CN112625990A - Recombinant escherichia coli for synthesizing 2' -fucosyllactose and construction method thereof - Google Patents

Abstract

The invention provides a recombinant escherichia coli for synthesizing 2' -fucosyllactose and a construction method thereof, wherein the recombinant escherichia coli is obtained by knocking out beta-galactosidase genes in the genome of the escherichia coli, synthesizing key enzyme genes of clavulanic acid from guanosine diphosphate rock sugar, strengthening and expressing key enzyme genes of a lactose permease gene and guanosine diphosphate rock sugar de-novo synthesis way, and expressing exogenous fucosyltransferase genes. The scheme of the invention can reduce lactose hydrolysis and improve the speed of transferring exogenous lactose into cells, thereby effectively improving the concentration of lactose in the cells; meanwhile, the consumption of the guanosine diphosphate rock sugar is reduced, and the intracellular guanosine diphosphate rock sugar concentration is effectively improved; the expression of exogenous fucosyltransferase gene is improved through codon optimization, and the synthesis of 2' -fucosyllactose is effectively promoted.

Description

Recombinant escherichia coli for synthesizing 2' -fucosyllactose and construction method thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to recombinant escherichia coli for synthesizing 2' -fucosyllactose and a construction method thereof.

Background

Breast milk is a complex mixture containing water, fat, protein, minerals, vitamins, polysaccharides, monosaccharides, disaccharides (lactose), oligosaccharides. Lactose is the main solid component of breast milk (-6.8% of total solids) and is widely present in animal milk. However, breast milk oligosaccharides with a more complex structure (-1% of total solids) are unique to humans. Currently, there are about 200 breast milk oligosaccharides with a well-established structure, and the degree of polymerization varies from 3 to 32. Structural features of breast milk oligosaccharides include lacto-N-acetylglucosamine backbone, straight chain β - (1,3/4) -glycosidic linkages, branched chain β - (1,6) -glycosidic linkages, lactose at the reducing end of the sugar chain and L-fucose or sialic acid at the non-reducing end.

2 '-fucosyllactose is the most abundant breast milk oligosaccharide in most breast feeding mothers' milk. In the milk of the secretory mother expressing the alpha- (1,2) -fucosyltransferase gene FUT2, the proportion of 2' -fucosyllactose in the total amount of oligosaccharides in breast milk can be as high as 30%. Therefore, 2 '-fucosyllactose is well studied, and 2' -fucosyllactose is found to have various beneficial functions. For example, 2' -fucosyllactose specifically inhibits the binding of campylobacter jejuni, typhoid bacillus, pathogenic escherichia coli, helicobacter pylori, norovirus to the corresponding host receptor to avoid infection. The 2' -fucosyllactose can inhibit the interaction of Pseudomonas aeruginosa with local epithelial cells, thereby preventing infection of gastrointestinal tract, urethra and respiratory system by Pseudomonas aeruginosa. In addition, 2' -fucosyllactose can inhibit lipopolysaccharide binding to receptor CD14 on the surface of cell membrane, thereby attenuating pathogenic escherichia coli type 1 pilus lipopolysaccharide-induced inflammation. It has recently been found that 2' -fucosyllactose also improves the learning and memory of rodents.

2' -fucosyllactose is good for health and is thus gradually used as a functional food material for infant formulas and medical nutritional products. However, the industrial production of 2 '-fucosyllactose is not straightforward, so that nowadays 2' -fucosyllactose is used in infant formula in amounts that are still lower than its natural content in breast milk. Early, 2' -fucosyllactose was chromatographically isolated from donated breast milk and could not be produced on a large scale due to limited material sources. 2' -fucosyllactose can be directly chemically synthesized, however, is limited by the chemical nature of the protecting group, cannot be scaled up, and chemical synthesis requires the use of toxic reagents and is less suitable for large-scale food production. Fucosyltransferase can catalytically synthesize 2' -fucosyllactose in vitro, however, the donor substrate, guanosine diphosphate fucose, is very expensive. Therefore, the enzymatic synthesis of 2' -fucosyllactose is not suitable for industrial production of several hundred tons per year. Microbial fermentation is currently the only technical route suitable for large-scale production. The technological advances of metabolic engineering, fermentation engineering, purification process and the like enable the 2' -fucosyllactose to be produced and sold in a large scale. During the fermentation process, the donor substrate molecule, guanosine diphosphate fucose, is synthesized de novo by the microorganism itself, while the acceptor substrate molecule, lactose, is generally added externally.

Therefore, the technical personnel in the field need to solve the problems by constructing the recombinant escherichia coli for synthesizing the 2 '-fucosyllactose, effectively promoting the fermentation and synthesis of the 2' -fucosyllactose and laying a solid foundation for future large-scale production.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a recombinant escherichia coli for synthesizing 2' -fucosyllactose and a construction method thereof, which overcome the problems of long test period, low success rate and the like of homologous recombination technology, optimize the expression of exogenous fucosyltransferase gene, and effectively promote the synthesis of 2' -fucosyllactose, thereby realizing the efficient fermentation synthesis of 2' -fucosyllactose.

In order to solve the above technical problems, in one aspect, the present invention provides a recombinant escherichia coli for synthesizing 2' -fucosyllactose, wherein the recombinant escherichia coli is obtained by knocking out a beta-galactosidase gene from a genome of an original strain of escherichia coli, synthesizing a key enzyme gene of kola acid from guanosine diphosphate fucose, intensively expressing a lactose permease gene and a key enzyme gene of a guanosine diphosphate fucose de-novo synthesis pathway, and expressing an exogenous fucosyltransferase gene.

Preferably, the beta-galactosidase gene is knocked out by using a CRISPR gene editing technology, and the key enzyme gene for synthesizing the kola acid from the guanosine diphosphate fucose is knocked out by using the CRISPR gene editing technology, namely, the undecadiene phosphate glucose phosphotransferase gene wcaJ is knocked out.

Preferably, when the gene wcaJ of undecadiene phosphate glucose phosphotransferase gene wcaJ is knocked out by using the CRISPR gene editing technology, the nucleotide sequence of the target sequence of gRNA1 of the knocked-out gene wcaJ is shown as SEQ ID No.1, the nucleotide sequence of the target sequence of gRNA2 of the knocked-out gene wcaJ is shown as SEQ ID No.2, the nucleotide sequence of an upstream primer identified by PCR is shown as SEQ ID No.3, and the nucleotide sequence of a downstream primer identified by PCR is shown as SEQ ID No. 4.

Preferably, when the CRISPR gene editing technology is used for knocking out the beta-galactosidase gene lacZ, the nucleotide sequence of the target sequence of the gRNA1 of the knocked-out gene lacZ is shown as SEQ ID No.5, the nucleotide sequence of the target sequence of the gRNA2 of the knocked-out gene lacZ is shown as SEQ ID No.6, the nucleotide sequence of the upstream primer identified by PCR is shown as SEQ ID No.7, and the nucleotide sequence of the downstream primer identified by PCR is shown as SEQ ID No. 8.

Preferably, the enhanced expression of the lactose permease gene and the key enzyme gene of the guanosine diphosphate rock sugar de novo synthesis pathway refers to the fact that a T7lac promoter is knocked in the upstream of a phosphomannose mutase gene manB of the guanosine diphosphate rock sugar de novo synthesis pathway by using a CRISPR gene editing technology.

Preferably, when a T7lac promoter is knocked in from the upstream of a phosphomannose mutase gene manB in a guanosine diphosphate fucose de novo synthesis pathway by using a CRISPR gene editing technology, the nucleotide sequence of a knocked-in fragment is shown as SEQ ID NO.9, the nucleotide sequence of a knocked-in position upstream gene is shown as SEQ ID NO.10, the nucleotide sequence of a knocked-in position downstream gene is shown as SEQ ID NO.11, the nucleotide sequence of an upstream primer identified by PCR is shown as SEQ ID NO.12, and the nucleotide sequence of a downstream primer identified by PCR is shown as SEQ ID NO. 13.

Preferably, the recombinant escherichia coli carries a plurality of recombinant plasmids for inducing high expression of proteins, wherein the recombinant plasmids respectively comprise genes manB, mannose-1-phosphate guanosine transferase genes manC, guanosine diphosphate mannose-4, 6-dehydratase genes gmd, guanosine diphosphate-L-fucose synthetase genes fcl, lactose permease genes lacY and exogenous fucosyltransferase genes.

Preferably, the exogenous fucosyltransferase wcfB gene is derived from bacteroides fragilis, and the codon-optimized wcfB gene sequence is shown in SEQ ID No. 14.

Preferably, the original strain is E.coli, preferably E.coli BL21(DE 3).

In the specific implementation process, the invention provides recombinant escherichia coli for synthesizing 2' -fucosyllactose, and the undecadiene phosphate glucose phosphotransferase gene wcaJ and the beta-galactosidase gene lacZ of the original strain genome are knocked out by adopting a CRISPR gene editing technology. At the same time, the T7lac promoter was knocked in upstream of the phosphomannose mutase gene manB of the de novo synthesis pathway of guanosine diphosphate rock sugar. And simultaneously overexpresses phosphomannose mutase gene manB, mannose-1-phosphoguanosine transferase gene manC, guanosine diphosphate mannose-4, 6-dehydratase gene gmd, guanosine diphosphate-L-fucose synthetase gene fcl, lactose permease gene lacY and codon optimized alpha-1, 2-fucose transferase gene wcFB.

The gene wcaJ, gene accession number GI: 8182600; gene lacZ, gene accession number GI: 8181469; gene manB, gene accession number GI: 946574; gene manC, gene accession number GI: 946580; gene gmd, gene accession number GI: 946562; gene fcl, gene accession number GI 946563; gene lacY, gene accession number GI: 949083; gene wcfB, gene accession No. GI: 3287270. The gene overexpression refers to the induction high expression of the protease molecules corresponding to each gene by adopting a recombinant plasmid expression vector.

On the other hand, the invention also provides a construction method of the recombinant Escherichia coli for synthesizing 2' -fucosyllactose, which comprises the following steps:

1) respectively preparing recombinant plasmids containing gene manB, gene manC, gene gmd, gene fcl, lactose permease gene lacY and codon optimized gene wcfB to obtain plasmids for constructing metabolic pathways;

2) the detection shows that the growth state of the original strain of escherichia coli BL21(DE3) is normal, the detection of the knock-in positions of target genes wcaJ and lacZ and a T7lac promoter and upstream and downstream sequences shows that the size of a PCR amplification band accords with the expectation, and the sequencing result is consistent with the NCBI sequence;

3) designing and preparing gRNA according to the insertion position of a target gene sequence and the sequence characteristics around the target gene sequence, cloning the gRNA and the Donor sequence to a gene editing vector Donor plasmid, and ensuring that the gRNA and the Donor sequence in the constructed vector are consistent with the target sequence through sequencing verification;

4) preparing escherichia coli BL21(DE3) electrotransformation competence, transforming Cas9 plasmid into BL21(DE3) competence, selecting spots to prepare BL21(DE3) -Cas9 electrotransformation competence, transforming Donor plasmid into BL21(DE3) -Cas9 electrotransformation competence, adding arabinose for induction, coating a plate, and carrying out gene editing strain screening experiment;

5) PCR amplification verifies that the edited BL21(DE3) is monoclonal, the size of a band successfully knocked out by the wcaJ gene is 771bp, and the size of a band successfully knocked out is 1049 bp; the size of a successful band of the lacZ gene knockout is 687bp, and the size of a successful band of the lacZ gene knockout is 983 bp; the size of a band of the T7lac promoter which is knocked in successfully is 1076bp, and no band exists when the promoter is not knocked in successfully; electrophorograms show that monoclonals with the wcaJ gene and the lacZ gene knocked out simultaneously and the T7lac promoter knocked in are successfully screened;

6) carrying out lysogenic treatment on the escherichia coli edited by the CRISPR in the step 5), and then converting the metabolic pathway construction plasmid obtained in the step 1) into lysogenic bacteria to obtain the recombinant escherichia coli capable of synthesizing 2' -fucosyllactose.

The recombinant escherichia coli is fermented and synthesized into the 2' -fucosyllactose, and the culture medium and the fermentation method are as follows:

LB medium (1L) tryptone, 10 g; yeast extract, 5 g; sodium chloride, 5 g. Preparing a solid culture medium, and then adding 15g of agar powder.

The recombinant E.coli of the present invention was cultured in 2mL of LB medium (kanamycin 50. mu.g/mL, streptomycin 50. mu.g/mL) at 37 ℃ and shaking table rotation speed 220rpm overnight for about 15 hours. Transferring 1mL of overnight-cultured seed solution into 50mL of LB medium (kanamycin 50. mu.g/mL, streptomycin 50. mu.g/mL, glucose added to a final concentration of 20 g/L), culturing at 30 ℃ and shaking table rotation speed of 250rpm for about 8h (bacterial solution OD)₆₀₀About 0.8). Lactose was added to a final concentration of 12g/L and IPTG was added at 0.3mM, and the fermentation was continued for 48h at 25 ℃ and a shaker speed of 250 rpm.

Compared with the prior art, the recombinant escherichia coli for synthesizing 2' -fucosyllactose and the construction method thereof disclosed by the invention have the positive effects that:

(1) the recombinant escherichia coli has a metabolic pathway for fermenting and synthesizing 2' -fucosyllactose, simultaneously overcomes the problems of long test period, low success rate and the like of a homologous recombination technology, and obtains an efficient genome editing scheme; except for synthesizing 2' -fucosyllactose, other characteristics of the original strain are not changed, and fermentation is not influenced; the adopted plasmid is a common escherichia coli plasmid, and the growth and metabolism of bacteria are not influenced.

(2) By improving the concentration of intracellular lactose and guanosine diphosphate fucose and optimizing the expression of exogenous fucosyltransferase gene, the synthesis of 2' -fucosyllactose is effectively promoted; the recombinant escherichia coli has clear construction thought, obvious actual effect and good application prospect.

(3) The recombinant escherichia coli takes glucose as a carbon source to synthesize the 2 '-fucosyllactose through fermentation, the cost is lower, the economic feasibility is higher, and the 2' -fucosyllactose with the concentration of 6-10 g/L can be obtained through fermentation for 48 hours.

(4) The recombinant escherichia coli is very stable in the amplification process and has great industrial application potential.

Drawings

FIG. 1 is an electrophoretogram of PCR-verified products of the wcaJ gene knockout;

(wcaJ-WT is an amplification product of wcaJ gene; wcaJ-Edit is an amplification product of wcaJ gene after knockout)

FIG. 2 is an electrophoretogram of PCR-verified products of lacZ gene knock-out;

(lacZ-WT is an amplification product of lacZ gene; lacZ-Edit is an amplification product of lacZ gene after knockout)

FIG. 3 is an electrophoretogram of PCR-verified products of knock-in of the T7lac promoter;

(7Lac-WT is an amplification product of a non-knock-in T7 Lac; 7Lac-Edit is an amplification product of a successful knock-in T7 Lac)

FIG. 4 is a mass spectrometric detection of the fermentation broth, wherein the mass peak of molecular weight 487.16 is 2' -fucosyllactose.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available. The materials, reagents, apparatus and methods used in the following examples are all conventional in the art and are commercially available.

Example 1

Obtaining of genes:

in this example, a gene manB derived from Escherichia coli MG1655 (gene accession number GI:946574), a gene manC (gene accession number GI:946580), a gene gmd (gene accession number GI:946562), a gene fcl (gene accession number GI:946563), a gene lacY (gene accession number GI:949083), and a gene wcFB derived from Bacteroides fragilis NCTC9343 (gene accession number GI:3287270) were obtained.

In the embodiment, Escherichia coli genes manB, manC, gmd, fcl and lacY are successfully obtained by extracting the genomic DNA of Escherichia coli MG 1655; the gene wcFB derived from the Bacteroides fragilis NCTC9343 is successfully obtained by artificially synthesizing a codon optimized gene and cloning the gene into an Escherichia coli plasmid. The successful acquisition of the gene lays a foundation for the construction of recombinant plasmids.

Example 2

Preparation of recombinant plasmid

The gene manC and gene manB derived from Escherichia coli MG1655 obtained in example 1 were subjected to PCR amplification using designed primers (the sequence of the upstream primer is shown in SEQ ID NO.15 and the sequence of the downstream primer is shown in SEQ ID NO. 16), the amplified fragments were subjected to gel-cutting purification and double-digested with restriction enzymes NcoI and HindIII, the digested fragments were ligated with plasmid pCOLADuet-1 which was also double-digested with NcoI and HindIII, and the vector: the target fragments were mixed at a molar ratio of 1:3, and after addition of T4DNA ligase, they were enzymatically ligated at 22 ℃ for 5 hours, and the ligation products were transformed into E.coli DH 5. alpha. competent cells and screened on kanamycin plates to obtain recombinant plasmid pCOLA-CB. The gene gmd and the gene fcl derived from Escherichia coli MG1655 obtained in example 1 were subjected to PCR amplification using designed primers (the sequence of the upstream primer is shown in SEQ ID NO.17 and the sequence of the downstream primer is shown in SEQ ID NO. 18), the amplified fragments were subjected to gel cutting purification and double-digested with NdeI and XhoI, the digested fragments were ligated to plasmid pCOLA-CB which was also double-digested with NdeI and XhoI, and the vector: the target fragments are mixed according to the molar ratio of 1:3, T4DNA ligase is added, then enzyme linkage is carried out for 5 hours at 22 ℃, the ligation product is transformed into escherichia coli DH5 alpha competent cells, and screening is carried out on a kanamycin plate, so as to obtain the recombinant plasmid pCOLA-CBGF.

The gene lacY derived from escherichia coli MG1655 obtained in example 1 was PCR-amplified using designed primers (the sequence of the upstream primer is shown in SEQ ID No.19 and the sequence of the downstream primer is shown in SEQ ID No. 20), the amplified fragment was gel-cut and purified, and double-cut with NdeI and XhoI, and the cut fragment was ligated to plasmid pCDFDuet-1, which was also double-cut with NdeI and XhoI, to vector: the target fragments are mixed according to the molar ratio of 1:3, T4DNA ligase is added, then enzyme linkage is carried out for 5h at 22 ℃, the ligation product is transformed into escherichia coli DH5 alpha competent cells, and screening is carried out on a streptomycin plate, so as to obtain the recombinant plasmid pCDF-lacY. The gene wcfB derived from bacteroides fragilis obtained in example 1 was PCR-amplified using designed primers (the sequence of the upstream primer is shown in SEQ ID No.21 and the sequence of the downstream primer is shown in SEQ ID No. 22), the amplified fragment was gel-cut and purified and double-cut with NcoI and BamHI, the cut fragment was ligated with the plasmid pCDF-lacY, which was also double-cut with NcoI and BamHI, and the vector: mixing the target fragments according to a molar ratio of 1:3, adding T4DNA ligase, carrying out enzymatic ligation for 5 hours at 22 ℃, transforming the ligation product to escherichia coli DH5 alpha competent cells, and screening on a streptomycin plate to obtain the recombinant plasmid pCDF-lacY-wcfB.

In this example, the recombinant plasmids pCOLA-CBGF and pCDF-lacY-wcfB were successfully prepared, which laid the foundation for the construction of the metabolic pathway related to 2' -fucosyllactose in Escherichia coli.

Example 3

Gene editing

In this example, wcaJ and lacZ gene knockout and T7lac promoter knock-in were achieved using CRISPR gene editing technology. The procedure of gene editing is described in detail below.

1) The detection shows that the growth state of the original strain of escherichia coli BL21(DE3) is normal, the detection of the knock-in positions of target genes wcaJ and lacZ and a T7lac promoter and upstream and downstream sequences shows that the size of a PCR amplification band accords with the expectation, and the sequencing result is consistent with the NCBI sequence;

2) designing and preparing gRNA according to the insertion position of a target gene sequence and the sequence characteristics around the target gene sequence, cloning the gRNA and the Donor sequence to a gene editing vector Donor plasmid, and ensuring that the gRNA and the Donor sequence in the constructed vector are consistent with the target sequence through sequencing verification;

3) preparing escherichia coli BL21(DE3) electrotransformation competence, transforming Cas9 plasmid into BL21(DE3) competence, selecting spots to prepare BL21(DE3) -Cas9 electrotransformation competence, transforming Donor plasmid into BL21(DE3) -Cas9 electrotransformation competence, adding arabinose for induction, coating a plate, and carrying out gene editing strain screening experiment;

5) PCR amplification verifies that the edited BL21(DE3) is monoclonal, the size of a band successfully knocked out by the wcaJ gene is 771bp, and the size of a band successfully knocked out is 1049 bp; the size of a successful band of the lacZ gene knockout is 687bp, and the size of a successful band of the lacZ gene knockout is 983 bp; the size of a band of the T7lac promoter which is knocked in successfully is 1076bp, and no band exists when the promoter is not knocked in successfully; electrophorograms show that monoclonals with both the wcaJ gene and the lacZ gene knocked out and the T7lac promoter knocked in were successfully screened.

In this example, the wcaJ gene of escherichia coli BL21(DE3) was knocked out by CRISPR gene editing technology (fig. 1 is an electrophoretogram of PCR-verified products of wcaJ gene knock-out), reducing the consumption of guanosine diphosphate fucose; realizing the knockout of lacZ gene of Escherichia coli BL21(DE3) (FIG. 2 is an electrophoretogram of PCR verification product of lacZ gene knockout), and reducing the hydrolysis of lactose in cells; realizes the knock-in of the T7lac promoter at the upstream of the gene manB of the genome of the escherichia coli (figure 3 is an electrophoresis chart of a PCR verification product of the knock-in of the T7lac promoter), promotes the genes manB, manC, gmd and fcl to express corresponding carbohydrases, and improves the content of guanosine diphosphate fucose in cells. The successful implementation of the three aspects can effectively promote the escherichia coli to synthesize the 2' -fucosyllactose.

Example 4

Construction of recombinant Escherichia coli for synthesizing 2' -fucosyllactose

Coli edited by CRISPR in example 3 was subjected to lysogenic treatment, and the metabolic pathway-constructing plasmid obtained in example 2 was transformed into lysogens to obtain recombinant escherichia coli capable of synthesizing 2' -fucosyllactose.

In the embodiment, the recombinant escherichia coli for synthesizing the 2' -fucosyllactose is successfully obtained, and a foundation is laid for further fermentation verification.

Example 5

Verification of recombinant Escherichia coli for synthesizing 2' -fucosyllactose

The strain was inoculated in 2mL of LB medium (kanamycin 50. mu.g/mL, streptomycin 50. mu.g/mL) at 37 ℃ and incubated overnight at 220rpm on a shaker for about 15 hours. Transferring 1mL of overnight-cultured seed solution into 50mL of LB medium (kanamycin 50. mu.g/mL, streptomycin 50. mu.g/mL, glucose added to a final concentration of 20 g/L), culturing at 30 ℃ and shaking table rotation speed of 250rpm for about 8h (bacterial solution OD)₆₀₀About 0.8). Lactose was added to a final concentration of 12g/L and IPTG was added at 0.3mM, and the fermentation was continued for 48h at 25 ℃ and a shaker speed of 250 rpm. Centrifuging the fermentation liquor at 8000rpm for 10min, collecting the supernatant, decocting the supernatant at 100 deg.C for 10min, centrifuging again, mixing 0.5mL of the supernatant with 0.5mL of purified water, filtering the sample with 0.22 μm filter membrane, and detecting by electrospray mass spectrometry (FIG. 4 is mass spectrometry detection fermentation liquor chromatogram, wherein the mass spectrum peak of molecular weight 487.16 is 2' -fucosyllactose).

In the embodiment, mass spectrometric detection of the shake flask fermentation broth shows that the recombinant escherichia coli successfully synthesizes 2' -fucosyllactose, which lays a foundation for further pilot scale amplification and even industrial production.

Although the present invention has been described in detail with reference to the above embodiments, it will be apparent to one skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for some of the features thereof. And such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

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<400> 14

atgctgtacg ttattctgcg ggggcgtctg gggaataatt tatttcagat tgcaaccgcc 60

gcctcactga cccagaactt tattttttgt accgttaata aggatcaaga gcggcaggtt 120

ttactgtata aagatagctt tttcaagaac atcaaggtta tgaaaggagt tccggatggt 180

attccatact ataaagaacc gtttcatgaa tttagcagaa ttccttatga ggaaggtaaa 240

gatctgatca ttgatggtta ttttcagagc gaaaaatact tcaaacgtag cgtggtttta 300

gacctgtacc gcataaccga tgaactgcgc aaaaagatat ggaacatatg tggtaatatc 360

cttgaaaagg gagaaaccgt tagcattcat gtgcggcgtg gcgactattt aaagctgccg 420

catgccttac cgttttgtgg taaaagctat tataagaacg caatccagta tatcggcgaa 480

gataaaattt ttatcatctg tagcgacgat atcgattggt gtaaaaagaa tttcatcggt 540

aaacgttact actttatcga aaataccacc ccgctgctgg acttatatat tcaaagcctg 600

tgcacccata atattatttc aaatagcagc ttcagctggt ggggagcatg gctgaatgaa 660

aattctaata agattgtgat cgccccacaa atgtggtttg gtattagtgt taaacttggt 720

gtttctgatc tgttacctgt gagctgggtt agactgccga ataattatac cctgggtcgc 780

tattgttttg ccttatataa agttgttgaa gactacctgc tgaatattct gcgtctgatt 840

tggaaacgta aaaagaatat gtaa 864

<210> 15

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cataccatgg cgcagtcgaa actctatcca g 31

<210> 16

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cccaagcttt ggggtaaggg aagatccgac 30

<210> 17

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cgccatatgt caaaagtcgc tctcatcacc 30

<210> 18

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ccgctcgagt tcctgacgta aaaacatcat t 31

<210> 19

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gaaagatctt atgtactatt taaaaaacac aaac 34

<210> 20

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

attggtacct taagcgactt cattcac 27

<210> 21

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ctttaataag gagatatacc atgggcatgc tgtacgttat tctgcgg 47

<210> 22

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

cgcgccgagc tcgaattcgg atccttacat attcttttta cgtttc 46

Claims (10)

1. A recombinant Escherichia coli for synthesizing 2' -fucosyllactose is characterized in that: the recombinant escherichia coli is obtained by knocking out a beta-galactosidase gene from a genome of an original escherichia coli strain, synthesizing a key enzyme gene of kola acid from guanosine diphosphate rock sugar, intensively expressing a key enzyme gene of a lactose permease gene and guanosine diphosphate rock sugar de-novo synthesis way, and expressing an exogenous fucosyltransferase gene.

2. The recombinant E.coli of claim 1, wherein: the beta-galactosidase gene is knocked out by using a CRISPR gene editing technology, and the key enzyme gene for synthesizing the kola acid from the guanosine diphosphate fucose is knocked out by using the CRISPR gene editing technology.

3. The recombinant E.coli of claim 2, wherein: when the CRISPR gene editing technology is utilized to knock out an undecadiene phosphate glucose phosphotransferase gene wcaJ, the nucleotide sequence of the target sequence of gRNA1 of the gene wcaJ is shown as SEQ ID No.1, the nucleotide sequence of the target sequence of gRNA2 of the gene wcaJ is shown as SEQ ID No.2, the nucleotide sequence of an upstream primer identified by PCR is shown as SEQ ID No.3, and the nucleotide sequence of a downstream primer identified by PCR is shown as SEQ ID No. 4.

4. The recombinant E.coli of claim 2, wherein: when the CRISPR gene editing technology is used for knocking out beta-galactosidase gene lacZ, the nucleotide sequence of the target sequence of gRNA1 of the knocked-out gene lacZ is shown as SEQ ID No.5, the nucleotide sequence of the target sequence of gRNA2 of the knocked-out gene lacZ is shown as SEQ ID No.6, the nucleotide sequence of an upstream primer identified by PCR is shown as SEQ ID No.7, and the nucleotide sequence of a downstream primer identified by PCR is shown as SEQ ID No. 8.

5. The recombinant E.coli of claim 1, wherein: the expression-enhanced lactose permease gene and the key enzyme gene of the guanosine diphosphate rock sugar de novo synthesis pathway refer to that a T7lac promoter is knocked in at the upstream of a phosphomannose mutase gene manB of the guanosine diphosphate rock sugar de novo synthesis pathway by using a CRISPR gene editing technology.

6. The recombinant E.coli of claim 5, wherein: when a T7lac promoter is knocked in at the upstream of a phosphomannose mutase gene manB in a guanosine diphosphate fucose de novo synthesis pathway by using a CRISPR gene editing technology, the nucleotide sequence of a knock-in fragment is shown as SEQ ID NO.9, the nucleotide sequence of a knock-in position upstream gene is shown as SEQ ID NO.10, the nucleotide sequence of a knock-in position downstream gene is shown as SEQ ID NO.11, the nucleotide sequence of an upstream primer identified by PCR is shown as SEQ ID NO.12, and the nucleotide sequence of a downstream primer identified by PCR is shown as SEQ ID NO. 13.

7. The recombinant E.coli of claim 1, wherein: the recombinant escherichia coli carries a plurality of recombinant plasmids for inducing high-expression proteins, and the recombinant plasmids respectively comprise genes manB, mannose-1-phosphate guanosine transferase genes manC, guanosine diphosphate mannose-4, 6-dehydratase genes gmd, guanosine diphosphate-L-fucose synthetase genes fcl, lactose permease genes lacY and exogenous fucosyltransferase genes.

8. The recombinant E.coli of claim 7, wherein: the exogenous fucosyltransferase wcfB gene is derived from bacteroides fragilis, and the codon-optimized wcfB gene sequence is shown in SEQ ID No. 14.

9. The recombinant E.coli of claim 1, wherein: the original strain is Escherichia coli, preferably Escherichia coli BL21(DE 3).

10. A construction method of recombinant Escherichia coli for synthesizing 2' -fucosyllactose is characterized by comprising the following steps:

1) respectively preparing recombinant plasmids containing gene manB, gene manC, gene gmd, gene fcl, lactose permease gene lacY and codon optimized gene wcfB to obtain plasmids for constructing metabolic pathways;

2) the detection shows that the growth state of the original strain of escherichia coli BL21(DE3) is normal, the detection of the knock-in positions of target genes wcaJ and lacZ and a T7lac promoter and upstream and downstream sequences shows that the size of a PCR amplification band accords with the expectation, and the sequencing result is consistent with the NCBI sequence;

3) designing and preparing gRNA according to the insertion position of a target gene sequence and the sequence characteristics around the target gene sequence, cloning the gRNA and the Donor sequence to a gene editing vector Donor plasmid, and ensuring that the gRNA and the Donor sequence in the constructed vector are consistent with the target sequence through sequencing verification;

4) preparing escherichia coli BL21(DE3) electrotransformation competence, transforming Cas9 plasmid into BL21(DE3) competence, selecting spots to prepare BL21(DE3) -Cas9 electrotransformation competence, transforming Donor plasmid into BL21(DE3) -Cas9 electrotransformation competence, adding arabinose for induction, coating a plate, and carrying out gene editing strain screening experiment;

5) PCR amplification verifies that the edited BL21(DE3) is monoclonal, the size of a band successfully knocked out by the wcaJ gene is 771bp, and the size of a band successfully knocked out is 1049 bp; the size of a successful band of the lacZ gene knockout is 687bp, and the size of a successful band of the lacZ gene knockout is 983 bp; the size of a band of the T7lac promoter which is knocked in successfully is 1076bp, and no band exists when the promoter is not knocked in successfully; electrophorograms show that monoclonals with the wcaJ gene and the lacZ gene knocked out simultaneously and the T7lac promoter knocked in are successfully screened;

6) carrying out lysogenic treatment on the escherichia coli edited by the CRISPR in the step 5), and then converting the metabolic pathway construction plasmid obtained in the step 1) into lysogenic bacteria to obtain the recombinant escherichia coli capable of synthesizing 2' -fucosyllactose.

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