CN110734889B

CN110734889B - Escherichia coli engineering strain for efficiently producing GDP-fucose

Info

Publication number: CN110734889B
Application number: CN201911092950.0A
Authority: CN
Inventors: 沐万孟; 张文立; 万李; 朱莺莺; 李雯
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-11-11
Filing date: 2019-11-11
Publication date: 2021-07-27
Anticipated expiration: 2039-11-11
Also published as: CN110734889A

Abstract

The invention provides an escherichia coli producing strain for efficiently producing GDP-fucose, belonging to the technical field of genetic engineering and microbial metabolic engineering. The invention takes glucose as a substrate, and relieves the metabolic pressure of a GDP-fucose synthesis pathway by combining and regulating the expression of phosphomannose mutase (ManB), GDP-mannose pyrophosphorylase (ManC), GDP-mannose 4, 6-dehydratase (Gmd) and GDP-fucose synthetase (Wcag) in the metabolic pathway. The invention also knocks out a gene UDP-glucose lipid carrier transferase (WcaJ) for decomposing GDP-fucose in escherichia coli through a CRISPR/Cas9 gene editing system and enhances the regeneration of cofactors in a metabolic pathway to further improve the accumulation of intracellular GDP-fucose.

Description

Escherichia coli engineering strain for efficiently producing GDP-fucose

Technical Field

The invention relates to an escherichia coli engineering strain for efficiently producing GDP-fucose, belonging to the technical field of genetic engineering and microbial metabolic engineering.

Background

GDP-fucose is a nucleotide sugar that is widely used as a glycosyl donor and a metabolic intermediate in various organisms and is an essential substance in the regulation of life metabolism. GDP-fucose is mainly used as an important glycosyl donor for protein glycosylation and is a precursor for the synthesis of other sugar nucleotides in eukaryotes and is mainly involved in the biosynthesis of fucosylated oligosaccharides in prokaryotes.

Human Milk Oligosaccharides (HMOs) are one of the most abundant solid components in human milk. The biological functions of the compound, such as improving intestinal flora, inhibiting pathogen adhesion, regulating immune response and promoting brain development, have wide prospects for development of infant development. In HMOs, the proportion of fucosylated oligosaccharides is about 50%, which requires GDP-fucose as a fucose donor and synthesizes fucosidyl bonds to lactose or more complex nascent oligosaccharides through functional expression of fucosyltransferases.

However, large-scale production of fucosylated HMOs can be a difficult task due to limitations in availability of GDP-fucose. As an essential precursor in fucosylation HMOs biosynthesis, the method for effectively producing GDP-fucose is significant.

Currently there are mainly two metabolic pathways that produce GDP-fucose, the de novo synthetic pathway and the salvage pathway, respectively. In the de novo synthesis pathway, fructose-6-phosphate is converted to GDP-mannose by three enzymes, mannose-6-phosphate isomerase (ManA), phosphomannose mutase (ManB) and GDP-mannose pyrophosphorylase (ManC). Where the enzyme reaction of ManC requires the cofactor guanosine 5' -triphosphate (GTP). The GDP-mannose is subjected to three-step reaction by two enzymes, namely GDP-mannose 4, 6-dehydratase (Gmd) and GDP-fucose synthetase (Wcag), to finally generate the target product GDP-fucose. Wherein the enzyme reaction of Wcag requires the participation of the cofactor NADPH. The salvage pathway requires the bifunctional enzyme fucokinase/fucose-1-phosphate guanylyltransferase (Fkp) from Bacteroides fragilis to directly convert the externally supplied fucose to GDP-fucose. This process also requires GTP as a cofactor.

The pentose phosphate pathway is an important pathway for providing intracellular NADPH, and glucose 6-phosphate dehydrogenase Zwf can promote the regeneration of NADPH. In addition, guanosine-inosine kinase Gsk can convert inosine and guanosine into GTP, an important cofactor in the reaction process, in the guanosine nucleotide biosynthesis pathway.

To date, several recombinant microorganisms have been developed for the biosynthesis of GDP-fucose. Cremoris NZ9000 expresses 4 key enzymes ManB, ManC, Gmd and WcaG, and GDP-fucose is synthesized by a one-pot method using mannose 6-phosphate as a substrate in a recent study. Despite the relatively high conversion efficiency of the substrate, regeneration of GTP is a great challenge for further production of GDP-fucose by a multi-enzyme cascade.

Lee et al have performed a series of work to increase the production of GDP-fucose based on the de novo pathway in engineered E.coli. By optimizing the carbon source, overexpressing key enzymes, enhancing NADPH or GTP regeneration as a cofactor and adjusting fed-batch fermentation mode, the yield of GDP-fucose was significantly increased from 55.2mg/L to 305.5 mg/L. However, the above yields are higher intracellular GDP-fucose accumulation when fed-batch fermentation is performed using a 2.5L fermentor and sufficient glucose supply is continued to increase cell density to a large extent, but the product accumulation in shake flasks is less than 10 mg/L.

Furthermore, in the above example, the investigator did not make modular adjustments to the de novo synthetic pathway, considering only a single variable and modulating the entire metabolic pathway in a linear fashion (simply overexpressing the enzymes involved in the GDP-fucose synthetic pathway), or a single enhanced regeneration of NADPH or GTP regeneration, which may lead to metabolic burden and flux imbalances, failing to achieve globally optimal yields.

Disclosure of Invention

Aiming at the problems of the prior art, the invention provides an escherichia coli engineering bacterium for efficiently producing GDP-fucose and a construction method thereof.

The first purpose of the invention is to provide an escherichia coli engineering bacterium for efficiently producing GDP-fucose, which can be used for regulating and controlling the expression of phosphomannose mutase ManB, GDP-mannose pyrophosphorylase ManC, GDP-mannose 4, 6-dehydratase Gmd and GDP-fucose synthetase Wcag.

In one embodiment of the invention, the engineered Escherichia coli also enhances the expression of glucose 6-phosphate dehydrogenase Zwf and guanosine-inosine kinase Gsk.

In one embodiment of the invention, the engineered escherichia coli also silences the expression of UDP-glucolipid carrier transferase WcaJ.

In one embodiment of the invention, the host bacterium of the engineering bacterium of Escherichia coli is Escherichia coli BL21(DE3), and the expression vectors are pACYCDuet-1, pETuet-1 and pCDFDuet-1.

In one embodiment of the invention, the Escherichia coli engineering bacteria express manC-manB as pACYCDuet-1, gmd-wcaG as pETuet-1 and gsk-zwf as pCDFDuet-1.

In one embodiment of the invention, the genes of ManB, ManC, Gmd, Wcag, Gsk and Zwf are derived from Escherichia coli (Escherichia coli) MG1655, and the nucleotide sequences are SEQ ID NO. 1-6 in sequence.

In one embodiment of the invention, the standard ribosome binding site and wild-type ribosome binding site are used to replace the ribosome binding site of the pACYCDuet-1-manC-manB and pETDuet-1-gmd-wcaG plasmids in E.coli engineering bacteria.

In one embodiment of the present invention, the nucleotide sequences of ribosome binding site RBS-32, RBS-ori, RBS-WT (manB), RBS-WT (manC), RBS-WT (gmd) and RBS-WT (wcaG) are SEQ ID Nos. 7 to 12 in that order.

The second purpose of the invention is to provide a method for improving the capability of producing GDP-fucose by escherichia coli, which is characterized in that the expression of phosphomannose mutase ManB, GDP-mannose pyrophosphorylase ManC, GDP-mannose 4, 6-dehydratase Gmd and GDP-fucose synthetase Wcag is controlled in a combined way; the expression is regulated by adopting a high copy expression element to regulate the expression of Gmd and Wcag, and adopting a low copy expression element to regulate the expression of ManB and ManC.

The third purpose of the invention is to provide a construction method of escherichia coli engineering bacteria for efficiently producing GDP-fucose, which comprises the following steps:

(1) constructing a pCDFDuet-1-gsk-zwf recombinant expression vector;

(2) constructing pACYCDuet-1-manC-manB, pETDuet-1-manC-manB, pCDFDuet-1-manC-manB, pACYCDuet-1-gmd-wcaG, pETDuet-1-gmd-wcaG and pCDFDuet-1-gmd-wcaG recombinant expression vectors, and screening out a recombinant plasmid combination with the highest GDP-fucose yield;

(3) replacing the ribosome binding sites of the selected pACYCDuet-1-manC-manB and pETDuet-1-gmd-wcaG plasmids with standard ribosome binding sites and wild-type ribosome binding sites, and comparing recombinant expression vectors with the highest GDP-fucose yield under the control of different RBSs;

(4) knocking out WcaJ gene in chromosome of escherichia coli BL21(DE3) by using CRISPR/Cas9 gene editing system;

(5) escherichia coli BL21(DE3) with the wcaJ gene removed is used as a host, and pCDFDuet-1-gsk-zwf and pACYCDuet-1-manC-manB and pETDuet-1-gmd-wcaG recombinant expression vectors are overexpressed.

The fourth purpose of the invention is to provide a fermentation method of escherichia coli engineering bacteria for efficiently producing GDP-fucose, which takes the genetic engineering bacteria as fermentation microorganisms and utilizes glucose as a carbon source to synthesize GDP-fucose.

The invention also claims the application of the strain in the preparation of GDP-fucose and derivatives thereof.

The invention has the beneficial effects that:

the invention utilizes genetic engineering technology to transform escherichia coli, regulates related genes of a GDP-fucose synthesis pathway through modular combination, regulates a Ribosome Binding Site (RBS) to regulate and control the translation strength, strengthens regeneration of cofactors related to a metabolic pathway, and knocks out related genes of a GDP-fucose catabolism pathway, thereby accurately regulating and controlling the carbon flux of the metabolic pathway, relieving metabolic pressure, improving the accumulation amount of intracellular GDP-fucose, accumulating 18mg/L GDP-fucose after fermenting for 20 hours, and compared with the yield of 10mg/L GDP-fucose obtained by shake flask fermentation in the prior art, the invention improves the yield of the GDP-fucose to 2 times of that in the prior art, and has important industrial application value.

Drawings

FIG. 1 is a diagram of the GDP-fucose metabolic pathway;

FIG. 2 is a flow chart of recombinant plasmid construction (taking pETDuet-1-manC-manB as an example);

FIG. 3 shows the result of agarose gel electrophoresis for PCR amplification of plasmid construction (taking manC and manB as examples);

FIG. 4 is a flow chart of the CRISPR/Cas9 gene editing system knocking out the wcaJ gene of the E.coli genome;

FIG. 5 is a gel electrophoresis of a knockout gene wcaJ nucleic acid;

FIG. 6 shows the protein expression levels of manC, manB, gmd, wcAG under the control of different RBSs;

FIG. 7 is the product GDP-fucose standard sample and the product sample mass spectrogram: wherein (1) a GDP-fucose standard; (2) (ii) a GDP-fucose fermentation product;

FIG. 8 shows the fermentation yield of GDP-fucose under different conditions: wherein (1): production of GDP-fucose by metabolic pathways under regulation by different copy number plasmids; (2): production of GDP-fucose by metabolic pathways under RBS modulation of varying intensities; (3): the yield of GDP-fucose of the bypass gene wcaJ is knocked out; (4): (iii) enhanced production of GDP-fucose following the cofactor regeneration pathway.

Detailed Description

The invention is further illustrated by the following specific examples.

Fermentation medium: 20g/L glucose, 13.5g/L potassium dihydrogen phosphate, 4.0g/L diammonium hydrogen phosphate, 1.7g/L citric acid, 1.4g/L magnesium sulfate heptahydrate and 10ml/L trace metal elements; the trace metal elements comprise 10g/L ferrous sulfate, 2.25g/L zinc sulfate heptahydrate, 1.0g/L anhydrous copper sulfate, 0.35g/L manganese sulfate monohydrate, 0.23g/L sodium borate decahydrate, 0.11g/L ammonium molybdate and 2.0g/L calcium chloride dihydrate.

LB solid medium: 10g/L peptone, 5g/L yeast extract powder, 10g/L sodium chloride and 15g/L agar powder.

LB liquid medium: 10g/L peptone, 5g/L yeast extract powder and 10g/L sodium chloride.

Protein expression detection:

the expression levels of overexpressed enzymes involved in the de novo pathway of GDP-fucose biosynthesis were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The overnight-cultured seed solution was inoculated into 25mL of LB medium, 2mL of the culture was taken 6 hours after induction with 1mM IPTG, and the cells were collected by centrifugation at 8,000 rpm. The cell pellet was resuspended in 50mM Tris-HCl (pH 7.5) and loading buffer was added, followed by 10 min boiling to rupture it. The supernatant was then collected by centrifugation at 12,000rpm for 10 minutes. SDS-PAGE analysis was performed on supernatant samples of different host strains. The gel was run at 80V until the sample completely passed through the concentrate. Then, the voltage was maintained at 120V. The gel was stained with Coomassie Brilliant blue for 3 hours and then destained with destaining solution. The gels were photographed using a Tanon gel imager and protein expression was analyzed.

Strain culture and fermentation:

each strain was inoculated into 4mL of LB medium. After overnight incubation, 0.5mL of the culture was inoculated into 25mL of the fermentation medium containing the corresponding plasmid antibiotic (final antibiotic concentrations: 100. mu.g/mL ampicillin, 25. mu.g/mL chloramphenicol, 30. mu.g/mL kanamycin and 50. mu.g/mL streptomycin.) and incubated at 200rpm and 37 ℃ in 250mL shake flasks. When the cell OD₆₀₀When the concentration reaches 0.6-0.8, isopropyl-beta-D-thiogalactopyranoside (IPTG) is added to the final concentration of 0.1 mM. Adding glucose as carbon source with final concentration of 20g/L, culturing at 25 deg.C, collecting fermentation broth after 20 hr, and detecting GDP-fucose yield.

And (3) detecting the synthesis of GDP-fucose:

to measure the yield of GDP-fucose after cell fermentation, 5mL of the culture was collected by centrifugation (8,000rpm, 15 minutes), and the collected cells were resuspended in an extraction buffer containing 50mM Tris-HCl (pH 7.5), 150mM sodium chloride, 10mM magnesium chloride, 5 mM. beta. -mercaptoethanol, and 5mM EDTA and then subjected to ultrasonication treatment. After centrifugation (8,000rpm, 10 minutes), the supernatant was analyzed by means of a High Performance Liquid Chromatography (HPLC) system (Agilent Technologies) and Inertsil ODS-SP column, UV wavelength 254 nm.

The column was eluted sequentially with 100% (v/v) solvent A (20 mM triethylamine acetate buffer pH 6.0) at a flow rate of 0.6mL/min for 10 minutes; a linear gradient of 0-4% solvent B (acetonitrile) for 10 min; eluent B linear gradient from 4% to 0 for 5 min; and 100% eluent A for 25 minutes (GDP-fucose product confirmation see FIG. 7).

Constructing detailed information of engineering bacteria:

example 1: construction of recombinant vectors

The specific steps are as follows (the construction process can refer to fig. 2):

genes manB, manC, Gmd, WcaG, Gsk and Zwf encoding manB, manC, Gmd, WcaG, Gsk and Zwf in Escherichia coli MG1655 were amplified by PCR and DNA fragment gel recovered by primers NdeI-manB-F/R, NcoI-manC-F/R, NcoI-Gmd-F/R, NdeI-WcaG-F/R, NcoI-Gsk-F/R and NdeI-Zwf-F/R (primer sequences are shown in Table 1) to obtain the relevant gene fragments (the PCR system is shown in Table 2, and the PCR electrophoresis results are shown in Table 3). And cloning manB, gmd and gsk to a first multiple cloning site of a corresponding Duet-1 plasmid through single enzyme digestion (a single enzyme digestion system is shown in table 3, and an enzyme linkage system is shown in table 4), and cloning manC, wcAG and zwf to a second cloning site of a corresponding Duet-1 plasmid through single enzyme digestion to finally obtain plasmids pET-manC-manB, pCDF-manC-manB, pACYC-manC-manB, pET-gmd-wcAG, pCDF-gmd-wcAG, pACYC-gmd-wcAG and pCDF-gsk-zwf.

TABLE 1 primer sequences

Note: the underlined part is the cleavage site.

TABLE 2 PCR System

PCR conditions were as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1kb/min, 32 cycles, storage at 4 ℃ and gel recovery of PCR products.

TABLE 3 enzyme digestion System

Enzyme cutting conditions are as follows: the enzyme digestion reaction was carried out at 37 ℃ for 40 min.

TABLE 4 enzyme-linked reaction System

Enzyme-linked conditions: 22 ℃ for 3-5 hours, or 16 ℃ overnight.

Example 2: replacement of the original ribosome binding site on the expression plasmid

In addition to the ribosome binding site (RBS-ori) of the expression plasmid itself, the present invention selects two RBSs, one is the standard ribosome binding site (RBS-32) reported in the literature, and the other is the wild-type ribosome binding site (RBS-WT) of manB, manC, gmd and wcaG carried by itself on the genome, and replaces the corresponding RBS with the original expression vector, thereby regulating the protein translation strength of each target gene (the different RBS sequences are shown in Table 5).

TABLE 5 RBS sequences

The constructed plasmids pACYC-manC-manB and pET-gmd-wcaG were used as templates, and the corresponding fragments and vectors (see the primer sequences in Table 6) were obtained using the primers manC- [ RBS-32] -F/R, manC- [ RBS-WT ] -F/R, manB- [ RBS-32] -F/R, manB- [ RBS-WT ] -F/R, gmd- [ RBS-WT ] -F/R, wcaG- [ RBS-32] -F/R, wcaG- [ RBS-WT ] -F/R, and the fragments and vectors were assembled using One-Step Cloning Kit (Vazyme) (see Table 7 in One-Step Cloning reaction system), and transformed into E.coli DH5 alpha, the plate is plated overnight, and after being transferred to 4mL LB medium overnight, the plasmid is collected and sequenced.

TABLE 6 primer sequences

Table 7 one-step cloning reaction system:

one-step cloning reaction conditions: 50 ℃ for 5 minutes.

And calculating the vector dosage and the insert dosage according to a formula by X/Y:

the optimum amount of the insertion vector used was ng (0.02 Xbase number of cloning vector) and (0.03pmol)

The optimum amount of insert used was (0.04 Xthe number of bases of insert) ng (0.06pmol)

Example 3: knockout of genomic Gene of Escherichia coli BL21(DE3)

The specific steps are as follows (the specific operation flow can refer to fig. 4):

(1) respectively amplifying an upstream fragment and a downstream fragment of the wcaJ gene by PCR by using primers of the awcaj-upflowlank-F/R and the awcaj-downflank-F/R (the sequences of PCR primers are shown in a table 8);

(2) performing fusion PCR amplification on the upstream and downstream fragments by using primers delta wcaJ-upflowank-F and delta wcaJ-downflank-R to obtain a complete template gene (the nucleic acid gel electrophoresis result of the PCR amplification product is shown in figure 5);

(3) carrying out PCR amplification on the existing pTargetF plasmid by using a primer N20-F/R to obtain a pTargetF plasmid with the target wcaJ;

(4) the pCas plasmid is transferred into Escherichia coli BL21(DE3) by electrotransfer, and 10mM arabinose is added to induce the expression of a lambda-Red Escherichia coli gene recombination system;

(5) the pTargetF plasmid was transformed into the above E.coli BL21(DE3), spread on LB plate containing kanamycin and spectinomycin, cultured at 30 ℃ for 12-20 hours, and the colony on the plate was verified to be knocked out;

(6) after successful verification, a colony with the target gene knocked out is inoculated to 2mL of LB culture medium, 0.5mM IPTG is added to induce the expression of sgRNA-pMB1 plasmid to cut the pTargetF plasmid so as to achieve the aim of removing the pTargetF plasmid;

(7) the bacteria without pTargetF plasmid is selected and inoculated into LB culture medium without resistance at 42 ℃, cultured overnight at 200rpm, and after a proper amount of bacterial liquid is taken and coated on an LB solid plate without resistance, whether pCas plasmid is removed or not is verified after bacterial colony growth.

TABLE 8 primer sequence Listing

Example 4: optimization of E.coli host by shake flask fermentation

(1) Screening of three different copy number recombinant plasmids

Plasmid pETDuet-1 has a higher copy number, plasmid pCDFDuet-1 has a medium copy number, and plasmid pACYCDuet-1 has a lower copy number. Wherein PBR332, CDF and p15A are replicons for expression plasmids pETDuet-1, pCDFDuet-1 and pACYCDuet-1, respectively, representing different copy numbers, which are 40, 20 and 10, respectively. By means of the steps in the embodiment 1, four key genes in a GDP-fucose synthesis pathway are expressed in a combined mode, 9 different engineering bacteria are obtained and are respectively represented as EWL 1-9. The yield of GDP-fucose of different engineering strains after fermentation is respectively 4.13mg/L, 2.03mg/L, 5.33mg/L, 3.4mg/L, 1.97mg/L, 3.24mg/L, 2.6mg/L, 0.63mg/L and 1 mg/L. The highest yield of 5.33mg/L was obtained for the engineered strain containing the recombinant plasmids pACYC-manC-manB and pET-gmd-wcaG (strain EWL3) (see FIG. 8(1) and Table 9 for GDP-fucose yield for each engineered strain). Thus, the upstream pathways expressing relatively low gene doses (genes manB and manC) while the downstream pathways expressing relatively high gene doses (genes gmd and wcAG) allow for higher GDP-fucose production.

(2) Selection of ribosome binding sites for different plasmids

The invention further adjusts the translation strength of the protein by regulating and controlling the RBS strength on the basis of regulating and controlling the metabolic pathway by plasmids with different copy numbers, thereby exploring the production potential of GDP-fucose of the metabolic pathway. First, the relative protein expression amounts of manB, manC, gmd and wcAG under the control of different RBSs were examined by protein expression gel electrophoresis experiments (see FIG. 6). Protein expression of EWL33, EWL03, EWL11 at 0 and 6 hours after the inducer addition was complete, respectively, is shown. The experimental results show that wild-type RBS has a relatively high protein translation strength, that the original RBS on the expression plasmid has a moderate translation strength, and that the selected standard-type RBS has a relatively lowest translation expression strength.

After replacing RBS-ori by RBS-32 and RBS-WT by the procedure described in example 2, we additionally obtained 9 strains under the control of different RBSs: EWL11, EWL12, EWL13, EWL21, EWL03, EWL23, EWL31, EWL32 and EWL 33. The results of the GDP-fucose production assay showed that the GDP-fucose production of the above strains were 4.9mg/L, 3.87mg/L, 2.13mg/L, 5.16mg/L, 5.33mg/L, 1.17mg/L, 6.77mg/L, 5.67mg/L and 2.1mg/L, respectively. It can be seen that the highest intracellular accumulation of GDP-fucose was obtained by controlling upstream genes (genes manB and manC) by RBS-32 whose translational strength was relatively low and controlling downstream genes (genes gmd and wcAG) by RBS-WT whose translational strength was relatively high. That is, E.coli hosts containing recombinant plasmids pACYC- [ RBS-32] -manC- [ RBS-32] -manB and pET- [ RBS-WT ] -gmd- [ RBS-WT ] -wcaG possessed the highest product accumulation, reaching 6.77mg/L (see FIG. 8(2) and Table 9 for GDP-fucose production of each engineered strain).

(3) The GDP-fucose yield is improved by knocking out GDP-fucose downstream catabolic gene wcaJ.

To increase the intracellular concentration of GDP-fucose, we knocked out the gene wcaJ encoding UDP-glucolipid carrier transferase using the CRISPR/Cas9 system by the procedure described in example 3, thereby blocking the metabolic flux from GDP-fucose to colanic acid. The results of the experiments showed that the GDP-fucose yield of the strain without deleting the wcaJ gene, that is, EWL31, was 6.77mg/L, whereas the accumulation amount of intracellular GDP-fucose reached 12.26mg/L when E.coli with deleting the wcaJ gene was used as the EWL34 of the host (see FIG. 8(3) and Table 9 for GDP-fucose yields of the respective engineered strains).

(4) The yield of GDP-fucose is increased by enhancing the recycling of the cofactor.

To further increase the intracellular concentration of GDP-fucose, two key enzymes involved in the NADPH regeneration pathway and the guanosine nucleotide biosynthesis pathway, glucose 6-phosphate dehydrogenase (Zwf) and guanosine-inosine kinase (Gsk), were overexpressed using the procedure described in example 1 to improve the balance of cofactor consumption and regeneration. The experimental results show that the obtained 3 strains are respectively expressed as EWL 35-37, the yield is respectively 15mg/L, 16.15mg/L and 18.33mg/L, namely the accumulation amount of GDP-fucose in host cells of over-expressed genes zwf and gsk reaches the highest and is 18.33mg/L (the yield of GDP-fucose of each engineering strain is shown in figure 8(4) and table 9).

TABLE 9 detailed information of various engineering bacteria and GDP-fucose production

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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ggcgatgtga ttaatcacaa aactgaaaac agctatgtgt atgctgaatc tggcctggtc 960

accaccgtcg gggtgaaaga tctggtagtg gtgcagacca aagatgcggt gctgattgcc 1020

gaccgtaacg cggtacagga tgtgaaaaaa gtggtcgagc agatcaaagc cgatggtcgc 1080

catgagcatc gggtgcatcg cgaagtgtat cgtccgtggg gcaaatatga ctctatcgac 1140

gcgggcgacc gctaccaggt gaaacgcatc accgtgaaac cgggcgaggg cttgtcggta 1200

cagatgcacc atcaccgcgc ggaacactgg gtggttgtcg cgggaacggc aaaagtcacc 1260

attgatggtg atatcaaact gcttggtgaa aacgagtcca tttatattcc gctgggggcg 1320

acgcattgcc tggaaaaccc ggggaaaatt ccgctcgatt taattgaagt gcgctccggc 1380

tcttatctcg aagaggatga tgtggtgcgt ttcgcggatc gctacggacg ggtgtaa 1437

<210> 3

<211> 1122

<212> DNA

<213> Escherichia coli MG1655

<400> 3

atgtcaaaag tcgctctcat caccggtgta accggacaag acggttctta cctggcagag 60

tttctgctgg aaaaaggtta cgaggtgcat ggtattaagc gtcgcgcatc gtcattcaac 120

accgagcgcg tggatcacat ttatcaggat ccgcacacct gcaacccgaa attccatctg 180

cattatggcg acctgagtga tacctctaac ctgacgcgca ttttgcgtga agtacagccg 240

gatgaagtgt acaacctggg cgcaatgagc cacgttgcgg tctcttttga gtcaccagaa 300

tataccgctg acgtcgacgc gatgggtacg ctgcgcctgc tggaggcgat ccgcttcctc 360

ggtctggaaa agaaaactcg tttctatcag gcttccacct ctgaactgta tggtctggtg 420

caggaaattc cgcagaaaga gaccacgccg ttctacccgc gatctccgta tgcggtcgcc 480

aaactgtacg cctactggat caccgttaac taccgtgaat cctacggcat gtacgcctgt 540

aacggaattc tcttcaacca tgaatccccg cgccgcggcg aaaccttcgt tacccgcaaa 600

atcacccgcg caatcgccaa catcgcccag gggctggagt cgtgcctgta cctcggcaat 660

atggattccc tgcgtgactg gggccacgcc aaagactacg taaaaatgca gtggatgatg 720

ctgcagcagg aacagccgga agatttcgtt atcgcgaccg gcgttcagta ctccgtgcgt 780

cagttcgtgg aaatggcggc agcacagctg ggcatcaaac tgcgctttga aggcacgggc 840

gttgaagaga agggcattgt ggtttccgtc accgggcatg acgcgccggg cgttaaaccg 900

ggtgatgtga ttatcgctgt tgacccgcgt tacttccgtc cggctgaagt tgaaacgctg 960

ctcggcgacc cgaccaaagc gcacgaaaaa ctgggctgga aaccggaaat caccctcaga 1020

gagatggtgt ctgaaatggt ggctaatgac ctcgaagcgg cgaaaaaaca ctctctgctg 1080

aaatctcacg gctacgacgt ggcgatcgcg ctggagtcat aa 1122

<210> 4

<211> 963

<212> DNA

<213> Escherichia coli MG1655

<400> 4

agtaaacaac gagtttttat tgctggtcat cgcgggatgg tcggttccgc catcaggcgg 60

cagctcgaac agcgcggtga tgtggaactg gtattacgca cccgcgacga gctgaacctg 120

ctggacagcc gcgccgtgca tgatttcttt gccagcgaac gtattgacca ggtctatctg 180

gcggcggcga aagtgggcgg cattgttgcc aacaacacct atccggcgga tttcatctac 240

cagaacatga tgattgagag caacatcatt cacgccgcgc atcagaacga cgtgaacaaa 300

ctgctgtttc tcggatcgtc ctgcatctac ccgaaactgg caaaacagcc gatggcagaa 360

agcgagttgt tgcagggcac gctggagccg actaacgagc cttatgctat tgccaaaatc 420

gccgggatca aactgtgcga atcatacaac cgccagtacg gacgcgatta ccgctcagtc 480

atgccgacca acctgtacgg gccacacgac aacttccacc cgagtaattc gcatgtgatc 540

ccagcattgc tgcgtcgctt ccacgaggcg acggcacaga atgcgccgga cgtggtggta 600

tggggcagcg gtacaccgat gcgcgaattt ctgcacgtcg atgatatggc ggcggcgagc 660

attcatgtca tggagctggc gcatgaagtc tggctggaga acacccagcc gatgttgtcg 720

cacattaacg tcggcacggg cgttgactgc actatccgcg agctggcgca aaccatcgcc 780

aaagtggtgg gttacaaagg ccgggtggtt tttgatgcca gcaaaccgga tggcacgccg 840

cgcaaactgc tggatgtgac gcgcctgcat cagcttggct ggtatcacga aatctcactg 900

gaagcggggc ttgccagcac ttaccagtgg ttccttgaga atcaagaccg ctttcggggg 960

taa 963

<210> 5

<211> 1305

<212> DNA

<213> Escherichia coli MG1655

<400> 5

atgaaatttc ccggtaaacg taaatccaaa cattacttcc ccgtaaacgc acgcgatccg 60

ctgcttcagc aattccagcc agaaaacgaa accagcgctg cctgggtagt gggtatcgat 120

caaacgctgg tcgatattga agcgaaagtg gatgatgaat ttattgagcg ttatggatta 180

agcgccgggc attcactggt gattgaggat gatgtagccg aagcgcttta tcaggaacta 240

aaacagaaaa acctgattac ccatcagttt gcgggtggca ccattggtaa caccatgcac 300

aactactcgg tgctcgcgga cgaccgttcg gtgctgctgg gcgtcatgtg cagcaatatt 360

gaaattggca gttatgccta tcgttacctg tgtaacactt ccagccgtac cgatcttaac 420

tatctacaag gcgtggatgg cccgattggt cgttgcttta cgctgattgg cgagtccggg 480

gaacgtacct ttgctatcag tccaggccac atgaaccagc tgcgggctga aagcattccg 540

gaagatgtga ttgccggagc ctcggcactg gttctcacct catatctggt gcgttgcaag 600

ccgggtgaac ccatgccgga agcaaccatg aaagccattg agtacgcgaa gaaatataac 660

gtaccggtgg tgctgacgct gggcaccaag tttgtcattg ccgagaatcc gcagtggtgg 720

cagcaattcc tcaaagatca cgtctctatc cttgcgatga acgaagatga agccgaagcg 780

ttgaccggag aaagcgatcc gttgttggca tctgacaagg cgctggactg ggtagatctg 840

gtgctgtgca ccgccgggcc aatcggcttg tatatggcgg gctttaccga agacgaagcg 900

aaacgtaaaa cccagcatcc gctgctgccg ggcgctatag cggaattcaa ccagtatgag 960

tttagccgcg ccatgcgcca caaggattgc cagaatccgc tgcgtgtata ttcgcacatt 1020

gcgccgtaca tgggcgggcc ggaaaaaatc atgaacacta atggagcggg ggatggcgca 1080

ttggcagcgt tgctgcatga cattaccgcc aacagctacc atcgtagcaa cgtaccaaac 1140

tccagcaaac ataaattcac ctggttaact tattcatcgt tagcgcaggt gtgtaaatat 1200

gctaaccgtg tgagctatca ggtactgaac cagcattcac ctcgtttaac gcgcggcttg 1260

ccggagcgtg aagacagcct ggaagagtct tactgggatc gttaa 1305

<210> 6

<211> 1476

<212> DNA

<213> Escherichia coli MG1655

<400> 6

atggcggtaa cgcaaacagc ccaggcctgt gacctggtca ttttcggcgc gaaaggcgac 60

cttgcgcgtc gtaaattgct gccttccctg tatcaactgg aaaaagccgg tcagctcaac 120

ccggacaccc ggattatcgg cgtagggcgt gctgactggg ataaagcggc atataccaaa 180

gttgtccgcg aggcgctcga aactttcatg aaagaaacca ttgatgaagg tttatgggac 240

accctgagtg cacgtctgga tttttgtaat ctcgatgtca atgacactgc tgcattcagc 300

cgtctcggcg cgatgctgga tcaaaaaaat cgtatcacca ttaactactt tgccatgccg 360

cccagcactt ttggcgcaat ttgcaaaggg cttggcgagg caaaactgaa tgctaaaccg 420

gcacgcgtag tcatggagaa accgctgggg acgtcgctgg cgacctcgca ggaaatcaat 480

gatcaggttg gcgaatactt cgaggagtgc caggtttacc gtatcgacca ctatcttggt 540

aaagaaacgg tgctgaacct gttggcgctg cgttttgcta actccctgtt tgtgaataac 600

tgggacaatc gcaccattga tcatgttgag attaccgtgg cagaagaagt ggggatcgaa 660

gggcgctggg gctattttga taaagccggt cagatgcgcg acatgatcca gaaccacctg 720

ctgcaaattc tttgcatgat tgcgatgtct ccgccgtctg acctgagcgc agacagcatc 780

cgcgatgaaa aagtgaaagt actgaagtct ctgcgccgca tcgaccgctc caacgtacgc 840

gaaaaaaccg tacgcgggca atatactgcg ggcttcgccc agggcaaaaa agtgccggga 900

tatctggaag aagagggcgc gaacaagagc agcaatacag aaactttcgt ggcgatccgc 960

gtcgacattg ataactggcg ctgggccggt gtgccattct acctgcgtac tggtaaacgt 1020

ctgccgacca aatgttctga agtcgtggtc tatttcaaaa cacctgaact gaatctgttt 1080

aaagaatcgt ggcaggatct gccgcagaat aaactgacta tccgtctgca acctgatgaa 1140

ggcgtggata tccaggtact gaataaagtt cctggccttg accacaaaca taacctgcaa 1200

atcaccaagc tggatctgag ctattcagaa acctttaatc agacgcatct ggcggatgcc 1260

tatgaacgtt tgctgctgga aaccatgcgt ggtattcagg cactgtttgt acgtcgcgac 1320

gaagtggaag aagcctggaa atgggtagac tccattactg aggcgtgggc gatggacaat 1380

gatgcgccga aaccgtatca ggccggaacc tggggacccg ttgcctcggt ggcgatgatt 1440

acccgtgatg gtcgttcctg gaatgagttt gagtaa 1476

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

gtcacacagg aaagtaccat 20

<210> 8

<211> 10

<212> DNA

<213> Artificial sequence

<400> 8

aagaaggaga 10

<210> 9

<211> 40

<212> DNA

<213> Artificial sequence

<400> 9

ttcggtcagg gccaactatt gcctgaaaaa gggtaacgat 40

<210> 10

<211> 50

<212> DNA

<213> Artificial sequence

<400> 10

gataaagaga acgtgttacg tcaatttata aatgatattc ggggataatt 50

<210> 11

<211> 50

<212> DNA

<213> Artificial sequence

<400> 11

acgcgaacgc gttgaaactg aataaattca aaaatacaga ggaataatac 50

<210> 12

<211> 50

<212> DNA

<213> Artificial sequence

<400> 12

tgctgaaatc tcacggctac gacgtggcga tcgcgctgga gtcataaatg 50

Claims

1. An Escherichia coli engineering bacterium for efficiently producing GDP-fucose is characterized in that the expression of phosphomannose mutase ManB, GDP-mannose pyrophosphorylase ManC, GDP-mannose 4, 6-dehydratase Gmd and GDP-fucose synthetase Wcag is controlled in a combined manner; the expression regulation adopts high-copy expression elements to regulate the expression of Gmd and Wcag, and adopts low-copy expression elements to regulate the expression of ManB and ManC; also strengthens the expression of glucose 6-phosphate dehydrogenase Zwf and guanosine-inosine kinase Gsk, silences the expression of UDP-glucose plasma carrier transferase WcaJ;

the host bacterium of the engineering bacterium is Escherichia coli BL21(DE3) and expressed by pACYCDuet-1manC-manB，Expression in pETuet-1gmd-wcaG，Expression with pCDFDuet-1gsk-zwf。

2. The engineered Escherichia coli as claimed in claim 1, wherein pACYCDuet-1-one in the engineered Escherichia coli is replaced by a standard ribosome binding site and a wild-type ribosome binding sitemanC-manBAnd pETDuet-1-gmd-wcaGThe ribosome binding site of the plasmid itself.

3. A method for improving the capability of Escherichia coli for producing GDP-fucose is characterized in that the expression of phosphomannose mutase ManB, GDP-mannose pyrophosphorylase ManC, GDP-mannose 4, 6-dehydratase Gmd and GDP-fucose synthetase Wcag is controlled in a combined way; the expression regulation adopts high-copy expression elements to regulate the expression of Gmd and Wcag, and adopts low-copy expression elements to regulate the expression of ManB and ManC; the method comprises the following steps:

(1) construction of pCDFDuet-1-gsk-zwfA recombinant expression vector;

(2) construction of pACYCDuet-1-manC-manB、pETDuet-1-manC-manB、pCDFDuet-1-manC-manB、pACYCDuet-1-gmd-wcaG、pETDuet-1-gmd-wcaG、pCDFDuet-1-gmd-wcaGRecombinant expression vector, screening out the recombinant plasmid combination with the highest GDP-fucose yield;

(3) substitution of standard ribosome binding site and wild-type ribosome binding site for pACYCDuet-1-manC-manBAnd pETDuet-1-gmd-wcaGThe ribosome binding site of the plasmid is selected, and recombinant expression vectors with the highest yield of GDP-fucose are screened under the control of different RBSs;

(4) knocking out Escherichia coli BL21(DE3) chromosome by using CRISPR/Cas9 gene editing systemwcaJA gene;

(5) to get out ofwcaJColi BL21(DE3) as host, over-expressing pCDFDuet-1-gsk-zwfAnd the screened pACYCDuet-1-manC-manBAnd pETDuet-1-gmd-wcaGA recombinant expression vector.

4. A method for producing GDP-fucose, characterized in that the Escherichia coli engineering bacteria of claim 1 are used as fermenting microorganisms, and GDP-fucose is synthesized by using glucose as a carbon source.

5. The use of the engineered Escherichia coli strain of claim 1 in the preparation of GDP-fucose and derivatives thereof.