CN114107157A - Construction and application of genetically engineered bacterium for producing N-acetylglucosamine - Google Patents
Construction and application of genetically engineered bacterium for producing N-acetylglucosamine Download PDFInfo
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- CN114107157A CN114107157A CN202111454285.2A CN202111454285A CN114107157A CN 114107157 A CN114107157 A CN 114107157A CN 202111454285 A CN202111454285 A CN 202111454285A CN 114107157 A CN114107157 A CN 114107157A
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- acetylglucosamine
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- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 title claims abstract description 52
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 title claims abstract description 51
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 title claims abstract description 50
- 229950006780 n-acetylglucosamine Drugs 0.000 title claims abstract description 50
- 241000894006 Bacteria Species 0.000 title claims abstract description 32
- 238000010276 construction Methods 0.000 title abstract description 9
- 241000588724 Escherichia coli Species 0.000 claims abstract description 24
- 238000000855 fermentation Methods 0.000 claims abstract description 23
- 230000004151 fermentation Effects 0.000 claims abstract description 23
- 238000010353 genetic engineering Methods 0.000 claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 108090001031 Glutamine-fructose-6-phosphate transaminase (isomerizing) Proteins 0.000 claims abstract description 8
- 108010054451 glucosamine acetyltransferase Proteins 0.000 claims abstract description 8
- 102000004894 Glutamine-fructose-6-phosphate transaminase (isomerizing) Human genes 0.000 claims abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 5
- 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 abstract description 5
- 239000008103 glucose Substances 0.000 claims abstract description 5
- 102000004190 Enzymes Human genes 0.000 claims abstract description 4
- 102100037579 D-3-phosphoglycerate dehydrogenase Human genes 0.000 claims abstract description 3
- 108010038555 Phosphoglycerate dehydrogenase Proteins 0.000 claims abstract description 3
- 230000037353 metabolic pathway Effects 0.000 claims abstract description 3
- 238000012258 culturing Methods 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000013604 expression vector Substances 0.000 claims description 10
- 239000013612 plasmid Substances 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 101150117187 glmS gene Proteins 0.000 claims description 7
- 101150100121 gna1 gene Proteins 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000012880 LB liquid culture medium Substances 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 239000011573 trace mineral Substances 0.000 claims description 4
- 235000013619 trace mineral Nutrition 0.000 claims description 4
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims description 3
- 102000018120 Recombinases Human genes 0.000 claims description 3
- 108010091086 Recombinases Proteins 0.000 claims description 3
- 235000019270 ammonium chloride Nutrition 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 230000006801 homologous recombination Effects 0.000 claims description 3
- 238000002744 homologous recombination Methods 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 230000037361 pathway Effects 0.000 claims description 3
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical group [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 3
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 3
- 235000011151 potassium sulphates Nutrition 0.000 claims description 3
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 claims description 2
- 230000015556 catabolic process Effects 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 9
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 229920002101 Chitin Polymers 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
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- 101150056133 GNPNAT1 gene Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
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- 101100456009 Bacillus subtilis (strain 168) manR gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
- AVVWPBAENSWJCB-GASJEMHNSA-N D-mannofuranose Chemical compound OC[C@@H](O)[C@H]1OC(O)[C@@H](O)[C@H]1O AVVWPBAENSWJCB-GASJEMHNSA-N 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
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- 101100459630 Escherichia coli (strain K12) nagC gene Proteins 0.000 description 1
- 206010016946 Food allergy Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102100023951 Glucosamine 6-phosphate N-acetyltransferase Human genes 0.000 description 1
- 241000208341 Hedera Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- 101150100212 Manf gene Proteins 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 101100031706 Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) nagF gene Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
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- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 150000002303 glucose derivatives Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 238000001823 molecular biology technique Methods 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 101150113739 nagR gene Proteins 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000007111 proteostasis Effects 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
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- 229910052725 zinc Inorganic materials 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
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Abstract
The invention discloses a construction method and application of a genetic engineering bacterium for producing N-acetylglucosamine, wherein the engineering bacterium is a recombinant escherichia coli constructed by introducing genes for coding glucosamine synthetase, glucosamine acetyltransferase and phosphoglycerate dehydrogenase into escherichia coli to express, and knocking out genes of metabolic pathway enzymes for decomposing N-acetylglucosamine in the escherichia coli; the constructed engineering strain is fermented and cultured by taking glucose as a substrate to synthesize the N-acetylglucosamine. The engineering bacteria synthesized by the method have high fermentation level of N-acetylglucosamine, and less accumulation of byproducts, and have industrial production potential.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for producing N-acetylglucosamine by applying genetic engineering transformation to escherichia coli to construct genetically engineered bacteria.
Background
N-acetylglucosamine (GlcNAc, CAS 134451-94-8) is a derivative of glucose, has reducibility, and is widely present in various organisms. Including bacteria, yeast, filamentous fungi, plants and animals, with the highest levels in the exoskeletons of marine life species animals. In the aspect of medicine, the N-acetylglucosamine can be used as an anti-inflammatory drug for treating rheumatic arthritis, can also improve the endoplasmic reticulum protein homeostasis and prolong the cell life. In food, N-acetylglucosamine is used as dietary supplement and has effects of protecting cartilage tissue and osteoarthritis. In addition, the compound can also be applied to cosmetics and feed additives, and has wide application.
The conventional production methods of N-acetylglucosamine mainly include a chitin hydrolysis method and an enzymatic conversion method, and the most important production method is the chitin hydrolysis method, but the methods have many problems. The raw materials of the hydrolysis method are shrimp shells or crab shells and the like, which may have allergens and easily cause allergic reactions of people with seafood allergy; secondly, as the demand of the market for N-acetylglucosamine is increasing, chitin as a limited resource may cause a phenomenon of short supply and short demand; moreover, in the hydrolysis process, a large amount of acid and alkali is used, which causes serious environmental pollution. The enzymolysis method has the problems of limited raw materials, high cost of the chitinase, long conversion time, low efficiency and the like, and is not suitable for industrial production.
Escherichia coli (e.coli) is a gram-negative, facultative anaerobic bacterium, and due to its short growth cycle, easy culture, strong metabolic plasticity, and abundant biochemical and physiological backgrounds, it becomes one of the best hosts for metabolic engineering and synthetic biology, and is also one of the most important organisms in industry. Therefore, the work of producing the N-acetylglucosamine by using escherichia coli fermentation is carried out at home and abroad, a series of metabolic engineering bacteria are constructed by using metabolic engineering, and the production process for producing the N-acetylglucosamine is obtained by optimizing fermentation parameters.
Disclosure of Invention
The invention aims to provide a construction method of a genetically engineered bacterium for producing N-acetylglucosamine and application of the genetically engineered bacterium in producing the N-acetylglucosamine.
In order to solve the technical problems, the invention provides a novel construction method and application of genetically engineered bacteria for producing N-acetylglucosamine, wherein the genetically engineered bacteria are stable in fermentation, long in period and high in product conversion rate. The genetic engineering bacteria firstly knock out an N-acetylglucosamine catabolism utilization path through RED homologous recombination technology, and then introduce the genes for expressing glucosamine synthetase, glucosamine acetyltransferase and phosphoglycerate dehydrogenase into escherichia coli for expression by virtue of an exogenous expression vector.
According to a preferred embodiment of the invention, said E.coli original strain
Is purchased from China center for culture collection and management of industrial microorganisms.
The glucosamine synthetase gene is derived from Escherichia coli W3110 and is synthesized by whole gene.
The glucosamine acetyltransferase gene is derived from saccharomycete S288C and is synthesized by whole gene.
The glucosamine synthetase and the glucosamine acetyltransferase gene are introduced into escherichia coli for expression, and the two genes are cloned to an expression vector and then expressed in the escherichia coli in a plasmid mode.
The construction method of the high-yield N-acetylglucosamine genetic engineering bacteria comprises the following specific steps: knocking out manXYZ and nagBACDE gene clusters in escherichia coli, respectively cloning glmS and GNA1 genes onto an expression vector, and transferring the expression vector into the escherichia coli with the manXYZ and nagBACDE gene clusters knocked out to obtain the high-yield N-acetylglucosamine genetic engineering bacteria. The construction method comprises the following more specific steps:
(1) knocking out gene clusters of manXYZ and nagBACDE of escherichia coli to obtain inactivated strains of the gene clusters manXYZ and nagBACDE;
(2) synthesizing a glmS gene (SEQ ID NO.18) encoding glucosamine synthetase and a GNA1 gene (SEQ ID NO.17) encoding glucosamine acetyltransferase all genes, and cloning to an expression vector;
(3) and (3) transforming the double-gene expression vector obtained in the step (2) into the strain obtained in the step (1) to obtain the N-acetylglucosamine production genetic engineering bacteria.
In the step (1), the knockout is realized by expressing RED recombinase by using pKD46 plasmid through a RED homologous recombination system.
In the step (2), the expression vector comprises: plasmid pBV 220.
The invention also discloses an application of the genetically engineered bacterium for producing N-acetylglucosamine, namely the genetically engineered bacterium for producing N-acetylglucosamine is used for producing N-acetylglucosamine, and the production method comprises the following steps:
(1) selecting a single colony of the N-acetylglucosamine-producing genetic engineering bacteria, placing the single colony in an LB liquid culture medium, and aerobically culturing at 30-35 ℃ and 220rpm for 8-10 hours; among them, it is preferable to culture at 31 to 33 ℃;
(2) inoculating the activated bacterial liquid into a seed culture medium, and aerobically culturing at 30-35 ℃ and 220rpm for 10-15 hours; wherein, the culture is preferably carried out at 31-33 ℃ for 12-14 hours;
(3) inoculating the cultured seed liquid into a fermentation culture medium, and performing fermentation culture at 30-35 ℃ and 220 rpm; among them, it is preferable to culture at 31 to 33 ℃;
(4) culturing until the thallus concentration OD600 is 0.5, heating to 35-40 deg.C, and continuing culturing until fermentation is finished. Wherein, the culture is preferably carried out at 36-38 ℃ for 22-25 hours;
the seed culture medium in the step (2) is an LB liquid culture medium, and the formula is as follows:
5g/L of yeast powder, 10g/L of tryptone and 10g/L of sodium chloride.
The formula of the fermentation medium in the step (3) is as follows:
1-5g/L potassium sulfate, 1-5g/L magnesium sulfate, 5-10g/L disodium hydrogen phosphate, 1-5g/L dipotassium hydrogen phosphate, 1-5g/L monopotassium phosphate, 4-8g/L ammonium chloride, 0.5-3g/L sodium chloride, 4-8g/L citric acid, 0.01-50mg/L trace elements and 1-100g/L glucose. The trace elements include: calcium, manganese, zinc, iron, aluminum and molybdenum.
The invention constructs a new metabolic pathway for producing N-acetylglucosamine in Escherichia coli (as shown in figure 1), enhances the expression of rate-limiting enzyme genes in the synthetic pathway of N-acetylglucosamine, blocks genes causing the consumption and backflow of N-acetylglucosamine, prevents the consumption and backflow of N-acetylglucosamine, and enables the engineering strain to accumulate N-acetylglucosamine. The method can obtain the genetic engineering escherichia coli which can synthesize the N-acetylglucosamine by using glucose, and meanwhile, the accumulation of byproducts is less, and the genetic engineering escherichia coli has the potential of industrial production.
Drawings
FIG. 1 is a schematic diagram showing the construction of the N-acetylglucosamine anabolism pathway in Escherichia coli.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the embodiments.
The experimental procedures used in the following examples are all conventional ones unless otherwise specified. Reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
Example 1 knock-out of E.coli
manXYZ and nagBACDE gene cluster
1. Coli according to E.coli
Genome sequence (GenBank No. CP060121) and plasmid pKD3, design primer:
man-P1：CCGAATTCCGATATCAAAGGAGGTAGCAAGT(SEQ ID NO.1)；
man-P2：CCGAGCTCCTATTAACAATAATACGGGAGA(SEQ ID NO.2)；
man-P3：CCCCTGCAGGGGCGTCTATCCTC(SEQ ID NO.3)；
man-P4：CAAGCTTCGATATCCTTCCGACAAGTTCAT(SEQ ID NO.4)；
man-P5：CCGAGCTCGTGTAGGCTGGAGCTGCTTC(SEQ ID NO.5)；
man-P6：CCCCTGCAGATGGGAATTAGCCATGGTCC(SEQ ID NO.6)；
nag-P1：CGGAATTCTGTTGGTTGTAGAGAGGTTGC(SEQ ID NO.7)；
nag-P2：AGCAGCTCCAGCCTACACCCCTGATTTCGTGATTGTTG(SEQ ID NO.8)；
nag-P3：GTGTAGGCTGGAGCTGCTTC(SEQ ID NO.9)；
nag-P4：ATGGGAATTAGCCATGCTCC(SEQ ID NO.10)；
nag-P5：ACCATGGCTAATTCCCATTGCTGGTTATGGGTGTTGTCT(SEQ ID NO.11)；
nag-P6：CCCAAGCTTGGAAACGGGTGTGTACTGTGGTGTGGCT(SEQ ID NO.12)；
coli E.coli by using primers man-P1, nag-P1 and the like
The genome sequence (GenBank No. CP060121) and the plasmid pKD3 are used as templates, commercial PCR reagents are utilized, and DNA fragments are obtained through PCR amplification and purified for standby.
And (3) PCR reaction system: 25. mu.L of Taq enzyme, 1.5. mu.L each of primers (10. mu. mol/L), and 1. mu.L of template.
And (3) PCR reaction process: 10min at 98 ℃, 15min at 55 ℃, 5sec at 72 ℃ and 30 cycles.
The obtained PCR product was subjected to agarose gel electrophoresis, and significant bands were present at about 2.0kb and 3.0 kb.
2. The RED recombinase expression plasmid vector pKD46 is transformed into Escherichia coli BL21(DE3) by electric shock transformation to obtain strain BL21(DE3)/pKD 46.
3. Competent cells were prepared by adding 1% L-arabinose to LB medium and shake-culturing the strain BL21(DE3)/pKD46 at 30 ℃ until OD600 reached 0.5. And (3) electrically transforming the DNA fragment prepared in the step (1) into the competent cell, and coating a chloramphenicol resistant plate to obtain a transformant.
4. Transformants were picked and identified by colony PCR with the following primers:
manF：GCTGTTAGGCGAGCAGGAAA(SEQ ID NO.13)；
manR：CCAGACATTGGCGAAGAAAA(SEQ ID NO.14)；
nagF：TTCGTGGGCGAGAATGGC(SEQ ID NO.15)；
nagR：CGATGATCTGACGGATAAAGT(SEQ ID NO.16)；
the colony of the selected transformant can be amplified by PCR to obtain a colony with a band of about 1.0kb, namely a manXYZ and nagBACDE double-gene cluster inactivated strain BL21(DE 3)/delta man and delta nag.
Example 2 construction of the glmS, GNA1 Gene Dual expression vector pBV220-glmS-GNA1
1. The GNA1 Gene was synthesized from the whole Gene sequence (NCBI Gene ID: 850529) with EcoR I and BamH I sites added at both ends, the specific sequence being shown below (SEQ ID NO. 17).
2. The glmS gene was synthesized from the entire genome sequence of E.coli W3110 with BamH I sites added to both ends, the specific sequence is shown below (SEQ ID NO. 18).
3. The GNA1 and glmS genes were ligated to EcoR I, BamH I and BgI II sites of pBV220 (purchased from vast Ling plasmid platform), respectively, using conventional molecular biology techniques to generate the vector pBV220-glmS-GNA 1.
Example 3 expression of the glmS, GNA1 Gene in BL21(DE3)/Δ man, Δ nag by plasmid
1. Coli strain DH5 α containing vector pBV220-glmS-GNA1 was cultured overnight in liquid LB medium and plasmid pBV220-glmS-GNA1 was extracted.
2. Culturing Escherichia coli strain BL21(DE 3)/delta man and delta nag, preparing competent cells, and electrically shocking and transforming plasmid vector pBV220-glmS-GNA1 into the strain to obtain genetic engineering strain BL21(DE 3)/delta man and delta nag/pBV220-glmS-GNA1, namely the genetic engineering strain capable of producing N-acetylglucosamine.
EXAMPLE 4 fermentation experiments with engineered strains
1. Seed and fermentation medium (1L):
seed culture medium: 5g of yeast powder, 10g of tryptone and 10g of sodium chloride;
fermentation medium: 2g of potassium sulfate, 3g of magnesium sulfate, 7g of disodium hydrogen phosphate, 4g of dipotassium hydrogen phosphate, 4g of monopotassium phosphate, 5g of ammonium chloride, 1g of sodium chloride, 5g of citric acid, 1mL of trace elements and 25mL of glucose.
Wherein, the microelements are: 60mg/L of calcium chloride; 10mg/L of manganese sulfate; 50mg/L of zinc sulfate; 5mg/L of ferrous sulfate; 5mg/L of aluminum trichloride; 3mg/L of sodium molybdate; boric acid 0.5 mg/L.
2. Fermentation process
(1) Picking single colonies of BL21(DE 3)/delta man,. DELTA.nag/pBV 220-glmS-GNA1-serA in 4mL LB tube, culturing at 32 ℃ and 220rpm for 8-10 hours;
(2) inoculating the activated bacterial liquid into 50mL of seed culture medium according to the proportion of 1%, and culturing for 13 hours at 32 ℃ and 220 rpm;
(3) inoculating the cultured seed solution to 50mL of fermentation medium according to the proportion of 1%, and performing fermentation culture at 32 ℃ and 220 rpm;
(4) culturing until the thallus concentration OD600 is 0.5, heating to 37 deg.C, and continuing culturing until fermentation is finished.
EXAMPLE 5 determination of N-acetylglucosamine concentration in fermentation broth
Diluting the fermentation liquor to a proper multiple, and detecting by adopting HPLC (high performance liquid chromatography), wherein the detection conditions are as follows:
and (3) detecting the column: hedera NH 2;
column temperature: 30 ℃;
mobile phase: a mixed solution (65: 35) of acetonitrile and phosphate buffer solution, wherein the flow rate is 1 mL/min;
detection wavelength: 195 nm;
through detection, after 24 hours of fermentation, the concentration of the N-acetylglucosamine in the fermentation liquid can reach 10 g/L.
The results show that the genetically engineered bacteria constructed by the metabolic engineering technology have the capability of producing the N-acetylglucosamine and have the potential of industrial production.
While the preferred embodiments of the present invention have been illustrated and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. A genetically engineered bacterium for producing N-acetylglucosamine, which is characterized in that: the engineering bacteria are recombinant escherichia coli constructed by introducing genes encoding glucosamine synthetase, glucosamine acetyltransferase and phosphoglycerate dehydrogenase into escherichia coli for expression and knocking out genes of metabolic pathway enzymes for decomposing N-acetylglucosamine in the escherichia coli.
2. The genetically engineered bacterium for producing N-acetylglucosamine according to claim 1, wherein: the glucosamine synthetase gene is derived from Escherichia coli W3110.
3. The genetically engineered bacterium for producing N-acetylglucosamine according to claim 1, wherein: the glucosamine acetyltransferase gene is derived from yeast S288C.
4. The genetically engineered bacterium for producing N-acetylglucosamine according to claim 1, wherein: the glmS gene of glucosamine synthetase shown as SEQ ID NO.18 and the GNA1 gene of glucosamine acetyltransferase shown as SEQ ID NO.17 are transformed into Escherichia coli with genes manXYZ and nagBACDE of N-acetylglucosamine catabolism utilization pathway enzyme knocked out through expression vectors for expression.
5. The genetically engineered bacterium for producing N-acetylglucosamine according to claim 4, wherein: the expression vector is plasmid pBV 220.
6. The genetically engineered bacterium for producing N-acetylglucosamine according to claim 4, wherein: the knockout is realized by expressing RED recombinase by pKD46 plasmid through a RED homologous recombination system.
7. The genetically engineered bacterium for producing N-acetylglucosamine according to claim 4, wherein: the host cell is Escherichia coli BL21(DE 3).
8. Use of the genetically engineered bacterium producing N-acetylglucosamine according to any one of claims 1 to 7 in the production of N-acetylglucosamine, wherein the method for producing N-acetylglucosamine comprises:
(1) selecting a single colony of the N-acetylglucosamine-producing genetic engineering bacteria, placing the single colony in an LB liquid culture medium, and aerobically culturing at 30-35 ℃ and 220rpm for 8-10 hours;
(2) inoculating the activated bacterial liquid into a seed culture medium, and aerobically culturing at 30-35 ℃ and 220rpm for 10-15 hours;
(3) inoculating the cultured seed liquid into a fermentation culture medium, and performing fermentation culture at 30-35 ℃ and 220 rpm;
(4) culturing until the bacterial concentration OD600When the temperature is 0.5 ℃, raising the temperature to 35-40 ℃ and continuing culturing until the fermentation is finished.
9. The genetically engineered bacterium for producing N-acetylglucosamine according to claim 12, wherein: the genetic engineering strain seed culture medium and the fermentation culture medium are LB liquid culture medium and salt culture medium, wherein the formula of the salt culture medium is potassium sulfate 1-5g/L, magnesium sulfate 1-5g/L, disodium hydrogen phosphate 5-10g/L, dipotassium hydrogen phosphate 1-5g/L, potassium dihydrogen phosphate 1-5g/L, ammonium chloride 4-8g/L, sodium chloride 0.5-3g/L, citric acid 4-8g/L, trace elements 0.01-50mg/L and glucose 1-100 g/L.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602627A (en) * | 2013-11-25 | 2014-02-26 | 武汉中科光谷绿色生物技术有限公司 | Novel N-acetylneuraminic acid-producing escherichia coli engineering bacteria as well as construction method and application thereof |
| CN104059872A (en) * | 2014-07-16 | 2014-09-24 | 华东理工大学 | High-yield N-acetylglucosamine metabolic engineering bacterium, as well construction method and applications thereof |
| CN104195094A (en) * | 2014-08-01 | 2014-12-10 | 张帆 | Bacillus subtilis for producing N-acetylglucosamine as well as construction method and application of bacillus subtilis |
| CN104293724A (en) * | 2014-09-22 | 2015-01-21 | 上海工业生物技术研发中心 | Genetically engineered bacteria for efficiently producing N-acetylglucosamine |
| CN104498517A (en) * | 2014-11-29 | 2015-04-08 | 滨州市金朗生物科技有限公司 | Construction and application methods of escherichia coli with high yield of producing N-acetylglucosamine |
| CN109929791A (en) * | 2019-04-15 | 2019-06-25 | 扬州日兴生物科技股份有限公司 | It is a kind of accumulate Glucosamine recombination bacillus coli and its application |
-
2021
- 2021-12-01 CN CN202111454285.2A patent/CN114107157A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602627A (en) * | 2013-11-25 | 2014-02-26 | 武汉中科光谷绿色生物技术有限公司 | Novel N-acetylneuraminic acid-producing escherichia coli engineering bacteria as well as construction method and application thereof |
| CN104059872A (en) * | 2014-07-16 | 2014-09-24 | 华东理工大学 | High-yield N-acetylglucosamine metabolic engineering bacterium, as well construction method and applications thereof |
| CN104195094A (en) * | 2014-08-01 | 2014-12-10 | 张帆 | Bacillus subtilis for producing N-acetylglucosamine as well as construction method and application of bacillus subtilis |
| CN104293724A (en) * | 2014-09-22 | 2015-01-21 | 上海工业生物技术研发中心 | Genetically engineered bacteria for efficiently producing N-acetylglucosamine |
| CN104498517A (en) * | 2014-11-29 | 2015-04-08 | 滨州市金朗生物科技有限公司 | Construction and application methods of escherichia coli with high yield of producing N-acetylglucosamine |
| CN109929791A (en) * | 2019-04-15 | 2019-06-25 | 扬州日兴生物科技股份有限公司 | It is a kind of accumulate Glucosamine recombination bacillus coli and its application |
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