CN103361289A - Strain for producing L-lysine and method for producing L-lysine by using same - Google Patents
Strain for producing L-lysine and method for producing L-lysine by using same Download PDFInfo
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- CN103361289A CN103361289A CN201310285525XA CN201310285525A CN103361289A CN 103361289 A CN103361289 A CN 103361289A CN 201310285525X A CN201310285525X A CN 201310285525XA CN 201310285525 A CN201310285525 A CN 201310285525A CN 103361289 A CN103361289 A CN 103361289A
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- 238000004519 manufacturing process Methods 0.000 title abstract description 12
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 title abstract description 11
- 235000019766 L-Lysine Nutrition 0.000 title abstract description 5
- 239000004472 Lysine Substances 0.000 title abstract description 5
- 238000000855 fermentation Methods 0.000 claims abstract description 41
- 230000004151 fermentation Effects 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 41
- 231100000350 mutagenesis Toxicity 0.000 claims abstract description 17
- 238000002703 mutagenesis Methods 0.000 claims abstract description 16
- 241000588724 Escherichia coli Species 0.000 claims abstract description 11
- 241000894006 Bacteria Species 0.000 claims description 62
- 239000002609 medium Substances 0.000 claims description 40
- 230000001580 bacterial effect Effects 0.000 claims description 36
- 238000011218 seed culture Methods 0.000 claims description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 36
- 239000001963 growth medium Substances 0.000 claims description 35
- 230000000968 intestinal effect Effects 0.000 claims description 35
- 239000007788 liquid Substances 0.000 claims description 34
- 238000003756 stirring Methods 0.000 claims description 31
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Inorganic materials [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims description 30
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 20
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 20
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 20
- 229930006000 Sucrose Natural products 0.000 claims description 19
- 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 claims description 19
- 229940041514 candida albicans extract Drugs 0.000 claims description 19
- 239000012138 yeast extract Substances 0.000 claims description 19
- 239000005720 sucrose Substances 0.000 claims description 18
- 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 14
- 239000011790 ferrous sulphate Substances 0.000 claims description 14
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 14
- 239000002054 inoculum Substances 0.000 claims description 14
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 14
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 14
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 14
- 239000000725 suspension Substances 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 13
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 11
- 150000002500 ions Chemical class 0.000 claims description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 108010046845 tryptones Proteins 0.000 claims description 8
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 235000005822 corn Nutrition 0.000 claims description 7
- 230000035772 mutation Effects 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 238000002513 implantation Methods 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 235000003276 Apios tuberosa Nutrition 0.000 claims description 4
- 244000105624 Arachis hypogaea Species 0.000 claims description 4
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 4
- 235000010744 Arachis villosulicarpa Nutrition 0.000 claims description 4
- 244000068988 Glycine max Species 0.000 claims description 4
- 235000010469 Glycine max Nutrition 0.000 claims description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 4
- 159000000007 calcium salts Chemical class 0.000 claims description 4
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 4
- 238000005468 ion implantation Methods 0.000 claims description 4
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 4
- 230000001678 irradiating effect Effects 0.000 claims description 4
- 159000000003 magnesium salts Chemical class 0.000 claims description 4
- 150000002696 manganese Chemical class 0.000 claims description 4
- 235000012054 meals Nutrition 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 159000000000 sodium salts Chemical class 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 238000012216 screening Methods 0.000 abstract description 8
- 230000008859 change Effects 0.000 abstract description 6
- 230000002068 genetic effect Effects 0.000 abstract description 5
- 238000004321 preservation Methods 0.000 abstract description 2
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- 229940023064 escherichia coli Drugs 0.000 abstract 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 39
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 16
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- 235000011114 ammonium hydroxide Nutrition 0.000 description 8
- 229930182817 methionine Natural products 0.000 description 7
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 6
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 6
- 229910019142 PO4 Inorganic materials 0.000 description 5
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 229940045641 monobasic sodium phosphate Drugs 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 5
- 239000010452 phosphate Substances 0.000 description 5
- 239000011591 potassium Substances 0.000 description 5
- 229910052700 potassium Inorganic materials 0.000 description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 5
- 230000004913 activation Effects 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
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- 229960002989 glutamic acid Drugs 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical group OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 2
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- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
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- 238000012360 testing method Methods 0.000 description 2
- AVIDUIVZJUIQGO-AKGZTFGVSA-N (2s)-2-amino-4-methylsulfanylpentanoic acid Chemical compound CSC(C)C[C@H](N)C(O)=O AVIDUIVZJUIQGO-AKGZTFGVSA-N 0.000 description 1
- VMSLCPKYRPDHLN-UHFFFAOYSA-N (R)-Humulone Chemical compound CC(C)CC(=O)C1=C(O)C(CC=C(C)C)=C(O)C(O)(CC=C(C)C)C1=O VMSLCPKYRPDHLN-UHFFFAOYSA-N 0.000 description 1
- 108091000044 4-hydroxy-tetrahydrodipicolinate synthase Proteins 0.000 description 1
- 241001112741 Bacillaceae Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 235000016068 Berberis vulgaris Nutrition 0.000 description 1
- 241000335053 Beta vulgaris Species 0.000 description 1
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- 241000186146 Brevibacterium Species 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 101100476962 Drosophila melanogaster Sirup gene Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- GMKMEZVLHJARHF-WHFBIAKZSA-N LL-2,6-diaminopimelic acid Chemical compound OC(=O)[C@@H](N)CCC[C@H](N)C(O)=O GMKMEZVLHJARHF-WHFBIAKZSA-N 0.000 description 1
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- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 1
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- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a strain for producing L-lysine, which is classified and named as Escherichia coli (E.coli) ((E.coli))Escherichiacoli) NT 1003. delta. Met. delta. Thr, which has been preserved in China Center for Type Culture Collection (CCTCC) at 30 months 5 in 2013, with the preservation number: CCTCC NO: and M2013239. The invention also provides a mutagenesis screening method of the strain and a method for producing L-lysine by using the strain. Escherichia coli (Escherichiacoli) The content of L-lysine in fermentation liquor after the NT1003 delta Met delta Thr strain is continuously passaged for 7 times is about 60g/L, no obvious change is caused, and the genetic stability is good.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a strain and produce the bacterial strain of 1B and the method for producing 1B thereof.
Background technology
1B (L-Lysine, Lys) is organism one of the indispensable amino acid of activity that sustains life, and is the second largest amino acid kind that is only second in the world monosodium glutamate, also is the important as precursors of the type materials such as bio-based nylon.The production method of 1B comprises extraction process, chemosynthesis/enzyme process and microbe fermentation method.Though traditional extraction process technique is simple, the raw material sources such as animal blood meal are limited, only limit to small-scale production; Synthesis method is produced the Methionin process need and is used hypertoxic raw material phosgene, and the possibility residual catalyst, and Product Safety is poor, and environmental issue is serious; The DL-Lys of chemosynthesis also needs through Enzymatic Resolution or transforms just can obtain 1B, has increased substantially production cost; Microbe fermentation method is the most important method of present industrial production Methionin, and fermentation raw material extensively is easy to get and low price, such as starch (tapioca (flour), W-Gum etc.), molasses (cane molasses, beet sirup etc.).
Microbe fermentation method is to utilize auxotrophic strain, by metabolic control fermentation, changes artificially and the pathways metabolism of controlling microorganism realizes the production of 1B.The approach of biosynthesizing Methionin has two kinds: a kind of is by AAA approach (such as yeast, Neurospora etc.), and another kind is by 2,6-diaminopimelate pathway (such as bacterium, blue-green algae, higher plant etc.).The former initiator is α-ketoglutaric acid, and the latter's initial substrate is aspartic acid.Threonine and methionine(Met) in the wild bacterial cell have feedback inhibition to E.C. 2.7.2.4., and Methionin has feedback inhibition simultaneously to E.C. 2.7.2.4. and dihydrodipicolinic acid synthase, thereby have limited the accumulation of Methionin in cell.Nineteen sixty Japan wood under wish that the usefulness uviolizing L-glutamic acid such as youth, middle mountain be clear hold in both hands bacillus and obtain a plant height Serine auxotroph variant, make Methionin production reach commercial production levels.US4066501 discloses the screening method of a kind of multiple resistance brevibacterium sp and side Bacillaceae bacterial strain, and it passes through repeatedly a-amino acid laurylamide (ALL), γ-methyl-Methionin (ML) and N
ω-carbobenzoxy-(Cbz)-Methionin, S-(2-amino-ethyl)-Cys (AEC) resistance screening are so that bacterial strain 1B output increased 50-60%.WO2009/014117 modifies and has reduced the activity of meso α-epsilon-diaminopymelic alpha acid route of synthesis enzyme by the genetically engineered approach, and imports the gene of coding diaminopimelate dehydrogenase, has obtained a kind of intestinal bacteria with 1B throughput.
The ion beam bioengineering technology has been widely used in the Biology Breeding aspect as a kind of brand-new selection by mutation technology.The Low Energy Nitrogen ionization can make structure and the permeability changes of microorganism wall/film, and cause gene damage when microorganism, and then makes microbial gene sequence and metabolism network noticeable change thereof, finally causes the microorganisms sudden change.This induced-mutation technique is applied to respectively L-glutamic acid, Pfansteihl (CN 102653724 A) and produces strain selection, so that L-glutamic acid, Pfansteihl improve respectively 35%, 46%.
Summary of the invention
The first purpose of the present invention provided a kind of can be simply, the bacterial strain of efficient, safety in production 1B.
The present invention's the second purpose provides a kind of acquisition methods of above-mentioned bacterial strains.
The present invention's the 3rd purpose provides a kind of method of utilizing above-mentioned bacterial strains to produce 1B.
In order to realize the first purpose of the present invention, the technical solution used in the present invention is as follows:
A kind of bacterial strain of producing 1B, its Classification And Nomenclature be intestinal bacteria (
Escherichia coli) NT1003 △ Met △ Thr, be preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO:M 2013239 on May 30th, 2013.
Morphology and the physiological and biochemical property of bacterial strain of the present invention are as follows:
Colony colour: canescence.
Aerobic mode: aerobic growth.
Bacterium colony size: 0.3 ~ 0.8 * 1 ~ 4 μ m
Suitable growth temperature: 34-40 ℃.
Suitable growth pH:6.8-7.2.
Thalli morphology: circular bacterium colony has metalluster.
Gramstaining: feminine gender.
The acquisition methods of above-mentioned intestinal bacteria NT1003 △ Met △ Thr, be with
E. coliA301 is starting strain, and by UV-LiCl complex mutation, low energy ion injects mutagenesis, just, multiple sieve and obtaining.
Specifically, the acquisition methods of described intestinal bacteria NT1003 △ Met △ Thr comprises the steps:
1) collecting cells
E. coliA301 is mixed and made into bacteria suspension with the sterilized water that adds lithium chloride, and bacterial concentration is 10
6Individual/mL;
2) UV-LiCl complex mutation
The bacteria suspension of step 1) is changed in the aseptic empty flat board that contains the magnetic force rotor, under stir speed (S.S.) 100-150 rpm, carry out uv irradiating mutagenesis, after get bacteria suspension and coat in the LB plate culture medium that adds lithium chloride, be inverted 34-40 ℃ of lower lucifuge and cultivate 1-2 d;
3) low energy ion injects mutagenesis
With step 2) in bacterial strain washing after cultivating, and make bacteria suspension and coat on the aseptic empty plate, after drying up, it is ion implantation to carry out nitrogen, rear wash-out, is applied on the LB plate culture medium, is inverted 34-40 ℃ of lower lucifuge and cultivates 1-2 d;
4) primary dcreening operation
The picking step 3) is cultivated the single bacterium colony that obtains, and is seeded on the slant medium to activate, and after 1-2 d is cultivated in 34-40 ℃ of lower inversion, moves to stir culture 8-12 h in the seed culture medium;
Some cultured bacterium colonies are inoculated into respectively in the 1L fermentor tank by certain inoculum size, pass into simultaneously air, stir fermentation 32-80 h, detect 1B content in the fermented liquid, just sift out 1B content greater than the bacterium colony of 55 g/L;
In the fermenting process, adopt ammoniacal liquor, 2 mol/L hydrochloric acid controlled fermentation liquid pH;
5) multiple sieve
Single colony inoculation that the step 4) primary dcreening operation is obtained activates to slant medium, after 1-2 d is cultivated in 34-40 ℃ of lower inversion, moves to stir culture 8-12 h in the seed culture medium;
Some cultured bacterium colonies are inoculated into respectively in the 5L fermentor tank by certain inoculum size, pass into simultaneously air, stir fermentation 32-80 h; Detect 1B content in the fermented liquid, filter out the high bacterium colony of 1B output;
Repeating step 1) to step 5), finishing screen selects that 1B content is 60 g/L and above aimed strain intestinal bacteria NT1003 △ Met △ Thr in the fermented liquid.
In the acquisition methods of above-mentioned intestinal bacteria NT1003 △ Met △ Thr, the addition of lithium chloride is the 3-5% of sterilized water quality in the described step 1), preferred 4%;
Described step 2) addition of lithium chloride is the 3-5% of LB plate culture medium quality in, preferred 4%; Described uv irradiating condition: 30W ultraviolet lamp; Irradiation distance 25cm; Irradiation time 30-110 s, preferred 50 s;
Low energy N+ ions condition in the described step 3): 2-4 Kv/cm * 3-9 min(is under the condition of 2-4 Kv/cm in strength of electric field, mutagenesis 3-9 min), be preferably: 3 Kv/cm * 7 min; Implantation dosage is 20 * 10
14-50 * 10
14Ions/cm
2Preferred 40 * 10
14Ions/cm
2
Described step 4) and 5) in, stirring velocity is 180-220 rpm in the seed culture; Inoculum size is 3-10% (v/v); Stirring velocity is 200-500 rpm in the fermentation.
Among the present invention, described LB substratum comprises following mass percent component: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, all the other are water, pH 6.8-7.2.
Described seed culture medium comprises following mass percent component: carbon source 0.1%-0.2%, nitrogenous source 3%-8%, inorganic salt 0.5%-1.3%, and all the other are water, pH 6.8-7.2.Wherein, described carbon source is glucose and/or sucrose; Described nitrogenous source is one or more mixing in peptone, yeast extract or the ammonium sulfate; Described inorganic salt are one or more mixing in sylvite, magnesium salts, molysite, manganese salt, sodium salt or the calcium salt.The preferred seed substratum, formed by following mass percent component: glucose 0.05%, sucrose 0.15%, peptone 3%, yeast extract 3%, ammonium sulfate 2%, dipotassium hydrogen phosphate 0.3%, sal epsom 0.1%, ferrous sulfate 0.05%, manganous sulfate 0.03%, SODIUM PHOSPHATE, MONOBASIC 0.25%, calcium carbonate 0.17%, all the other are water, pH 6.8-7.2.
Fermention medium in the described fermentor tank comprises following mass percent component: carbon source 1.5%-20%, nitrogenous source 0.5%-2%, and inorganic salt 0.1%-1%, all the other are water, pH 6.8-7.2; Wherein, described carbon source is glucose and/or sucrose; Described nitrogenous source is one or more mixing in groundnut meal, soybean cake powder, corn steep liquor, yeast extract or the ammonium sulfate; Described inorganic salt are one or more mixing in sylvite, magnesium salts, molysite, manganese salt, sodium salt or the calcium salt.Fermenting process adopts ammoniacal liquor, 2 mol/L hydrochloric acid controlled fermentation liquid pH.Preferred fermention medium, formed by following mass percent component: glucose 6%, sucrose 14%, corn steep liquor 1%, groundnut meal 0.4%, soybean cake powder 0.4%, ammonium sulfate 0.2%, dipotassium hydrogen phosphate 0.08%, sal epsom 0.02%, ferrous sulfate 0.04%, manganous sulfate 0.05%, SODIUM PHOSPHATE, MONOBASIC 0.15%, calcium carbonate 0.13%, all the other are water, pH 6.8-7.2.Fermenting process adopts ammoniacal liquor, 2 mol/L hydrochloric acid controlled fermentation liquid pH.
A kind of method of utilizing intestinal bacteria NT1003 △ Met △ Thr to produce 1B comprises the steps:
1) slant culture
NT1003 △ Met △ Thr is inoculated in the slant medium, culture temperature 34-40 ℃, incubation time 1-2 d;
2) seed culture
The bacterial strain NT1003 △ Met △ Thr of step 1) slant culture is inoculated in the seed culture medium, and culture temperature is 34-40 ℃, stir culture 8-12 h;
3) fermentation culture
With step 2) bacterial strain NT1003 △ Met △ Thr after the seed culture moves on in the 5L fermentor tank by certain inoculum size, and liquid amount is 2-3L in the 5L fermentor tank, and culture temperature is 34-40 ℃; Pass into simultaneously air, to keep dissolved oxygen environment suitable in the fermentation system, stir fermentation 32-80 h.
Utilizing bacterial strain NT1003 △ Met △ Thr to produce in the method for 1B, described slant medium is the LB substratum, and it is the same specifically to fill a prescription.
Described step 2) in, stirring velocity is 180-220 rpm; The seed culture based formulas is the same.
In the described step 3), stirring velocity is 200-500 rpm; Inoculum size is 3-10% (v/v); Fermentative medium formula is the same.
Produce a preferred implementation of 1B as intestinal bacteria NT1003 △ Met △ Thr of the present invention, the prescription of described slant medium: LB substratum, the mass percent component is: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, and all the other are water, pH 6.8-7.2;
The prescription of described seed culture medium: sucrose 2%, peptone 2%, ammonium sulfate 2%, yeast extract 1%, dipotassium hydrogen phosphate 0.4%, sal epsom 0.3%, ferrous sulfate 0.02%, manganous sulfate 0.07%, all the other are water, pH 6.8-7.2;
The prescription of described fermention medium: glucose 20%, corn steep liquor 1.5%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 0. 5%, manganous sulfate 0. 2%, ferrous sulfate 0.4%, sal epsom 0.1%, all the other are water, pH 6.8-7.2.
Beneficial effect of the present invention:
The present invention adopts Low energy N+ ions and UV-LiCl complex mutation is combined, final screening obtains the intestinal bacteria NT1003 △ Met △ Thr that 1B is produced in a strain, 1B content is about 100 g/L in 7 secondary fermentation liquid of bacterial strain continuous passage, without noticeable change, genetic stability is good; Utilize this bacterial strain to produce 1B, can improve by a relatively large margin 1B content in the tunning.
Description of drawings
Fig. 1 is the mutagenesis screening schema of intestinal bacteria NT1003 △ Met △ Thr.
Embodiment
Biomaterial of the present invention, its Classification And Nomenclature be intestinal bacteria (
Escherichia coli) NT1003 △ Met △ Thr, on May 30th, 2013 be preserved in Chinese Typical Representative culture collection center (be called for short CCTCC, the address is: China. Wuhan. Wuhan University), deposit number: CCTCC NO:M 2013239.
The below enumerates embodiment the present invention is further specified, but does not therefore limit content of the present invention.
Embodiment 1: the present embodiment explanation will E. coliA301
As starting strain mutagenesis screening intestinal bacteriaNT1003 △ Met △ Thr
Method.
In the present embodiment:
1) LB substratum is comprised of following mass percent component: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, all the other are water, pH 6.8-7.2.
2) seed culture medium, formed by following mass percent component: glucose 0.05%, sucrose 0.15%, peptone 3%, yeast extract 3%, ammonium sulfate 2%, dipotassium hydrogen phosphate 0.3%, sal epsom 0.1%, ferrous sulfate 0.05%, manganous sulfate 0.03%, SODIUM PHOSPHATE, MONOBASIC 0.25%, calcium carbonate 0.17%, all the other are water, pH 6.8-7.2.
3) fermention medium, formed by following mass percent component: glucose 6%, sucrose 14%, corn steep liquor 1%, groundnut meal 0.4%, soybean cake powder 0.4%, ammonium sulfate 0.2%, dipotassium hydrogen phosphate 0.08%, sal epsom 0.02%, ferrous sulfate 0.04%, manganous sulfate 0.05%, SODIUM PHOSPHATE, MONOBASIC 0.15%, calcium carbonate 0.13%, all the other are water, pH 6.8-7.2.Fermenting process adopts ammoniacal liquor, 2 mol/L hydrochloric acid controlled fermentation liquid pH.
With recombination bacillus coli
E. coliA301 (Chen Kequan etc., the impact that nitrogenous source produces L-arginine to the restructuring Escherichia coli fermentation, biological processing, November in 2011, the 6th phase of the 9th volume P11-14) was starting strain.This starting strain is the disclosed bacterial strain of formerly open source literature that the applicant has, and by voluntarily preservation of applicant, the applicant freely sends this starting strain to the public in this statement from present patent application day.
As shown in Figure 1, it is as follows to obtain the concrete steps of intestinal bacteria NT1003 △ Met △ Thr:
1) collecting cells
Get 37 ℃ of constant temperature culture 1 d's
E. coliThe fresh inclined-plane of A301 adds sterilized water 15 mL, scrapes to wash bacterial strain and change in the 250 mL triangular flasks with granulated glass sphere, adds the sterilized water that contains 4% lithium chloride in the triangular flask to make bacteria suspension, adjusts bacterial concentration 10
6Individual/mL;
2) UV-LiCl complex mutation
Get the bacteria suspension of 15 mL step 1), change in the aseptic empty flat board that contains the magnetic force rotor, under 100 rpm stirring velocitys, place that the 25cm place carries out uv irradiating mutagenesis under the 30W ultraviolet lamp, irradiation time is 50s, get bacteria suspension after mutagenesis finishes and coat in the LB plate culture medium that contains 4% lithium chloride, be inverted 37 ℃ of lower lucifuges and cultivate 1d;
3) low energy ion injects mutagenesis
With sterilized water washing step 2) in intestinal bacteria 2 times after cultivating, and make bacteria suspension, get 0.1 mL bacteria suspension and evenly coat on the aseptic empty plate, dry up with sterile wind, at 3 Kv/cm * 7min, implantation dosage is 40 * 10
14Ions/cm
2It is ion implantation under the condition intestinal bacteria to be carried out nitrogen, ion implantation complete after, take out plate, under aseptic condition, with 1 mL sterilized water wash-out, be applied on the LB plate culture medium, 37 ℃ of lower lucifuges are inverted cultivation 1d;
4) primary dcreening operation
Some single colony inoculation that step 3) mutagenesis is obtained activates to slant medium, after 1d is cultivated in 37 ℃ of lower inversions, activate bacterial strain to its seed culture medium with transfering loop difference picking one ring, stirring velocity 200 rpm, after cultivating 10h, be linked in the fermention medium in the 1L fermentor tank by the inoculum size of 10% (v/v) again, pass into simultaneously air, stirring velocity 400 rpm, 80 h ferment.Detect 1B content in the fermented liquid, primary dcreening operation goes out in the fermented liquid 1B content greater than single bacterium colony of 55 g/L.
5) multiple sieve
Single colony inoculation of step 4) primary dcreening operation is activated to slant medium, after 1 d is cultivated in 37 ℃ of lower inversions, activate bacterial strain to seed culture medium with transfering loop picking one ring, stirring velocity 200 rpm, after cultivating 10h, be linked in the fermention medium in the 5L fermentor tank by the inoculum size of 10% (v/v), pass into simultaneously air, stirring velocity 400 rpm, 80 h ferment; Detect 1B content in the fermented liquid, sift out again the high single bacterium colony of 1B content in the fermented liquid.
Again step 5) is sieved again the single bacterium colony that obtains and be mixed with bacteria suspension, repeating step 1) to step 5), measuring in the fermented liquid 1B content when SBA-40E bio-sensing instrument is 60 g/L and when above, and corresponding bacterial strain is aimed strain intestinal bacteria NT1003 △ Met △ Thr.
Embodiment 2
Mutagenesis screening method according to embodiment 1 obtains intestinal bacteria NT1003 △ Met △ Thr.
Wherein, the lithium chloride addition is 3% of LB plate culture medium quality; Time of ultraviolet irradiation is 110s; The Low energy N+ ions condition is 4 Kv/cm * 3 min, and implantation dosage is 50 * 10
14Ions/cm
2.
Embodiment 3
Mutagenesis screening method according to embodiment 1 obtains intestinal bacteria NT 1003.
Wherein, the lithium chloride addition is 5% of LB plate culture medium quality; Time of ultraviolet irradiation is 30s; The Low energy N+ ions condition is 42 Kv/cm * 9 min, and implantation dosage is 20 * 10
14Ions/cm
2.
Morphology and the physiological and biochemical property of the intestinal bacteria NT 1003 that above-described embodiment 1-3 obtains are as follows:
Colony colour: canescence.
Aerobic mode: aerobic growth.
Bacterium colony size: 0.3 ~ 0.8 * 1 ~ 4 μ m.
Suitable growth temperature: 34-40 ℃.
Suitable growth pH:6.8-7.2.
Thalli morphology: circular bacterium colony has metalluster.
Gramstaining: feminine gender.
The inheritance stability property testing of the intestinal bacteria NT1003 △ Met △ Thr of embodiment 1-3
In the fermention medium take dextrose plus saccharose as carbon source, the genetic stability of the mutant strain intestinal bacteria NT1003 △ Met △ Thr that detection embodiment 1-3 obtains, the result is as shown in table 1 in the strain passage fermentation test.1B content is measured by SBA-40E bio-sensing instrument in the fermented liquid.
The genetic stability test of table 1 intestinal bacteria NT1003 △ Met △ Thr
Can find out that from experimental result go down to posterity through 7 times, the 1B stable yield of mutant strain intestinal bacteria NT1003 △ Met △ Thr has preferably genetic stability.
Embodiment 4: the method that present embodiment explanation intestinal bacteria NT1003 △ Met △ Thr produces 1B
The culture medium prescription that present embodiment is used:
Slant medium: the LB substratum, the mass percent component is: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, all the other are water, pH 6.8-7.2.
Seed culture medium: glucose 0.1%, sucrose 0.1%, peptone 2%, ammonium sulfate 1%, dipotassium hydrogen phosphate 0.1%, sal epsom 0.03%, ferrous sulfate 0.05%, manganous sulfate 0.02%, SODIUM PHOSPHATE, MONOBASIC 0.05%, calcium carbonate 0.05%, all the other are water, pH 6.8-7.2.
Fermention medium: glucose 1%, sucrose 0.5%, yeast extract 0.5%, ammonium sulfate 1%, potassium primary phosphate 0.05%, manganous sulfate 0.02%, sal epsom 0.02%, sodium-chlor 0.01%, all the other are water, pH 6.8-7.2.
1) slant culture
The intestinal bacteria NT1003 △ Met △ Thr that embodiment 1 is obtained is inoculated in the slant medium, activation 1 d under 37 ℃ of conditions;
2) seed culture
With step 2) the bacterial strain NT1003 △ Met △ Thr of slant culture is inoculated in the seed culture medium, and liquid amount is 20 mL in the 500 mL triangular flasks, and culture temperature is 34 ℃, cultivates 12 h under the 180 rpm shaking speed;
3) fermentation culture
The bacterial strain NT1003 △ Met △ Thr of step 3) seed culture is inoculated in the fermention medium, inoculum size is 3% (v/v), liquid amount is 2 L in the 5 L fermentor tanks, culture temperature is 34 ℃, pass into simultaneously air, to keep dissolved oxygen environment suitable in the fermentation system, stirring velocity is at 400 rpm, fermenting process adopts ammoniacal liquor, 2 mol/L hydrochloric acid controlled fermentation liquid pH, and fermentation time is 32 h.Record through SBA-40E type bio-sensing instrument that 1B content is 60 g/L in the fermented liquid.
Embodiment 5: present embodiment explanation intestinal bacteriaNT1003 △ Met △ Thr
Produce the method for 1B
The culture medium prescription that present embodiment is used:
Slant medium: the LB substratum, the mass percent component is: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, all the other are water, pH 6.8-7.2.
Seed culture medium: sucrose 2%, peptone 3%, ammonium sulfate 5%, potassium primary phosphate 0.1%, sal epsom 0.3%, ferrous sulfate 0.5%, calcium carbonate 0.5%, all the other are water, pH 6.8-7.2.
Fermention medium: sucrose 10%, ammonium sulfate 1.5%, potassium primary phosphate 0. 5%, manganous sulfate 0. 3%, sal epsom 0.4%, sal epsom 0.3%, calcium carbonate 0.5%, all the other are water, pH 6.8-7.2.
1) slant culture
The intestinal bacteria that embodiment 1 is obtained are inoculated in NT1003 △ Met △ Thr in the slant medium, activation 1 d under 40 ℃ of conditions;
2) seed culture
With step 2) the bacterial strain NT1003 △ Met △ Thr of slant culture is inoculated in the seed culture medium, and liquid amount is 30 mL in the 500 mL triangular flasks, and culture temperature is 40 ℃, cultivates 8 h under the 220 rpm shaking speed;
3) fermentation culture
The bacterial strain NT1003 △ Met △ Thr of step 3) seed culture is inoculated in the fermention medium, inoculum size is 10% (v/v), liquid amount is 3 L in the 5 L fermentor tanks, culture temperature is 40 ℃, pass into simultaneously air, to keep dissolved oxygen environment suitable in the fermentation system, stirring velocity is at 400 rpm, fermenting process adopts ammoniacal liquor, 2 mol/L hydrochloric acid controlled fermentation liquid pH, and fermentation time is 80 h.Record through SBA-40E type bio-sensing instrument that 1B content is 57 g/L in the fermented liquid.
Embodiment 6: present embodiment explanation intestinal bacteriaNT1003 △ Met △ Thr
The technique of fermentation production of L-lysine.
The culture medium prescription that present embodiment is used:
Slant medium: the LB substratum, the mass percent component is: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, all the other are water, pH 6.8-7.2.
Seed culture medium: sucrose 2%, ammonium sulfate 5%, yeast extract 1%, potassium primary phosphate 0.1%, sal epsom 0.3%, ferrous sulfate 0.5%, calcium carbonate 0.5%, all the other are water, pH 6.8-7.2.
Fermention medium: sucrose 20%, ammonium sulfate 1.5%, potassium primary phosphate 0. 5%, manganous sulfate 0. 3%, sal epsom 0.4%, ferrous sulfate 0.3%, calcium carbonate 0.1%, all the other are water, pH 6.8-7.2.
1) slant culture
The intestinal bacteria NT1003 △ Met △ Thr that embodiment 1 is obtained is inoculated in the slant medium, activation 2 d under 38 ℃ of conditions;
2) seed culture
With step 2) the bacterial strain NT1003 △ Met △ Thr of slant culture is inoculated in the seed culture medium, and liquid amount is 30 mL in the 500 mL triangular flasks, and culture temperature is 38 ℃, cultivates 8 h under the 200 rpm shaking speed;
3) fermentation culture
The bacterial strain NT1003 △ Met △ Thr of step 3) seed culture is inoculated in the fermention medium, inoculum size is 8% (v/v), liquid amount is 3 L in the 5 L fermentor tanks, culture temperature is 38 ℃, pass into air in the fermentor tank, to keep dissolved oxygen environment suitable in the fermentation system, stirring velocity is at 400 rpm, fermenting process adopts ammoniacal liquor, 2 mol/L hydrochloric acid controlled fermentation liquid pH, and fermentation time is 72 h.Record through SBA-40E type bio-sensing instrument that 1B content is 62 g/L in the fermented liquid.
Embodiment 7: present embodiment explanation intestinal bacteriaNT1003 △ Met △ Thr
Produce the method for 1B
The culture medium prescription that present embodiment is used:
Slant medium: the LB substratum, the mass percent component is: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, all the other are water, pH 6.8-7.2.
Seed culture medium: sucrose 2%, peptone 2%, ammonium sulfate 2%, yeast extract 1%, dipotassium hydrogen phosphate 0.4%, sal epsom 0.3%, ferrous sulfate 0.02%, manganous sulfate 0.07%, all the other are water, pH 6.8-7.2.
Fermention medium: glucose 10%, sucrose 10%, corn steep liquor 1.5%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 0. 5%, manganous sulfate 0. 2%, ferrous sulfate 0.4%, sal epsom 0.1%, all the other are water, pH 6.8-7.2.
1) slant culture
The intestinal bacteria NT1003 △ Met △ Thr that embodiment 1 is obtained is inoculated in the slant medium, activation 2 d under 37 ℃ of conditions;
2) seed culture
With step 2) the bacterial strain NT1003 △ Met △ Thr of slant culture is inoculated in the seed culture medium, and liquid amount is 50 mL in the 500 mL triangular flasks, and culture temperature is 37 ℃, cultivates 10 h under the 200 rpm shaking speed;
3) fermentation culture
The bacterial strain NT1003 △ Met △ Thr of step 3) seed culture is inoculated in the fermention medium, inoculum size is 10% (v/v), liquid amount is 2 L in the 5 L fermentor tanks, culture temperature is 37 ℃, pass into air in the fermentor tank, to keep dissolved oxygen environment suitable in the fermentation system, stirring velocity is at 500 rpm, fermenting process adopts ammoniacal liquor, 2 mol/L hydrochloric acid controlled fermentation liquid pH, and fermentation time is 80 h.Record through SBA-40E type bio-sensing instrument that 1B content is 58g/L in the fermented liquid.
Claims (10)
1. the bacterial strain of 1B is produced in a strain, it is characterized in that, its Classification And Nomenclature be intestinal bacteria (
Escherichia coli) NT1003 △ Met △ Thr, be preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO:M 2013239 on May 30th, 2013.
2. the acquisition methods of intestinal bacteria NT1003 △ Met △ Thr claimed in claim 1 is characterized in that, be with
E. coliA301 is starting strain, and by UV-LiCl complex mutation, low energy ion injects mutagenesis, sieves just, again to obtain.
3. method according to claim 2 is characterized in that, comprises the steps:
1) collecting cells
E. coliA301 is mixed and made into bacteria suspension with the sterilized water that adds lithium chloride, and bacterial concentration is 10
6Individual/mL;
2) UV-LiCl complex mutation
The bacteria suspension of step 1) is changed in the aseptic empty flat board that contains the magnetic force rotor, under 100-150 rpm stirring velocity, carry out uv irradiating mutagenesis, after get bacteria suspension and coat in the LB plate culture medium that adds lithium chloride, be inverted 34-40 ℃ of lower lucifuge and cultivate 1-2 d;
3) low energy ion injects mutagenesis
With step 2) in bacterial strain washing after cultivating, and make bacteria suspension and coat on the aseptic empty plate, after drying up, it is ion implantation to carry out nitrogen, rear wash-out, is applied on the LB plate culture medium, is inverted 34-40 ℃ of lower lucifuge and cultivates 1-2 d;
4) primary dcreening operation
Step 3) is cultivated the single colony inoculation that obtains to slant medium, activate, after 1-2 d is cultivated in 34-40 ℃ of lower inversion, move to stir culture 8-12 h in the seed culture medium;
Some cultured bacterium colonies are inoculated into respectively in the 1L fermentor tank by certain inoculum size, pass into simultaneously air, stir fermentation 32-80 h, detect 1B content in the fermented liquid, just sift out 1B content greater than the bacterium colony of 55 g/L;
5) multiple sieve
Single colony inoculation that the step 4) primary dcreening operation is obtained activates to slant medium, after 1-2 d is cultivated in 34-40 ℃ of lower inversion, moves to stir culture 8-12 h in the seed culture medium;
Some cultured bacterium colonies are inoculated into respectively in the 5L fermentor tank by certain inoculum size, pass into simultaneously air, stir fermentation 32-80 h; Detect 1B content in the fermented liquid, filter out the high bacterium colony of 1B output;
Repeating step 1) to step 5), finishing screen is selected in the fermented liquid 1B stable content at 60 g/L and above aimed strain intestinal bacteria NT1003 △ Met △ Thr.
4. method according to claim 3 is characterized in that, the addition of lithium chloride is the 3-5% of sterilized water quality in the described step 1);
Described step 2) addition of lithium chloride is the 3-5% of LB plate culture medium quality in;
Described step 4) and 5) in, stirring velocity is 180-220 rpm in the seed culture; Inoculum size is 3-10% (v/v); Stirring velocity is 200-500 rpm in the fermentation.
5. method according to claim 3 is characterized in that, described step 2) the medium ultraviolet illuminate condition: the 30W ultraviolet lamp; Irradiation distance 25cm; Irradiation time 30-110 s.
6. method according to claim 3 is characterized in that, Low energy N+ ions condition in the described step 3): 2-4 Kv/cm * 3-9 min, implantation dosage are 20 * 10
14-50 * 10
14Ions/cm
2
7. method according to claim 3 is characterized in that, described LB substratum comprises following mass percent component: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%, and all the other are water, pH 6.8-7.2;
Described seed culture medium comprises following mass percent component: carbon source 0.1%-0.2%, nitrogenous source 3%-8%, inorganic salt 0.5%-1.3%, and all the other are water, pH 6.8-7.2; Wherein, described carbon source is glucose and/or sucrose; Described nitrogenous source is one or more mixing in peptone, yeast extract or the ammonium sulfate; Described inorganic salt are one or more mixing in sylvite, magnesium salts, molysite, manganese salt, sodium salt or the calcium salt;
Described fermention medium comprises following mass percent component: carbon source 1.5%-20%, nitrogenous source 0.5%-2%, and inorganic salt 0.1%-1%, all the other are water, pH 6.8-7.2; Wherein, described carbon source is glucose and/or sucrose; Described nitrogenous source is one or more mixing in groundnut meal, soybean cake powder, corn steep liquor, yeast extract or the ammonium sulfate; Described inorganic salt are one or more mixing in sylvite, magnesium salts, molysite, manganese salt, sodium salt or the calcium salt.
8. a method of utilizing intestinal bacteria NT1003 △ Met △ Thr claimed in claim 1 to produce 1B is characterized in that, comprises the steps:
1) slant culture
NT1003 △ Met △ Thr is inoculated in the slant medium, culture temperature 34-40 ℃, incubation time 1-2 d;
2) seed culture
The bacterial strain NT1003 △ Met △ Thr of step 1) slant culture is inoculated in the seed culture medium, and culture temperature is 34-40 ℃, stir culture 8-12 h;
3) fermentation culture
With step 2) bacterial strain NT1003 △ Met △ Thr after cultivating is inoculated in the fermention medium, and culture temperature is 34-40 ℃; Pass into simultaneously air, stir fermentation 32-80 h.
9. method according to claim 8 is characterized in that, the slant medium in the described step 1) is the LB substratum, and concrete prescription is described with claim 7;
Described step 2) in, stirring velocity is 180-220 rpm; The seed culture based formulas is with claim 7;
In the described step 3), stirring velocity is 200-500 rpm; Fermentative medium formula is with claim 7.
10. method according to claim 9, it is characterized in that, the prescription of described seed culture medium: sucrose 2%, peptone 2%, ammonium sulfate 2%, yeast extract 1%, dipotassium hydrogen phosphate 0.4%, sal epsom 0.3%, ferrous sulfate 0.02%, manganous sulfate 0.07%, all the other are water, pH 6.8-7.2;
The prescription of described fermention medium: glucose 20%, corn steep liquor 1.5%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 0. 5%, manganous sulfate 0. 2%, ferrous sulfate 0.4%, sal epsom 0.1%, all the other are water, pH 6.8-7.2.
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| CN104726512A (en) * | 2013-12-24 | 2015-06-24 | 中粮生物化学（安徽）股份有限公司 | Method for preparing lysine through fermenting |
| 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 |
| CN104726381A (en) * | 2015-04-09 | 2015-06-24 | 南京工业大学 | Strain for producing L-lysine and method for producing L-lysine by using same |
| CN107446909A (en) * | 2017-09-29 | 2017-12-08 | 南京工业大学 | Immobilization method of escherichia coli and method for producing L-lysine by feeding and fermenting immobilized escherichia coli |
| CN107446909B (en) * | 2017-09-29 | 2020-09-29 | 南京工业大学 | Immobilization method of escherichia coli and method for producing L-lysine by feeding and fermenting immobilized escherichia coli |
| CN109055449A (en) * | 2018-08-21 | 2018-12-21 | 南京工业大学 | Method for producing 1, 5-pentanediamine by fermenting bean dreg hydrolysate |
| CN110964670A (en) * | 2019-12-26 | 2020-04-07 | 江南大学 | Escherichia coli capable of producing L-lysine and application thereof |
| CN112322672A (en) * | 2020-11-16 | 2021-02-05 | 中海油常州涂料化工研究院有限公司 | Method for preparing pentanediamine through multi-bacterium combined fermentation |
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