A kind of Escherichia coli with high fucose production and its construction method and application

Abstract

The present application relates to the field of microbial genetic engineering technology, and in particular to a high-fucose-producing Escherichia coli and its construction method and application. The present application provides a high-fucose-producing Escherichia coli, wherein the Escherichia coli knocks out the transporter of 2'-fucosyllactose and overexpresses the transporter of fucose; the transporter of 2'-fucosyllactose includes SetA and / or MdfA; the transporter of fucose includes FucP and / or EmrD. The present application provides a high-fucose-producing Escherichia coli, wherein an exogenous FUC transporter FucP with a strong promoter is integrated into the genome of Escherichia coli or an endogenous transporter EmrD replaced with a strong promoter, and the 2'-FL transporter SetA and / or MdfA are knocked out, which can reduce the residual amount of 2'-FL and increase the yield of FUC, and the effect is very significant and has a high application value.

Description

Technical Field

[0001] The present application relates to the technical field of microbial genetic engineering, and in particular to a high-fucose-producing Escherichia coli and a construction method and application thereof. Background Art

[0002] Fucose (Fucose, FUC) is a widely existing natural sugar, also known as 6-deoxy-galactose. L-fucose is one of the five monosaccharides that constitute breast milk oligosaccharides and one of the eight essential sugars for the human body. It is widely present in animals, plants, and algae, and plays an important physiological function. L-fucose can regulate intestinal flora and intestinal health; it can bind to viruses, bacteria, and toxins to prevent them from infecting cells, thereby enhancing the body's resistance. L-fucose has the function of moisturizing the skin, delaying skin aging, stimulating fibroblast proliferation, and preventing radiation, ultraviolet rays, etc. from damaging fibroblasts. At present, the commercial production of FUC mainly includes methods such as alginate hydrolysis to extract fucoidan and then purify it, chemical methods to convert hexose as raw materials, and enzymatic hydrolysis of extracellular polysaccharides of fucose-producing bacteria. These L-fucose production methods have problems such as low economic benefits, unfriendly to the environment, and limited application scenarios. Therefore, the invention of a new L-fucose production method has potential application value. The biosynthesis method only requires cheap carbon sources and renewable donors within cells as raw materials, and can achieve high economic output at a low environmental cost, so it has broader application prospects.

[0003] The precursors of Fuc synthesis in microorganisms include GDP-L-fucose, 2'-fucosyllactose (2'-FL) synthesized in the cell itself, and exogenously added lactose as a medium. The synthesis process is that first, fructose-6-phosphate synthesizes GDP-L-fucose under the action of genes manA, manB, manC, gmd and wcaG, and extracellular lactose is transported into the cell by β-galactoside permease LacY, and then reacts with GDP-L-fucose to synthesize 2'-FL. 2'-FL is decomposed into fucose and lactose under the action of α-L-fucosidase. However, the current microbial synthesis yield of Fuc is still low and cannot meet the needs of large-scale industrial production. Summary of the invention

[0004] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide an Escherichia coli that reduces the 2'-FL transport efficiency to increase the FUC level and a construction method and application thereof.

[0005] To achieve the above purpose, the technical solution adopted by this application is:

[0006] In a first aspect, the present application provides a high-fucose-producing Escherichia coli, in which the expression level or binding efficiency of a 2'-fucosyllactose transporter is reduced and the expression level of a fucose transporter is increased.

[0007] As a preferred embodiment of the Escherichia coli described in the present application, the Escherichia coli has a knockout or knockdown of a 2'-fucosyllactose transporter and overexpresses a fucose transporter.

[0008] As a preferred embodiment of the Escherichia coli described in the present application, the transport protein of 2'-fucosyllactose includes SetA and / or MdfA; the transport protein of fucose includes FucP and / or EmrD.

[0009] According to the inventor's previous research, 2'-FL is quickly excreted from the cell after synthesis and cannot be hydrolyzed into fucose by α-L-fucosidase. At the same time, fucose accumulates in the cell in a short period of time after production, which brings survival pressure to the cell, which greatly limits the accumulation of FUC. On this basis, the present application provides a high-fucose-producing Escherichia coli, in which an exogenous FUC transporter FucP with a strong promoter is integrated into the genome of Escherichia coli or replaced with an endogenous transporter EmrD with a strong promoter, and the 2'-FL transporter SetA and / or MdfA are knocked out, which can reduce the residual amount of 2'-FL and increase the yield of FUC, with a very significant effect and high application value.

[0010] As a preferred embodiment of the Escherichia coli described in the present application, the amino acid sequence of the 2'-fucosyllactose transporter SetA is shown as SEQ ID NO.1; the amino acid sequence of the 2'-fucosyllactose transporter MdfA is shown as SEQ ID NO.2.

[0011] As a preferred embodiment of the Escherichia coli described in the present application, the fucose transporter FucP is an exogenous transporter derived from Escherichia coli K-12, and the amino acid sequence of the transporter FucP is shown in SEQ ID NO.3.

[0012] As a preferred embodiment of the Escherichia coli described in the present application, the fucose transporter EmrD is an endogenous transporter, and the amino acid sequence of the transporter EmrD is shown in SEQ ID NO.4.

[0013] As a preferred embodiment of the Escherichia coli described in the present application, the Escherichia coli is prepared by using Escherichia coli FUC001 as a chassis strain, knocking out or knocking down the 2'-fucosyllactose transporter, and replacing the integrated exogenous fucose transporter or the natural promoter of the endogenous fucose transporter with a strong promoter.

[0014] As a preferred embodiment of the Escherichia coli described in the present application, the strong promoter is at least one of a T7 promoter, a CaMV promoter, a SV40 promoter and a SFFV promoter.

[0015] In a second aspect, the present application provides a method for constructing the above-mentioned Escherichia coli, comprising the following steps:

[0016] S1. knocking out or knocking down the 2'-fucosyllactose transporter in Escherichia coli FUC001 using gene editing technology to obtain Escherichia coli A; the 2'-fucosyllactose transporter includes SetA and / or MdfA;

[0017] S2. Replace the natural promoter of the gene encoding the fucose transporter in the Escherichia coli A obtained in step S1 with a strong promoter to obtain Escherichia coli with high fucose production; the fucose transporter includes FucP and / or EmrD.

[0018] The present application utilizes gene editing technology to knock out the transporter of 2'-FL to reduce the residual amount of 2'-FL, thereby increasing the accumulation of FUC. The present application utilizes gene editing technology to replace the natural promoter of the transporter encoding gene with a strong promoter to increase the expression level of the endogenous transporter, thereby increasing the transport of LNnT to the extracellular space to increase the extracellular concentration of FUC.

[0019] As a preferred embodiment of the construction method described in the present application, when the fucose transporter is FucP, the operation of step S2 is as follows: construct an expression cassette with a strong promoter coding sequence and a FucP protein coding sequence, clone the expression cassette fragment into an expression vector and transform it into the Escherichia coli A obtained in step S1 to obtain Escherichia coli with high fucose production.

[0020] As a preferred embodiment of the construction method described in the present application, the Escherichia coli FUC001 is mainly prepared by the following steps:

[0021] A1, knocking out the β-galactosidase gene lacZ, the fucose isomerase / fucokinase gene cluster fucIk, and the 11-carbonyl glucose phosphotransferase gene wcaJ of Escherichia coli BL21star (DE3) by gene editing technology to obtain Escherichia coli I;

[0022] A2, transferring the plasmid carrying the coding sequences of mannose phosphate mutant enzyme manB, mannose-1-phosphate guanosyltransferase manC, GDP-D-mannose-4,6-dehydratase Gmd and GDP-L-fucose synthase wcaG into the Escherichia coli I obtained in step A1 to obtain Escherichia coli II;

[0023] A3. The plasmid carrying the coding sequences of α-1,2-fucosyltransferase wbgL and α-L-fucosidase AfcA is transferred into the Escherichia coli II obtained in step A2 to obtain Escherichia coli FUC001.

[0024] In a third aspect, the present application provides the use of the above-mentioned Escherichia coli in producing fucose.

[0025] In a fourth aspect, the present application provides a method for producing fucose, which is mainly obtained by fed-batch fermentation of the above-mentioned Escherichia coli.

[0026] Compared with the prior art, this application has the following beneficial effects:

[0027] The present application provides an Escherichia coli with high fucose production, in which an exogenous FUC transporter FucP with a strong promoter is integrated into the genome of the Escherichia coli or an endogenous transporter EmrD replaced with a strong promoter, and 2'-FL transporters SetA and / or MdfA are knocked out, so that the residual amount of 2'-FL can be reduced from 10-12 g / L to less than 2 g / L, and the yield of FUC can be increased from 50.1 g / L to 66.5 g / L, the effect is very significant, and the application value is high. DETAILED DESCRIPTION

[0028] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with specific embodiments.

[0029] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0030] In the following examples, the gene information used is shown in Table 1.

[0031] Table 1 Genes and their information used in the following examples

[0032]

[0033] In the following examples and comparative examples, unless otherwise specified, the methods used to transfer plasmids into competent cells or to transform them were all chemical transformations; the resistance plates used were kanamycin resistance plates; and the positive transformants screened were identified by PCR detection and cloned fragment sequencing.

[0034] In the following examples and comparative examples, unless otherwise specified, the cloning refers to cloning using a non-ligase-dependent single-fragment one-step cloning kit, and the operation is performed according to the kit instructions. The non-ligase-dependent single-fragment one-step cloning kit is provided by Nanjing Novezan Biotech Co., Ltd., and the product number is C112-02.

[0035] The trace element solution is mainly composed of 13.74g / L nitrilotriacetic acid sodium salt, 5.6g / L ammonium ferric citrate, 0.9g / LZnSO2·7H2O, 0.2g / L CoCl2·6H2O, 1.0g / L MnCl2·4H2O, 0.10g / L CuCl2·2H2O, 0.2g / LH3BO3, and 0.2g / L Na2MoO4·2H2O, and is filtered and sterilized with a filter membrane with a pore size of 0.22μm.

[0036] The fermentation medium used in the fed-batch fermentation experiment contained 10 g / L initial glycerol, 4.0 g / L (NH4)2SO4, 9.2 g / L K2HPO4, 8.2 g / L KH2PO4, 0.3 g / L citric acid, 6.0 g / L tryptone, 2.0 g / L yeast extract, 10 mg / L thiamine, 2.0 g / L MgSO4·7H2O, 0.02 g / L CaCl2 and 10 mL / L trace element solution.

[0037] The feed solution used in the fed-batch fermentation experiment included 800 g / L carbon source and 5 g / L MgSO4·7H2O. The carbon source was glucose and glycerol. The mass ratio of glucose to glycerol was glucose:glycerol=4:6.

[0038] pEcCas vector, pSPIN plasmid, pCDFuet plasmid, pRSFDuet plasmid, pETD plasmid and pEcgRNA plasmid were all provided by Addgene.

[0039] The techniques not described in detail in the following embodiments and effect examples are all commonly used techniques in the art, and reference may be made to "Handbook of Molecular Biology Experiments" (Ma Wenli, People's Military Medical Publishing House), "Molecular Biology Experiments (Second Edition)" (Zhejiang University Press), and "Cell Biology Experiments" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0040] Example 1

[0041] This example provides a high-fucose-producing chassis strain FUC001 and a construction method thereof, wherein the construction method comprises the following steps:

[0042] 1.1 The lacZ, fucIK and wcaj genes in Escherichia coli star BL21 (DE3) were knocked out using the improved CRISPR-Cas9 technology to obtain strain A; the knockout vector used was the pEcCas vector containing Cas9 and λ-Red recombinase, and the pEcgRNA containing the sgRNA sequence and the N20 specific sequence was used for targeted gene editing;

[0043] 1.2 Amplify manB, manC, GDP-mannose 4,6-dehydrogenase gmd and wcaG from Escherichia coli str.K-12 substr.MG1655 by PCR respectively, and insert them into the vector pRSFDuet-1 in sequence through the corresponding restriction sites to obtain the recombinant plasmid pRSFDuet-manC-manB-gmd-wcaG, transform it into the strain A obtained in step 1.1, and obtain positive transformants by resistance plate screening. The obtained positive transformants are strain B;

[0044] 1.3 Based on the wbgL sequence in Escherichia coli O126, the sequence of the α-1,2-fucosyltransferase gene wbgL* was obtained through codon optimization and chemical synthesis. The wbgL gene fragment was obtained by PCR amplification using wbgL-F / R as the primer pair (see Table 1), and inserted into the vector pETDuet-1 through the corresponding restriction sites to obtain the recombinant vector pETDuet-wbgL;

[0045] 1.4 The afcA specific sequence was obtained by codon optimization and chemical synthesis. The expression cassette fragment containing afcA was cloned into the pETDuet-wbgL plasmid obtained in step 1.3 to obtain the pETDuet-wbgL-afcA plasmid, which was transformed into the strain B obtained in step 1.2. The positive transformants were obtained by resistance plate screening. The obtained positive transformants were strain C, and the obtained strain C was FUC001;

[0046] The primer sequences used in the above steps are shown in Table 2.

[0047] Table 2 Primers used to construct E. coli FUC001

[0048]

[0049]

[0050] Example 2

[0051] This embodiment provides a high-fucose-producing Escherichia coli and a construction method thereof, wherein the construction method comprises the following steps:

[0052] 2.1 Entrust a biological company to construct the pCDFuet plasmid with the T7-FucP expression cassette, and use PCR technology (primers FUCP-F / FUCP-R) to obtain the T7-FucP expression cassette fragment;

[0053] 2.2 The rhaA-N32 specific sequence was cloned into the pSPIN plasmid to obtain the pSPIN-rhaA(N32) plasmid, and the T7-FucP expression cassette fragment obtained in step 2.2 was cloned into the pSPIN-rhaA(N32) plasmid to obtain pSPIN-rhaA(N32)-FucP, which was transformed into the strain FUC001 obtained in Example 1 to obtain the strain FUC021;

[0054] 2.3 The setA gene in the strain FUC021 obtained in step 2.2 was knocked out using the improved CRISPR-Cas9 technology to obtain the strain FUC023; the knockout vector used was a pEcCas vector containing Cas9 and λ-Red recombinase, and pEcgRNA containing an sgRNA sequence and an N20-specific sequence was used for targeted gene editing;

[0055] The primers used in the above steps are shown in Table 3.

[0056] Table 3 Primers used to construct E. coli FUC023

[0057]

[0058] Example 3

[0059] This example provides a high-fucose-producing Escherichia coli and a construction method thereof. The construction method is similar to that of Example 2, except that in step 2.3, the knocked-out setA gene is replaced with mdfA, and the remaining steps and operations remain unchanged. The engineered bacteria prepared in step 2.3 are named FUC024, and the primers used in the construction of FUC024 are shown in Table 4.

[0060] Table 4 Primers used to construct E. coli FUC024

[0061]

[0062] Example 4

[0063] This embodiment provides a high-fucose-producing Escherichia coli and a construction method thereof, wherein the construction method comprises the following steps:

[0064] 4.1 Design the homologous fragment of EmrD promoter knockout, add T7 promoter and lactose operator lacO sequence to the primer, and obtain the homologous fragment of T7-lacO-EmrD promoter by PCR;

[0065] 4.2 Obtain the EmrD-N20 fragment by PCR, and clone EmrD-N20 into pEcgRNA using T4 DNA ligase to obtain the pEcgRNA-EmrD plasmid;

[0066] 4.3 The homologous fragment of the T7-lacO-EmrD promoter obtained in step 4.1 and the pEcgRNA-EmrD plasmid obtained in step 4.2 were co-transformed into the strain FUC001 obtained in Example 1 to obtain strain FUC022;

[0067] 4.4 According to the operation of step 2.3 in Example 2, the setA gene in the strain FUC022 obtained in step 4.3 was knocked out to obtain strain FUC029;

[0068] The primers used in the above steps are shown in Table 5.

[0069] Table 5 Primers used to construct E. coli FUC029

[0070]

[0071]

[0072] Example 5

[0073] This example provides a high-fucose-producing Escherichia coli and a construction method thereof. The construction method is similar to that of Example 4, except that in step 4.3, the knocked-out setA gene is replaced with mdfA, and the remaining steps and operations remain unchanged. The engineered bacteria prepared in step 2.3 are named FUC030.

[0074] Example 6

[0075] This embodiment provides a high-fucose-producing Escherichia coli and a construction method thereof, wherein the construction method comprises the following steps:

[0076] 6.1 According to the operation of Example 2, the setA gene in the strain FUC001 obtained in Example 1 was knocked out to obtain the strain FUC011;

[0077] 6.2 Knock out the mdfA gene in the strain FUC011 obtained in step 6.1 according to the procedure of Example 3 to obtain strain FUC017;

[0078] 6.3 According to the procedure of Example 2, the FucP gene was integrated into the FUC017 obtained in step 6.2 to obtain strain FUC035.

[0079] Example 7

[0080] This embodiment provides a high-fucose-producing Escherichia coli and a construction method thereof, wherein the construction method comprises the following steps:

[0081] 7.1 According to the operation of Example 4, the natural promoter of the EmrD gene in FUC017 obtained in step 6.2 of Example 6 was replaced with the T7 promoter to obtain strain FUC036.

[0082] Comparative Examples 1-5

[0083] Comparative Examples 1-5 respectively provide a high-fucose-producing Escherichia coli and a construction method thereof, wherein the construction method is to knock out the mdfA, mdtM, ​​ompC, ompF or proP gene in the strain FUC001 obtained in Example 1 according to the operation of step 2.3 of Example 2. Specifically, the mdfA gene was knocked out in Comparative Example 1, and the resulting strain was named FUC012; the mdtM gene was knocked out in Comparative Example 2, and the resulting strain was named FUC013; the ompC gene was knocked out in Comparative Example 3, and the resulting strain was named FUC014; the ompF gene was knocked out in Comparative Example 4, and the resulting strain was named FUC015; the proP gene was knocked out in Comparative Example 5, and the resulting strain was named FUC016, and the primers used are shown in Table 6.

[0084] Table 6 Primers used to construct E. coli FUC014-FUC016

[0085]

[0086] Comparative Examples 6-9

[0087] Comparative Examples 6-9 respectively provide a high-fucose-producing Escherichia coli and a construction method thereof, wherein the construction method is to knock out the mdtM, ​​ompC, ompF or proP gene in the strain FUC021 obtained in Example 2 according to the operation of step 2.3 of Example 2. Specifically, in Comparative Example 6, the mdtM gene is knocked out, and the resulting strain is named FUC025; in Comparative Example 7, the ompC gene is knocked out, and the resulting strain is named FUC026; in Comparative Example 8, the ompF gene is knocked out, and the resulting strain is named FUC027; in Comparative Example 9, the proP gene is knocked out, and the resulting strain is named FUC028, and the primers used are shown in Table 6.

[0088] Comparative Examples 10-13

[0089] Comparative Examples 10-13 respectively provide a high-fucose-producing Escherichia coli and a construction method thereof, wherein the construction method is to knock out the mdtM, ​​ompC, ompF or proP gene in the strain FUC022 obtained in Example 4 according to the operation of step 2.3 of Example 2. Specifically, in Comparative Example 10, the mdtM gene is knocked out, and the resulting strain is named FUC031; in Comparative Example 11, the ompC gene is knocked out, and the resulting strain is named FUC032; in Comparative Example 12, the ompF gene is knocked out, and the resulting strain is named FUC033; in Comparative Example 13, the proP gene is knocked out, and the resulting strain is named FUC034, and the primers used are shown in Table 6.

[0090] Effect example

[0091] 1. The E. coli of Examples 1-9 and Comparative Examples 1-13 were subjected to a fed-batch fermentation experiment, and the yield of fucose in the fermentation broth was detected by high performance liquid chromatography.

[0092] 1. Fed-batch fermentation experiment

[0093] (1) Escherichia coli was cultured in a 1 L shake flask containing 150 mL of LB medium for 6 h to obtain a secondary seed solution;

[0094] (2) 150 mL of the seed solution obtained in step (1) was transferred into a 5 L fermentation tank containing 2.5 L of fermentation medium, 28% (v / v) NH4OH was automatically added to adjust the pH to 6.8, the dissolved oxygen was maintained at 30-50% by automatically controlling the stirring speed, the culture temperature was 37°C, the ventilation rate was 2 VVM, and the rotation speed was 900 rpm;

[0095] (3) When the initial glycerol in the fermentation medium was completely consumed, the feed solution was added to the fermenter at a constant rate of 4 g / L / h, the fermentation temperature was adjusted to 29.5°C, and the fermentation was continued for 4 h;

[0096] (4) adding isopropyl β-d-1-thiogalactopyranoside to a final concentration of 0.2 mM and 40 g of lactose, supplementing with 17.78 g / L / h of glycerol solution, culturing for 90 h before fermentation is terminated to obtain a fermentation broth.

[0097] 2. Post-treatment of fermentation broth and HPLC detection

[0098] 1 mL of fermentation broth was placed in a boiling water bath for 10 min to kill the strain, and then centrifuged at 12000 g for 10 min. The supernatant was filtered using a 0.22 μm filter, and the filtrate was used to detect FUC and 2'-FL.

[0099] HPX.87H chromatographic column (Bio-Rad) and high performance liquid chromatograph (LC-16, Shimadzu, Japan) were used to detect FUC and 2'-FL in the sample to be tested, and the FUC and 2'-FL content in the sample to be tested was calculated according to the external standard method. The chromatographic conditions were 5 mM sulfuric acid aqueous solution as the mobile phase, flow rate of 0.5 mL / min, column temperature of 60 °C, and injection volume of 10 μL.

[0100] Take 0-2.0g / L gradient concentration of FUC standard, and draw a standard curve with FUC concentration as the horizontal axis and liquid chromatography peak area as the vertical axis; 2'-FL standard curve is drawn according to the FUC method.

[0101] The calculated FUC and 2'-FL concentrations in different fermentation broths were converted to the yields obtained by fermentation in a 5 L fermenter. The results are shown in Table 4.

[0102] Table 4 Calculation results of FUC and 2'-FL production of different Escherichia coli

[0103]

[0104] As shown in Table 1, the present invention knocks out the potential 2'-FL transporter SetA, MdfA, MdtM, OmpC, OmpF, and ProP of the chassis strain FUC001 (FUC011-FUC016), and it is found that the strains with SetA and MdfA knocked out have the least 2'-FL residue. At the same time, the present invention also replaces the natural promoter of the potential endogenous transporter of FUC and the FucP protein from Escherichia coli K12 with the strong promoter T7 promoter (FUC021-FUC022), and it is found that enhancing the expression level of FucP and EmrD can improve the level of FUC. According to the above results, knocking out the 2'-FL transporter and enhancing the expression level of FucP and EmrD are combined respectively, and it is finally found that the combination of knocking out SetA and / or MdfA and enhancing the expression level of FucP or EmrD can greatly reduce the residue of 2'-FL to improve the level of FUC, thereby achieving the purpose of high-yield fucose.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A high-fucose-producing Escherichia coli, characterized in that: The Escherichia coli knocks out a 2'-fucosyllactose transporter and overexpresses a fucose transporter; The transport protein of 2'-fucosyllactose is SetA and / or MdfA; the transport protein of fucose is FucP and / or EmrD; The amino acid sequence of the 2'-fucosyllactose transporter SetA is shown in SEQ ID NO.1; the amino acid sequence of the 2'-fucosyllactose transporter MdfA is shown in SEQ ID NO.2; The fucose transporter FucP is an exogenous transporter derived from Escherichia coli K-12, and the amino acid sequence of the transporter FucP is shown in SEQ ID NO.3; The fucose transporter EmrD is an endogenous transporter, and the amino acid sequence of the transporter EmrD is shown in SEQ ID NO.4; The Escherichia coli uses Escherichia coli FUC001 as a base strain, and the preparation method of the Escherichia coli FUC001 comprises the following steps: A1, knocking out the β-galactosidase gene lacZ, the fucose isomerase / fucokinase gene cluster fucIk, and the 11-carbonyl glucose phosphotransferase gene wcaJ of Escherichia coli BL21star (DE3) by gene editing technology to obtain Escherichia coli I; A2, transferring the plasmid carrying the coding sequences of mannose phosphate mutant enzyme manB, mannose-1-phosphate guanosyltransferase manC, GDP-D-mannose-4,6-dehydratase Gmd and GDP-L-fucose synthase wcaG into the Escherichia coli I obtained in step A1 to obtain Escherichia coli II; A3, transferring the plasmid carrying the coding sequences of α-1,2-fucosyltransferase wbgL and α-L-fucosidase AfcA into the Escherichia coli II obtained in step A2 to obtain Escherichia coli FUC001; The accession number of the phosphomannose mutant enzyme manB is NP_416552.1; The accession number of the mannose-1-phosphate guanosine transferase manC is NP_416553.1; The accession number of the GDP-D-mannose-4,6-dehydratase Gmd is NP_416557.1; The accession number of the GDP-L-fucose synthase wcaG is NP_416556.1; The accession number of the α-1,2-fucosyltransferase wbgL is ADN43847.1; The accession number of the α-L-fucosidase AfcA is AAQ72464.

1.

2. The Escherichia coli according to claim 1, characterized in that The Escherichia coli strain uses Escherichia coli FUC001 as a chassis strain, and the 2'-fucosyllactose transporter is knocked out, and the natural promoter of the integrated exogenous fucose transporter or the endogenous fucose transporter is replaced with a strong promoter.

3. The method for constructing Escherichia coli according to claim 1 or 2, characterized in that: The following steps are involved: S1. knocking out the 2'-fucosyllactose transporter in Escherichia coli FUC001 using gene editing technology to obtain Escherichia coli A; the 2'-fucosyllactose transporter is SetA and / or MdfA; S2. Replace the natural promoter of the gene encoding the fucose transporter in the Escherichia coli A obtained in step S1 with a strong promoter to obtain Escherichia coli with high fucose production; the fucose transporter is FucP and / or EmrD.

4. The construction method according to claim 3, characterized in that: When the fucose transporter is FucP, the operation of step S2 is as follows: construct an expression cassette with a strong promoter coding sequence and a FucP protein coding sequence, clone the expression cassette fragment into an expression vector and transform it into the Escherichia coli A obtained in step S1 to obtain an Escherichia coli with high fucose production.

5. Use of the Escherichia coli as claimed in claim 1 or 2 in producing fucose.

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