CN116004488A - Recombinant strain and method for producing glycollic acid - Google Patents
Recombinant strain and method for producing glycollic acid Download PDFInfo
- Publication number
- CN116004488A CN116004488A CN202210672569.7A CN202210672569A CN116004488A CN 116004488 A CN116004488 A CN 116004488A CN 202210672569 A CN202210672569 A CN 202210672569A CN 116004488 A CN116004488 A CN 116004488A
- Authority
- CN
- China
- Prior art keywords
- escherichia coli
- recombinant
- strain
- coli strain
- genes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 12
- 241000588724 Escherichia coli Species 0.000 claims abstract description 32
- 101150030625 fucO gene Proteins 0.000 claims abstract description 17
- 101100098786 Bacillus subtilis (strain 168) tapA gene Proteins 0.000 claims abstract description 14
- 101100321116 Escherichia coli (strain K12) yqhD gene Proteins 0.000 claims abstract description 14
- 101150081631 aldA gene Proteins 0.000 claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 229960005091 chloramphenicol Drugs 0.000 claims description 11
- 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 claims description 11
- 238000000034 method Methods 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 8
- 241000219194 Arabidopsis Species 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 7
- 238000005516 engineering process Methods 0.000 claims description 7
- 230000006801 homologous recombination Effects 0.000 claims description 7
- 238000002744 homologous recombination Methods 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 241001646716 Escherichia coli K-12 Species 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 3
- 241000589232 Gluconobacter oxydans Species 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 239000013598 vector Substances 0.000 claims description 2
- 230000005526 G1 to G0 transition Effects 0.000 abstract description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 117
- 239000012634 fragment Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 description 7
- 230000015556 catabolic process Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- WGCNASOHLSPBMP-UHFFFAOYSA-N Glycolaldehyde Chemical compound OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000013600 plasmid vector Substances 0.000 description 4
- 229920003023 plastic Polymers 0.000 description 4
- 239000004033 plastic Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 101150113187 yqhD gene Proteins 0.000 description 3
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 101000693878 Ideonella sakaiensis (strain NBRC 110686 / TISTR 2288 / 201-F6) Poly(ethylene terephthalate) hydrolase Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 101000693873 Unknown prokaryotic organism Leaf-branch compost cutinase Proteins 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 229910021654 trace metal Inorganic materials 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 description 1
- 108010053754 Aldehyde reductase Proteins 0.000 description 1
- 102100027265 Aldo-keto reductase family 1 member B1 Human genes 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- AEMRFAOFKBGASW-UHFFFAOYSA-M Glycolate Chemical compound OCC([O-])=O AEMRFAOFKBGASW-UHFFFAOYSA-M 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 229940061720 alpha hydroxy acid Drugs 0.000 description 1
- 150000001280 alpha hydroxy acids Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000013064 chemical raw material Substances 0.000 description 1
- 231100000481 chemical toxicant Toxicity 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000003144 genetic modification method Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of biology, in particular to a recombinant escherichia coli strain and a method for producing glycollic acid. The recombinant escherichia coli strain provided by the invention has the fucO and yqhD genes knocked out and expresses the Gox0313 and aldA genes. The strain can efficiently utilize EG, and can utilize 6.84g/LEG within 120h, and OD of strain in stationary phase 600 Is maintained at about 6 to 7. The glycolic acid yield is rapidly increased within 24 hours before the culture, and reaches 5.02g/L within 120 hours.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recombinant escherichia coli strain and a method for producing glycollic acid.
Background
Ethylene Glycol (EG) is one of the main products of complete degradation of PET and is also an important organic chemical raw material. For biodegradable PET, under ideal conditions, the microorganism can secrete PET degrading enzymes, so that EG is produced by complete degradation of PET, EG can be further metabolized by other microorganisms, growth is provided or used for producing high-value chemicals, and finally, the environment-friendly concept of green chemistry is realized. The laboratory has constructed artificial mixed bacteria system to degrade PET in early stage, and the experimental result shows that EG is used as one of PET degradation products and has certain inhibiting effect on PET hydrolase. Related studies have also reported that EG is identified as a competitive inhibitor of PET hydrolase. Therefore, the design and construction of the recombinant mode biological high-efficiency utilization EG can effectively promote PET enzymolysis, and the use of EG as a raw material for producing high-value chemicals such as glycollic acid and the like can also widen the application field of EG.
The biological method can avoid the problems of difficult separation and purification caused by poor selectivity and a plurality of byproducts of the chemical method. In addition, glycolic acid has been widely used in the preparation of biodegradable materials, and EG is converted into glycolic acid, so that the conversion of PET waste into biodegradable materials can be further realized, and a new scheme is provided for the development of recycling economy. The research aims at constructing a recombinant escherichia coli which can efficiently utilize EG and convert EG into glycolic acid which is a high value-added product, and further realizing the reutilization of EG which is a plastic degradation product.
At present, most of reported microorganisms with EG utilization capacity are not model organisms, the genetic background and the genetic modification method are not clear, the model organisms utilizing EG are used for producing glycollic acid, and meanwhile, the model organisms are applied to industrial application of degrading plastics and converting the plastics into glycollic acid, so that an effective solution can be provided for recycling waste plastics. Compared with other bacteria, the escherichia coli MG1655 contains an endogenous pathway of glycolaldehyde metabolism, the key for realizing the utilization of EG and the efficient conversion of EG is to construct an oxidation pathway of EG to glycolaldehyde, and the escherichia coli has a definite genetic background and a simple genetic operation method and is a potential chassis microorganism for converting EG into high-value chemicals.
Incomplete oxidation of EG can produce glycolic acid, which is one of the inexpensive raw materials for the production of glycolic acid. Glycolate is an alpha-hydroxy acid, both alcoholic and acidic in nature, and has been widely used in chemical cleaning, synthetic biodegradable materials, textile industry, food processing and pharmaceutical industry. In addition, it is a very important intermediate in organic synthesis, and participates in long chain polymerization, esterification, oxidation/reduction, and other reactions. Currently, glycolic acid is produced by a series of chemical reactions, mostly by means of fossil resources and toxic chemicals. It is known that most microorganisms capable of producing glycolic acid using EG are non-model organisms, and it is difficult to perform chassis modification. Therefore, development of model organisms to produce glycolic acid efficiently is of great industrial value.
Disclosure of Invention
In view of this, the present invention provides a recombinant E.coli strain and a method for producing glycolic acid. The strain can efficiently utilize EG, convert the EG into glycolic acid, and the glycolic acid yield reaches 5.02g/L within 120 h.
In order to achieve the above object, the present invention provides the following technical solutions:
a recombinant escherichia coli strain, which has fucO (aldehyde reductase) and yqhD genes (alcohol dehydrogenase genes) knocked out and expresses Gox0313 (alcohol dehydrogenase gene), aldA gene (aldehyde dehydrogenase gene).
In the invention, the chassis strain is escherichia coli K12 series strain, and in some implementations, the chassis strain is MG1655.
In the present invention, the Gox0313 gene is derived from Gluconobacter oxydans Gluconobacter oxydans. The gene is subjected to codon optimization aiming at escherichia coli MG1655, and an RBS sequence is added at the front end of the gene, and the amino acid sequence of the gene is shown as SEQ ID NO. 1. In some embodiments, the invention introduces BamHI and EcoRI cleavage sites at both ends of the Gox0313 gene, respectively, through which the Gox0313 gene is inserted into a plasmid vector.
In the invention, the aldA gene is an endogenous gene of escherichia coli MG1655, and in some specific embodiments, an RBS sequence is added at the 5' end of the aldA gene, and the nucleotide sequence of the aldA gene is shown as SEQ ID NO. 2. In some embodiments, the invention introduces XbaI and SalI cleavage sites at both ends of the aldA gene, respectively, through which the aldA gene is inserted into a plasmid vector.
In the present invention, the recombinant E.coli strain has a genome which integrates chloramphenicol tag and Arabidopsis resistance tag genes. The invention utilizes lambda-Red homologous recombination technology to integrate the chloramphenicol tag and the Arabidopsis resistance tag into the positions of the fucO and yqhD genes in the genome while knocking out the fucO and yqhD genes.
In the invention, knockout of fucO and yqhD genes and insertion of chloramphenicol and Arabidopsis resistance tags are realized by using lambda-Red homologous recombination technology.
The invention also provides application of the recombinant escherichia coli strain in production of glycolic acid.
The invention also provides a construction method of the recombinant escherichia coli strain, which comprises the following steps:
taking an escherichia coli K12 series strain as chassis bacteria, knocking out fucO and yqhD genes by adopting a lambda-Red homologous recombination technology, and integrating chloramphenicol tags and Arabic resistance tags into genome of the chassis bacteria to obtain intermediate strains;
connecting the optimized Gox0313 and aldA genes to a PTrc99a vector to obtain a recombinant plasmid;
and transforming the recombinant plasmid into the intermediate strain to obtain the recombinant escherichia coli strain.
The invention also provides a method for producing glycollic acid, which takes EG as a carbon source, ferments the recombinant escherichia coli strain disclosed by the invention, and marks the strain FYGA.
In the invention, the fucO and yqhD genes are knocked out by utilizing the lambda-Red homologous recombination technology. Firstly, constructing fragment 1 containing a fucO gene knockout fragment and a resistance tag sequence, wherein the nucleotide sequence of the fragment 1 is shown as SEQ ID NO. 3; fragment 2 containing yqhD gene knockout fragment and resistance tag sequence, its nucleotide sequence is shown in SEQ ID NO. 4. The kind of the resistance tag sequence is not particularly limited in the present invention, and a person skilled in the art may select the resistance tag according to practical situations, and in the specific embodiment of the present invention, the resistance tag is a chloramphenicol tag or an arabica resistance tag. The chloramphenicol tag and the Arabidopsis resistance tag are integrated into the genome of Chassis bacteria by lambda-Red homologous recombination technology, respectively replacing the fucO and yqhD genes.
The recombinant escherichia coli strain FYGA provided by the invention has the fucO and yqhD genes knocked out and expresses Gox0313 and aldA genes. The strain can efficiently utilize EG, can utilize 6.84g/L EG within 120h, and has OD at strain stabilization stage 600 Is maintained at about 6 to 7. The glycolic acid yield is rapidly increased within 24 hours before the culture, and reaches 5.02g/L within 120 hours.
Drawings
FIG. 1 shows a schematic diagram of recombinant E.coli producing glycolic acid using EG;
FIG. 2 shows an EG pathway map for E.coli;
FIG. 3 shows EG concentration changes during degradation of EG by recombinant strain FYGA;
FIG. 4 shows the change in concentration of glycolic acid produced by recombinant strain FYGA using EG;
FIG. 5 shows OD during EG degradation by recombinant strain FYGA 600 。
Detailed Description
The invention provides a recombinant escherichia coli strain and a method for producing glycollic acid. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
The lambda-Red homologous recombination technology is adopted in the escherichia coli MG1655 to knock out the fucO and yqhD genes in the genome, the fucO gene is knocked out to introduce an Arabic resistance tag for substitution, the yqhD gene is knocked out to introduce a chloramphenicol tag for substitution, and the resistance tag is not required to be knocked out. The final knockout fucO gene and yqhD gene strains were both arabidopsis and chloramphenicol resistant. The sequence of the knockdown fragment is shown in SEQ ID NO. 3-4.
The exogenous gene Gox0313 is synthesized by Nanjing Jinsri biotechnology Co., ltd, codon optimization is carried out for chassis cell escherichia coli MG1655, RBS sequence is added in front of synthesizing exogenous gene, bamHI and EcoRI restriction sites are respectively introduced at two ends of synthesized fragment, gox0313 gene and plasmid vector pTrc99a are assembled through BamHI and EcoRI double restriction and T4 connection, thus obtaining recombinant plasmid pEc01004. The method comprises the steps of amplifying an aldA gene fragment in an escherichia coli genome by using primers aldA-F and aldA-R through PCR, adding a RBS sequence and enzyme digestion sites XbaI and SalI, wherein the used primer sequence is shown in table 1, assembling the aldA gene fragment and a plasmid vector pEc01004 through double enzyme digestion of XbaI and SalI and T4 connection, obtaining a recombinant plasmid pEc01005, taking escherichia coli MG1655 with fucO and yqhD genes knocked out as chassis cells, transforming the plasmid pEc01005 into the chassis cells to collect recombinant strain FYGA, wherein the recombinant strain has Arabic resistance, chloramphenicol resistance and ampicillin resistance, and the recombinant escherichia coli has a glycolic acid production schematic diagram by using EG and an escherichia coli path diagram shown in figures 1 and 2 respectively.
TABLE 1 primer list required for constructing recombinant plasmid pEc01005
Shake flask fermentation experiments were performed on recombinant strains in M9 medium with 10g/L EG as the sole carbon source. Determination of EG concentration Change during fermentation, glycolic acid concentration Change and OD during FYGA degradation of EG 600 The values and results are shown in FIGS. 3 to 5.
At the 5 th hour of shake flask fermentation, total RNA of the strain FYGA is extracted, cDNA is subjected to reverse transcription, qPCR reaction is performed, and exogenous genes and endogenous gene transcription levels in a path for producing glycollic acid by using EG in the recombinant strain are determined by taking an endogenous gene gapA of escherichia coli MG1655 as a reference gene.
Wherein, the culture medium comprises the following components:
m9 medium: 200mL of 5 XM 9 salt solution, 2mL of 1M MgSO4 solution, 100 mu L of CaCl2 solution, 1mL of trace metal solution and 700mL of 2g/L yeast extract powder solution are measured, 10g/L EG solution is added as a carbon source, the mixture is fully and uniformly mixed and dissolved after ultrasonic treatment, the volume is fixed to 1L, and the mixture is sub-packaged and sterilized.
5×m9 salt solution: weigh 6.78g Na 2 HPO 4 、3g KH 2 PO 4 、0.5g NaCl、1g NH 4 Cl was dissolved well and fixed to 200mL.
Trace metal solution: 1.6g FeCl was weighed out 3 、0.2g CoCl 2 ·H 2 O、0.1gCuCl 2 、0.13g ZnCl 2 、0.2g Na 2 MoO 4 、0.05g H 3 BO 3 Ultrasonic dissolving in 0.1M HCl, and fixing volume to 1L.
As can be seen from FIGS. 3 to 5, recombinant strain FYGA was able to efficiently utilize EG as compared to the control group. The OD of the strain at the stationary phase can be utilized at 6.84g/LEG within 120h 600 Is maintained at about 6 to 7. The glycolic acid yield is rapidly increased within 24 hours before the culture, and reaches 5.02g/L within 120 hours.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> university of Tianjin
<120> a recombinant strain and a method for producing glycolic acid
<130> MP22009248
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1048
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
aagttaagag gcaagaatgg cggataccat gctggcggcg gtggtgcgcg aatttggcaa 60
accgctgagc attgaacgcc tgccgattcc ggatattaaa ccgcatcaga ttctggtgaa 120
agtggatacc tgcggcgtgt gccataccga tctgcatgcg gcgcgcggcg attggccgag 180
caaaccgaac ccgccgttta ttccgggcca tgaaggcgtg ggccatattg tggcggtggg 240
cagccaagtg ggcgattttg tgaaaaccgg cgatgtggtg ggcgtgccgt ggctgtatag 300
cgcgtgcggc cattgcgaac attgcctggg cggctgggaa accctgtgcg aaaaacaaga 360
tgataccggc tataccgtga acggctgctt tgcggaatat gtggtggccg atccgaacta 420
tgtggcgcat ctgccgagca ccattgatcc gctgcaagcg agcccggtgc tgtgcgcggg 480
cctgaccgtg tataaaggcc tgaagatgac cgaagcccgc ccgggtcagt gggtggcggt 540
gagcggtgtg ggcggcctgg gtcagatggc ggtgcagtat gcggtggcga tgggcatgaa 600
cgtggtggcg gtggatattg atgatgaaaa actggcgacc gcgaaaaaac tgggcgcgag 660
cctgaccgtg aacgcgaaag ataccgatcc ggcgcgcttt attcaacagc agattggcgg 720
cgcgcacggc gccctggtga ccgcggtggg ccgcacggcg ttcagccaag cgatgggcta 780
tgcgcgccgc ggcggcacca ttgtgctgaa cggcctgccg ccgggcgatt ttccggtgag 840
catttttgat atggtgatga acggcaccac cattcgcggc agcattgtgg gcacccgcct 900
ggatatgatt gaagcgatgg atttttttgc gcgcggcaaa gtgaaaagcg tggtgacccc 960
gggcaaactg gaaaacatta acaccatttt tgatgattta cagaacggcc gcctggaagg 1020
ccgcaccgtg ctggattttc gcagctaa 1048
<210> 2
<211> 1462
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
aacaaaatga ggaggtactg agatgtcagt acccgttcaa catcctatgt atatcgatgg 60
acagtttgtt acctggcgtg gagacgcatg gattgatgtg gtaaaccctg ctacagaggc 120
tgtcatttcc cgcatacccg atggtcaggc cgaggatgcc cgtaaggcaa tcgatgcagc 180
agaacgtgca caaccagaat gggaagcgtt gcctgctatt gaacgcgcca gttggttgcg 240
caaaatctcc gccgggatcc gcgaacgcgc cagtgaaatc agtgcgctga ttgttgaaga 300
agggggcaag atccagcagc tggctgaagt cgaagtggct tttactgccg actatatcga 360
ttacatggcg gagtgggcac ggcgttacga gggcgagatt attcaaagcg atcgtccagg 420
agaaaatatt cttttgttta aacgtgcgct tggtgtgact accggcattc tgccgtggaa 480
cttcccgttc ttcctcattg cccgcaaaat ggctcccgct cttttgaccg gtaataccat 540
cgtcattaaa cctagtgaat ttacgccaaa caatgcgatt gcattcgcca aaatcgtcga 600
tgaaataggc cttccgcgcg gcgtgtttaa ccttgtactg gggcgtggtg aaaccgttgg 660
gcaagaactg gcgggtaacc caaaggtcgc aatggtcagt atgacaggca gcgtctctgc 720
aggtgagaag atcatggcga ctgcggcgaa aaacatcacc aaagtgtgtc tggaattggg 780
gggtaaagca ccagctatcg taatggacga tgccgatctt gaactggcag tcaaagccat 840
cgttgattca cgcgtcatta atagtgggca agtgtgtaac tgtgcagaac gtgtttatgt 900
acagaaaggc atttatgatc agttcgtcaa tcggctgggt gaagcgatgc aggcggttca 960
atttggtaac cccgctgaac gcaacgacat tgcgatgggg ccgttgatta acgccgcggc 1020
gctggaaagg gtcgagcaaa aagtggcgcg cgcagtagaa gaaggggcga gagtggcgtt 1080
cggtggcaaa gcggtagagg ggaaaggata ttattatccg ccgacattgc tgctggatgt 1140
tcgccaggaa atgtcgatta tgcatgagga aacctttggc ccggtgctgc cagttgtcgc 1200
atttgacacg ctggaagatg ctatctcaat ggctaatgac agtgattacg gcctgacctc 1260
atcaatctat acccaaaatc tgaacgtcgc gatgaaagcc attaaagggc tgaagtttgg 1320
tgaaacttac atcaaccgtg aaaacttcga agctatgcaa ggcttccacg ccggatggcg 1380
taaatccggt attggcggcg cagatggtaa acatggcttg catgaatatc tgcagaccca 1440
ggtggtttat ttacagtctt aa 1462
<210> 3
<211> 2296
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atgacttagc cgccacgctg gcggtcaaag tgacacaggc cgggcatcag gcaacgattg 60
tttcgacaga taaaggctac tgtcagttac tttcaccgac attacgtatt cgtgattact 120
tccagaaacg ttggctggat gcgccattta tcgataaaga atttggcgtt caaccgcagc 180
agttgcccga ttactgggga cttgcgggga tcagcagttc aaaggtaccg ggtgttgcgg 240
gaatcggacc aaaaagcgcc acgcagctgc tggtcgagtt tcagagtctg gaagggatat 300
atgagaatct ggatgcggtt gccgaaaagt ggcgcaaaaa attagaaacc cataaagaga 360
tggcgtttct gtgccgcgat attgcccgct tacaaaccga tttgcatatc gacggcaatt 420
tacagcaatt gcggttggta cggtaacggc gagccggata cgccgcaaac gtcgtatccg 480
gcattatcac atcagcgcat cgcggaaccc ctatttgttt atttttctaa atacattcaa 540
atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 600
agagtatgaa atctaacaat gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg 660
taggcatagg cttggttatg ccggtactgc cgggcctctt gcgggatatc gtccattccg 720
acagcatcgc cagtcactat ggcgtgctgc tagcgctata tgcgttgatg caatttctat 780
gcgcacccgt tctcggagca ctgtccgacc gctttggccg ccgcccagtc ctgctcgctt 840
cgctacttgg agccactatc gactacgcga tcatggcgac cacacccgtc ctgtggatcc 900
tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct 960
atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt 1020
gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct 1080
tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct 1140
tcctaatgca ggagtcgcat aagggagagc gtcgaccgat gcccttgaga gccttcaacc 1200
cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct 1260
tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg 1320
accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc 1380
acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg 1440
ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc 1500
gaggctggat ggccttcccc attatgattc ttctcgcctc cggcggcatc gggatgcccg 1560
cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag cttcaaggat 1620
cgctcgcggc tcttaccagc ctaacttcga tcactggacc gctgatcgtc acggcgattt 1680
atgccgcctc ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc gccctatacc 1740
ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg acctaacatc 1800
cttctccttg ttgctttacg aaattactct tcaattcgta acccataggt tttgaatttc 1860
tccagcacta cggcaatctc ttcatcgctc agcactggca ccgggtccgt aatcgccagg 1920
gtcgtcaggt aaagttgcgc cagcacttca acttcatgcg ccagccataa cgctttttcc 1980
agattcacct cacaagcgat aagcccatga tgttgtaaca aagttgcctt acgatttttg 2040
agagccagcg caacatgttc agaaagttcg cgtgttccaa aggtcgcata aggcgcgcaa 2100
ggaatagaat taccgccagc cgccgcaatc atgtagtgaa tagcggggat cgatcggtta 2160
agaatggaaa ctgccgtgca atgaacggca tgattgtgaa caaccgcgtt ggcatccggt 2220
ctgctttgat aggctgccat atggaaacgc cattcgcttg aggggagctt tccttcctca 2280
tgtttaccgt tgccat 2296
<210> 4
<211> 1765
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
cattgagacg caaccctgcc agcggcacct ctgacgtggc ataggtttcg cactcaaacg 60
gcaacggcac cgtcagcagc aggtattcat tggcatcata acgaaacacg cgttcattga 120
tataaccgat tttatgcccg gaaaagagaa ttatgatgcc aggctcgtac atcaccggtg 180
tacgtgcgaa aggcgtctcg ccatacaaca aacgcacatc gggcaacagt cctgacaaac 240
tattttcttt atttttcagt ttattaactt tatccgccag caagcggcaa atctcttcac 300
gtttcatatc gcgtaatttc ttaggaataa tgcggcaatt tgattgtgcg caattttgta 360
gcatttctcc agcactctgg agaaataggc aagacattgg cagaaatgag cattgagagc 420
cagggcgctg gcgatcacaa tgaaaaacat caggcagatc gttctctgcc ctcatattgg 480
cccagcaaag ggagcaagta cgcggaaccc ctatttgttt atttttctaa atacattcaa 540
atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 600
agagtatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta 660
aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc 720
tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct 780
ttattcacat tcttgcccgc ctgatgaacg ctcacccggg gtttcgtatg gccatgaaag 840
acggtgagct ggtgatctgg gatagtgttc acccttgtta caccgttttc catgagcaaa 900
ctgaaacgtt ttcgtccctc tggagtgaat accacgacga tttccggcag tttctccaca 960
tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta 1020
ttgagaatat gttttttgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa 1080
acgtggccaa tatggacaac ttcttcgccc ccgttttcac gatgggcaaa tattatacgc 1140
aaggcgacaa ggtgctgatg ccgctggcga tccaggttca tcatgccgtt tgtgatggct 1200
tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg 1260
cgtaagcttt ttacgcctca aactttcgtt ttcgggcatt tcgtccagac ttaagttcac 1320
aacacctcac cggagcctgc tccggtgagt tcatataaag gaggaacgta tggctaatcc 1380
aaccgttatt aagctacagg atggcaatgt catgccccag ctgggactgg gcgtctggca 1440
agcaagtaat gaggaagtaa tcaccgccat tcaaaaagcg ttagaagtgg gttatcgctc 1500
gattgatacc gccgcggcct acaagaacga agaaggtgtc ggcaaagccc tgaaaaatgc 1560
ctcagtcaac agagaagaac tgttcatcac cactaagctg tggaacgacg accacaagcg 1620
cccccgcgaa gccctgctcg acagcctgaa aaaactccag cttgattata tcgacctcta 1680
cttaatgcac tggcccgttc ccgctatcga ccattatgtc gaagcatgga aaggcatgat 1740
cgaattgcaa aaagagggat taatc 1765
Claims (11)
1. A recombinant escherichia coli strain, characterized in that the fucO and yqhD genes are knocked out and the Gox0313, aldA genes are expressed.
2. The recombinant E.coli strain according to claim 1, wherein the chassis strain is E.coli K12 series strain.
3. The recombinant escherichia coli strain of claim 2, wherein the escherichia coli K12 series strain is MG1655.
4. The recombinant escherichia coli strain according to claim 1, wherein the Gox0313 gene is derived from gluconobacter oxydans.
5. The recombinant escherichia coli strain according to claim 1, wherein the nucleotide sequence of the Gox0313 gene is shown as SEQ ID No. 1.
6. The recombinant escherichia coli strain of claim 1, wherein the aldA gene has a nucleotide sequence as shown in SEQ ID No. 2.
7. Recombinant E.coli strain according to any one of claims 1 to 6, characterized in that its genome also integrates chloramphenicol-and Arabidopsis resistance-tagged genes.
8. The recombinant escherichia coli strain of claim 7, wherein the chloramphenicol tag and the arabidopsis resistance tag are integrated into the genome in place of the fucO and yqhD genes, respectively.
9. Use of a recombinant escherichia coli strain according to any one of claims 1-8 for the production of glycolic acid.
10. The method for constructing a recombinant E.coli strain according to any one of claims 1 to 8, comprising:
taking an escherichia coli K12 series strain as chassis bacteria, knocking out fucO and yqhD genes by adopting a lambda-Red homologous recombination technology, and integrating chloramphenicol tags and Arabic resistance tags into genome of the chassis bacteria to obtain intermediate strains;
connecting the optimized Gox0313 and aldA genes to a PTrc99a vector to obtain a recombinant plasmid;
and transforming the recombinant plasmid into the intermediate strain to obtain the recombinant escherichia coli strain.
11. A process for producing glycolic acid, characterized in that the recombinant escherichia coli strain according to any one of claims 1 to 8 is fermented with EG as a carbon source.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210672569.7A CN116004488A (en) | 2022-06-15 | 2022-06-15 | Recombinant strain and method for producing glycollic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210672569.7A CN116004488A (en) | 2022-06-15 | 2022-06-15 | Recombinant strain and method for producing glycollic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116004488A true CN116004488A (en) | 2023-04-25 |
Family
ID=86034088
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210672569.7A Pending CN116004488A (en) | 2022-06-15 | 2022-06-15 | Recombinant strain and method for producing glycollic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116004488A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120122169A1 (en) * | 2009-07-30 | 2012-05-17 | Metabolic Explorer | Mutant yqhd enzyme for the production of a biochemical by fermentation |
| US20130316416A1 (en) * | 2012-02-23 | 2013-11-28 | Massachusetts Institute Of Technology | Engineering microbes and metabolic pathways for the production of ethylene glycol |
| CN105586304A (en) * | 2014-11-18 | 2016-05-18 | 北京化工大学 | Recombinant strain for producing glycolate based polymers and application of recombinant strain |
| CN107312737A (en) * | 2017-07-25 | 2017-11-03 | 北京理工大学 | A kind of recombination bacillus coli, preparation method and the method for synthesizing 3,4 dihydroxy butyric acid |
| KR20180042660A (en) * | 2016-10-18 | 2018-04-26 | 건국대학교 산학협력단 | A recombinat organism enhanced furfural tolerance and method for production of isobutanol using the same |
| WO2019046946A1 (en) * | 2017-09-07 | 2019-03-14 | The Governing Council Of The University Of Toronto | Production of glycolate from ethylene glycol and related microbial engineering |
| CN113481138A (en) * | 2021-07-26 | 2021-10-08 | 天津大学 | Engineering strain, preparation method thereof and method for producing glycollic acid by efficiently utilizing ethylene glycol |
-
2022
- 2022-06-15 CN CN202210672569.7A patent/CN116004488A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120122169A1 (en) * | 2009-07-30 | 2012-05-17 | Metabolic Explorer | Mutant yqhd enzyme for the production of a biochemical by fermentation |
| US20130316416A1 (en) * | 2012-02-23 | 2013-11-28 | Massachusetts Institute Of Technology | Engineering microbes and metabolic pathways for the production of ethylene glycol |
| CN105586304A (en) * | 2014-11-18 | 2016-05-18 | 北京化工大学 | Recombinant strain for producing glycolate based polymers and application of recombinant strain |
| KR20180042660A (en) * | 2016-10-18 | 2018-04-26 | 건국대학교 산학협력단 | A recombinat organism enhanced furfural tolerance and method for production of isobutanol using the same |
| CN107312737A (en) * | 2017-07-25 | 2017-11-03 | 北京理工大学 | A kind of recombination bacillus coli, preparation method and the method for synthesizing 3,4 dihydroxy butyric acid |
| WO2019046946A1 (en) * | 2017-09-07 | 2019-03-14 | The Governing Council Of The University Of Toronto | Production of glycolate from ethylene glycol and related microbial engineering |
| CN113481138A (en) * | 2021-07-26 | 2021-10-08 | 天津大学 | Engineering strain, preparation method thereof and method for producing glycollic acid by efficiently utilizing ethylene glycol |
Non-Patent Citations (2)
| Title |
|---|
| LAURA SALUSJÄRVI等: "Biotechnological production of glycolic acid and ethylene glycol: current state and perspectives", APPL MICROBIOL BIOTECHNOL., vol. 103, no. 6, 1 February 2019 (2019-02-01), pages 1 - 5, XP036746619, DOI: 10.1007/s00253-019-09640-2 * |
| 马宁;朱康佳;毛银;邓禹;: "代谢工程改造大肠杆菌提高乙醇酸产率", 生物工程学报, no. 02, 10 October 2017 (2017-10-10) * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kohlstedt et al. | From lignin to nylon: cascaded chemical and biochemical conversion using metabolically engineered Pseudomonas putida | |
| Rathnasingh et al. | Development and evaluation of efficient recombinant Escherichia coli strains for the production of 3‐hydroxypropionic acid from glycerol | |
| Park et al. | Low‐pH production of d‐lactic acid using newly isolated acid tolerant yeast Pichia kudriavzevii NG7 | |
| TWI453284B (en) | Methods for the synthesis of olefins acid and derivatives | |
| Henard et al. | Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts | |
| US10584359B2 (en) | Genetically recombinant Saccharomyces cerevisiae for degrading kitchen waste | |
| US20090023182A1 (en) | Complementary metabolizing organisms and methods of making same | |
| KR20180002704A (en) | Genetically Engineered Microorganisms for the Production of Chlorisimide-Induced Products | |
| CN106086102B (en) | Engineering bacterium for producing trans-4-hydroxy-L-proline and construction method and application thereof | |
| Cao et al. | Unlocking nature’s biosynthetic potential by directed genome evolution | |
| CN105543214B (en) | Utilize the metabolic engineering coli strain construction method of acetic acid production succinic acid and application | |
| CN104685059A (en) | Biosynthetic pathways, recombinant cells, and methods | |
| Bayer et al. | Biosensor and chemo-enzymatic one-pot cascade applications to detect and transform PET-derived terephthalic acid in living cells | |
| Li et al. | Bio-refinery of xylose processing wastes for green polymalic acid production and l-malic acid recovery by engineered Aureobasidium pullulans in a non-waste-disposal system | |
| CN104630100A (en) | Reconstructed Klebsiella pneumoniae and application of reconstructed Klebsiella pneumoniae in production of R-acetoin | |
| CN116004488A (en) | Recombinant strain and method for producing glycollic acid | |
| US9783581B2 (en) | Method for producing plastic raw material from blue-green algae | |
| CN114134092B (en) | Recombinant microorganism capable of efficiently utilizing methanol and application thereof | |
| JP2014023528A (en) | Modified microorganism and method for producing 1,4-butanediol using it | |
| CN110218691A (en) | One plant of genetic engineering bacterium for synthesizing altheine and its construction method and application | |
| KR101028039B1 (en) | Microorganism Enhanced Resistance of Succinic Acid and Method for Preparing Succinic Acid Using the Same | |
| AU2008346589B2 (en) | Clostridium sartagoformum for the generation of biogas | |
| CN115927152A (en) | Synthesis and accumulation of vanillin in genetic engineering bacterium escherichia coli | |
| Szymanowska-Powałowska et al. | Microbial purification of postfermentation medium after 1, 3-PD production from raw glycerol | |
| JP6778870B2 (en) | Cyanobacteria mutant strain and succinic acid and D-lactic acid production method using it |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |