CN112662604A - Recombinant escherichia coli for synthesizing 3-fucosyllactose and construction method thereof - Google Patents

Abstract

The invention provides a recombinant escherichia coli for synthesizing 3-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 3-fucosyllactose is effectively promoted.

Description

Recombinant escherichia coli for synthesizing 3-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 3-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.

3-fucosyllactose is a commonly occurring and abundant oligosaccharide in breast milk of lactating mothers. The proportion of 3-fucosyllactose in the total amount of oligosaccharide in breast milk can be up to 5%. Therefore, the study of 3-fucosyllactose is more thorough, and 3-fucosyllactose is found to have various beneficial functions. For example, 3-fucosyllactose specifically inhibits the binding of pathogenic e.coli, norovirus, to the corresponding host receptor to avoid infection. The 3-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. The 3-fucosyllactose can also specifically proliferate intestinal specific beneficial bacteria so as to regulate the structure and function of intestinal flora. In addition, 3-fucosyllactose enhances intestinal mucus barrier function by up-regulating the expression of genes involved in intestinal goblet cell secretion.

The 3-fucosyllactose is beneficial to health, and related test data show that the 3-fucosyllactose meets the requirements of food safety regulations and is expected to be used as a functional food raw material of infant formula foods and medical nutritional products. However, industrial production of 3-fucosyllactose is not easy. Early, 3-fucosyllactose was chromatographically isolated from donated breast milk and could not be produced on a large scale due to limited material sources. 3-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 catalyze the synthesis of 3-fucosyllactose in vitro, however, the donor substrate, guanosine diphosphate fucose, is very expensive. Therefore, the enzymatic synthesis of 3-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 progress of metabolic engineering, fermentation engineering, purification process and the like makes the large-scale production of the 3-fucosyllactose gradually become practical. 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 3-fucosyllactose, effectively promoting the fermentation and synthesis of the 3-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 3-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 3-fucosyllactose, thereby realizing the high-efficiency fermentation synthesis of 3-fucosyllactose.

In order to solve the above technical problems, in one aspect, the present invention provides a recombinant escherichia coli for synthesizing 3-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 cafF gene is derived from Ackermansia muciniphila, and the codon-optimized cafF 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 a recombinant escherichia coli for synthesizing 3-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, 3-fucose transferase gene cafF.

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 cafF, gene accession number GI: 187425317. 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 the 3-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 cafF 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 3-fucosyllactose.

The recombinant escherichia coli is fermented and synthesized into the 3-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 invention discloses a recombinant escherichia coli for synthesizing 3-fucosyllactose and a construction method thereof, and the construction method has the positive effects that:

(1) the recombinant escherichia coli has a metabolic pathway for fermenting and synthesizing 3-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 3-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 3-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 ferment and synthesize the 3-fucosyllactose, the cost is lower, the economic feasibility is higher, and the 3-fucosyllactose with the concentration of 1.0-1.5 g/L can be obtained after 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 chromatogram of an ion chromatography detection fermentation broth, wherein the chromatographic peak at 11.159 minutes is 3-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 cafF derived from Ackermanella viscocata ATCC BAA-835 (Gene accession number GI: 187425317) 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 cafF derived from Ackermansia muciniphila ATCC BAA-835 was successfully obtained by artificially synthesizing a codon-optimized gene and cloning it 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 T4 DNA 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, T4 DNA 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, T4 DNA 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 cafF derived from akkermansia muciniphila obtained in example 1 was amplified by PCR 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 purified by gel cutting and double-digested with NcoI and BamHI, the digested fragment was ligated with the plasmid pCDF-lacY, which was also double-digested with NcoI and BamHI, and the vector: the target fragments are mixed according to the molar ratio of 1:3, T4 DNA 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-cafF.

In this example, the recombinant plasmids pCOLA-CBGF and pCDF-lacY-cafF were successfully prepared, which laid the foundation for the construction of the metabolic pathway related to the 3-fucosyllactose of E.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 3-fucosyllactose.

Example 4

Construction of recombinant Escherichia coli for synthesizing 3-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 3-fucosyllactose.

In the embodiment, the recombinant escherichia coli for synthesizing the 3-fucosyllactose is successfully obtained, and a foundation is laid for further fermentation verification.

Example 5

Verification of 3-fucosyllactose synthesized by recombinant escherichia coli

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 liquid at 8000rpm for 10min, collecting supernatant, decocting the supernatant at 100 deg.C for 10min, centrifuging again, mixing 0.5mL supernatant with 0.5mL purified water, filtering the sample with 0.22 μm filter membrane, and detecting by high performance liquid chromatography (FIG. 4 is ion chromatography detection fermentation liquid chromatogram, wherein the chromatographic peak of 11.159 min is 3-fucosyllactose).

In the embodiment, the high performance liquid chromatography detection of the shake flask fermentation liquid shows that the recombinant escherichia coli successfully synthesizes the 3-fucosyllactose, and lays a foundation for further pilot scale-up and 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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atgaagaccc tgaaaattag ctttctgcag agcaccccgg actttggtcg tgaaggcatg 60

ctgcagctgc tgaaaagtcg gtaccacgtt gtggaagacg acagcgattt tgattacctg 120

gtggcaaccc cgtggttcta tgttaaccgg gaagcttttt atgatttttt agaacgggcc 180

ccgggtcata ttaccgttat gtatggttgt catgaagcaa ttgcaccgga ttttatgtta 240

tttgattact atatcggtct tgacacagtg ccgggtagcg atcgtaccgt gaaactgcct 300

tatttacgtc atcatctgga agaagttcac ggcggaaagg aaggtttaga tgcgcatgcg 360

ctgctggcaa gcaaaaccgg attttgtaac tttatatacg caaaccgtaa gtctcatccg 420

aatcgtgatg caatgtttca taaactgagc gcatttcgtt ttgttaatag tctgggtccg 480

catttaaata ataccccggg tgatggccat cgcgccgaag attggtatgc cagcagcatc 540

cgtatgaaaa agccgtacaa atttagcatt gcctttgaga atgcatggta tccgggttac 600

accagcgaaa aaattgttac cagcatgctg gcaggtacca ttccgattta ttggggtaat 660

ccggatatta gccgtgaatt taatagcgca agctttatta attgtcatga ttttccgacc 720

ctggatgatg cagcagcata tgttaaaaaa gttgatgaag atgataatct gtggtgtgaa 780

attatgagcc gtccgtggaa aaccccggaa caggaagcac gttttctgga agaaaccgaa 840

cgtgaaaccg caaaactgta taaaattttt gatcagagcc cggaagaagc acgtcgtaaa 900

ggtgatggta cctgggttag ctattatcag cgttttctga aacgtggtca tcgtatgcag 960

ctggcatggc gtcgtctgaa aaatcgtctg cgtcgttaa 999

<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> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ctttaataag gagatatacc atgggcatga agaccctgaa aattagcttt c 51

<210> 22

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

cgcgccgagc tcgaattcgg atccttaacg acgcagacga tttttcagac 50

Claims (10)

1. A recombinant Escherichia coli for synthesizing 3-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 cafF gene is derived from akkermansia muciniphila, and the sequence of the codon-optimized cafF gene 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 3-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 cafF 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 3-fucosyllactose.

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