CN1472309A - Acetic acid resistant mutant strain of Escherichia coli DH5α and its breeding method and application - Google Patents
Acetic acid resistant mutant strain of Escherichia coli DH5α and its breeding method and application Download PDFInfo
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- CN1472309A CN1472309A CNA031294553A CN03129455A CN1472309A CN 1472309 A CN1472309 A CN 1472309A CN A031294553 A CNA031294553 A CN A031294553A CN 03129455 A CN03129455 A CN 03129455A CN 1472309 A CN1472309 A CN 1472309A
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- acetate
- acetic acid
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 title claims abstract description 35
- 238000009395 breeding Methods 0.000 title claims abstract 3
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 title claims 3
- 230000001580 bacterial effect Effects 0.000 claims abstract description 20
- 241000588724 Escherichia coli Species 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 11
- 229940023064 escherichia coli Drugs 0.000 claims abstract 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 30
- 241000894006 Bacteria Species 0.000 claims description 30
- 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 23
- 238000000034 method Methods 0.000 claims description 23
- 239000008103 glucose Substances 0.000 claims description 22
- 230000008569 process Effects 0.000 claims description 10
- 238000010790 dilution Methods 0.000 claims description 8
- 239000012895 dilution Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 230000005855 radiation Effects 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 150000003863 ammonium salts Chemical class 0.000 claims description 2
- 230000035772 mutation Effects 0.000 claims description 2
- 159000000000 sodium salts Chemical class 0.000 claims description 2
- 230000002269 spontaneous effect Effects 0.000 claims description 2
- 239000012531 culture fluid Substances 0.000 claims 1
- 238000002955 isolation Methods 0.000 claims 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 19
- 230000012010 growth Effects 0.000 abstract description 16
- 230000008901 benefit Effects 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 241000894007 species Species 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 12
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 11
- 239000000243 solution Substances 0.000 description 9
- 235000019156 vitamin B Nutrition 0.000 description 9
- 239000011720 vitamin B Substances 0.000 description 9
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 239000001632 sodium acetate Substances 0.000 description 8
- 235000017281 sodium acetate Nutrition 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 229910017053 inorganic salt Inorganic materials 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- 230000000968 intestinal effect Effects 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 235000006180 nutrition needs Nutrition 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 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
- 239000002253 acid Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 101150099105 alien gene Proteins 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 238000012269 metabolic engineering Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012807 shake-flask culturing Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- 101100084022 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) lapA gene Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
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- 239000011521 glass Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 101150009573 phoA gene Proteins 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- -1 pyruvic acid Chemical class 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
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- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种大肠杆菌(Escherichiacoli)DH5α的耐乙酸突变株CGMCC No.0941及其选育方法和应用。本发明对DH5α进行诱变，在存在乙酸抑制压力的条件下选育其耐乙酸突变株，以提高外源基因的表达水平。本发明的菌种可用于外源基因的表达。本发明的DH5α的耐乙酸突变菌的优点在于生长优势明显，在耐乙酸能力增强的同时乙酸的产生也减少，容易实现高密度培养，外源基因表达效率提高。The invention discloses an acetic acid-resistant mutant strain CGMCC No.0941 of Escherichia coli (Escherichiacoli) DH5α, a breeding method and application thereof. In the invention, DH5α is mutated, and its acetic acid-resistant mutant strain is bred under the condition of acetic acid inhibitory pressure, so as to increase the expression level of exogenous genes. The bacterial species of the present invention can be used for the expression of foreign genes. The acetic acid-resistant mutant of DH5α of the present invention has the advantages of obvious growth advantage, the acetic acid production is reduced while the acetic acid-resistant ability is enhanced, high-density culture is easy to achieve, and the expression efficiency of exogenous genes is improved.
Description
Technical field
The present invention relates to a kind of intestinal bacteria, particularly a kind of mutant strain of bacillus coli DH 5 alpha and selection thereof and application.
Background technology
Intestinal bacteria (Esherichia coli) have that growth is fast, cultivation is with low cost, genetic background is clear, easily realize advantage such as high-density culture, are one of most widely used heterologous gene expression systems.It is exactly can accumulate some metabolic by-prodss in culturing process that yet there is a bigger problem in this expression system, mainly be acetate, not only cause the waste of carbon source, and can suppress the growth of thalline and reduce expression of exogenous gene efficient, seriously influence the throughput of intestinal bacteria when high-density culture and (seen document: Trends in Biotechnology, 1996,14:98～105).
In order to solve acetate to colibacillary inhibition problem, some researchs in past mainly concentrate on the following aspects: (1) medium optimization; (2) fermenting process control; (3) seed selection acetate generates approach-phosphotransacetylase (PTA) or E.C. 2.7.2.1 (ACK) deletion mutantion strain; (4) metabolic engineering method.The method of the inhibition of these several solution acetate mainly concentrates in the generation that reduces acetate in the culturing process, and growth and the raising expression of exogenous gene efficient of improving thalline has all been obtained certain effect, but the part that also comes with some shortcomings.When being optimized substratum, when selecting other carbon source such as glycerine place of glucose for use, tend to increase the cost of cultivation, its effect also has than big-difference with bacterial strain uses therefor is different.Restricted stream adds glucose can effectively be controlled acetate with control ratio growth velocity subcritical value generation in the fermenting process, but can make thalline be in the semistarvation attitude, be with the sacrifice specific growth rate, the prolongation incubation time is realized, is unfavorable for the expression of exogenous gene relevant with growth especially.Fermentation-separation coupling operation can be removed when by product produces, but higher to equipment requirements, investment strengthens, and is difficult to use in scale operation.PTA and ACK deletion mutantion strain though can reduce the generation of acetate, tend to cause cell growth to slow down, and accumulate other organic acid such as pyruvic acid, succsinic acid, and this can suppress expression of exogenous gene equally.It is the basic method that solves the acetate problem that the metabolic engineering method is transformed coli strain, need to consider the structure and the regulation and control characteristics of colibacillary whole metabolism network comprehensively, remain further to be studied, existing work mostly is guides metabolism stream into the more weak metabolite of restraining effect or polysaccharide, and disappearance PTA and ACK etc.
The anti-acetate intestinal bacteria of seed selection mutant bacteria also can be used as a kind of effective means to strengthen tolerance and the raising expression of exogenous gene efficient of bacterial strain to acetate, the contriver had once screened the mutant strain of the anti-acetate JL3 of intestinal bacteria JM101, not only improved the growth of acetate rejection condition hypothallus, and reduced the accumulation of acetate and improved expression efficiency (the microorganism journal of external source beta-galactosidase gene, 2001,41:223～228).Bacillus coli DH 5 alpha (the HanahanD of bibliographical information, J.Mol.Biol., 1983,166:557～580) owing to its transformation efficiency height, be one of host bacterium the most frequently used in the genetically engineered, yet compare with some other coli strain, DH5 α consumption of glucose in minimum medium is slow, energy for growth is poor, and very responsive to acetate, be difficult for realizing high-density culture, so there is more problem in the high-density culture of DH5 α.
Summary of the invention
One of technical issues that need to address of the present invention are the mutant strains of anti-acetate that discloses a kind of bacillus coli DH 5 alpha, to overcome the above-mentioned defective that prior art exists;
Two of the technical issues that need to address of the present invention provide the selection of described mutant strain.
Design of the present invention is such:
DH5 α is carried out mutagenesis, and its mutant strain of anti-acetate of seed selection under the condition that has acetate inhibition pressure is to improve the expression of exogenous gene level.
Bacillus coli DH 5 alpha is a kind of bacterial classification that colon bacillus belongs to that belongs to, DH5 α is carried out mutagenesis or utilizes its spontaneous mutation, under the condition that has acetate inhibition pressure, carry out seed selection, can obtain the said mutant strain of anti-acetate of the present invention (Escherichia coli) DA19.This bacterial classification on June 6th, 2003 in China Committee for Culture Collection of Microorganisms's preservation, preserving number is CGMCC No.0941.
This bacterial classification has following characteristic:
This bacterial classification is identical with size with the bacterium DH5 α colony shape that sets out; The same glucose, glycerine, the sodium acetate of can utilizing with the bacterium that sets out is carbon source, can not utilize lactose, sucrose, starch as carbon source, and be identical with the nutritional needs of the bacterium DH5 α that sets out, is vitamins B
1The nutritional needs type; The plasmid pUC18 or the pUC19 that will contain the beta-galactosidase gene of lacZ promotor and regulation and control thereof change in the cell, show blue look, the same α-Hu Bu phenomenon that still exists with the bacterium that sets out at the LB medium agar flat-plate bacterial colony that contains IPTG and X-gal.
1. morphological specificity:
(1) form of cell and size: shaft-like, 0.5～3 μ m.
(2) formation of sporozoite: can not form spore.
(3) gramstaining: feminine gender.
2. the growth conditions on various substratum:
(1) the LB agar plate is cultivated (30 ℃, 48 hours)
Colony shape: circular (Circular), level and smooth (Smooth), expansion (Spread)
Size: 1～3mm;
Tone: ivory buff.
(2) the LB agar slant is cultivated (30 ℃, 24 hours)
Well-grown, lawn smooth (Smooth).
(3) LB liquid culture (30 ℃, 10 hours)
Well-grown, the nutrient solution muddiness, placing for some time can sedimentation (Sediment).
(4) generate ammonia by peptone: the positive;
(5) to the demand of oxygen: amphimicrobian;
(6) generate acid by sugar
Positive: glucose, glycerine, fructose;
Negative: sucrose, lactose;
(7) growth pH scope: 6.0～8.0;
(8) growth optimal temperature: 28～37 ℃;
(9) nutritional needs type test: vitamins B
1Auxotrophy.
The method that above-mentioned characteristic can adopt the outstanding Bacteria Identification handbook of uncle (Bergey ' s Manual ofDeterminative Bacteriology) to introduce is differentiated, mutant strain DA19 of the present invention is that with the difference of the bacterium DH5 α maximum of setting out its anti-acetate ability is strong, the cell concentration of growing in containing the minimum medium of acetate is much higher than its bacterium DH5 α that sets out, and is the thalline yield Y of benchmark with the glucose that consumes
X/GBe more than 2 times of bacterium DH5 α that set out.
The selection of the mutant strain of the anti-acetate DA19 of bacillus coli DH 5 alpha of the present invention in turn includes the following steps:
(1) DH5 α bacterial classification is cultivated, obtained DH5 α bacterium liquid;
(2) use
60Co carries out radiation to DH5 α bacterium liquid, and dosage is 200-900Gy;
(3) be that seed carries out batch culture to the DH5 α bacterium liquid after the radiation, incubation time 16-40h;
(4) cultured continuously progressively improves thinning ratio in the culturing process, be lower than 0.10h with initial thinning ratio
-1, final thinning ratio is higher than 0.3h
-1Fed-batch medium A, final OD
600Be not less than 0.3;
(5) continue cultured continuously, progressively improve thinning ratio in the culturing process, with initial thinning ratio 0.05-0.12h
-1, final thinning ratio is not less than 0.25h
-1Fed-batch medium B;
(6) carry out single bacterium colony with the minimum medium flat board that contains glucose and acetate after cultured continuously finishes and separate, can seed selection arrive bacterial classification of the present invention;
Said culture medium A or B are carbon source with glucose, and A contains the acetate of 50-70mmol/L and the peptone of 5g/L, and B contains the acetate of 100-150mmol/L and the peptone of 2.5g/L.Preferred acetate is a kind of in sodium salt, sylvite or the ammonium salt of acetate, changes to avoid directly adding the pH that acetate causes.
According to optimized technical scheme of the present invention,, use once more the nutrient solution after the fed-batch medium B end
60Co carries out radiation, and adopts the method for step (3), (4), (5) and (6) to cultivate, so that the anti-acetate ability of the bacterial classification that is obtained further to be provided.
Bacterial classification of the present invention can be used for expression alien gene.
The advantage of the mutant bacteria of anti-acetate of DH5 α of the present invention is that growth vigor is obvious, also reduces in the generation of acetate simultaneously of anti-acetate ability enhanced, realizes high-density culture easily, and exogenous gene expression efficient improves.
In order to understand content of the present invention better, be described further by following examples.
Embodiment
Embodiment 1
DH5 α bacterial classification one ring that the picking inclined-plane is preserved, [1L contains 30mL YPS substratum in the access 250mL Erlenmeyer flask: Tryptone (Britain Oxiod company) 10g, yeast extract (Britain Oxiod company) 5g, NaCl 10g, pH7.2], 30 ℃, 250r/min shaking table overnight incubation gets first order seed, in the 30mL YPS substratum, the same terms is cultivated 10h as secondary seed to switching first order seed 1mL in the 250mL Erlenmeyer flask.Get secondary seed 10ml in the centrifugal 15min of 5000rpm, abandon supernatant, add the 10ml stroke-physiological saline solution, mixing is with dosage 700Gy's
60Behind the Co radiation 40min, [1L contains 250ml MY substratum: yeast extract (Britain Oxiod company) 4g, glucose 2g, Na in the access 500mL micro glass reactor
2HPO
412H
2O15.12g, KH
2PO
43g, NaCl 0.5g, NH
4Cl 1g, MgSO
47H
2O 0.5g, CaCl
20.011g, 1% vitamins B
10.2mL, glucose 1.8g, Polypepton (big five nutrition of Japan) 5g, sodium acetate 5g, pH7.0], extract nutrient solution simultaneously out.Progressively improve thinning ratio in the cultured continuously, reach 0.351h during 180h
-1, OD
600Nm reaches 0.393.The substratum that add stream this moment switches to substratum B[1L and contains: glucose 1.6g, Polypepton2.5g, sodium acetate 10g, pH7.0, inorganic salt and vitamins B
1Identical with culture medium A], thinning ratio drops to 0.1h
-1, proceed cultured continuously.Progressively improve thinning ratio in the culturing process, 310h reaches 0.263h
-1Get the final nutrient solution in the reactor, suitable dilution is coated with the MAA flat board and [contains glucose 2g among the 1L, sodium acetate 5g, agar 20g, inorganic salt and vitamins B
1Identical with culture medium A], bacterium colony is occurred containing the 5g/L sodium acetate than morning and big single bacterium colony carrying out further anti-acetate ability evaluation, glucose concn is [to contain inorganic salt and vitamins B among the 1L in the 2.2g/L MA substratum
1Identical with culture medium A] shake-flask culture 16h, seed selection to mutant strain DA series see Table 1 with DH5 α cultivation results, as shown in Table 1, mutant strain DA series is because the increase of anti-acetate ability, the cell concentration of growing in the MA substratum is much higher than its bacterium DH5 α that sets out, and is the thalline yield Y of benchmark with the glucose that consumes
X/GImproved more than 2 times.
Table 1
Strain name DH5 α DA19 DA42 DA50 DA52 DA53
Cell concn/(g/L) 0.178 0.517 0.499 0.492 0.466 0.481
Y
X/G a/(g/g) 0.063 0.216 0.207 0.204 0.191 0.199
When the cultured continuously of fed-batch medium B finishes among the embodiment 1, get nutrient solution 10ml, use dosage 700Gy
60Co is irradiation once more, carries out cultured continuously again, and method is with embodiment 1, but fed-batch medium C[1L contains: glucose 1.6g, sodium acetate 10g, pH7.0, inorganic salt and vitamins B
1Identical with culture medium A], initial thinning ratio is 0.082h
-1, progressively improving thinning ratio, 176h reaches 0.158h
-1, this moment OD
600Nm is 0.030, finishes cultured continuously.Get the final nutrient solution in the reactor, suitably dilution is coated with MAB flat board [sodium acetate 10g among the 1L, other component content is identical with the MAA flat board], and choosing colony occurs early and big single bacterium colony.Containing the 10g/L sodium acetate, glucose concn is [to contain inorganic salt and vitamins B among the 1L in the 5g/L MA substratum
1Identical with culture medium A] shake-flask culture 20h, seed selection to mutant strain DB series and embodiment 1 in the cultivation results of the DA19 that obtains see Table 2, because the anti-acetate ability of mutant strain DB series further strengthens, the cell concentration of growing in the MA substratum is the thalline yield Y of benchmark than DA19 height with the glucose that consumes
X/GAlso increase.
Table 2
Strain name DA19 DB1 DB7 DB8 DB15 DB18
Cell concn/(g/L) 0.330 0.449 0.440 0.494 0.503 0.465
Y
X/G a/(g/g) 0.124 0.131 0.136 0.142 0.152 0.151
Embodiment 3
DH5 α that will cultivate as method as described in the embodiment 1 and DA19 first order seed are transferred 1mL respectively in two 500mL Erlenmeyer flasks in the 100mL YPS substratum, and the same terms is cultivated the secondary seed of 10h as fermentor cultivation.Secondary seed is inoculated in the M substratum of 5L fermentor tank respectively, and [amount that contains glucose among the 1L sees Table 3, inorganic salt and vitamins B
1Content identical with culture medium A among the embodiment 1], inoculation back volume 3.2L, 30 ℃, mixing speed and air flow are adjusted according to the variation of dissolved oxygen, more than 20%, control pH6.9～7.0 generate situation with growth and the acetate of investigating in minimum medium with dissolved oxygen in the process that guarantees to cultivate, cultivation results sees Table 3, wherein Y
X/GBe to be the thalline yield of benchmark with the glucose that consumes, Y
A/XIt is the product acetate yield of unit thalline.As shown in Table 3, in minimum medium, the growth of DA19 improves greatly, Y
X/GBe much higher than the bacterium DH5 α that sets out, can reach higher cell concentration.
Table 3
The initial DH5 α DA19 of Portugal
The dense bacterium of grape sugar is dense
1Acetate
1Y
A/X 1,2Y
X/G 1Bacterium is dense
1Acetate
1Y
A/X 1,2Y
X/G 1
Degree/(g/L)/(mmol/)/(mmol/(g/g)/(g/L)/(mmol/L)/(mmol/L (g/g)
g/L) L) /g) g)
2
3 0.454 3.65 8.04 0.203 0.890 1.89 2.63 0.404
10
3 0.468 3.74 8.52 0.050 1.97 19.5 9.89 0.192
10
4，5 - - - - 4.14 4.84 1.17 0.404
102
4,593.2 3.56 0.2571. of----26.2 are peak value; 2. be that the maximum time unit of acetic acid concentration thalline produces the acetate yield; 3. regulate pH with 2mol/LNaOH; 4. use 1mol/L NH
3H
2O regulates pH; 5.1L [1L trace element mixing solutions contains FeSO to add 0.5mL trace element mixing solutions in the substratum
47H
2O 40g, MnSO
4NH
2O 10g, AlCl
36H
2O 10g, CoCl
24g, ZnSO
47H
2O 2g, Na
2MoO
42H
2O 2g, CuCl
22H
2O 1g, H
3BO
40.5g].
Embodiment 4
The plasmid that will have phoA promoter regulation Urogastron (EGF) genetic expression changes set out bacterium DH5 α and mutant bacteria DA19 over to, among the DB15, the secondary seed 1ml that cultivates as the described condition of embodiment 1 inserts 250ml respectively and shakes in the 30ml YPS10G substratum in the bottle [1L YPS substratum contains glucose 10g], 30 ℃, the 250r/min shaking table is cultivated, the centrifugal 10min of 12000rpm, detect the content of EGF in the supernatant liquor with electrophoresis method, Coomassie brilliant blue dyeing, the results are shown in Table 4, as shown in Table 4, the EGF (Y of the EFG level of the mutant strain of anti-acetate DA19 and DB15 expression and unit thalline expression
EGF/X) all be higher than DH5 α, show that the mutant strain of anti-acetate expression EGF efficient improves.
Table 4
Strains DH5α DA19 DB15
EGF/(mg/L) 3.98 7.54 5.72
Y
EGF/X/ (mg/g) mutant strain of the anti-acetate series growth of 4.04 6.22 5.68DH5 α improves, anti-acetate ability strengthens, the thalline yield improves, acetate produces minimizing in culturing process simultaneously, easily realize high-density culture, be used for expression alien gene efficient and improve.According to mutant bacteria disclosed by the invention and embodiment, relevant technologies personnel are the anti-acetate mutant strain of seed selection DH5 α easily, is applied in the production of highly dense fermentation and exogenous genes products.
Claims (6)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102870710A (en) * | 2012-09-14 | 2013-01-16 | 吴江市水产养殖有限公司 | Radiation breeding aid for fishes |
| CN105002128A (en) * | 2015-08-31 | 2015-10-28 | 农业部沼气科学研究所 | Zymomonas mobilis with resistance to acetic acid of high concentration and application thereof |
-
2003
- 2003-06-23 CN CN 03129455 patent/CN1227355C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102870710A (en) * | 2012-09-14 | 2013-01-16 | 吴江市水产养殖有限公司 | Radiation breeding aid for fishes |
| CN105002128A (en) * | 2015-08-31 | 2015-10-28 | 农业部沼气科学研究所 | Zymomonas mobilis with resistance to acetic acid of high concentration and application thereof |
| CN105002128B (en) * | 2015-08-31 | 2018-09-25 | 农业部沼气科学研究所 | A kind of zymomonas mobilis of resisting high-concentration acetic acid and its application |
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