CN102191289B - Fermentation preparation method of lysine - Google Patents
Fermentation preparation method of lysine Download PDFInfo
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- CN102191289B CN102191289B CN2011100656996A CN201110065699A CN102191289B CN 102191289 B CN102191289 B CN 102191289B CN 2011100656996 A CN2011100656996 A CN 2011100656996A CN 201110065699 A CN201110065699 A CN 201110065699A CN 102191289 B CN102191289 B CN 102191289B
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- 238000000855 fermentation Methods 0.000 title claims abstract description 44
- 230000004151 fermentation Effects 0.000 title claims abstract description 44
- 239000004472 Lysine Substances 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims description 11
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 title claims description 6
- 241000894006 Bacteria Species 0.000 claims abstract description 40
- 108091000044 4-hydroxy-tetrahydrodipicolinate synthase Proteins 0.000 claims abstract description 33
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 14
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 14
- 239000002157 polynucleotide Substances 0.000 claims abstract description 14
- 238000012258 culturing Methods 0.000 claims abstract description 3
- 239000002245 particle Substances 0.000 claims description 26
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 23
- 229930195722 L-methionine Natural products 0.000 claims description 23
- 239000000654 additive Substances 0.000 claims description 12
- 239000013612 plasmid Substances 0.000 claims description 11
- 241000186031 Corynebacteriaceae Species 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims description 8
- 230000008676 import Effects 0.000 claims description 8
- 238000005507 spraying Methods 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 239000004459 forage Substances 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 5
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 229960000304 folic acid Drugs 0.000 claims description 4
- 235000019152 folic acid Nutrition 0.000 claims description 4
- 239000011724 folic acid Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 claims description 3
- 229930182817 methionine Natural products 0.000 claims description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 2
- 229930003756 Vitamin B7 Natural products 0.000 claims description 2
- 239000011735 vitamin B7 Substances 0.000 claims description 2
- 235000011912 vitamin B7 Nutrition 0.000 claims description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 abstract description 16
- 238000000034 method Methods 0.000 abstract description 9
- 239000000047 product Substances 0.000 abstract description 8
- 235000019766 L-Lysine Nutrition 0.000 abstract description 6
- 239000013067 intermediate product Substances 0.000 abstract description 4
- 150000001413 amino acids Chemical class 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 230000000996 additive effect Effects 0.000 description 4
- 238000007599 discharging Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000000968 intestinal effect Effects 0.000 description 4
- 235000018977 lysine Nutrition 0.000 description 4
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- 150000007523 nucleic acids Chemical group 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 102000000818 NADP Transhydrogenases Human genes 0.000 description 2
- 108010001609 NADP Transhydrogenases Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 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
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
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- 230000029087 digestion Effects 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
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- 235000011187 glycerol Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 102200033071 rs104894957 Human genes 0.000 description 2
- 102200057450 rs515726129 Human genes 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- ZKGFMZJIENXHDL-UHFFFAOYSA-N 1,2-dihydropyridine-2,3-dicarboxylic acid Chemical compound OC(=O)C1NC=CC=C1C(O)=O ZKGFMZJIENXHDL-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- STPNLYLAJRZZGA-UHFFFAOYSA-N N1C(C=CC=C1)C(=O)O.N1=C(C=CC=C1)C(=O)O.N1C=CC=C1 Chemical compound N1C(C=CC=C1)C(=O)O.N1=C(C=CC=C1)C(=O)O.N1C=CC=C1 STPNLYLAJRZZGA-UHFFFAOYSA-N 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
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- 239000000706 filtrate Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
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- 231100000350 mutagenesis Toxicity 0.000 description 1
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- -1 pH7.3 Substances 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a fermentation method of L-lysine. The method comprises the following steps: introducing the polynucleotide for coding dihydrodipicolinate synthase variant in the bacteria generating L-lysine to ensure that the obtained bacteria express the dihydrodipicolinate synthase variant; and culturing the obtained bacteria under fermentation conditions. In addition, the invention also provides an intermediate product used in the fermentation method, a product which is further produced by utilizing the fermentation method and the like.
Description
Technical field
The invention belongs to the amino acid fermentation field; Particularly; The present invention relates to the fermentation process of L-Methionin, it comprises that the polynucleotide with coding dihydrodipicolinic acid synthase variant import the bacterium that produces L-Methionin, thereby makes the bacterium that is obtained express said dihydrodipicolinic acid synthase's variant; Under fermentation conditions cultivate the bacterium that obtains.In addition, the present invention also provides intermediate product used in the said fermentation process and has utilized said fermentation process further to produce product etc.
Background technology
L-Methionin is important amino acid starting material, can be used as seasonings, food, fodder additives use, also can be used as the effective or adjunct ingredient in healthcare products, the medicine, is widely used in grocery trade, feed industry, pharmacy industry and other chemical industry.Current, the production of L-Methionin mainly is through the fermentative prodn of mikrobe, as utilizing coryneform bacteria production.
The mikrobe that is used for fermentative prodn can be a wild-type microorganisms, but more be higher auxotrophy, resistance anomaly and the metabolism anomaly mikrobe of output that obtains through mutagenesis or genetically engineered.The mikrobe of the character improvement that obtains for genetically engineered, wherein vital is exactly the excellent gene of character.
Dihydrodipicolinic acid synthase's variant is an important enzyme on the L-Methionin pathways metabolism.(can be although wild-type pyrrole dihydrodipicolinic acid synthase and part variant thereof are disclosed referring to NCBI (http://www.ncbi.nlm.nih.gov) albumen and gene accession number AAC75531.1; Also can be referring to Chinese patent ZL94194962), but but report not of the research of other variants of this enzyme not have to enlighten yet and on new site, suddenlys change.
The inventor has found new dihydrodipicolinic acid synthase unexpectedly through long-term and arduous research, and it has also obtained higher lysine production when importing engineering bacterium fermentation.
Summary of the invention
The technical problem that the present invention will solve is to provide the fermentation process of L-Methionin; It comprises that the polynucleotide with coding dihydrodipicolinic acid synthase variant import the bacterium that produces L-Methionin, thereby makes the bacterium that is obtained express said dihydrodipicolinic acid synthase's variant.In addition, the present invention also provides intermediate product used in the said fermentation process and has utilized said fermentation process further to produce product etc.
Particularly, in first aspect, the invention provides the fermentation process of L-Methionin, it comprises:
(1) bacterium that the polynucleotide of dihydrodipicolinic acid synthase's variant import to produce L-Methionin of will encoding; Thereby make the bacterium that is obtained express said dihydrodipicolinic acid synthase's variant, wherein said dihydrodipicolinic acid synthase's variant is replaced by other natural amino acids on the position of wild-type dihydrodipicolinic acid synthase's M13, V60 or A83; With
(2) bacterium of culturing step (1) acquisition under fermentation conditions.
Wherein, wild-type dihydrodipicolinic acid synthase is that those skilled in the art know, and its sequence is shown in NCBI (http://www.ncbi.nlm.nih.gov) albumen and gene accession number AAC75531.1.
In the fermentation process of preferred first aspect present invention, said polynucleotide encoding dihydrodipicolinic acid synthase variant, like its nucleotide sequence shown in SEQ ID No:2.
In the fermentation process of preferred first aspect present invention; By other natural amino acid replacements, preferred replacement is selected from M13K, V60L and A83G to said dihydrodipicolinic acid synthase's variant on the position of wild-type dihydrodipicolinic acid synthase's M13, V60 and A83.In embodiment of the present invention, the aminoacid sequence of said dihydrodipicolinic acid synthase's variant is shown in SEQ ID No:1.
Those skilled in the art can derive its coding nucleotide sequence according to the aminoacid sequence of dihydrodipicolinic acid synthase's variant, and preferably codon optimized nucleotide sequence is like what optimize to the used nectar numeral service condition of fermentation.In embodiment of the present invention, said dihydrodipicolinic acid synthase's variant is by the polynucleotide encoding shown in SEQ ID No:2.
Said polynucleotide can be imported into the bacterium that produces L-Methionin through various modes well-known to those skilled in the art, as long as can make the bacterium that produces L-Methionin express said pyridine nucleotide transhydrogenase variant.Said polynucleotide can directly be imported into, and for example utilize transfered cells such as microsome, particle gun; Also can be imported into indirectly, for example can be through being structured in transfered cell on the plasmid vector.The said polynucleotide that import can be incorporated on the genome of cell and express, and expression also can dissociate.Usually, be not suitable as the cloning host bacterium owing to produce the bacterium of L-Methionin itself, therefore preferred said polynucleotide are to import the bacterium that produces L-Methionin through shuttle plasmid.Wherein, the said shuttle plasmid shuttle plasmid of intestinal bacteria and the bacterium that produces L-Methionin preferably.So just, can in escherichia coli host, carry out DNA reorganization operation easily.
The bacterium that produces L-Methionin has many kinds, and those skilled in the art knew comprises escherich's bacillus, coryneform bacteria and Serratia, preferably coryneform bacteria.In embodiment of the present invention, the bacterium of said product L-Methionin is Corynebacterium glutamicum.
In the fermentation process of preferred first aspect present invention, the leavening temperature of said fermentation condition is 28-35 ℃, is preferably 29-33 ℃, more preferably 30-32 ℃, and as 30 ℃.
Also in the fermentation process of preferred first aspect present invention, the substratum of said fermentation condition comprises sugar, NH
4Cl, CaCl
2, KH
2PO
4, peptone, MgSO
4, FeSO
4, MnSO
4, vitamin H, and folic acid.In embodiment of the present invention, the prescription of said substratum is: 40g sucrose, 20g NH
4Cl, 2g CaCl
2, 1gKH
2PO
4, 1g peptone, 500mg MgSO
47H
2O, 15mg FeSO
47H
2O, 10mgMnSO
47H
2O, 200 μ g vitamin Hs and 50 μ g folic acid, pH7.3, water complement to 1 liter.
In second aspect, the invention provides the preparation method of L-lysine additives for forage, it comprises:
(1) step (2) in the fermentation process of embodiment of the present invention first aspect; With
(2) collecting fermented liquid carries out membrane sepn successively and filtrating is carried out spraying drying;
(3) particle that step (2) spraying drying is obtained sieves;
(4) particle diameter that screening is obtained is more than or equal to the grain breakage of 1.5mm, and the particle diameter that itself and screening are obtained mixes less than the particle of 1.5mm, carries out spraying drying once more; With
(5) particle that step (4) spraying drying is obtained sieves, and collection cut size is less than the particle of 1.5mm.
Wherein, for the shape that makes final granulated fodder additive is more even, fluid bed dryer inner exhaust gas temperature is unsuitable too high, should not be higher than 85 ℃ usually, preferably remains on 80 ± 3 ℃.
The preparation method of second aspect present invention can also be in the step in the fermentation process of embodiment of the present invention first aspect (2) before, comprises the step (1) in the fermentation process of embodiment of the present invention first aspect.But; This is not preferred usually; Therefore the preparation method of second aspect present invention does not comprise the step (1) in the fermentation process of embodiment of the present invention first aspect; And comprising the step of under fermentation conditions cultivating the bacterium (the especially bacterium of high yield L-Methionin) that produces L-Methionin, this also contains within the scope of the invention.
Because the preparation method of second aspect present invention can produce the granulated fodder additive of National standard, therefore preferably wherein do not comprise the process of dressing, corresponding can reducing cost, the business promotion of being more convenient for.
In the third aspect, the invention provides the intermediate product that uses among the product that obtains according to the present invention and the present invention.
Particularly, the invention provides the L-lysine additives for forage that the preparation method according to second aspect present invention obtains, preferably wherein do not contain seed dressing agent (like, Schardinger dextrins etc.).
In addition, the invention provides dihydrodipicolinic acid synthase's variant, replaced by other natural amino acids on the position of its M13, V60 or A83 the wild-type dihydrodipicolinic acid synthase.By other natural amino acid replacements, preferred replacement is selected from M13K, V60L and A83G to preferred said dihydrodipicolinic acid synthase's variant on the position of wild-type dihydrodipicolinic acid synthase's M13, V60 and A83.In embodiment of the present invention, the aminoacid sequence of said dihydrodipicolinic acid synthase's variant is shown in SEQ IDNo:1.
Correspondingly, the invention provides the polynucleotide of coding dihydrodipicolinic acid synthase variant, preferably its nucleotide sequence is shown in SEQ ID No:2.
The present invention has following beneficial effect: the fermentation yield of Methionin effectively improves; The fermented liquid steady quality; Variant enzyme and wild-type enzyme textural difference are little, can degrade no potential safety hazard smoothly; Fermented liquid can directly be used to produce granulated fodder additive; The step of producing granulated fodder additive is easy, need not to introduce the requirement that seed dressing agent can be up to state standards.
For the ease of understanding, below will the present invention be described in detail through concrete embodiment.What need particularly point out is that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.According to the argumentation of this specification sheets, many variations of the present invention, change all are conspicuous concerning one of ordinary skill in the art.
In addition, the present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included this paper in and carried out reference, just looks like that repeated description is the same excessively in this article for their full text.
Embodiment
Below further specify content of the present invention through embodiment.As do not specialize; Conventional means that used technique means is well known to those skilled in the art among the embodiment and commercially available common instrument, reagent can be referring to the references such as manufacturers instruction of " molecular cloning experiment guide (the 3rd edition) " (Science Press), " microbiology experiment (the 4th edition) " (Higher Education Publishing House) and corresponding instrument and reagent.
The preparation of embodiment 1 dihydrodipicolinic acid synthase's variant gene
Sequence according to our design; Entrust Shanghai to give birth to worker's Bioisystech Co., Ltd's composite coding dihydrodipicolinic acid synthase variant gene and be built among intestinal bacteria-coryneform bacteria shuttle plasmid pMS2 (can available from the U.S. representative microbial preservation center (ATCC), goods number ATCC 67189) through commercial sources.Clone's process is carried out with reference to the operational guidance of " molecular cloning experiment guide " and used commercialization reagent, and concise and to the point process is following:
Pass through automatic dna synthesizer; The nucleic acid fragment of synthesizing dihydro pyridine dicarboxylic acid synthetic enzyme variant gene; With T4 polynucleotide kinase (available from TaKaRa company) 5 ' end of these nucleic acid fragments is carried out phosphorylation; Wait then behind these 5 nucleic acid fragments of mixed in molar ratio in 65 ℃ of sex change 5 minutes, annealing is cooled to 16 ℃, adds T4 dna ligase (available from TaKaRa company) and connects 12 hours.Then; Get the above-mentioned connection product of 1 μ L and in 50 μ L reaction volumes, carry out pcr amplification; Wherein forward primer (has been introduced EcoR I restriction enzyme site) shown in the SEQ ID No:3 of sequence table, reverse primer (introduced Xba I restriction enzyme site) shown in the SEQ ID No:4 of sequence table; Reaction conditions is: with 94 ℃ of sex change 4 minutes, extend with 94 ℃ of sex change 30 seconds, 63 ℃ of annealing 60 seconds and 72 ℃ then and carried out 35 circulations in 30 seconds, extended 4 minutes with 72 ℃ at last and be cooled to 4 ℃.
The above-mentioned PCR product of agarose gel electrophoresis reclaims the fragment of about 0.9kb size, with EcoR I and this fragment of Xba I double digestion, and be connected with the T4DNA ligase enzyme through the pMS2 of these two endonuclease digestions plasmid, be transformed among the intestinal bacteria Top10F '.Choose positive colony; Extracting goes out plasmid wherein; Through sequence verification; The corresponding nucleotide sequence shown in the SEQ ID No:1 of sequence table, sequence coding the complete ORF of the dihydrodipicolinic acid synthase's variant shown in SEQ ID No:2, by company plasmid that builds (called after pMS2-dap) and corresponding intestinal bacteria transformant (called after E.coli-cispnt) are returned.
Embodiment 2 coryneform fermenting experiments
Changing the pMS2-dap plasmid coryneform bacteria engineering bacteria of L-fermenting lysine over to through electrotransformation (can be available from the U.S. representative microbial preservation center (ATCC); Goods number ATCC 31269) in; Its concise and to the point process is: coryneform bacteria shaking culture in 50mL LB liquid nutrient medium is reached 0.7 to OD500, and centrifugal collection thalline is resuspended in thalline in 10% (V/V) glycerine solution of 200 μ L precoolings after 10% (V/V) glycerine solution washing with 0 ℃ of precooling; Add the pMS2-dap plasmid; Be transferred to after mixing in the 0.1cm electric shock cup, the condition that continues 5ms in 1.8kV shocks by electricity, and adds the liquid LB substratum that 1mL contains 0.5% (M/M) glucose then immediately; Bathed 5 minutes in 42 ℃ of temperature, be coated on then on the solid LB substratum that contains 100 μ g/mL penbritins and 35 μ g/mL kantlex and cultivated 36 hours in 30 ℃.After the conversion bacterial strain that grows extracts total DNA; With above-mentioned forward primer and reverse primer pcr amplification; Agarose gel electrophoresis find the to have an appointment fragment of 0.9kb size shows the gene construct shown in the SEQ ID No:1 of sequence table has been imported in the coryneform bacteria engineering bacteria.Simultaneously pMS2 plasmid electricity is transformed into the coryneform bacteria engineering bacteria of L-fermenting lysine, forms the negative control bacterium.
The positive coryneform bacteria engineering bacteria that above-mentioned electricity is transformed and negative control bacterium respectively in liquid LB substratum shaking culture reach 0.5 to OD500, (every liter of culture medium prescription is the inoculum size access fermenting lysine substratum with 5%: 40g sucrose, 20g NH
4Cl, 2g CaCl
2, 1g KH
2PO
4, 1g peptone, 500mgMgSO
47H
2O, 15mg FeSO
47H
2O, 10mg MnSO
47H
2O, 200 μ g vitamin Hs and 50 μ g folic acid are adjusted to pH7.3 with Tris-HCl) in cultivated 72 hours with 30 ℃ of vibrations (150rpm).Centrifugal collection medium supernatant (that is, fermented liquid) is with the L-Methionin in Paper Chromatography separation and the quantitative culture medium.The result finds; The content of L-Methionin has reached 19.6g/L in the fermention medium of positive coryneform bacteria engineering bacteria; And the content of L-Methionin is merely 12.0g/L in the fermention medium of negative control bacterium; Show the gene construct that has imported shown in the SEQ ID No:1 of sequence table, output has improved 63.3%, and the output that is higher than the engineering bacteria that imports the wild-type pyridine nucleotide transhydrogenase in the prior art improves ratio.
The preparation of embodiment 3L-lysine additives for forage
Carry out membrane sepn with the fermented liquid of embodiment 2 preparation through Suntar-III type ultra-filtration membrane (can available from SanDa film Science ltd).Then, will filtrate directly with vaporific spray in the fluid bed dryer and keep exhaust temperature 80 ± 3 ℃ constant, collect the particle of discharging.Particle to discharging sieves; Send into crusher in crushing for particle diameter more than or equal to the particle of 1.5mm; Then the particle after the fragmentation and the particle diameter that obtains of screening are mixed less than the particle of 1.5mm and also spray in the fluid bed dryer once more and keep exhaust temperature constant at 80 ± 3 ℃; Collect the particle of discharging and sieve out the particle of particle diameter, be the L-lysine additives for forage less than 1.5mm.Particle diameter is greater than the particle of 1.5mm once more after the fragmentation, and the particle of the discharging that obtains with the filtrating drying of next batch mixes.
More than the L-lysine additives for forage of preparation is through detecting water cut＜1%; Grain diameter does not have obviously visible dust less than 1.5mm; The mixovariation coefficient is 7～9; Tap density is 550～630g/L; Do not contain other additives, reached national standard fully.
Claims (10)
1.L-the fermentation process of Methionin, it comprises:
(1) bacterium that the polynucleotide of dihydrodipicolinic acid synthase's variant import to produce L-Methionin of will encoding; Thereby make the bacterium that is obtained express said dihydrodipicolinic acid synthase's variant, wherein said dihydrodipicolinic acid synthase's variant nucleotide sequence coded by shown in SEQ ID No:2; With
(2) bacterium of culturing step (1) acquisition under fermentation conditions.
2. the described fermentation process of claim 1, wherein said polynucleotide are to import the bacterium that produces L-Methionin through shuttle plasmid.
3. the described fermentation process of claim 1, wherein said bacterium is a coryneform bacteria.
4. the described fermentation process of claim 1, the leavening temperature of wherein said fermentation condition is 28-35 ℃.
5. the described fermentation process of claim 4, the leavening temperature of wherein said fermentation condition is 29-33 ℃.
6. the described fermentation process of claim 5, the leavening temperature of wherein said fermentation condition is 30-32 ℃.
7. the described fermentation process of claim 6, the leavening temperature of wherein said fermentation condition is 30 ℃.
8. the described fermentation process of claim 1, the substratum of wherein said fermentation condition comprises sugar, NH
4Cl, CaCl
2, KH
2PO
4, peptone, MgSO
4, FeSO
4, MnSO
4, vitamin H, and folic acid.
9.L-the preparation method of lysine additives for forage, it comprises:
(1) step (2) of arbitrary described fermentation process of enforcement claim 1-8; With
(2) collecting fermented liquid carries out membrane sepn successively and filtrating is carried out spraying drying;
(3) particle that step (2) spraying drying is obtained sieves;
(4) particle diameter that screening is obtained is more than or equal to the grain breakage of 1.5mm, and the particle diameter that itself and screening are obtained mixes less than the particle of 1.5mm, carries out spraying drying once more; With
(5) particle that step (4) spraying drying is obtained sieves, and collection cut size is less than the particle of 1.5mm.
10. the polynucleotide of coding dihydrodipicolinic acid synthase variant, its nucleotide sequence is shown in SEQ ID No:2.
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| US20160002684A1 (en) * | 2013-02-08 | 2016-01-07 | Ningxia Eppen Biotech Co., Ltd | Method for Producing L-Lysine by Modifying Aconitase Gene and/or Regulatory Elements thereof |
| CN103981230B (en) * | 2013-02-08 | 2019-03-19 | 内蒙古伊品生物科技有限公司 | The method of the bacterial fermentation production L-lysine of reduction and/or enzymatic activity reduction is expressed with aconitase |
| CN103146772B (en) * | 2013-02-08 | 2014-06-18 | 宁夏伊品生物科技股份有限公司 | Method for fermenting production of L-lysine through using aconitase expression weakened and/or enzymatic activity reduced bacteria |
| CN109370882B (en) * | 2014-12-17 | 2021-08-20 | 宁夏伊品生物科技股份有限公司 | Apparatus and components for use in fermentation of lysine |
| CN109370886B (en) * | 2014-12-17 | 2021-08-31 | 宁夏伊品生物科技股份有限公司 | Feeding fermentation equipment |
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| KR101052573B1 (en) * | 2004-04-02 | 2011-07-29 | 씨제이제일제당 (주) | Process for preparing granular animal feed additive with uniform content and granular animal feed additive produced thereby |
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