CN102234666B - Fed-batch fermentation preparation of lysine - Google Patents
Fed-batch fermentation preparation of lysine Download PDFInfo
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- CN102234666B CN102234666B CN2011101514954A CN201110151495A CN102234666B CN 102234666 B CN102234666 B CN 102234666B CN 2011101514954 A CN2011101514954 A CN 2011101514954A CN 201110151495 A CN201110151495 A CN 201110151495A CN 102234666 B CN102234666 B CN 102234666B
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- 238000000855 fermentation Methods 0.000 title claims abstract description 23
- 230000004151 fermentation Effects 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims description 4
- 239000004472 Lysine Substances 0.000 title description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 title description 4
- 102000000818 NADP Transhydrogenases Human genes 0.000 claims abstract description 36
- 108010001609 NADP Transhydrogenases Proteins 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 32
- 241000894006 Bacteria Species 0.000 claims abstract description 29
- 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 22
- 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 claims description 21
- 239000008103 glucose Substances 0.000 claims description 21
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 18
- 229930195722 L-methionine Natural products 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 18
- 241000186031 Corynebacteriaceae Species 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 12
- 108091033319 polynucleotide Proteins 0.000 claims description 12
- 102000040430 polynucleotide Human genes 0.000 claims description 12
- 239000002157 polynucleotide Substances 0.000 claims description 12
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- 229960000304 folic acid Drugs 0.000 claims description 11
- 235000019152 folic acid Nutrition 0.000 claims description 11
- 239000011724 folic acid Substances 0.000 claims description 11
- 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 10
- 229930003756 Vitamin B7 Natural products 0.000 claims description 10
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- 235000011912 vitamin B7 Nutrition 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 9
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 238000009923 sugaring Methods 0.000 claims description 8
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 7
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 7
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- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- 235000019270 ammonium chloride Nutrition 0.000 claims description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract 2
- 238000012258 culturing Methods 0.000 abstract 2
- 229910052757 nitrogen Inorganic materials 0.000 abstract 1
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 abstract 1
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- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 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 description 4
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- 230000005611 electricity Effects 0.000 description 3
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- 230000008676 import Effects 0.000 description 3
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
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- 108010061982 DNA Ligases Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
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- 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
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
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- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 235000019766 L-Lysine Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
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- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
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- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
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- 230000026731 phosphorylation Effects 0.000 description 1
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- 239000013600 plasmid vector Substances 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for fed-batch fermentation of L-lysine, which comprises the following steps of: introducing engineering bacteria for expressing pyridine nucleotide transhydrogenase variants into a first fermentation tank for culturing, and inoculating the obtained culture solution to a second fermentation tank for culturing; then, continuously feeding sugar in batches to the second fermentation tank; and then, continuously feeding sugar and nitrogen sources in batches to the second fermentation tank.
Description
Technical field
The invention belongs to the amino acid fermentation field; Particularly; The present invention relates to flow the method that adds fermentation L-Methionin; It comprises that the engineering bacteria of expressing the pyridine nucleotide transhydrogenase variant is inserted first fermentor tank is inoculated in second fermentor cultivation in cultivation and with the nutrient solution that obtains, then to the second fermentor tank continuous flow sugaring, afterwards again to second sugaring of fermentor tank continuous flow and the nitrogenous source.In addition, the present invention also provides the product of said method production 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.
Pyridine nucleotide transhydrogenase is an important enzyme on the L-Methionin pathways metabolism.(can be although the subunit of wild-type pyridine nucleotide transhydrogenase is disclosed referring to NCBI (http://www.ncbi.nlm.nih.gov) albumen and gene accession number NP416120.1 and AAC74674.1; Also can be referring to Chinese patent ZL94194707), but but report not of the research of this enzyme variants.
The inventor is through long-term and arduous research; Except having found the new pyridine nucleotide transhydrogenase that significantly improves enzymic activity unexpectedly; The inventor has studied the method for the engineering bacterium fermentation that is fit to contain this enzyme in more detail, in actual production, has increased the output of Methionin.
Summary of the invention
The technical problem that the present invention will solve is that the stream that provides new adds the method for fermentation L-Methionin; It comprises comprising that the engineering bacteria of expressing the pyridine nucleotide transhydrogenase variant is inserted first fermentor tank is inoculated in second fermentor cultivation in cultivation and with the nutrient solution that obtains; Then to the second fermentor tank continuous flow sugaring, afterwards again to second sugaring of fermentor tank continuous flow and the nitrogenous source.In addition, the present invention also provides the product of said method production etc.
Particularly, in first aspect, the invention provides the method that continuous flow adds fermentation L-Methionin, it comprises:
(1) engineering bacteria that will express the pyridine nucleotide transhydrogenase variant inserts first fermentor tank in 31-38 ℃ of cultivation 10-20 hour, and wherein said pyridine nucleotide transhydrogenase variant improves with respect to the activity of wild-type pyridine nucleotide transhydrogenase;
(2) nutrient solution that step (1) is obtained is inoculated in second fermentor tank with the inoculum size of 3-7% (volume), cultivates 3-8 hour in 36-40 ℃;
(3) to the second fermentor tank continuous flow sugaring, wherein the dosage that per hour flows of sugar is the 0.2-0.35% (weight) that cultivates liquid measure in second fermentor tank, carries out 10-18 hour; With
(4) to second sugaring of fermentor tank continuous flow and the nitrogenous source; Wherein the dosage that per hour flows of sugar is the 0.3-0.5% (weight) that cultivates liquid measure in second fermentor tank; And the dosage that per hour flows of nitrogenous source is the 0.1-0.25% (weight) that cultivates liquid measure in second fermentor tank; Carried out preferred 50-55 hour 30-65 hour.
In this article, " first " and " second " is the fermentor tank of modifying for the district office when modifying fermentor tank, and promptly first fermentor tank is different with second fermentor tank.In this article, inoculum size have those skilled in the art can the conventional implication of understanding, specifically when representing, refer to the percentage of bacterial culture fluid (the bacterium liquid of access) volume with respect to the culture volume that is inserted with volume percent.
Preferably in the method for first aspect present invention, the culture medium prescription in first fermentor tank is: contain glucose 500-750 kilogram, cane molasses 50-300 kilogram, steeping water 400-600 kilogram, KH in per 18 cubic metres of substratum
2PO
4The 30-80 kilogram, MgSO
47H
2O 3-15 kilogram, FeSO
47H
2O 0.1-1 kilogram, MnSO
47H
2O 0.1-1 kilogram, vitamin H 5-30 gram and folic acid 3-10 gram.In embodiment of the present invention, the culture medium prescription in first fermentor tank is: contain 600 kilograms of glucose, 200 kilograms of cane molasses, 520 kilograms of steeping waters, KH in per 18 cubic metres of substratum
2PO
445 kilograms, MgSO
47H
27 kilograms of O, FeSO
47H
20.5 kilogram of O, MnSO
47H
20.5 kilogram of O, vitamin H 12 grams and folic acid 5 grams.
Preferably in the method for first aspect present invention, the culture medium prescription of second fermentor tank is: contain glucose 10000-15000 kilogram, cane molasses 50-3000 kilogram, steeping water 10000-15000 kilogram, KH in per 315 cubic metres of substratum
2PO
4The 700-1200 kilogram, MgSO
47H
2O 5-220 kilogram, FeSO
47H
2The O5-25 kilogram, MnSO
47H
2O 5-220 kilogram, vitamin H 150-300 gram and folic acid 50-120 gram.In embodiment of the present invention, the culture medium prescription of second fermentor tank is: contain 12000 kilograms of glucose, 1000 kilograms of cane molasses, 12000 kilograms of steeping waters, KH in per 315 cubic metres of substratum
2PO
41000 kilograms, MgSO
47H
2100 kilograms of O, FeSO
47H
2O 12, MnSO
47H
2O 120 kg, vitamin H 200 grams and folic acid 85 grams.
The part culture condition of step (3) and (4) (as, temperature, pH etc.) with can be the same or different of step (2).Temperature in preferred steps (3) and (4) is identical with the temperature in the step (2), promptly is 36-40 ℃, preferably is 38-39 ℃.In the step (2), do not flow add operation; And in step (3) and (4), pH maintains between 6.5 to 7.8, and this can add alkali through stream simply or acid realizes.
In this article, the stream dosage has those skilled in the art institute can the conventional implication of understanding, and specifically the time as expressed in weight percent, the weight that refers to the adding material accounts for the percentage of the weight that is added into material (like, nutrient solution).Preferably in the method for first aspect present invention, the sugar in step (3) and (4) is glucose.In step (3), the dosage that per hour flows of glucose is the 0.2-0.35% (weight) that cultivates liquid measure in second fermentor tank, is preferably 0.27-0.33% (weight).In step (4), the dosage that per hour flows of glucose is the 0.3-0.5% (weight) that cultivates liquid measure in second fermentor tank, is preferably 0.42-0.48% (weight).
Preferably in the method for first aspect present invention, the nitrogenous source in the step (4) is inorganic nitrogen-sourced, and preferably ammonium sulfate or ammonium chloride are like ammonium sulfate.In step (4), the dosage that per hour flows of ammonium sulfate is the 0.1-0.25% (weight) that cultivates liquid measure in second fermentor tank, is preferably 0.17-0.23% (weight).
In the present invention; The wild-type pyridine nucleotide transhydrogenase is that those skilled in the art know; Comprise α subunit and β subunit, wherein the sequence of α subunit and β subunit is respectively shown in NCBI (http://www.ncbi.nlm.nih.gov) albumen and gene accession number NP416120.1 and AAC74674.1.The measuring method of enzymic activity is existing known, measures enzymic activity as adopting the method described in the specific embodiment of the invention, so just can confirm whether the activity of pyridine nucleotide transhydrogenase variant has raising.
In the method for preferred first aspect present invention; Said polynucleotide encode successively pyridine nucleotide transhydrogenase α subunit variant and pyridine nucleotide transhydrogenase β subunit variant, the polynucleotide of the pyridine nucleotide transhydrogenase α subunit variant of promptly encoding are positioned at the upper reaches of the polynucleotide of coding pyridine nucleotide transhydrogenase β subunit variant.In embodiment of the present invention, the nucleotide sequence of said polynucleotide is shown in SEQ ID No:1.
Wherein, by other natural amino acid replacements, preferred replacement is selected from M102L, L127R and Q322K to said pyridine nucleotide transhydrogenase α subunit variant on the position of M102, L127 or Q322, and most preferably its aminoacid sequence is shown in SEQ ID No:2.Those skilled in the art can derive its coding nucleotide sequence according to the aminoacid sequence of pyridine nucleotide transhydrogenase α subunit 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 pyridine nucleotide transhydrogenase α subunit variant is by the polynucleotide encoding shown in SEQ ID No:3.
In addition, by other natural amino acid replacements, preferred replacement is selected from A398S and D400H to wherein said pyridine nucleotide transhydrogenase β subunit variant on the position of A398 or D400, and most preferably its aminoacid sequence is shown in SEQ ID No:4.Those skilled in the art can derive its coding nucleotide sequence according to the aminoacid sequence of pyridine nucleotide transhydrogenase α subunit 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 pyridine nucleotide transhydrogenase β subunit variant is by the polynucleotide encoding shown in SEQ ID No:5.
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.In the method for preferred first aspect present invention, engineering bacteria is a coryneform bacteria.Because coryneform bacteria itself is not suitable as the cloning host bacterium, therefore preferred said polynucleotide are to import coryneform through shuttle plasmid.Wherein, preferably intestinal bacteria and coryneform shuttle plasmid of said shuttle plasmid.So just, can in escherichia coli host, carry out DNA reorganization operation easily.In embodiment of the present invention, engineering bacteria is Corynebacterium glutamicum.
In second aspect, the invention provides the product that obtains according to the present invention.Particularly, the invention provides the fermented liquid of the method preparation of first aspect present invention, wherein the content of L-Methionin is not less than 90g/L, preferably is not less than 120g/L.This fermented liquid can become the L-lysine additives for forage by convection drying.
The present invention has following beneficial effect: fermentation process is fit to express the engineering bacteria generation Methionin of pyridine nucleotide transhydrogenase variant; The fermentation yield of Methionin has obtained effective raising in suitability for industrialized production; The time of fermentative prodn is short, has improved yield of unit time; Fermentation needs the parameter of control less, has simplified operation, industrialized standardized production preferably; Variant enzyme and wild-type enzyme textural difference are little, can degrade no potential safety hazard smoothly.
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 pyridine nucleotide transhydrogenase variant gene construct
Sequence according to our design; Entrust Shanghai to give birth to the pyridine nucleotide transhydrogenase variant gene construct of two pyridine nucleotide transhydrogenase subunits of worker's Bioisystech Co., Ltd's composite coding variant 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 pyridine synthesis Nucleotide transhydrogenase variant gene construct; 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 T4DNA ligase enzyme (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:6 of sequence table, reverse primer (introduced Xba I restriction enzyme site) shown in the SEQ ID No:7 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 2.9kb 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 two pyridine nucleotide transhydrogenase subunit variants shown in SEQ ID No:2 and SEQ ID No:4, by company plasmid that builds (called after pMS2-cispnt) and corresponding intestinal bacteria transformant (called after E.coli-cispnt) are returned.
Embodiment 2 colibacillary determinations of activity and coryneform fermenting experiment
(can be according to existing transhydrogenase activity determination method referring to .Cloning andexpression of the transhydrogenase gene of Escherichia coli.J.Bacteriol. such as Clarke DM; 162:367-373); Conversion there is the E.coli-cispnt bacterial strain of pMS2-cispnt plasmid measure the transhydrogenase activity respectively with the negative Top10F ' bacterial strain of conduct contrast; The specific enzyme activity of the E.coli-cispnt bacterial strain of finding has improved 163% than the specific enzyme activity of negative Top10F ' bacterial strain, improves ratio far above the specific enzyme activity of expressing with the wild-type pyridine nucleotide transhydrogenase in the prior art.
Changing the pMS2-cispnt 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; Centrifugal collection thalline; After 10% (V/V) glycerine solution washing with 0 ℃ of precooling thalline is resuspended in 10% (V/V) glycerine solution of 200 μ L precoolings, adds the pMS2-cispnt plasmid, be transferred to after mixing in the 0.1cm electric shock cup; The condition that continues 5ms in 2kV shocks by electricity; Add 1mL then immediately and contain the liquid LB substratum of 0.5% (M/M) glucose, bathed 5 minutes, 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 ℃ in 42 ℃ of temperature.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 2.9kb 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 14.8g/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 23.3%, 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 stream of embodiment 3L-Methionin adds fermentation instance 1
The coryneform bacteria engineering bacteria that the conversion of embodiment 2 is had a pMS2-cispnt plasmid inserts 20 cubic metres of fermentor tanks with 0.5% inoculum size, and (culture medium prescription wherein is: 600 kilograms of glucose, 200 kilograms of cane molasses, 520 kilograms of steeping waters, KH
2PO
445 kilograms, MgSO
47H
27 kilograms of O, FeSO
47H
20.5 kilogram of O, MnSO
47H
20.5 kilogram of O, vitamin H 12 gram and folic acid 5 grams, water is settled to 18 cubic metres), in 35 ℃ of saturated aerobic culture 15 hours, improve cell density.
Then, (culture medium prescription wherein is: 12000 kilograms of glucose, 1000 kilograms of cane molasses, 12000 kilograms of steeping waters, KH with 350 cubic metres of fermentor tanks of the direct injection of the nutrient solution of 20 cubic metres of fermentor tanks
2PO
41000 kilograms, MgSO
47H
2100 kilograms of O, FeSO
47H
212 kilograms of O, MnSO
47H
2The O 120 kg, vitamin H 200 gram and folic acid 85 grams, water is settled to 315 cubic metres), in 38 ℃ of saturated aerobic culture 5 hours.Then, per hour stream adds 1000 kilograms of glucose, continues 15 hours, during exhaust vapour to keep volume; Afterwards, per hour stream adds 1500 kilograms of glucose and 650 kilograms of ammonium sulfate, continuing fermentation 50 hours, during exhaust vapour to keep volume.During stream adds, add NaOH and concentrated hydrochloric acid pH is maintained between 6.5 to 7.8, add alkali when promptly being lower than lower bound, be higher than height and add acid in limited time.The stream added-time, can emit 1 cubic metre of 100 kilograms of glucose of nutrient solution dissolving or ammonium sulfate, stream adds.Fermentation finishes, and thin-layer chromatography detects and wherein produces L-Methionin 97g/L, reaches the standard of industrial application.
The stream of embodiment 4L-Methionin adds fermentation instance 2
Basically with embodiment 3, different was, the nutrient solution of 20 cubic metres of fermentor tanks is directly injected 350 cubic metres of fermentor tanks, in 39 ℃ of saturated aerobic culture 3 hours.Then, per hour stream adds 1100 kilograms of glucose, continues 15 hours; Afterwards, per hour stream adds 1500 kilograms of glucose and 700 kilograms of ammonium sulfate, and continuing fermentation 55 hours, thin-layer chromatography detect and wherein produce L-Methionin 128g/L.
The stream of embodiment 5L-Methionin adds fermentation instance 3
Basically with embodiment 3, different was, the coryneform bacteria engineering bacteria that the conversion of embodiment 2 is had a pMS2-cispnt plasmid inserts 20 cubic metres of fermentor tanks with 0.5% inoculum size, in 31 ℃ of saturated aerobic culture 18 hours.Final thin-layer chromatography detects and wherein produces L-Methionin 103g/L.
Claims (12)
1. stream adds the method for fermentation L-Methionin, and it comprises:
(1) engineering bacteria that will express the pyridine nucleotide transhydrogenase variant inserts first fermentor tank in 31-38 ℃ of cultivation 10-20 hour; The coded polynucleotide of wherein said pyridine nucleotide transhydrogenase variant encode successively pyridine nucleotide transhydrogenase α subunit variant and pyridine nucleotide transhydrogenase β subunit variant; The aminoacid sequence of wherein said pyridine nucleotide transhydrogenase α subunit variant is shown in SEQ ID No:2; With, the aminoacid sequence of wherein said pyridine nucleotide transhydrogenase β subunit variant is shown in SEQ ID No:4;
(2) nutrient solution that step (1) is obtained is inoculated in second fermentor tank with the inoculum size of 3-7% (volume), cultivates 3-8 hour in 36-40 ℃;
(3) to the second fermentor tank continuous flow sugaring, wherein the dosage that per hour flows of sugar is the 0.2-0.35% (weight) that cultivates liquid measure in second fermentor tank, carries out 10-18 hour, and wherein sugar is glucose; With
(4) to second sugaring of fermentor tank continuous flow and the nitrogenous source; Wherein the dosage that per hour flows of sugar is the 0.3-0.5% (weight) that cultivates liquid measure in second fermentor tank; And the dosage that per hour flows of nitrogenous source is the 0.1-0.25% (weight) that cultivates liquid measure in second fermentor tank; Carried out 30-65 hour, wherein sugar be glucose and wherein nitrogenous source be ammonium sulfate or ammonium chloride.
2. the described method of claim 1, wherein the culture medium prescription in first fermentor tank is: contain glucose 500-750 kilogram, cane molasses 50-300 kilogram, steeping water 400-600 kilogram, KH in per 18 cubic metres of substratum
2PO
4The 30-80 kilogram, MgSO
47H
2O 3-15 kilogram, FeSO
47H
2O 0.1-1 kilogram, MnSO
47H
2O0.1-1 kilogram, vitamin H 5-30 gram and folic acid 3-10 gram.
3. the described method of claim 2, wherein the culture medium prescription in first fermentor tank is: contain 600 kilograms of glucose, 200 kilograms of cane molasses, 520 kilograms of steeping waters, KH in per 18 cubic metres of substratum
2PO
445 kilograms, MgSO
47H
27 kilograms of O, FeSO
47H
20.5 kilogram of O, MnSO
47H
20.5 kilogram of O, vitamin H 12 grams and folic acid 5 grams.
4. the described method of claim 1, wherein the culture medium prescription of second fermentor tank is: contain glucose 10000-15000 kilogram, cane molasses 50-3000 kilogram, steeping water 10000-15000 kilogram, KH in per 315 cubic metres of substratum
2PO
4The 700-1200 kilogram, MgSO
47H
2O 5-220 kilogram, FeSO
47H
2O 5-25 kilogram, MnSO
47H
2O 5-220 kilogram, vitamin H 150-300 gram and folic acid 50-120 gram.
5. the described method of claim 4, wherein the culture medium prescription of second fermentor tank is: contain 12000 kilograms of glucose, 1000 kilograms of cane molasses, 12000 kilograms of steeping waters, KH in per 315 cubic metres of substratum
2PO
41000 kilograms, MgSO
47H
2100 kilograms of O, FeSO
47H
2O 12, MnSO
47H
2O 120 kg, vitamin H 200 grams and folic acid 85 grams.
6. the described method of claim 1, wherein the temperature in step (3) and (4) is identical with temperature in the step (2).
7. the described method of claim 6, wherein temperature in step (3) and (4) and the temperature in the step (2) are 38-39 ℃.
8. the described method of claim 1, wherein engineering bacteria is a coryneform bacteria.
9. the described method of claim 1, the nucleotide sequence of the coded polynucleotide of wherein said pyridine nucleotide transhydrogenase variant is shown in SEQ ID No:1.
10. the described method of claim 1 wherein in the step (4), was carried out 50-55 hour.
11. the fermented liquid of the arbitrary described method preparation of claim 1-10, wherein the content of L-Methionin is not less than 90g/L.
12. the described fermented liquid of claim 11, wherein the content of L-Methionin is not less than 120g/L.
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| CN102533889B (en) * | 2012-01-13 | 2014-07-16 | 中粮生物化学（安徽）股份有限公司 | Method for continuously fermenting lysine |
| CN102763771B (en) * | 2012-06-07 | 2013-10-16 | 宁夏伊品生物科技股份有限公司 | Fermentation and coating of lysine and xylanase |
| CN104278062A (en) * | 2013-07-01 | 2015-01-14 | 中粮生物化学（安徽）股份有限公司 | Method for processing molasses, obtained molasses clear liquid, lysine fermentation medium and lysine production method |
| CN104726512B (en) * | 2013-12-24 | 2018-08-07 | 中粮生物化学（安徽）股份有限公司 | A kind of method of preparing lysine through fermentation |
| CN104726510B (en) * | 2013-12-24 | 2018-07-17 | 中粮生物化学（安徽）股份有限公司 | A kind of method of preparing lysine through fermentation |
| CN105779523B (en) * | 2014-12-17 | 2019-01-08 | 内蒙古伊品生物科技有限公司 | Fed-batch cultivation and its equipment |
| 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 |
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