CN113481138A - Engineering strain, preparation method thereof and method for producing glycollic acid by efficiently utilizing ethylene glycol - Google Patents
Engineering strain, preparation method thereof and method for producing glycollic acid by efficiently utilizing ethylene glycol Download PDFInfo
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- CN113481138A CN113481138A CN202110843255.4A CN202110843255A CN113481138A CN 113481138 A CN113481138 A CN 113481138A CN 202110843255 A CN202110843255 A CN 202110843255A CN 113481138 A CN113481138 A CN 113481138A
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- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 title claims abstract description 162
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 10
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 26
- 101150081631 aldA gene Proteins 0.000 claims abstract description 15
- 241000588724 Escherichia coli Species 0.000 claims description 30
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 10
- 241000589232 Gluconobacter oxydans Species 0.000 claims description 9
- 238000000855 fermentation Methods 0.000 claims description 8
- 230000004151 fermentation Effects 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims 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 description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 241000221198 Basidiomycota Species 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 3
- 241000186016 Bifidobacterium bifidum Species 0.000 claims 2
- 229920003023 plastic Polymers 0.000 abstract description 6
- 239000004033 plastic Substances 0.000 abstract description 6
- 239000007857 degradation product Substances 0.000 abstract description 3
- 239000002699 waste material Substances 0.000 abstract description 3
- MMXZSJMASHPLLR-UHFFFAOYSA-N pyrroloquinoline quinone Chemical compound C12=C(C(O)=O)C=C(C(O)=O)N=C2C(=O)C(=O)C2=C1NC(C(=O)O)=C2 MMXZSJMASHPLLR-UHFFFAOYSA-N 0.000 description 17
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 14
- 101150030625 fucO gene Proteins 0.000 description 12
- 239000002609 medium Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 241000589776 Pseudomonas putida Species 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 3
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 3
- 101100098786 Bacillus subtilis (strain 168) tapA gene Proteins 0.000 description 3
- 101100321116 Escherichia coli (strain K12) yqhD gene Proteins 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 2
- 239000005977 Ethylene Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108020005199 Dehydrogenases Proteins 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 241000320117 Pseudomonas putida KT2440 Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000003144 genetic modification method Methods 0.000 description 1
- -1 lanthanide metals Chemical class 0.000 description 1
- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 150000003138 primary alcohols Chemical class 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
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Abstract
The invention relates to the technical field of engineering strains, in particular to an engineering strain, a preparation method thereof and a method for producing glycollic acid by efficiently utilizing ethylene glycol. The engineering strain is obtained by transferring a Gox0313 gene into a chassis strain and overexpressing an aldA gene. The engineering strain can efficiently utilize the ethylene glycol to produce the glycolic acid, and provides an effective solution for the reutilization of waste plastic degradation products.
Description
Technical Field
The invention relates to the technical field of engineering strains, in particular to an engineering strain, a preparation method thereof and a method for producing glycollic acid by efficiently utilizing ethylene glycol.
Background
Ethylene Glycol (EG) is an important depolymerizing monomer in the degradation process of PET plastics, and the metabolism of EG is more diverse than that of TPA (TPA), a degradation product. Currently, the pathway responsible for the conversion of EG to glycolic acid has been identified in a variety of organisms. These initial steps are catalyzed by dehydrogenases with broad specificity, involved in the metabolism of short-chain alcohols and aldehydes. In Pseudomonas putida, the initial reaction is catalyzed by periplasmic alcohol dehydrogenase, pedE, and pedH, which are isoenzymes whose activities depend on pyrroloquinoline quinone (PQQ) and require catalysis by metal ions to function, and are pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs), in which pedE uses calcium ions as a cofactor and pedH uses lanthanide metals as a cofactor, and it has been reported that PQQ-ADHs are successfully expressed in Escherichia coli.
At present, most of the reported microorganisms with glycol utilization capability are not model organisms, and the genetic background and the genetic modification method are not clear. Therefore, there is a need to construct a model organism that can efficiently utilize ethylene glycol to produce glycolic acid, while being applicable to industrial applications where plastics are degraded and converted to glycolic acid, providing an effective solution for the reuse of waste plastics.
Disclosure of Invention
In view of the above, the invention provides an engineering strain, a preparation method thereof and a method for producing glycolic acid by efficiently utilizing ethylene glycol. The engineering strain can efficiently utilize ethylene glycol to produce glycolic acid, and provides an effective solution for recycling waste plastic degradation products.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an engineering strain, which is obtained by transferring a Gox0313 gene into chassis bacteria and overexpressing an aldA gene.
In the present invention, the pedE and the PQQ enzyme cluster, which are cofactors of calcium ions, are selected as foreign genes to be introduced into E.coli. Medium-chain alcohol dehydrogenase Gox0313 has been identified in Gluconobacter oxydans, and has been successfully used in Escherichia coli to realize heterologous expression and protein purification, and Gox0313 has a wide substrate spectrum and can oxidize various primary alcohols. In addition, Escherichia coli MG1655 having propylene glycol-utilizing ability was obtained through laboratory evolution and identified to have a mutation of the propylene glycol oxidoreductase gene fucO into fucO (I7L/L8V). The invention screens the pedE and PQQ genes from pseudomonas putida, the Gox0313 gene from gluconobacter oxydans and the mutant enzyme fucO (I7L/L8V) of Escherichia coli MG1655, respectively, and introduces the genes into wild Escherichia coli MG1655 to obtain Escherichia coli MG1655-Gox0313 capable of efficiently utilizing glycol, and obtains engineering Escherichia coli MG 1655-Go031x 3-aldA capable of highly producing glycollic acid by over-expressing the glycolaldehyde gene aldA endogenously utilized by the Escherichia coli.
Preferably, the G0x0313 gene is derived from Gluconobacter oxydans.
Preferably, the bacteria of the bottom plate is Escherichia coli.
In a particular embodiment provided by the invention, the basidiomycete is escherichia coli MG 1655.
The invention also provides a construction method of the engineering strain, which comprises transferring the Gox0313 gene into the Chassis bacteria and overexpressing the aldA gene.
Preferably, the Gox0313 gene is derived from Gluconobacter oxydans.
Preferably, the bacteria of the bottom plate is Escherichia coli.
In a particular embodiment provided by the invention, the basidiomycete is escherichia coli MG 1655.
The invention also provides a method for producing glycolic acid by efficiently utilizing ethylene glycol, which comprises the following steps:
and (3) performing fermentation culture on the engineering strain by using ethylene glycol as a carbon source.
Preferably, the shake flask fermentation conditions are: m9 medium without glucose supplemented with 10g/L ethylene glycol as sole carbon source at 37 ℃ and 220 rpm.
The invention provides an engineering strain, a preparation method thereof and a method for producing glycolic acid by efficiently utilizing ethylene glycol. The engineering strain is obtained by transferring a Gox0313 gene into a chassis strain and overexpressing an aldA gene. The invention has the technical effects that:
the invention constructs the escherichia coli capable of producing the glycollic acid by depolymerizing the monomer ethylene glycol with the plastic. The wild escherichia coli MG1655 cannot utilize ethylene glycol as a unique carbon source, the escherichia coli MG1655 is used as a chassis cell, heterologous ethylene glycol dehydrogenase from pseudomonas putida and gluconobacter oxydans and mutant enzyme of the escherichia coli MG1655 are screened and are respectively introduced into the escherichia coli MG1655, the escherichia coli MG1655-Gox0313 capable of efficiently utilizing the ethylene glycol is obtained, the glycolaldehyde gene aldA is utilized through overexpression of escherichia coli endogenesis, the utilization rate of the ethylene glycol is improved, and meanwhile, the engineering escherichia coli MG1655-Gox0313-aldA capable of highly producing glycolic acid is obtained.
Drawings
FIG. 1 shows the growth of wild-type E.coli MG1655 in LB medium supplemented with 10g/L of EG and the use of EG;
FIG. 2 shows the effect of introduction of one or more of the genes pedE, PQQ, aldA into E.coli MG1655 on the ethylene glycol utilization;
FIG. 3 shows the effect of knockout of endogenous genes fucO and/or yqhD on ethylene glycol utilization;
FIG. 4 shows the effect of Gox0313, fucO (I7L/L8V), fucO (I7L/L8V)/aldA gene introduced into E.coli MG1655 on the ability to utilize ethylene glycol;
FIG. 5 shows the growth of E.coli MG1655 in M9 medium lacking glucose and the use of EG by introducing the Gox0313 gene;
FIG. 6 shows the metabolic pathway of ethylene glycol;
FIG. 7 shows the effect of the introduction of Gox0313 and Gox0313/aldA genes into E.coli MG1655 on the ability to utilize ethylene glycol, and the growth of MG1655-Gox 0313-aldA;
FIG. 8 shows the glycolic acid production rates of MG1655-Gox0313, MG1655-Gox0313-aldA strains.
Detailed Description
The invention discloses an engineering strain, a preparation method thereof and a method for efficiently utilizing glycol to produce glycolic acid, and a person skilled in the art can use the contents to reference the contents and properly improve the process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The gene sequence used by the invention is as follows:
pedE:
GCCACTGCGCCAGCAGCGCCGGCTGCGCCGGCGGGTAAGAGCGTTACCTGGGAGGACATCGCCAATGACCACTTGACCACTAAGGACGTTTTACAGTATGGTATGGGTACGAACGCTCAACGCTGGTCCCCGCTGGCGCAAGTTAACGACCAGAACGTGTTTAAACTAACCCCGGCGTGGTCTTACAGCTTTGGTGACGAGAAACAGCGCGGTCAAGAAAGCCAGGCAATTGTTTCTGATGGTGTTGTCTATGTTACCGGCTCGTACAGCCGTGTCTTCGCCTTGGATGCCAAAACCGGCAAGAGACTGTGGACCTATAACCATCGTCTGCCGGACAACATCCGCCCGTGCTGTGATGTGGTCAACCGTGGTGCGGCGATTTTTGGCGACAAAATTTATTTCGGCACGCTGGACGCGCGCCTTATCGCCCTGGATAAACACACCGGTAAAGTGGTGTGGAATAAAAAGTTTGGCGACCACAGCGCGGGCTACACCATGACCGGGGCACCGGTTCTGATCAAAGACAAGACCTCGGGCAAGGTGTTGCTGATCCACGGTTCTTCCGGTGATGAATTTGGTGTGGTGGGTCAACTGTTCGCTCGTGACCCGGACACTGGCGAAGAGGTTTGGATGCGTCCGTTTGTAGAAGGTCATATGGGTCGCCTGAATGGTAAGGACAGCACCCCGACCGGTGATGTTAAAGCACCGTCCTGGCCTGATGACCCGACGACCGAAACCGGCAAGGTAGAGGCATGGTCTCACGGTGGTGGTGCTCCGTGGCAGTCAGCGAGCTTTGACCCGGAAACCAATACCATTATCGTAGGTGCCGGTAACCCGGGACCGTGGAACACTTGGGCTCGTACCAGTAAAGACGGTAACCCGCATGACTTCGATAGCCTGTATACCAGCGGCCAGGTCGGCGTGGATCCAAGCACCGGTGAGGTGAAATGGTTTTACCAGCATACGCCGAATGATGCGTGGGACTTCTCCGGCAACAACGAACTGGTTTTGTTCGATTACAAAGACAAAAACGGCAATGTGGTTAAAGCGACCGCGCATGCAGATCGTAATGGTTTTTTCTACGTTGTTGACCGTAATAATGGTAAGTTGCAAAACGCGTTTCCGTTCGTGGACAACATTACTTGGGCGAGCCATATCGATCTGAAAACGGGGCGTCCGGTCGAGAACCCGGGACAACGTCCGGCGAAGCCGCTGCCGGGTGAAACGAAGGGCAAGCCGGTGGAGGTCAGCCCGCCTTTCCTGGGCGGCAAGAACTGGAACCCAATGGCATACAGCCAGGATACCGGCCTGTTCTACATTCCGGGTAACCAATGGAAAGAGGAATATTGGACGGAAGAGGTGAATTATAAGAAGGGTAGCGCGTACCTGGGGATGGGCTTTCGTATTAAACGTATGTATGATGATCACGTGGGCACCCTGCGCGCAATGGATCCAACGACCGGTAAGCTTGTTTGGGAGCACAAAGAGCACCTGCCGTTGTGGGCAGGCGTCCTGGCGACCAAGGGCAACTTGGTGTTCACCGGCACTGGCGACGGCTTCTTTAAAGCGTTTGACGCGAAGACTGGCAAGGAGCTTTGGAAATTCCAAACCGGTTCCGGAATCGTCAGCCCGCCGATTACCTGGGAGCAGGATGGCGAGCAGTACATCGGTGTGACCGTTGGTTACGGCGGCGCTGTGCCGTTGTGGGGTGGTGATATGGCAGAACTGACCAAACCGGTTGCCCAAGGTGGCTCCTTCTGGGTTTTCAAAATCCCGTCTTGGGACAATAaGACAGCACAGAGATAA
PQQ:
CCATGGATATGTGGAAGAAACCTGCTTTTATCGATTTACGTCTCGGTCTGGAAGTGACGCTGTACATTTCTAACCGCTAATCGCCCCGCCCGCCGTTCGCGCGGGCATCTTTTTACATTACCCGGTCCGTCTTCATGTTCATTAAAGTCCTCGGTTCCGCCGCCGGCGGCGGTTTCCCGCAATGGAACTGCAACTGCGCCAACTGCCAGGGTCTGCGCGACGGCACGATCCAGGCCAGCGCCCGCACCCAGTCGTCGATCATCGTCAGCGATAACGGTCAGGAGTGGGTGCTGTGCAACGCCTCGCCGGATATCAGTCAGCAGATTGCCCACACGCCCGAGTTAAATAAAGCCGGCGTGCTGCGCGGAACGCATATCGGCGGCATTATTCTCACCGACAGCCAGATCGACCACACCACCGGGTTACTGAGCCTGCGCGAAGGCTGCCCGCACCAGGTGTGGTGCACCCCGGAGGTCCATGAGGATCTCTCCACCGGTTTCCCGGTGTTTACCATGCTGCGACACTGGAACGGCGGCCTGGTGCATCACCCCATCGCGCCGCAGCAGCCTTTTACCGTTGACGCCTGTCCGGATTTGCAGTTTACCGCGGTGCCTATCGCCAGCAACGCGCCGCCCTATTCGCCGTATCGCGACCGGCCGCTGCCGGGCCATAACGTGGCGCTGTTTATCGAAAACCGCCGCAACGGGCAGACGCTGTTCTACGCACCGGGGCTCGGTGAACCGGATAAGACCATTCTGCCATGGCTGCAAAAAGCGGATTGTCTGCTGATCGATGGCACCGTCTGGCAGGATGACGAACTGCAGGCCGCCGGCGTTGGGCGCAATACCGGCCGCGATATGGGCCACCTGGCGCTCGGTGATGAGCACGGGATGATGGCCCTGCTGGCCTCCCTGTCGGCAAAACGTAAAATTCTTATTCATATCAATAACACCAACCCGATCCTTAACGAGCGGTCTCCCCAGCGCCAGGCGCTGACGCAACAGGGGATTGAAGTGAGCTGGGACGGGATGGCTATCACCCTTCAGGATACCGCATGCTGATCACCGACACGCTGTCGCCGCAGGCCTTTGAAGAGGCCCTGCGGGCCAAAGGCGCCTTCTACCATATTCACCACCCTTACCACATTGCCATGCATAACGGCGACGCGACCCGCGAGCAAATTCAGGGCTGGGTGGCGAACCGGTTCTACTACCAGACCACCATTCCGCTGAAAGACGCGGCGATCATGGCCAACTGCCCGGACGCGCAGACCCGGCGCAAATGGGTGCAGCGGATCCTCGACCACGATGGCAGCCATGGCGAGGACGGCGGGATTGAAGCCTGGCTGCGGCTGGGAGAAGCGGTCGGTCTGAGCCGCGACGATCTGCTCAGCGAGCGCCACGTCTTGCCCGGCGTGCGCTTCGCGGTGGATGCCTATCTTAACTTCGCCCGTCGCGCATGCTGGCAGGAGGCGGCCTGCAGCTCTCTGACCGAGCTGTTCGCCCCGCAGATCCATCAGTCGCGCCTCGACAGCTGGCCGCAGCACTACCCGTGGATCAAAGAGGAAGGCTATTTTTATTTCCGCAGCCGTCTGAGCCAGGCCAACCGCGACGTGGAGCATGGCCTGGCGCTGGCGAAGGCCTACTGCGACAGCGCTGAAAAACAGAACCGGATGCTGGAGATCCTGCAGTTTAAGCTCGACATTTTATGGTCGATGCTCGACGCCATGACCATGGCCTATGCCCTGCAGCGCCCGCCCTATCATACGGTCACCGACAAGGCGGCCTGGCACACGACCCGACTGGTGTAACCATGCAAAAAACCTCCATCGTCGCCTTTCGTCGCGGCTACCGTCTGCAGTGGGAAGCTGCCCAGGAGAGCCATGTGATCCTCTATCCGGAGGGAATGGCTAAACTCAATGAGACCGCCGCGGCGATCCTCGAGCTGGTCGATGGCCGGCGCGACGTCGCGGCGATTATCGCCGTGCTCAACGAACGCTTCCCGGAAGCCGGTGGCGTCGATGACGACGTCATCGAGTTCCTGCATATAGCCTGTCAACAGAAGTGGATCACCTGCCGTGAGCCAGAGTAAACCCGCCGTCAATCCGCCGCTGTGGCTGCTGGCGGAGCTAACCTACCGCTGCCCGCTGCAGTGCCCCTACTGTTCGAATCCGCTGGACTTCGCCCAACAGGACAAGGAGCTGACCACCGAACAATGGATCGAGGTCTTTCGCCAGGCGCGGGCGATGGGCAGCGTACAGCTGGGCTTTTCCGGCGGCGAGCCGCTGACCCGTAAAGATCTGCCAGAGCTGATCCGCGCCGCGCGCGACCTCGGATTCTATACCAACCTGATCACCTCCGGCATAGGGCTGACGGAGAGCAAACTCGACGCCTTCAGCGAGGCCGGGCTGGACCATATCCAGATTAGCTTCCAGGCCAGCGATGAGGTGCTCAACGCTGCGCTGGCCGGTAACAAAAAAGCCTTCCAGCAGAAGCTGGCGATGGCCAGGGCGGTAAAAGCGCGCGACTACCCGATGGTGCTCAACTTCGTTCTTCACCGGCACAACATCGATCAGCTGGATAAAATTATCGCGCTGTGCATTGAGCTGGAAGCGGATGACGTTGAGCTGGCCACCTGTCAGTTCTACGGTTGGGCGTTCCTCAATCGCGAGGGGCTGCTGCCGACCCGGGAACAGATCGCCCGCGCCGAGCAGGTGGTCGCCGACTACCGGCAGAAAATGGCCGCCAGCGGCAATCTCACCAACCTGCTGTTTGTCACCCCGGACTATTACGAAGAGCGGCCGAAAGGCTGCATGGGCGGCTGGGGATCGATTTTCCTCAGCGTCACCCCGGAAGGCACTGCGTTGCCGTGCCACAGCGCGCGCCAGCTGCCGGTGGCGTTTCCGTCGGTGCTGGAGCAGAGCCTGGAGTCGATCTGGTACGACTCGTTCGGCTTCAATCGCTACCGCGGGTATGACTGGATGCCGGAGCCGTGCCGCTCCTGTGATGAAAAAGAGAAAGACTTCGGCGGCTGCCGCTGCCAGGCCTTTATGCTGACCGGCAGCGCCGATAACGCCGACCCGGTGTGCAGCAAATCGCCGCATCATCACAAAATCCTCGACGCCCGGCGCGAAGCGGCCTGCAGCGACATAAAAGTCAGCCAGCTGCAGTTCCGCAACCGTACCCGCTCGCAGCTGATCTACAAAACCCGGGACCTGTAATGACGCTGGCGACCCGCACCGTCACGCTGCCGGGCGGCCTGCAGGCCACCCTGGTCCATCAGCCGCATGCCGATCGCGCCGCGGCCCTGGCGCGGGTCGCCGCCGGCAGCCACCACGAACCATCGCGCTTTCCCGGTCTGGCGCACCTGCTGGAGCATCTGCTGTTTTACGGCGGTGAGCGCTACCGGGATGATGACCGGCTGATGGGCTGGGTGCAGCGCCAGGGCGGAAGCGTGAATGCCACCACCCTGGCCCGCCACAGCGCGTTCTTTTTCGAGGTCGCCGCCGATGGTCTGGCTGACGGTGTCGCGCGTCTACAGGAGATGCTGAAGGCGCCGCGGCTGCTCAGGGAAGATATTCAGCGTGAAGTGGCGGTGATTGACGCCGAGTACCGCCTGATCCAGCAGCATGAGCCGTCGCGCCGGGAAGCCGCCGTGCGCCACGCCGCCAGCGCGCCCGCGGCCTTTCGCCGCTTTCAGGTAGGCAGTGCCGACGCGCTGGCGGGCGATCGCCCTGCACTGCAGGCGGCGTTACGTGATTTTCACCGCACCCATTACGTCGCCCGGCGGATGCAGCTCTGGCTGCAAGGGCCGCAGTCGCTGGAGGCGCTCGGCGAGCTGGCGGCCCGTTTCGCCACCGGGCTTGCCGCAGGCGAGGCTCCGCCGCCGGCGCCACCGCTGCGCCTGGGCGAATCCACTGCACTGCAGCTGGCGGTCTCCAGCCAGCCCGCGCTGTGGCGCTGCCCGCTGATCGCCTTAAGTGACAACGTCACGTTATTGCGCGAGTTTTTGCTTGATGAAGCCCCTGGCAGCCTGATGGCCGGGCTGCGCCAGCGTGGCCAGGCGCAAGAGGTGGCGCTGAGCTGGCTGTATCAGGATCGGCACGTCGGCTGGCTGGCGCTGGTCTTCGCCAGCGACCGGCCGGAACAGGTCGACCAGCAGATAACCCACTGGCTGCAGGCGCTACGGCAGACGACGCCAGACCAGCAGCAACACTACTATCAGCTGTCCTGGCGCCGTTTTCAAACGCTGTCGCCCCTCGATCAGCTGCGCCAGCGGGCATTCGGCTTTGCCCCCGGCGGGCCGCCCGTCGGGTTCGCCGATTTTTGCGCCGCCCTGCAGGCCGCCCCCACGGTCACCCTGGCCTGCCAGACCATTTCCCCAGGGGAGCCTGTTGCCACCCAGGGCTTTAGCCTGCCGCTTAGCCGGTGGTGGCGCCGCCCGGTCTCTGACCCGGCGCTGGAATTCGCTTTTTATCCGCAAGCCGCTGGCGGCCTCGCCGCGGAAAACCCGGAGAAAGCCGCGCCGCTGCTCCACCTCCCGTCACCGGGCGAGCCGCCGAGGCTCCTGCTCCGACCGCCCTTCTACTGCTCGCCCGATATGGCCGAAGGGCTGGCGCGCGGGGAACAGCTGCGCCCGCTGCTCGCCGCGCTGCGCCACGCCGGGGGCCGCGGCGAGTGGCATCGCGTCGACGGCAGCTGGCAGTTGCTCCTGCAGTGGCCAGCGTCCGGCCGACGGCCGGTGGCGATTCTGCAGGCCATTATGCGGCAGCTCGCGCTCCCGGTCGCCCCGCTGGCCCCGCCGCCGGAGAGTATTGCTATCCGTCATCTCATGGCTTTGCTCCCCGATCGGTTGGGCGCATCAGAGCTTCAGGAAGGTTGGCTGGCGGCCCTGACCGGCGGCAGCGCAGAGGATGCGCAGTGGATCGCGCGCCAGCTGAGCCTGCTTAGCGCCCGGGTTAACCCGCCGGGCAACCTCTCTGGCACCTGCCGCCGCGGCGTCGAACGGCTGACTTTCCCCGGGGGCGATACAGCGCTGCTGGTCTTTATTCCGCTGCCGGAAGGCGCCTCGCTGGCGGCCCTGCGGGTGCTGGCGCAGCGCTGTGAGCCACTCTTTTTCCAGCGCCTGCGGGTGGAGCAGCAGATCGGCTATGTGGTGAGCTGCCGCTATCAGCGCGTCGCCGATCGCGACGGGCTGCTGATGGCGCTCCAGTCCCCGGATCGCCGCGCCGTGGCGCTGCTGCGCTGTTGCAACACCTTTCTGCGCCAGCTGCCGCCGCTGGATGAAACGACCTTCAGGCCGCTACAGCAGCGCCTGGCCGCACAGGTTCGCGCCCGGACGCCGCCCGAGGCGCAAGCGCTGGCCGCCCTGCGTCAGAAGTATGGTTTAGCGGAGCTGACGCCGCAGGCGGCTGACGCGCTGCGCGTTGAAGAGGTCGACGACCTGGCCTACGAGATGTCCCGCCGTCGCCGCCGCTGGCGGGTACTGTTCACTGCCGGGGATTAACTCGAG
yqhD:
TTAGCGGGCGGCTTCGTATATACGGCGGCTGACATCCAACGTAATGTCATGATTTTCGCCCAGTTGGGTCATGCCGTGCTCTTCCAGTTTTTTCAGCAAAGCCGGGATGGAGCTGCCGTCCAGACCGTAGTCGGAGAGGTGGGTCGGCACGCCTAATTGCTCAAAGAAATTGCGGGTTGCGGCAATCGCGGCGTCAATACGCTCATCATCGGAACCTTCAGTGATGTTCCAGACGCGTTCAGCATATTGCAGCAGCTTAGCGCGCTTGGTATCGCGTTTTTCATTCCACAGTGCAGGCAGGACGATAGCCAGTGTTTGCGCGTGATCCAGACCGTGCATCGCAGTCAGTTCGTGGCCCAGCATATGCGTTGCCCAGTCCTGCGGTACGCCAGCGCCAATCAAACCGTTCAGCGCCTGAGTCGCCGCCCACATGACGTTGGCGCGCACATCGTAGTTTTCTGGCTCTTTCAGGGCTTTCGGACCATCTTCGATTAGCGTCAGCAAAATGCCTTCTGCGAAACGGTCCTGAATTTTGGCATCAACCGGTTTGGTAACATACTGTTCCACGGTGTGTACAAAGGCGTCCACTACGCCGTTAGCCACCTGACGCGGCGGCAGGGTGTAGGTATAAACCGGATCGAGCACGGCAAATACCGGCTGAACATGGGCAGAATGGAACGCCTGCTTGTCGCCTGTGGTTTTACGGGAGATCACCGCGCCTGCGTTGGATTCTGAACCGGTTGCTGGCAGCGTCAGCACACAGCCCATCGGGATGGCGCTTTTAATCTCTTTACCGCCCGTTTGCAGAATGTGCCACGGATCGATATTTTCCGGATAGTTAGCCGCTGCGGCGATAAATTTGGTGCCGTCCAGTACAGAACCGCCGCCAACCGCCAGCAGGAAAGTCACTTTCTGTTCGCGAACCAGTTTCACGGCGTTCATCAGCGTTTCATAAGCCGGGTTTGGCTCAATACCGCCAAATTCCAGCACGTCCATGCCTTTCAGGGCATCCAGAACTTGATCGAGAACGCCGGTTTTTTTCACGCTGCCGCCGCCGTAGGTAATCAATACGCGAGCATCGTGAGGAATTTGTTCGCGTAAACCAGCGATTGCGCCTTTACCAAACAGAATGCGGGTTGGGGTGTGCAGATTAAAGTTGTTCAT
fucO:
ATGGCTAACAGAATGATTCTGAACGAAACGGCATGGTTTGGTCGGGGTGCTGTTGGGGCTTTAACCGATGAGGTGAAACGCCGTGGTTATCAGAAGGCGCTGATCGTCACCGATAAAACGCTGGTGCAATGCGGCGTGGTGGCGAAAGTGACCGATAAGATGGATGCTGCAGGGCTGGCATGGGCGATTTACGACGGCGTAGTGCCCAACCCAACAATTACTGTCGTCAAAGAAGGGCTCGGTGTATTCCAGAATAGCGGCGCGGATTACCTGATCGCTATTGGTGGTGGTTCTCCACAGGATACTTGTAAAGCGATTGGCATTATCAGCAACAACCCGGAGTTTGCCGATGTGCGTAGCCTGGAAGGGCTTTCCCCGACCAATAAACCCAGTGTACCGATTCTGGCAATTCCTACCACAGCAGGTACTGCGGCAGAAGTGACCATTAACTACGTGATCACTGACGAAGAGAAACGGCGCAAGTTTGTTTGCGTTGATCCGCATGATATCCCGCAGGTGGCGTTTATTGACGCTGACATGATGGATGGTATGCCTCCAGCGCTGAAAGCTGCGACGGGTGTCGATGCGCTCACTCATGCTATTGAGGGGTATATTACCCGTGGCGCGTGGGCGCTAACCGATGCACTGCACATTAAAGCGATTGAAATCATTGCTGGGGCGCTGCGAGGATCGGTTGCTGGTGATAAGGATGCCGGAGAAGAAATGGCGCTCGGGCAGTATGTTGCGGGTATGGGCTTCTCGAATGTTGGGTTAGGGTTGGTGCATGGTATGGCGCATCCACTGGGCGCGTTTTATAACACTCCACACGGTGTTGCGAACGCCATCCTGTTACCGCATGTCATGCGTTATAACGCTGACTTTACCGGTGAGAAGTACCGCGATATCGCGCGCGTTATGGGCGTGAAAGTGGAAGGTATGAGCCTGGAAGAGGCGCGTAATGCCGCTGTTGAAGCGGTGTTTGCTCTCAACCGTGATGTCGGTATTCCGCCACATTTGCGTGATGTTGGTGTACGCAAGGAAGACATTCCGGCACTGGCGCAGGCGGCACTGGATGATGTTTGTACCGGTGGCAACCCGCGTGAAGCAACGCTTGAGGATATTGTAGAGCTTTACCATACCGCCTGGTAA
fucO(I7L/L8V):
ATGATGGCTAACAGAATGCTGGTGAACGAAACGGCATGGTTTGGTCGGGGTGCTGTTGGGGCTTTAACCGATGAGGTGAAACGCCGTGGTTATCAGAAGGCGCTGATCGTCACCGATAAAACGCTGGTGCAATGCGGCGTGGTGGCGAAAGTGACCGATAAGATGGATGCTGCAGGGCTGGCATGGGCGATTTACGACGGCGTAGTGCCCAACCCAACAATTACTGTCGTCAAAGAAGGGCTCGGTGTATTCCAGAATAGCGGCGCGGATTACCTGATCGCTATTGGTGGTGGTTCTCCACAGGATACTTGTAAAGCGATTGGCATTATCAGCAACAACCCGGAGTTTGCCGATGTGCGTAGCCTGGAAGGGCTTTCCCCGACCAATAAACCCAGTGTACCGATTCTGGCAATTCCTACCACAGCAGGTACTGCGGCAGAAGTGACCATTAACTACGTGATCACTGACGAAGAGAAACGGCGCAAGTTTGTTTGCGTTGATCCGCATGATATCCCGCAGGTGGCGTTTATTGACGCTGACATGATGGATGGTATGCCTCCAGCGCTGAAAGCTGCGACGGGTGTCGATGCGCTCACTCATGCTATTGAGGGGTATATTACCCGTGGCGCGTGGGCGCTAACCGATGCACTGCACATTAAAGCGATTGAAATCATTGCTGGGGCGCTGCGAGGATCGGTTGCTGGTGATAAGGATGCCGGAGAAGAAATGGCGCTCGGGCAGTATGTTGCGGGTATGGGCTTCTCGAATGTTGGGTTAGGGTTGGTGCATGGTATGGCGCATCCACTGGGCGCGTTTTATAACACTCCACACGGTGTTGCGAACGCCATCCTGTTACCGCATGTCATGCGTTATAACGCTGACTTTACCGGTGAGAAGTACCGCGATATCGCGCGCGTTATGGGCGTGAAAGTGGAAGGTATGAGCCTGGAAGAGGCGCGTAATGCCGCTGTTGAAGCGGTGTTTGCTCTCAACCGTGATGTCGGTATTCCGCCACATTTGCGTGATGTTGGTGTACGCAAGGAAGACATTCCGGCACTGGCGCAGGCGGCACTGGATGATGTTTGTACCGGTGGCAACCCGCGTGAAGCAACGCTTGAGGATATTGTAGAGCTTTACCATACCGCCTGGTAA
Gox0313:
ATGGCGGATACCATGCTGGCGGCGGTGGTGCGCGAATTTGGCAAACCGCTGAGCATTGAACGCCTGCCGATTCCGGATATTAAACCGCATCAGATTCTGGTGAAAGTGGATACCTGCGGCGTGTGCCATACCGATCTGCATGCGGCGCGCGGCGATTGGCCGAGCAAACCGAACCCGCCGTTTATTCCGGGCCATGAAGGCGTGGGCCATATTGTGGCGGTGGGCAGCCAAGTGGGCGATTTTGTGAAAACCGGCGATGTGGTGGGCGTGCCGTGGCTGTATAGCGCGTGCGGCCATTGCGAACATTGCCTGGGCGGCTGGGAAACCCTGTGCGAAAAACAAGATGATACCGGCTATACCGTGAACGGCTGCTTTGCGGAATATGTGGTGGCCGATCCGAACTATGTGGCGCATCTGCCGAGCACCATTGATCCGCTGCAAGCGAGCCCGGTGCTGTGCGCGGGCCTGACCGTGTATAAAGGCCTGAAGATGACCGAAGCCCGCCCGGGTCAGTGGGTGGCGGTGAGCGGTGTGGGCGGCCTGGGTCAGATGGCGGTGCAGTATGCGGTGGCGATGGGCATGAACGTGGTGGCGGTGGATATTGATGATGAAAAACTGGCGACCGCGAAAAAACTGGGCGCGAGCCTGACCGTGAACGCGAAAGATACCGATCCGGCGCGCTTTATTCAACAGCAGATTGGCGGCGCGCACGGCGCCCTGGTGACCGCGGTGGGCCGCACGGCGTTCAGCCAAGCGATGGGCTATGCGCGCCGCGGCGGCACCATTGTGCTGAACGGCCTGCCGCCGGGCGATTTTCCGGTGAGCATTTTTGATATGGTGATGAACGGCACCACCATTCGCGGCAGCATTGTGGGCACCCGCCTGGATATGATTGAAGCGATGGATTTTTTTGCGCGCGGCAAAGTGAAAAGCGTGGTGACCCCGGGCAAACTGGAAAACATTAACACCATTTTTGATGATTTACAGAACGGCCGCCTGGAAGGCCGCACCGTGCTGGATTTTCGCAGCTAA
aldA:
ATGTCAGTACCCGTTCAACATCCTATGTATATCGATGGACAGTTTGTTACCTGGCGTGGAGACGCATGGATTGATGTGGTAAACCCTGCTACAGAGGCTGTCATTTCCCGCATACCCGATGGTCAGGCCGAGGATGCCCGTAAGGCAATCGATGCAGCAGAACGTGCACAACCAGAATGGGAAGCGTTGCCTGCTATTGAACGCGCCAGTTGGTTGCGCAAAATCTCCGCCGGGATCCGCGAACGCGCCAGTGAAATCAGTGCGCTGATTGTTGAAGAAGGGGGCAAGATCCAGCAGCTGGCTGAAGTCGAAGTGGCTTTTACTGCCGACTATATCGATTACATGGCGGAGTGGGCACGGCGTTACGAGGGCGAGATTATTCAAAGCGATCGTCCAGGAGAAAATATTCTTTTGTTTAAACGTGCGCTTGGTGTGACTACCGGCATTCTGCCGTGGAACTTCCCGTTCTTCCTCATTGCCCGCAAAATGGCTCCCGCTCTTTTGACCGGTAATACCATCGTCATTAAACCTAGTGAATTTACGCCAAACAATGCGATTGCATTCGCCAAAATCGTCGATGAAATAGGCCTTCCGCGCGGCGTGTTTAACCTTGTACTGGGGCGTGGTGAAACCGTTGGGCAAGAACTGGCGGGTAACCCAAAGGTCGCAATGGTCAGTATGACAGGCAGCGTCTCTGCAGGTGAGAAGATCATGGCGACTGCGGCGAAAAACATCACCAAAGTGTGTCTGGAATTGGGGGGTAAAGCACCAGCTATCGTAATGGACGATGCCGATCTTGAACTGGCAGTCAAAGCCATCGTTGATTCACGCGTCATTAATAGTGGGCAAGTGTGTAACTGTGCAGAACGTGTTTATGTACAGAAAGGCATTTATGATCAGTTCGTCAATCGGCTGGGTGAAGCGATGCAGGCGGTTCAATTTGGTAACCCCGCTGAACGCAACGACATTGCGATGGGGCCGTTGATTAACGCCGCGGCGCTGGAAAGGGTCGAGCAAAAAGTGGCGCGCGCAGTAGAAGAAGGGGCGAGAGTGGCGTTCGGTGGCAAAGCGGTAGAGGGGAAAGGATATTATTATCCGCCGACATTGCTGCTGGATGTTCGCCAGGAAATGTCGATTATGCATGAGGAAACCTTTGGCCCGGTGCTGCCAGTTGTCGCATTTGACACGCTGGAAGATGCTATCTCAATGGCTAATGACAGTGATTACGGCCTGACCTCATCAATCTATACCCAAAATCTGAACGTCGCGATGAAAGCCATTAAAGGGCTGAAGTTTGGTGAAACTTACATCAACCGTGAAAACTTCGAAGCTATGCAAGGCTTCCACGCCGGATGGCGTAAATCCGGTATTGGCGGCGCAGATGGTAAACATGGCTTGCATGAATATCTGCAGACCCAGGTGGTTTATTTACAGTCTTAA
the reagents or apparatus used in the present invention are commercially available.
The invention is further illustrated by the following examples:
example 1 wild-type E.coli MG1655 unable to utilize EG
The wild-type E.coli MG1655 was cultured in LB medium to which 10g/L of EG was added as a sole carbon source, and shake flask fermentation experiments at 37 ℃ and 220rpm demonstrated that the wild-type E.coli MG1655 could grow in LB medium containing 10g/L of EG, but could not effectively utilize EG in the medium (FIG. 1).
Example 2 screening of ethylene glycol-utilizing foreign Gene to obtain recombinant Escherichia coli having ethylene glycol-utilizing ability
The pedE and PQQ genes derived from Pseudomonas putida KT2440 were introduced into Escherichia coli MG1655, and the culture was carried out by culturing with M9 containing no glucose, and the medium was supplemented with 10g/L of ethylene glycol as a substrate. Experiments prove that the Escherichia coli only introduced with the pedE and PQQ genes cannot utilize ethylene glycol, the concentration of the ethylene glycol is slightly increased in the culture process, and the concentration change possibly caused by the volatilization of a culture medium in the shaking table fermentation process is considered. In order to prevent accumulation of glycolaldehyde and inhibit glycol utilization, the endogenous gene aldA was overexpressed upon introduction of exogenous genes pedE and PQQ, and the concentration of glycol was reduced, but the effect was not significant (fig. 2). Since the endogenous genes of ethylene glycol, fucO and yqhD, may cause transformation of glycolaldehyde into ethylene glycol, further knocking out the endogenous genes, the concentration of ethylene glycol was found to be reduced to a minimum of 7.23g/L (FIG. 3).
Example 3 construction of heterologous pathway for ethylene glycol utilization in E.coli MG1655
The exogenous pathway for introducing Pseudomonas putida into Escherichia coli could not completely utilize ethylene glycol in the medium, and the Gox0313 gene derived from Gluconobacter oxydans and the mutant enzyme fucO (I7L/L8V) of Escherichia coli MG1655 itself were introduced into Escherichia coli MG1655, respectively, and cultured by M9 culture without glucose, and the medium was supplemented with 10g/L of ethylene glycol as a substrate. Wherein, the Escherichia coli introduced with the Gox0313 gene of gluconobacter oxydans can fully utilize 10g/L glycol and OD within 72h600Eventually reaching 11. On the other hand, the ethylene glycol utilization ability of E.coli introduced with mutant enzyme fucO (I7L/L8V) was poor, and on the basis, the ethylene glycol utilization ability of E.coli over-expressing endogenous gene aldA was not improved (FIG. 4, FIG. 5). The mutant enzyme fucO (I7L/L8V) is reported in the literature to be greatly influenced by oxygen flux, and the oxygen concentration needs to be strictly controlled in the fermentation process, so that the cost is increased for the degradation and utilization of EG.
Example 4 overexpression of endogenous genes to optimize glycolic acid production
In order to obtain an engineered escherichia coli strain capable of utilizing ethylene glycol to highly produce glycolic acid, an endogenous gene aldA is overexpressed in an MG1655-Gox0313 strain, the rate of utilizing ethylene glycol by the MG1655-Gox0313-aldA strain is higher than that of the MG1655-Gox0313 strain during 0-46h of culture, the content of ethylene glycol in a culture medium of the MG1655-Gox0313-aldA strain is basically kept stable after 46h of culture, and the yield of glycolic acid is measured, so that the yield of the MG1655-Gox0313 strain reaches 1.19g/L and the yield of the MG1655-Gox0313-aldA strain reaches 3.45g/L which is 2.9 times that of the yield of glycolic acid after 96h of fermentation (fig. 7 and fig. 8).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. An engineering strain is characterized in that the engineering strain is obtained by transferring a Gox0313 gene into a chassis strain and overexpressing an aldA gene.
2. The engineered strain of claim 1, wherein the Gox0313 gene is derived from gluconobacter oxydans.
3. The engineered strain of claim 1, wherein the basidiomycete is escherichia coli.
4. The engineered strain of any one of claims 1 to 3, wherein the basidiomycete is Escherichia coli MG 1655.
5. A construction method of an engineering strain is characterized by comprising the steps of transferring a Gox0313 gene into chassis bacteria and over-expressing an aldA gene.
6. The method for constructing a recombinant vector as claimed in claim 5, wherein the Gox0313 gene is derived from Gluconobacter oxydans.
7. The method according to claim 5, wherein the Bacillus bifidus is Escherichia coli.
8. The method according to any one of claims 5 to 7, wherein the Bacillus bifidus is Escherichia coli MG 1655.
9. A method for producing glycolic acid by efficiently utilizing ethylene glycol is characterized by comprising the following steps:
performing fermentation culture on the engineering strain of any one of claims 1 to 4 by using ethylene glycol as a carbon source.
10. The method of claim 9, wherein the shake flask fermentation conditions are: m9 medium without glucose supplemented with 10g/L EG as the sole carbon source at 37 ℃ and 220 rpm.
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| CN111194352A (en) * | 2017-09-07 | 2020-05-22 | 多伦多大学管理委员会 | Production of glycolate from ethylene glycol and related microbial engineering |
| CN112779197A (en) * | 2019-11-08 | 2021-05-11 | 中国科学院上海高等研究院 | Method for producing ethylene glycol and glycollic acid by using escherichia coli and genetic engineering bacteria |
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| CN111194352A (en) * | 2017-09-07 | 2020-05-22 | 多伦多大学管理委员会 | Production of glycolate from ethylene glycol and related microbial engineering |
| US20200277635A1 (en) * | 2017-09-07 | 2020-09-03 | The Governing Council Of The University Of Toronto | Production of glycolate from ethylene glycol and related microbial engineering |
| CN112779197A (en) * | 2019-11-08 | 2021-05-11 | 中国科学院上海高等研究院 | Method for producing ethylene glycol and glycollic acid by using escherichia coli and genetic engineering bacteria |
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| CN116004488A (en) * | 2022-06-15 | 2023-04-25 | 天津大学 | Recombinant strain and method for producing glycollic acid |
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