leucine dehydrogenase that cofactor is NADH
LeuDH converts in introduction rod bacillus, it is thus achieved that the genetic engineering bacterium that intracellular redox equilibrium changes, and cultivates this bacterial strain and can be used in
Produce Valine.In the fermentation liquid of shake flask fermentation, the Valine yield of starting strain CGMCC1.299 is 1.2g/L, and this
The Valine yield of the bar bacterium that invention provides is 3.8g/L, than starting strain output increased 216%.Fermentation liquid at 50L tank
In, the Valine yield of starting strain CGMCC1.299 is 2.9g/L, and the Valine of the bar bacterium that the present invention provides produces
Amount is 9.1g/L, and ratio starting strain output increased 213.7%, pole significantly improves Valine yield (P ＜ 0.01).On Zong He
Stating fermentation test result, bar bacterium all pole that deposit number is CGMCC No.7041 that the present invention provides significantly improves L-figured silk fabrics
Propylhomoserin yield (P ＜ 0.01).
Biological deposits explanation
Classification And Nomenclature: Beijing corynebacterium Corynebacterium pekinense is in December in 2012 is deposited on the 26th
State's Microbiological Culture Collection administration committee common micro-organisms center, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica, deposit number be its deposit number be CGMCC No.7041.
Accompanying drawing explanation
Fig. 1 shows Valine biosynthesis route map；
Fig. 2 shows the schematic diagram of recombiant plasmid pXMJ19-leuDH.
Detailed description of the invention
The invention discloses a kind of albumen, encode the DNA molecular of this albumen, conversion host containing this DNA molecular should
Convert host for the method producing Valine.Those skilled in the art can use for reference present disclosure, is suitably modified technique ginseng
Number realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art,
They are considered as being included in the present invention.Method and the application of the present invention are described by preferred embodiment, relevant
Method described herein and application substantially can be modified or suitably in without departing from present invention, spirit and scope by personnel
Change and combination, realize and apply the technology of the present invention.
The present invention provide albumen, encode the DNA molecular of this albumen, this conversion of conversion host containing this DNA molecular
Host's agents useful for same in the method producing Valine all can be buied by market.Wherein, the deposit number of starting strain is
CGMCC1.299。
Below in conjunction with embodiment, the present invention it is expanded on further:
The structure of embodiment 1 expression plasmid pXMJ19-leuDH
1. with the genomic DNA of bacillus megaterium Bacillus megaterium ATCC14945 as template,
With the primer of leuF and leuR composition to carrying out PCR amplification, obtain pcr amplification product:
LeuF:5-' GAAGCTTATGAAAATTTTTGACTAT-3 ' (underscore is HindIII enzyme action recognition site)
LeuR:5-' CGAATTCTTAGCGCGTATGACGTCTAC-3 ' (underscore is EcoRI enzyme action recognition site)
2. the PCR primer of recycling step 1 link carrier pEASYTM-T5 (purchased from Beijing full formula limited public affairs of gold biotechnology
Department), obtain recombiant plasmid pT5-leuDH, order-checking obtains leuDH gene order, as shown in SEQ ID No.1；
3. with two kinds of restricted enzyme (HindIII and EcoRI) double digestion plasmid pT5-leuDH and carrier pXMJ19,
Cut after glue reclaims and respectively obtain leuDH fragment (about 1100bp) and pXMJ19 carrier framework (about 6600bp)；
4. two double digestion products of step 3 gained are attached, convert escherichia coli, through mould containing 17 μ g/mL chlorine
The LB culture medium flat plate screening of element obtains positive colony, extracts recombiant plasmid pXMJ19-leuDH, its structure after amplification cultivation
Schematic diagram is as shown in Figure 2.
The structure of embodiment 2 engineering bacteria CGMCC1.299/pXMJ19-leuDH
1. prepare the electroporated competent cell of bar bacterium；
The most electroporated: the pXMJ19-leuDH plasmid of 3-8 μ L to be joined in competent cell, places 10min on ice；
Proceed to 1mm electric shock cup, 1.8 or 2.1kV electric shock 5-7ms；
3. add LB culture medium 1mL, 33 DEG C, 150rpm cultivate 1h,；Coat after concentration containing 25 μ g/mL kanamycin
On brain heart culture medium flat plate, 33 DEG C of constant temperature culture 36-48h；
4. screening positive clone, (has the spy of about 1.1kb by the primer formed with leuF/leuR to carrying out PCR qualification
Different band for the positive), obtain recombinant bacterium.
By named for recombinant bacterium Beijing corynebacterium Corynebacterium pekinense, this bacterial strain is in 2012 12
Within 26th, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) moon, deposit number is
CGMCC No.7041。
The bar bacterium fermenting and producing Valine that deposit number is CGMCC No.7041 that embodiment 3 present invention provides
1. culture medium
Seed activation culture medium: yeast extract 1%, peptone 1%, sodium chloride 0.5%, glucose 0.5%, agar
2%, pH7.2；
Seed culture medium: Semen Maydis pulp 2.5%, glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, potassium dihydrogen phosphate
0.1%, carbamide 0.1%, CaCO30.5%, pH7.2；
Fermentation medium: Semen Maydis pulp 0.5%, glucose 12.0%, ammonium sulfate 4.0%, magnesium sulfate 0.04%, biphosphate
Potassium 0.1%, CaCO34%, VH50ug/L, VB1·HCl100μg/L,pH7.2；
2. shake flask fermentation produces Valine
(1) seed culture: bar bacterium inclined-plane seed 1 ring that the picking present invention provides is connected to equipped with 20mL seed culture medium
In 500mL triangular flask, 33 DEG C, 220r/min shaken cultivation 16-22h；
(2) fermentation culture: 2mL seed liquor is seeded to equipped with in the 500mL triangular flask of 20mL fermentation medium, 33 DEG C,
220r/min shaken cultivation 72h；IPTG(final concentration of 10 μm o l/L are added during 24h) induction；
(3) take 1mL fermentation liquid centrifugal (12000rpm, 2min), collect supernatant, with HPLC test bar bacillus with compare bacterium
Valine content in fermentation liquid, its concentration is as shown in table 1.
The comparative result of Valine content in the fermentation liquid of table 1 shake flask fermentation
As shown in data in table 1, the Valine yield of starting strain CGMCC1.299 is 1.2g/L, and the present invention provides
The Valine yield of bar bacterium be 3.8g/L, ratio starting strain output increased 216%, pole significantly improves Valine and produces
Amount (P ＜ 0.01).
3.50L tank fermenting and producing Valine
(1) secondary seed is cultivated: picking bar bacterium inclined-plane seed 2-3 ring is connected to the 2L tri-equipped with 100mL seed culture medium
In the bottle of angle, 33 DEG C, 220r/min shaken cultivation about 12-16h；Proceeded in the 5L triangular flask containing 1L culture fluid again, 33 DEG C,
220r/min continues to cultivate 16-20h；
(2) 50L fermentation: 3L seed liquor is seeded to equipped with in the 50L fermentation tank of 22L fermentation medium, 31 DEG C of cultivations
72h；During regulate pH7.0 with ammonia, maintain remaining sugar concentration to be 2-3% with the glucose of 80% (m/v), relative dissolved oxygen controls
Between 10-20%, 24h adds the final concentration of 10 μm o l/L of IPTG(constantly) induction；
(3) taking 1mL fermentation liquid centrifugal (12000rpm, 2min), collect supernatant, HPLC test bar bacillus sends out with compareing bacterium
Valine content in ferment liquid, its concentration is as shown in table 2.
The comparative result of Valine content in the fermentation liquid of table 250L tank
As shown in table 2, the Valine yield of starting strain CGMCC1.299 is 2.9g/L, and the bar that the present invention provides
The Valine yield of bacterium is 9.1g/L, ratio starting strain output increased 213.7%, and pole significantly improves Valine yield (P
＜ 0.01).
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (4)
1. the leucine dehydrogenase as shown in SEQ ID No.1 generates the application of valine in Corynebacterium glutamicum, and it is special
Levying and be, the carrier framework of employing is pXMJ19.
2. there is DNA molecular and the carrier framework of the leucine dehydrogenase of coding aminoacid sequence as shown in SEQ ID No.1
The bar bacterium of the recombinant vector of pXMJ19, it is characterised in that described bar bacterium is Beijing corynebacterium MHZ-1010
(Corynebacterium pekinense), deposit number is CGMCC No.7041；
The DNA molecular of the leucine dehydrogenase of described coding aminoacid sequence as shown in SEQ ID No.1, its nucleotide sequence
As shown in SEQ ID No.2.
3. the bar bacterium described in claim 2 is for the purposes of fermenting and producing Valine.
4. the method for use bar bacterium fermenting and producing Valine described in claim 2, it is characterised in that take described bar bacterium
In seed culture medium after expanding propagation, it is placed in fermentation medium and cultivates 50～120h in 28 DEG C～37 DEG C, to obtain final product.
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| CN113278655B (en) * | 2020-05-13 | 2022-05-17 | 安徽华恒生物科技股份有限公司 | Recombinant microorganism for producing L-valine and construction method and application thereof |
| CN113278568B (en) * | 2020-05-27 | 2022-10-21 | 安徽华恒生物科技股份有限公司 | Recombinant escherichia coli for producing L-valine and application thereof |
| CN113278641B (en) * | 2020-05-27 | 2022-06-21 | 安徽华恒生物科技股份有限公司 | Recombinant escherichia coli for producing L-valine as well as construction method and application thereof |
| CN113913478B (en) * | 2021-11-26 | 2023-06-02 | 江南大学 | Method for producing L-valine by fermenting Brevibacterium flavum |
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