A recombinant strain and method for producing glycolic acid

Abstract

The present invention relates to the field of biotechnology, and in particular to a recombinant Escherichia coli strain and a method for producing glycolic acid. The recombinant Escherichia coli strain provided by the present invention has its fucO and yqhD genes knocked out, and expresses Gox0313 and aldA genes. The strain can efficiently utilize EG, and can utilize 6.84 g / LEG within 120 hours, and the OD of the strain in the stable period is 2.34~3.34 g / LEG. 600 The maintenance rate was about 6 to 7. In the first 24 hours of culture, the production of glycolic acid increased rapidly, and within 120 hours, the production of glycolic acid reached 5.02 g / L.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant Escherichia coli strain and a method for producing glycolic acid. Background Art

[0002] 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, microorganisms can secrete PET-degrading enzymes to completely degrade PET and produce EG. EG can be further metabolized by other microorganisms to provide growth or be used to produce high-value chemicals, ultimately realizing the environmental protection concept of green chemistry. Our laboratory previously constructed an artificial mixed bacteria system to degrade PET. The experimental results showed that EG, as one of the products of PET degradation, has a certain inhibitory effect on PET hydrolase. Related studies have also reported that EG has been identified as a competitive inhibitor of PET hydrolase. Therefore, designing and constructing recombinant model organisms to efficiently utilize EG can effectively promote PET enzymatic hydrolysis, and using EG as a raw material to produce high-value chemicals such as glycolic acid can also broaden the application field of EG.

[0003] The biological method for preparing glycolic acid can avoid the problems of poor selectivity and difficulty in separation and purification caused by the chemical method due to the large number of by-products. In addition, glycolic acid has been widely used in the preparation of biodegradable materials. The conversion of EG into glycolic acid can further realize the conversion of PET waste into biodegradable materials, providing a new solution for the development of circular economy. This study aims to construct a recombinant Escherichia coli that can efficiently utilize EG and convert it into a high-value-added product, glycolic acid, so as to realize the reuse of EG, a plastic degradation product.

[0004] At present, most of the reported microorganisms with the ability to utilize EG are not model organisms, and their genetic background and genetic modification methods are not clear. Using EG model organisms to produce glycolic acid and applying it to industrial applications of plastic degradation and conversion into glycolic acid can provide an effective solution for the recycling of waste plastics. Compared with other bacteria, Escherichia coli MG1655 contains an endogenous pathway for glycolaldehyde metabolism. The key to its utilization of EG and efficient conversion of EG lies in the construction of an oxidation pathway from EG to glycolaldehyde. Escherichia coli has a clear genetic background and simple genetic manipulation methods, and is a potential chassis microorganism for the conversion of EG into high-value chemicals.

[0005] Incomplete oxidation of EG can generate glycolic acid, which is one of the cheap raw materials for producing glycolic acid. Glycolate is an α-hydroxy acid that has both the properties of alcohol and acid. It has been widely used in chemical cleaning, synthetic biodegradable materials, textile industry, food processing and pharmaceutical industry. In addition, it is also a very important intermediate in organic synthesis, participating in reactions such as long-chain polymerization, esterification and oxidation / reduction. At present, glycolic acid is mainly produced by a series of chemical reactions using fossil resources and toxic chemicals. Most of the microorganisms known to produce glycolic acid using EG are non-model organisms, and it is difficult to carry out chassis modification. Therefore, the development of model organisms to efficiently produce glycolic acid has great industrial value. Summary of the invention

[0006] In view of this, the present invention provides a recombinant Escherichia coli strain and a method for producing glycolic acid. The strain can efficiently utilize EG and convert it into glycolic acid, and the glycolic acid production reaches 5.02 g / L within 120 hours.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] A recombinant Escherichia coli strain, in which fucO (aldehyde reductase) and yqhD genes (alcohol dehydrogenase gene) are knocked out, and Gox0313 (alcohol dehydrogenase gene) and aldA gene (aldehyde dehydrogenase gene) are expressed.

[0009] In the present invention, the chassis strain is an Escherichia coli K12 series strain. In some specific implementations, the chassis strain is MG1655.

[0010] In the present invention, the Gox0313 gene is derived from Gluconobacter oxydans. The gene is codon-optimized for Escherichia coli MG1655, and an RBS sequence is added to the front end of the gene, and its amino acid sequence is shown in SEQ ID NO. 1. In some specific embodiments, the present invention introduces BamHI and EcoRI restriction sites at both ends of the Gox0313 gene, and inserts the Gox0313 gene into a plasmid vector through the two sites.

[0011] In the present invention, the aldA gene is an endogenous gene of Escherichia coli MG1655. In some specific embodiments, the present invention adds an RBS sequence at the 5' end of the aldA gene, and its nucleotide sequence is shown in SEQ ID NO. 2. In some specific embodiments, the present invention introduces XbaI and SalI restriction sites at both ends of the aldA gene, respectively, and inserts the aldA gene into a plasmid vector through these two sites.

[0012] In the present invention, the genome of the recombinant Escherichia coli strain also integrates chloramphenicol tag and Abraham resistance tag genes. The present invention utilizes λ-Red homologous recombination technology to knock out the fucO and yqhD genes while integrating the chloramphenicol tag and Abraham resistance tag into the positions of the fucO and yqhD genes in the genome.

[0013] In the present invention, the knockout of fucO and yqhD genes and the insertion of chloramphenicol tag and Abraxine resistance tag are achieved by using λ-Red homologous recombination technology.

[0014] The present invention also provides the use of the recombinant Escherichia coli strain in producing glycolic acid.

[0015] The present invention also provides a method for constructing a recombinant Escherichia coli strain, comprising the following steps:

[0016] Using Escherichia coli K12 series strains as the base bacteria, the fucO and yqhD genes were knocked out by λ-Red homologous recombination technology, and the chloramphenicol tag and the Abraxine resistance tag were respectively integrated into the genome of the base bacteria to replace the fucO and yqhD genes, thus obtaining the intermediate strains.

[0017] The optimized Gox0313 and aldA genes were connected to the PTrc99a vector to obtain a recombinant plasmid;

[0018] The recombinant plasmid is transformed into the intermediate strain to obtain the recombinant Escherichia coli strain.

[0019] The present invention also provides a method for producing glycolic acid, using EG as a carbon source to ferment the recombinant Escherichia coli strain of the present invention, which is marked as strain FYGA.

[0020] In the present invention, the fucO and yqhD genes are knocked out using λ-Red homologous recombination technology. First, a fragment 1 containing a fucO gene knockout fragment and a resistance tag sequence is constructed, and its nucleotide sequence is shown in SEQ ID NO.3; a fragment 2 containing a yqhD gene knockout fragment and a resistance tag sequence, and its nucleotide sequence is shown in SEQ ID NO.4. The present invention does not specifically limit the type of the resistance tag sequence, and those skilled in the art can choose it according to actual conditions. In a specific embodiment of the present invention, the resistance tag is a chloramphenicol tag and an Abra resistance tag. Through λ-Red homologous recombination technology, the chloramphenicol tag and the Abra resistance tag replace the fucO and yqhD genes respectively and are integrated into the genome of the bottom plate bacteria.

[0021] The recombinant Escherichia coli strain FYGA provided by the present invention has its fucO and yqhD genes knocked out and expresses Gox0313 and aldA genes. The strain can efficiently utilize EG and can utilize 6.84 g / L EG within 120 hours. The OD of the strain in the stable period is 600 The maintenance rate was about 6 to 7. In the first 24 hours of culture, the production of glycolic acid increased rapidly, and within 120 hours, the production of glycolic acid reached 5.02 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram showing the production of glycolic acid by recombinant Escherichia coli using EG;

[0023] Figure 2 Shown is the pathway diagram of EG utilization by E. coli;

[0024] Figure 3 The changes of EG concentration during the degradation of EG by the recombinant strain FYGA are shown;

[0025] Figure 4 The concentration changes of glycolic acid produced by the recombinant strain FYGA using EG are shown;

[0026] Figure 5 The OD value of the recombinant strain FYGA during the degradation of EG 600 . DETAILED DESCRIPTION

[0027] The present invention provides a recombinant Escherichia coli strain and a method for producing glycolic acid. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0028] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0029] The present invention will be further described below in conjunction with embodiments:

[0030] Example 1

[0031] The fucO and yqhD genes in the genome of Escherichia coli MG1655 were knocked out by using λ-Red homologous recombination technology. The fucO gene was knocked out to introduce an Abraxa resistance tag instead, and the yqhD gene was knocked out to introduce a chloramphenicol tag instead, without knocking out the resistance tag. The strains with the fucO and yqhD genes finally knocked out have both Abraxa resistance and chloramphenicol resistance. The sequences of the knocked-out fragments are shown in SEQ ID NO.3-4.

[0032] The exogenous gene Gox0313 was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and codon optimization was performed for the chassis cell Escherichia coli MG1655. An RBS sequence was added in front of the synthesized exogenous gene, and BamHI and EcoRI restriction sites were introduced at both ends of the synthetic fragment. The Gox0313 gene and plasmid vector pTrc99a were double-digested with BamHI and EcoRI and assembled with T4 ligation to obtain the recombinant plasmid pEc01004. In the Escherichia coli genome, primers aldA-F and aldA-R were used to amplify the aldA gene fragment by PCR, and the primers were added with RBS sequences and restriction sites XbaI and SalI. The primer sequences used are shown in Table 1. The aldA gene fragment and the plasmid vector pEc01004 were double-digested with XbaI and SalI and assembled with T4 ligation to obtain the recombinant plasmid pEc01005. The Escherichia coli MG1655 after knocking out the fucO and yqhD genes was used as the chassis cell, and the plasmid pEc01005 was transformed into the chassis cell to obtain the recombinant strain FYGA. The recombinant strain has Abraxine resistance, chloramphenicol resistance and ampicillin resistance. The schematic diagram of the recombinant Escherichia coli using EG to produce glycolic acid and the pathway diagram of Escherichia coli using EG are shown in Figure 1 and Figure 2 .

[0033] Table 1 List of primers required for constructing recombinant plasmid pEc01005

[0034]

[0035] The recombinant strain was fermented in M9 medium with 10 g / L EG as the sole carbon source. The changes in EG concentration and glycolic acid concentration during the fermentation process and the OD value during the degradation of EG by FYGA were measured. 600 The results are as follows Figures 3 to 5 shown.

[0036] At the 5th hour of shake flask fermentation, total RNA of strain FYGA was extracted, and qPCR reaction was performed after reverse transcription of cDNA. The endogenous gene gapA of Escherichia coli MG1655 was used as the reference gene to determine the transcription levels of exogenous genes and endogenous genes in the pathway of glycolic acid production using EG in the recombinant strain.

[0037] The culture medium consists of:

[0038] M9 medium: Measure 200mL 5×M9 salt solution, 2mL 1M MgSO4 solution, 100μL CaCl2 solution, 1mL trace metal solution, 700mL 2g / L yeast extract solution, add 10g / L EG solution as the carbon source, mix thoroughly after ultrasonication, make up to 1L, and sterilize after aliquoting.

[0039] 5×M9 salt solution: weigh 6.78gNa 2 HPO 4 , 3g KH 2 PO 4 , 0.5 g NaCl, 1 g NH 4 The Cl was fully dissolved and the volume was made up to 200 mL.

[0040] Trace metal solution: weigh 1.6 g FeCl 3 , 0.2 g CoCl 2 ·H 2 O, 0.1 gCuCl 2 , 0.13g ZnCl 2 , 0.2gNa 2 MoO 4 , 0.05 g H 3 BO 3 Dissolve in 0.1M HCl by ultrasonication and make up to 1L.

[0041] Depend on Figures 3 to 5 It can be seen that compared with the control group, the recombinant strain FYGA can efficiently utilize EG. It can utilize 6.84g / LEG within 120h, and the OD of the strain in the stable period is 600 The maintenance rate was about 6 to 7. In the first 24 hours of culture, the production of glycolic acid increased rapidly, and within 120 hours, the production of glycolic acid reached 5.02 g / L.

[0042] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Tianjin University <120> A recombinant strain and method for producing glycolic acid <130> MP22009248 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1048 <212> DNA <213> 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 <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 <400> 3 atgacttagc cgccacgctg gcggtcaaag tgacacaggc cgggcatcag gcaacgattg 60 tttcgacaga taaaggctac tgtcagttac tttcaccgac attacgtatt cgtgattact 120 tccagaaacg ttggctggat gcgccatta 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 gcgggatac 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 <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 tcatataag gaggaacgta tggctaatcc 1380 aaccgttatt aagctacagg atggcaatgt catgccccag ctgggactgg gcgtctggca 1440 agcaagtaat gaggagta tcaccgccat tcaaaagcg ttagaagtgg gttatcgctc 1500 gattgatacc gccgcggcct acagaacga agaaggtgtc gggcaagccc tgaaaaatgc 1560 ctcagtcaac aggagaac tgttcatcac cactaagctg tggaacgacg accacaagcg 1620 cccccgcgaa gccctgctcg acagcctgaa aaaactccag cttgattata tcgaccta 1680 cttaatgcac tggcccgttc ccgctatcga ccattatgtc gaagcatgga aaggcatgat 1740 Cgaattgcaa aaagggatt taatc 1765

Claims

1. A recombinant Escherichia coli strain, characterized in that: That f and yD The gene was knocked out and expressed Gox0313, aldA Gene; The chassis strain is Escherichia coli MG1655; The nucleotide sequence of the Gox0313 gene is shown in SEQ ID NO.1; Said aldA The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

2. The recombinant Escherichia coli strain according to claim 1, characterized in that Its genome also integrates chloramphenicol tag and Abraxine resistance tag genes.

3. The recombinant Escherichia coli strain according to claim 2, characterized in that The chloramphenicol tag and the Abraxane resistance tag are respectively replaced f and yD The gene is integrated into the genome.

4. Use of the recombinant Escherichia coli strain according to any one of claims 1 to 3 in the production of glycolic acid.

5. The method for constructing a recombinant Escherichia coli strain according to any one of claims 1 to 3, characterized in that: include: Using Escherichia coli MG1655 as the base strain, λ-Red homologous recombination technology was used to knock out f and yD gene, replacing the chloramphenicol tag and the Abraxine resistance tag respectively f and yD The gene was integrated into the genome of the bottom plate bacteria to obtain an intermediate strain; The optimized Gox0313、aldA The gene was connected to the PTrc99a vector to obtain a recombinant plasmid; The recombinant plasmid is transformed into the intermediate strain to obtain the recombinant Escherichia coli strain.

6. A method for producing glycolic acid, characterized in that: The recombinant Escherichia coli strain according to any one of claims 1 to 3 is fermented using ethylene glycol as a carbon source.

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