CN118931812B

CN118931812B - A kind of Escherichia coli with high fucose production and its construction method and application

Info

Publication number: CN118931812B
Application number: CN202411004466.9A
Authority: CN
Inventors: 吴金勇; 夏子涵; 陈祥松; 袁丽霞; 姚建铭
Original assignee: Hefei Zhongke Health Biotechnology Research Institute Co ltd
Current assignee: Hefei Weihe Hexagonal Biotechnology Co.,Ltd.
Priority date: 2024-07-25
Filing date: 2024-07-25
Publication date: 2025-03-04
Anticipated expiration: 2044-07-25
Also published as: CN118931812A

Abstract

本申请涉及微生物基因工程技术领域，尤其是涉及一种高产岩藻糖的大肠杆菌及其构建方法和应用。本申请提供了一种高产岩藻糖的大肠杆菌，所述大肠杆菌敲除2’‑岩藻糖基乳糖的转运蛋白和过表达岩藻糖的转运蛋白；所述2’‑岩藻糖基乳糖的转运蛋白包括SetA和/或MdfA；所述岩藻糖的转运蛋白包括FucP和/或EmrD。本申请提供了一种高产岩藻糖的大肠杆菌，在大肠杆菌的基因组中整合了带有强启动子的外源FUC转运蛋白FucP或替换为强启动子的内源转运蛋白EmrD，同时敲除了2’‑FL转运蛋白SetA和/或MdfA，能够使2’‑FL的残留量下降，使FUC的产量提高，效果十分显著，具有较高的应用价值。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

Escherichia coli for high-yield fucose and construction method and application thereof

Technical Field

The application relates to the technical field of microbial genetic engineering, in particular to escherichia coli for high-yield fucose, and a construction method and application thereof.

Background

Fucose (Fucose, FUC) is a widely existing natural sugar, also called 6-deoxy-galactose, and L fucose is one of five monosaccharides constituting breast milk oligosaccharide, is one of eight essential sugars for human body, and is widely existing in animals, plants, and algae, exerting important physiological functions. L-fucose can regulate intestinal flora, regulate intestinal health, and can be combined with virus, bacteria and toxin to prevent cell infection, thereby enhancing body resistance. The L-fucose has the functions of moisturizing skin, delaying skin aging, stimulating fibroblast proliferation, and preventing injury to fibroblasts caused by radiation, ultraviolet rays and the like. At present, commercial production of FUC mainly comprises methods of extracting fucoidan by alginic acid hydrolysis and purifying again, converting hexose by chemical method as raw material, and hydrolyzing bacterial extracellular polysaccharide of producing fucose by enzyme, etc., and the L-fucose production methods have the problems of low economic benefit, unfriendly environment, limited application scene, etc., so the novel L-fucose production method has potential application value. The biosynthesis method only needs to take cheap carbon sources and renewable donors in cells as raw materials, and obtains high economic output at lower environmental cost, thus having wider application prospect.

The synthetic precursors of Fuc in microorganisms include intracellular self-synthesized GDP-L-fucose, 2 '-fucosyllactose (2' -FL) and exogenously added lactose as vehicles. The synthesis process includes synthesizing GDP-L-fucose with fructose-6-phosphate under the action of gene manA, manB, manC, gmd and wcaG, transferring extracellular lactose to cell with beta-galactoside permease LacY, and synthesizing 2' -FL with GDP-L-fucose. 2' -FL is decomposed into fucose and lactose by the action of alpha-L-fucosidase. However, the microbial synthesis yield of Fuc is still low at present, and the requirement of mass industrial production cannot be met.

Disclosure of Invention

The application aims to overcome the defects of the prior art and provide the escherichia coli for reducing the 2' -FL transfer efficiency to improve the FUC level, and the construction method and the application thereof.

In order to achieve the above purpose, the technical scheme adopted by the application is as follows:

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

As a preferred embodiment of the E.coli of the present application, the E.coli knocks out or knocks down a transporter of 2' -fucosyllactose and a transporter over-expressing fucose.

As a preferred embodiment of the E.coli of the present application, the transporter of 2' -fucosyllactose comprises SetA and/or MdfA, and the transporter of fucose comprises FucP and/or EmrD.

According to previous studies of the inventors, it was found that 2' -FL is excreted out of cells faster after synthesis, cannot be catalyzed and hydrolyzed into fucose by α -L-fucosidase, and accumulation in cells in a short time after fucose production brings survival pressure to cells, which greatly limits accumulation of FUC. On the basis, the application provides the escherichia coli with high fucose yield, the exogenous FUC transport protein FucP with the strong promoter or the endogenous transport protein EmrD replaced by the strong promoter is integrated in the genome of the escherichia coli, and meanwhile, the 2'-FL transport protein SetA and/or the MdfA are knocked out, so that the residual quantity of the 2' -FL can be reduced, the yield of FUC is improved, the effect is very obvious, and the escherichia coli has higher application value.

As a preferred embodiment of the escherichia coli, the amino acid sequence of the transport protein SetA of the 2 '-fucosyllactose is shown as SEQ ID NO.1, and the amino acid sequence of the transport protein MdfA of the 2' -fucosyllactose is shown as SEQ ID NO. 2.

As a preferred embodiment of the escherichia coli, 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 as SEQ ID No. 3.

As a preferred embodiment of the E.coli of 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.

As a preferred embodiment of the escherichia coli, the escherichia coli is prepared by taking escherichia coli FUC001 as a chassis strain and replacing a natural promoter of an integrated exogenous fucose transporter or endogenous fucose transporter with a strong promoter by knocking out or knocking down the 2' -fucosyl lactose transporter.

As a preferred embodiment of the E.coli of the present application, the strong promoter is at least one of T7 promoter, caMV promoter, SV40 promoter and SFFV promoter.

In a second aspect, the application provides a construction method of the escherichia coli, comprising the following steps:

S1, knocking out or knocking down a transport protein of 2 '-fucosyllactose in escherichia coli FUC001 by utilizing a gene editing technology to obtain escherichia coli A, wherein the transport protein of 2' -fucosyllactose comprises SetA and/or MdfA;

S2, replacing a natural promoter of a fucose transporter coding gene in the escherichia coli A obtained in the step S1 with a strong promoter to obtain the escherichia coli with high fucose yield, wherein the fucose transporter comprises FucP and/or EmrD.

The application uses gene editing technology to knock out the transport protein of 2'-FL to reduce the residual quantity of 2' -FL, thereby improving the accumulation of FUC. According to the application, the natural promoter of the transporter encoding gene is replaced by a strong promoter through a gene editing technology, so that the expression level of the endogenous transporter can be improved, and the LNnT is transported to the outside of cells to improve the extracellular concentration of FUC.

As a preferred embodiment of the construction method of the present application, when the fucose transporter is FucP, the operation of step S2 is to construct an expression cassette having a strong promoter coding sequence and a FucP protein coding sequence, clone the expression cassette fragment into the expression vector and transform into E.coli A obtained in step S1, thereby obtaining E.coli with high fucose yield.

As a preferred embodiment of the construction method of the present application, the E.coli FUC001 is prepared mainly by the steps of:

a1, knocking out a beta-galactosidase gene lacZ of escherichia coli BL21star (DE 3), a fucose isomerase/fucokinase gene cluster fucIk and an undecyl phosphate glucose phosphotransferase gene wcaJ by a gene editing technology to obtain escherichia coli I;

A2, transferring plasmids with phosphomannose mutase manB, mannose-1-guanosine phosphate transferase manC, GDP-D-mannose-4, 6-dehydratase Gmd and GDP-L-fucose synthetase wcaG coding sequences into the escherichia coli I obtained in the step A1 to obtain escherichia coli II;

A3, transferring the plasmid with the alpha-1, 2-fucosyltransferase wbgL and the alpha-L-fucosidase AfcA coding sequences into the escherichia coli II obtained in the step A2 to obtain escherichia coli FUC001.

In a third aspect, the application provides the use of the above E.coli for the production of fucose.

In a fourth aspect, the application provides a process for the production of fucose, mainly obtained by fed-batch fermentation of E.coli as described above.

Compared with the prior art, the application has the following beneficial effects:

The application provides a high-yield fucose escherichia coli, wherein an exogenous FUC transport protein FucP with a strong promoter or an endogenous transport protein EmrD replaced by the strong promoter is integrated in the genome of the escherichia coli, meanwhile, 2'-FL transport protein SetA and/or MdfA are knocked out, the residual quantity of 2' -FL can be reduced from 10-12g/L to within 2g/L, the yield of FUC is improved from 50.1g/L to 66.5g/L, the effect is very remarkable, and the high-yield fucose escherichia coli has high application value.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present application, the present application will be further described with reference to the following specific examples.

In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.

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

TABLE 1 Gene used in the following examples and information thereof

In the following examples and comparative examples, unless otherwise specified, the plasmid used was transformed into competent cells or transformed by chemical transformation, the resistance plate used was a kanamycin resistance plate, and positive transformants obtained by selection were identified by PCR detection and sequencing of cloned fragments.

In the following examples and comparative examples, unless specifically indicated, cloning was performed using a single-piece, one-step cloning kit that was not ligase dependent, and was operated according to the kit instructions, and was supplied by Nanjinouzan Biotechnology Co., ltd, cat No. C112-02.

The microelement solution mainly comprises 13.74g/L nitrilotriacetic acid sodium salt and 5.6g/L ferric ammonium citrate 、0.9g/L ZnSO₂·7H₂O、0.2g/L CoCl₂·6H₂O、1.0g/L MnCl₂·4H₂O、0.10g/L CuCl2·2H₂O、0.2g/L H₃BO₃、0.2g/L Na₂MoO₄·2H₂O, and is filtered and sterilized by a filter membrane with the pore diameter of 0.22 mu m.

The fermentation medium used in the fed-batch fermentation experiments contained 10g/L of initial glycerol 、4.0g/L(NH₄)₂SO₄、9.2g/L K₂HPO₄、8.2g/L KH₂PO₄、0.3g/L citric acid, 6.0g/L of tryptone, 2.0g/L of yeast extract, 10mg/L of thiamine, 2.0g/L of MgSO ₄·7H₂O、0.02g/L CaCl₂ and 10mL/L of trace element solution.

The feed solution used in the fed-batch fermentation experiment included 800g/L of a carbon source, glucose and glycerol in a mass ratio of glucose to glycerol=4:6, and 5g/L of MgSO ₄·7H₂ O.

PEcCas vector, pSPIN plasmid, pCDFuet plasmid, pRSFDuet plasmid, pETD plasmid and pEcgRNA plasmid are all provided by addgene.

The techniques described in detail in the following examples and effect examples are common in the art, and reference may be made to "molecular biology laboratory Manual (Ma Wenli, civil military press)", "molecular biology experiment (second edition)", "Zhejiang university press)", and "cell biology experiment" (Yang Hongbing, hou Lixia, zhang Yuxi, higher education press).

Example 1

The embodiment provides a chassis strain FUC001 for high-yield fucose and a construction method thereof, wherein the construction method comprises the following steps:

1.1, using improved CRISPR-Cas9 technology to knock out lacZ, fucIK and wcaj genes in escherichia coli star BL21 (DE 3) to obtain a strain A, wherein the adopted knock-out vector is pEcCas vector containing Cas9 and lambda-Red recombinase, and pEcgRNA containing sgRNA sequence and N20 specific sequence is used for targeted gene editing;

1.2 amplifying manB, manC, GDP-mannose 4, 6-dehydrogenase gmd and wcaG from escherichia coli str.K-12 substrestring.MG1655 by PCR respectively, sequentially inserting the same into a vector pRSFDuet-1 by corresponding enzyme cutting sites to obtain a recombinant plasmid pRSFDuet-manC-manB-gmd-wcaG, transforming the recombinant plasmid into the strain A obtained in the step 1.1, and screening the recombinant plasmid by a resistance flat plate to obtain a positive transformant, wherein the obtained positive transformant is the strain B;

1.3 obtaining a sequence of alpha-1, 2-fucosyltransferase gene wbgL by codon optimization and chemical synthesis according to wbgL sequences in escherichia coli O126, obtaining wbgL gene fragments by PCR amplification with wbgL-F/R as a primer pair (see table 1), and inserting the wbgL gene fragments into a vector pETDuet-1 through corresponding cleavage sites to obtain a recombinant vector pETDuet-wbgL;

1.4, obtaining afcA specific sequence through codon optimization and chemical synthesis, cloning afcA-containing expression cassette fragment into pETDuet-wbgL plasmid obtained in step 1.3 to obtain pETDuet-wbgL-afcA plasmid, transforming into strain B obtained in step 1.2, screening by a resistance plate to obtain a positive transformant, wherein the obtained positive transformant is strain C, and the obtained strain C is FUC001;

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

TABLE 2 primers used in the construction of E.coli FUC001

Example 2

The embodiment provides escherichia coli for high fucose production and a construction method thereof, wherein the construction method comprises the following steps of:

2.1 entrusting biological company to construct pCDFuet plasmid with T7-FucP expression cassette, obtaining T7-FucP expression cassette fragment by PCR technique (primer FUCP-F/FUCP-R);

2.2 cloning rhaA-N32 specific sequence into pSPIN plasmid to obtain pSPIN-rhaA (N32) plasmid, cloning the T7-FucP expression cassette fragment obtained in step 2.2 into pSPIN-rhaA (N32) plasmid to obtain pSPIN-rhaA (N32) -FucP, and transforming into strain FUC001 obtained in example 1 to obtain strain FUC021;

2.3 knocking out setA genes in the strain FUC021 obtained in the step 2.2 by utilizing an improved CRISPR-Cas9 technology to obtain a strain FUC023, wherein the adopted knocking-out vector is a pEcCas vector containing Cas9 and lambda-Red recombinase, and pEcgRNA containing sgRNA sequences and N20 specific sequences is used for targeted gene editing;

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

TABLE 3 primers used in the construction of E.coli FUC023

Example 3

The embodiment provides a high-fucose-yield escherichia coli and a construction method thereof, wherein the construction method is similar to that of the embodiment 2, except that in the step 2.3, the knocked-out setA gene is replaced by mdfA, the rest steps and operations are unchanged, the engineering bacteria prepared in the step 2.3 are named FUC024, and primers adopted in the process of constructing the FUC024 are shown in Table 4.

TABLE 4 primers used in the construction of E.coli FUC024

Example 4

4.1 designing EmrD homologous fragments of the promoter knockout, adding a T7 promoter and lactose operon lacO sequence into the primer, and obtaining the homologous fragments of the T7-lacO-EmrD promoter through PCR;

4.2 cloning EmrD-N20 into pEcgRNA by PCR to give a fragment EmrD-N20 using T4 DNA ligase to give a pEcgRNA-EmrD plasmid;

4.3 Co-transformation of the homologous fragment of the T7-lacO-EmrD promoter from step 4.1 and the pEcgRNA-EmrD plasmid from step 4.2 into the strain FUC001 from example 1 to give strain FUC022;

4.4 the setA gene in the strain FUC022 obtained in step 4.3 was knocked out according to the procedure of step 2.3 in example 2, to obtain strain FUC029;

the primers used in the above steps are shown in Table 5.

TABLE 5 primers used in the construction of E.coli FUC029

Example 5

The embodiment provides a high-fucose-yield escherichia coli and a construction method thereof, wherein the construction method is similar to that of the embodiment 4, and the difference is that in the step 4.3, the knocked setA gene is replaced by mdfA, the rest steps and the operation are unchanged, and the engineering bacteria prepared in the step 2.3 are named FUC030.

Example 6

6.1 the setA gene of strain FUC001 obtained in example 1 was knocked out according to the procedure of example 2 to give strain FUC011;

6.2 the mdfA gene in strain FUC011 obtained in step 6.1 was knocked out according to the procedure of example 3 to obtain strain FUC017;

6.3 the FucP gene was integrated into the FUC017 obtained in step 6.2 according to the procedure of example 2, resulting in strain FUC035.

Example 7

7.1 substitution of the T7 promoter with the natural promoter of EmrD gene in FUC017 obtained in step 6.2 of example 6 was performed according to the procedure of example 4 to obtain the strain FUC036.

Comparative examples 1 to 5

Comparative examples 1 to 5 respectively provide a high-yield fucose E.coli and a construction method thereof, in which mdfA, mdtM, ompC, ompF or proP gene in the strain FUC001 obtained in example 1 was knocked out, respectively, according to the procedure of step 2.3 of example 2. Specifically, comparative example 1 knocked out mdfA gene, resulting strain was designated FUC012, comparative example 2 knocked out mdtM gene, resulting strain was designated FUC013, comparative example 3 knocked out ompC gene, resulting strain was designated FUC014, comparative example 4 knockout ompF gene, resulting strain was designated FUC015, comparative example 5 knocked out proP gene, resulting strain was designated FUC016, and the primers used are shown in table 6.

TABLE 6 primers used in the construction of E.coli FUC014-FUC016

Comparative examples 6 to 9

Comparative examples 6 to 9 respectively provide a high-yield fucose E.coli and a construction method thereof, in which mdtM, ompC, ompF or proP gene in the strain FUC021 obtained in example 2 was knocked out, respectively, by the procedure of step 2.3 of example 2. Specifically, comparative example 6 knockdown mdtM gene, resulting strain was designated FUC025, comparative example 7 knockdown ompC gene, resulting strain was designated FUC026, comparative example 8 knockdown ompF gene, resulting strain was designated FUC027, comparative example 9 knockdown proP gene, resulting strain was designated FUC028, and the primers used are shown in table 6.

Comparative examples 10 to 13

Comparative examples 10 to 13 respectively provided a high-yield fucose E.coli and a construction method thereof, in which mdtM, ompC, ompF or proP gene in the strain FUC022 obtained in example 4 was knocked out, respectively, according to the procedure of step 2.3 of example 2. Specifically, comparative example 10 knockdown mdtM gave the strain designated FUC031, comparative example 11 knockdown ompC gene, the strain designated FUC032, comparative example 12 knockdown ompF gene, the strain designated FUC033, and comparative example 13 knockdown proP gene, the primers used are shown in Table 6.

Effect example

1. The E.coli of examples 1-9 and comparative examples 1-13 were subjected to fed-batch fermentation experiments, and the production of fucose in the fermentation broth was detected by high performance liquid chromatography.

1. Batch fed fermentation experiments

(1) E.coli is cultivated in a 1L shake flask containing 150mL LB culture medium for 6h, and secondary seed liquid is obtained;

(2) Transferring 150mL of the seed solution obtained in the step (1) into a 5L fermentation tank containing 2.5L fermentation medium, automatically adding 28% (v/v) NH ₄ OH to adjust the pH to 6.8, and maintaining dissolved oxygen at 30-50% by automatically controlling the stirring speed, wherein the culture temperature is 37 ℃, the aeration rate is 2VVM, and the rotating speed is 900rpm;

(3) After the initial glycerol in the fermentation medium is completely consumed, feeding the feed solution into the fermentation tank at a constant rate of 4g/L/h, and fermenting for 4h at a fermentation temperature of 29.5 ℃;

(4) Adding isopropyl beta-d-1-thiopyran galactoside to make the final concentration of the isopropyl beta-d-1-thiopyran galactoside be 0.2mM and 40g lactose, supplementing 17.78g/L/h glycerol solution, and after culturing for 90 hours, ending fermentation to obtain fermentation liquor.

2. Post-treatment of fermentation broth and high performance liquid chromatography detection

1ML of the fermentation broth was boiled in water for 10min to kill the strain, and then centrifuged at 12000g for 10min, and the obtained supernatant was filtered using a 0.22 μm filter, and the obtained filtrate was used for detection of FUC and 2' -FL.

FUC and 2'-FL were detected in the sample to be measured using hpx.87h column (berle), high performance liquid chromatograph (LC-16, shimadzu), and the FUC and 2' -FL contents in the sample to be measured were calculated according to the external standard method. The chromatographic conditions were 5mM aqueous sulfuric acid solution as mobile phase, flow rate of 0.5mL/min, column temperature of 60℃and sample injection amount of 10. Mu.L.

And drawing a standard curve by taking FUC standard substances with gradient concentration of 0-2.0g/L and taking FUC concentration as an abscissa and liquid chromatography peak area as an ordinate, and drawing a 2' -FL standard curve according to the FUC method.

The calculated FUC and 2' -FL concentrations in the different fermentation broths were converted to the 5L fermentors and the results are shown in Table 4.

TABLE 4 FuC and 2' -FL yield calculations for different E.coli

As shown in Table 1, the present invention found minimal residues of 2'-FL from strains knockout SetA and MdfA by knocking out the potential 2' -FL transporter SetA, mdfA, mdtM, ompC, ompF, proP of Chassis strain FUC001 (FUC 011-FUC 016). Meanwhile, the invention also discovers that the enhancement of the expression level of FucP and EmrD can improve the level of FUC by replacing the natural promoters of FUC potential endogenous transport proteins and FucP proteins derived from escherichia coli K12 with a strong promoter T7 promoter (FUC 021-FUC 022). According to the results, the expression levels of the knockdown 2'-FL transporter and the enhancement FucP and EmrD are respectively combined, and finally, the combination of the knockdown SetA and/or MdfA and the enhancement FucP or EmrD expression levels is found to be capable of greatly reducing the residue of 2' -FL so as to improve the FUC level and achieve the purpose of high-yield fucose.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the scope of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present application.

Claims

1. A high-fucose-producing Escherichia coli, characterized in that the Escherichia coli has a knockout of 2'-fucosyllactose transporter and an overexpressed 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 is based on Escherichia coli FUC001, wherein 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 it comprises the following steps:

S1. Using gene editing technology to knock out the 2'-fucosyllactose transporter in Escherichia coli FUC001 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: constructing an expression cassette with a strong promoter coding sequence and a FucP protein coding sequence, cloning the expression cassette fragment into an expression vector and transforming it into the Escherichia coli A obtained in step S1 to obtain Escherichia coli with high fucose production.

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