CN112662604B - 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 a beta-galactosidase gene, synthesizing a key enzyme gene of a colanic acid from guanosine diphosphate fucose, strengthening and expressing a lactose permease gene, a key enzyme gene of a guanosine diphosphate fucose head synthesis pathway and expressing an exogenous fucosyltransferase gene in a genome of the escherichia coli. The scheme of the invention can reduce lactose hydrolysis and improve the transfer rate of exogenous lactose into cells, thereby effectively improving the lactose concentration in the cells; meanwhile, the consumption of guanosine diphosphate fucose is reduced, and the concentration of guanosine diphosphate fucose in cells is effectively improved; the expression of the exogenous fucosyltransferase gene is improved by 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, proteins, minerals, vitamins, polysaccharides, monosaccharides, disaccharides (lactose), oligosaccharides. Lactose is the major solid component of breast milk (6.8% of total solids) and is widely present in animal milk. However, the structurally complex breast milk oligosaccharides (-1% of total solids) are unique to humans. Currently, there are about 200 structurally sound breast milk oligosaccharides with a degree of polymerization ranging from 3 to 32. The structural features of breast milk oligosaccharides include a lactose-N-acetylglucosamine backbone, linear beta- (1, 3/4) -glycosidic linkages, branched beta- (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 milk oligosaccharide which is ubiquitous and relatively abundant in breast milk of lactating mothers. The proportion of 3-fucosyllactose to the total amount of breast milk oligosaccharides may be up to 5%. Thus, the study of 3-fucosyllactose was more thorough, and 3-fucosyllactose was found to have various beneficial functions. For example, 3-fucosyllactose specifically inhibits pathogenic E.coli, norovirus binding to the corresponding host receptor and thereby avoids infection. 3-fucosyllactose inhibits the interaction of Pseudomonas aeruginosa with local epithelial cells, thereby avoiding infection of gastrointestinal tract, urinary tract and respiratory system by Pseudomonas aeruginosa. 3-fucosyllactose can also specifically proliferate intestinal specific beneficial bacteria to modulate the structure and function of the intestinal flora. In addition, 3-fucosyllactose enhances intestinal mucus barrier function by up-regulating expression of genes related to intestinal goblet cell secretion.

3-fucosyllactose is beneficial for health and related test data show that it meets the requirements of food safety regulations and is expected to be used as a functional food raw material for infant formulas and medical nutritional products. However, the industrial production of 3-fucosyllactose is not easy. Early, 3-fucosyllactose was obtained by chromatographic separation from donated breast milk, and it was not amenable to mass production due to limited material sources. 3-fucosyllactose can be directly chemically synthesized, however, it is limited by the chemical nature of the protecting group, cannot be scaled up, and chemical synthesis requires the use of toxic reagents, which is not suitable for large-scale food production. Fucosyltransferases can catalyze the synthesis of 3-fucosyllactose in vitro, but the donor substrate guanosine diphosphate fucose is very expensive. Therefore, enzymatic synthesis of 3-fucosyllactose is not applicable to industrial production of hundreds of tons per year. Microbial fermentation is currently the only technical route suitable for mass production. The technical progress of metabolic engineering, fermentation engineering, purification technology and the like makes the mass production of 3-fucosyl lactose gradually become reality. During fermentation, the donor substrate molecule guanosine diphosphate fucose is synthesized de novo by the microorganism itself, whereas the acceptor substrate molecule lactose is generally added externally.

Therefore, constructing a recombinant escherichia coli for synthesizing 3-fucosyllactose, effectively promoting the fermentation synthesis of 3-fucosyllactose, and laying a solid foundation for future mass production is a problem to be solved by the technicians in the field.

Disclosure of Invention

In view of the above, the technical problem to be solved by the 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 the homologous recombination technology, optimize the expression of an exogenous fucosyltransferase gene, and effectively promote the synthesis of 3-fucosyllactose, thereby realizing the efficient fermentation synthesis of 3-fucosyllactose.

In order to solve the technical problems, on the one hand, the scheme of the invention provides a recombinant escherichia coli for synthesizing 3-fucosyllactose, wherein the recombinant escherichia coli is obtained by knocking out a beta-galactosidase gene, synthesizing a key enzyme gene of a colanic acid from guanosine diphosphate fucose, strengthening and expressing a lactose permease gene, a key enzyme gene of a guanosine diphosphate fucose head synthesis pathway and expressing an exogenous fucosyltransferase gene in a genome of an original escherichia coli strain.

Preferably, the beta-galactosidase gene is knocked out by using a CRISPR gene editing technology, and the key enzyme gene for synthesizing the colanic acid from guanosine diphosphate fucose is knocked out by using the CRISPR gene editing technology.

Preferably, when the CRISPR gene editing technology is utilized to knock out the undecadiene phosphate glucose phosphotransferase gene wcaJ, the nucleotide sequence of the target sequence of the gcaJ gene gcaJ is shown as SEQ ID NO.1, the nucleotide sequence of the target sequence of the gcaJ gene gca2 is shown as SEQ ID NO.2, the nucleotide sequence of the PCR identified upstream primer is shown as SEQ ID NO.3, and the nucleotide sequence of the PCR identified downstream primer is shown as SEQ ID NO. 4.

Preferably, when the CRISPR gene editing technology is utilized to knock out the beta-galactosidase gene lacZ, the nucleotide sequence of the target sequence of the gRNA1 of the knock-out gene lacZ is shown as SEQ ID NO.5, the nucleotide sequence of the target sequence of the gRNA2 of the knock-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 fucose de novo synthesis pathway means that a T7lac promoter is knocked in upstream of a mannosyl phosphate mutase gene manB of the guanosine diphosphate fucose de novo synthesis pathway by using a CRISPR gene editing technology.

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

Preferably, the recombinant E.coli carries a plurality of recombinant plasmids for inducing a high expression protein, the recombinant plasmids respectively comprising a gene manB, a mannose-1-phosphate guanyltransferase gene manC, a guanyldiphosphate mannose-4, 6-dehydratase gene gmd, a guanyldiphosphate-L-fucose synthase gene fcl, a lactose permease gene lacY, and an exogenous fucosyltransferase gene.

Preferably, the exogenous fucosyltransferase cafF gene is derived from Alkermansia 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 a specific implementation process, the invention provides a recombinant escherichia coli for synthesizing 3-fucosyllactose, which adopts a CRISPR gene editing technology to knock out an original strain genome undecadiene phosphate glucose phosphotransferase gene wcaJ and a beta-galactosidase gene lacZ. Meanwhile, the T7lac promoter was knocked in upstream of the mannosyl mutase gene manB of guanosine diphosphate fucose from the head synthesis pathway. And, simultaneously, the phosphomannose mutase gene manB, the mannose-1-phosphate guanylate transferase gene manC, the guanosine diphosphate mannose-4, 6-dehydratase gene gmd, the guanosine diphosphate-L-fucose synthase gene fcl, the lactose permease gene lacY, the codon-optimized alpha-1, 3-fucose transferase gene cafF are overexpressed.

The gene wcaJ, the 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, accession number GI:946563; gene lacY, accession number GI 949083; gene cafF, gene accession number GI 187425317. The gene overexpression is to realize the induction high expression of 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 recombinant escherichia coli for synthesizing 3-fucosyllactose, which comprises the following steps:

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

2) Detection shows that the growth state of the original strain of the escherichia coli BL21 (DE 3) is normal, and detection of the knock-in position of target genes wcaJ and lacZ and the T7lac promoter and the sequence at the upstream and downstream thereof shows that the size of PCR amplified bands accords with the expectation, and the sequencing result accords with NCBI sequence;

3) According to the insertion position of the target gene sequence and the sequence characteristics around the target gene sequence, designing and preparing gRNA, 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 (DE 3) electrotransformation competence, transforming Cas9 plasmid into BL21 (DE 3) competence, picking spots to prepare BL21 (DE 3) -Cas9 electrotransformation competence, transforming Donor plasmid into BL21 (DE 3) -Cas9 electrotransformation competence, adding arabinose to induce and then coating a plate, and carrying out a gene editing strain screening experiment;

5) The size of a successful band of the wcaJ gene knocked out is 771bp, and the size of a non-knocked-out band is 1049bp after PCR amplification verification and editing; the successful band size of the lacZ gene knockout is 687bp, and the unsuccessful band size of the lacZ gene knockout is 983bp; the size of the band which is knocked in successfully by the T7lac promoter is 1076bp, and no band which is knocked in successfully is not formed; the electropherogram shows that monoclonals in which the wcaJ gene and the lacZ gene are knocked out simultaneously and the T7lac promoter is knocked in are successfully screened;

6) And 5) carrying out a lysogeny treatment on the E.coli edited by CRISPR in the step 5), and then converting the metabolic pathway construction plasmid obtained in the step 1) into a lysogeny bacterium to obtain the recombinant E.coli capable of synthesizing 3-fucosyl lactose.

The invention discloses a method for synthesizing 3-fucosyllactose by fermenting recombinant escherichia coli, which comprises the following steps:

LB medium (1L) tryptone, 10g; yeast extract, 5g; sodium chloride, 5g. And preparing a solid culture medium, and 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. 1mL of the overnight cultured seed solution was transferred to 50mL of LB medium (kanamycin 50. Mu.g/mL, streptomycin 50. Mu.g/mL, glucose with a final concentration of 20 g/L) and cultured at 30℃and shaking table rotation speed of 250rpm for about 8 hours (bacterial liquid OD) ₆₀₀ About 0.8). Lactose at a final concentration of 12g/L and 0.3mM IPTG were added and the shaking table was rotated at 25℃and 250rpm to continue the induction fermentation for 48 hours.

The recombinant escherichia coli for synthesizing the 3-fucosyllactose and the construction method thereof have the positive effects compared with the prior art that:

(1) The recombinant escherichia coli has a metabolic pathway for synthesizing 3-fucosyllactose by fermentation, and meanwhile, the problems of long test period, low success rate and the like of the homologous recombination technology are overcome, and a high-efficiency genome editing scheme is obtained; other characteristics of the original strain are not changed except for synthesizing 3-fucosyllactose, and fermentation is not affected; the adopted plasmid is a common escherichia coli plasmid, and does not influence the growth and metabolism of bacteria.

(2) The synthesis of 3-fucosyl lactose is effectively promoted by increasing the concentration of intracellular lactose and guanosine diphosphate fucose and simultaneously optimizing the expression of an exogenous fucosyl transferase gene; the recombinant escherichia coli disclosed by the invention is clear in construction thought, remarkable in actual effect and good in application prospect.

(3) The recombinant escherichia coli is fermented and synthesized into the 3-fucosyl lactose by taking glucose as a carbon source, so that the cost is lower, the economic feasibility is higher, and the 3-fucosyl lactose with the concentration of 1.0-1.5 g/L can be obtained after 48 hours of fermentation.

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

Drawings

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

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

FIG. 2 is an electrophoretogram of a PCR-verified product of lacZ gene knockout;

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

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

( 7Lac-WT is the amplification product of the non-knocked-in T7 Lac; 7 Lac-edition is an amplified product of successful T7Lac knock-in )

FIG. 4 is a graph of ion chromatography detection of fermentation broth, wherein the 11.159 minute chromatographic peak is 3-fucosyllactose.

Detailed Description

The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The raw materials and reagents used in the invention are all commercially available. The materials, reagents, instruments and methods used in the examples below are all conventional in the art and are commercially available.

Example 1

Gene acquisition:

in this example, 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) and gene cafF (gene accession number GI: 187425317) derived from Acremonium mucin ATCC BAA-835 were obtained from Escherichia coli MG 1655.

In this example, the E.coli genes manB, manC, gmd, fcl and lacY were successfully obtained by extracting the genomic DNA of E.coli MG 1655; the gene cafF derived from Acremonium muciniphilum ATCC BAA-835 was successfully obtained by artificially synthesizing the codon optimized gene and cloning it into E.coli plasmid. Successful acquisition of the gene lays a foundation for construction of recombinant plasmids.

Example 2

Preparation of recombinant plasmids

PCR amplification of the gene manC and the gene manB derived from E.coli MG1655 obtained in example 1 was performed using the designed primers (the upstream primer sequence is shown in SEQ ID NO.15, the downstream primer sequence is shown in SEQ ID NO. 16), the amplified fragments were subjected to gel cutting purification, double restriction enzymes NcoI and HindIII were used for double restriction, the digested fragments were ligated with plasmid pCOLADuet-1 which was also subjected to double restriction with NcoI and HindIII, and the vector was ligated: the target fragments are mixed according to the mol ratio of 1:3, added with T4 DNA ligase, and then subjected to enzyme ligation for 5 hours at 22 ℃, the ligation products are transformed into competent cells of escherichia coli DH5 alpha, and screening is carried out on a kanamycin plate to obtain the recombinant plasmid pCOLA-CB. The gene gmd derived from E.coli MG1655 and gene fcl obtained in example 1 were amplified by PCR using the designed primers (the upstream primer sequence is shown in SEQ ID NO.17 and the downstream primer sequence is shown in SEQ ID NO. 18), the amplified fragments were subjected to gel-cutting purification, double-digested fragments were digested with NdeI and XhoI, and the digested fragments were ligated with plasmid pCOLA-CB which was also digested with NdeI and XhoI, and the vector was ligated: the target fragments are mixed according to the mol ratio of 1:3, added with T4 DNA ligase, and then subjected to enzyme ligation for 5 hours at 22 ℃, the ligation products are transformed into competent cells of escherichia coli DH5 alpha, and screening is carried out on a kanamycin plate to obtain recombinant plasmid pCOLA-CBGF.

The gene lacY derived from E.coli MG1655 obtained in example 1 was amplified by PCR using a designed primer (the upstream primer sequence is shown in SEQ ID NO.19, the downstream primer sequence is shown in SEQ ID NO. 20), the amplified fragment was subjected to gel cutting purification, double digestion was performed with NdeI and XhoI, the digested fragment was ligated with plasmid pCDFDuet-1 which was also subjected to double digestion with NdeI and XhoI, and the vector was: the target fragments are mixed according to the mol ratio of 1:3, added with T4 DNA ligase, and then subjected to enzyme ligation for 5 hours at 22 ℃, the ligation products are transformed into competent cells of escherichia coli DH5 alpha, and screening is carried out on a streptomycin plate to obtain the recombinant plasmid pCDF-lacY. PCR amplification of the gene cafF derived from Alkermansia muciniphila obtained in example 1 was performed using a designed primer (the upstream primer sequence is shown in SEQ ID NO.21, the downstream primer sequence is shown in SEQ ID NO. 22), the amplified fragment was subjected to gel cutting purification, double cleavage with NcoI and BamHI, and the digested fragment was ligated with plasmid pCDF-lacY which was also subjected to double cleavage with NcoI and BamHI, and the vector was: the target fragments are mixed according to the mol ratio of 1:3, added with T4 DNA ligase, and then subjected to enzyme ligation for 5 hours at 22 ℃, the ligation products are transformed into competent cells of escherichia coli DH5 alpha, and screening is carried out on a streptomycin plate to obtain the recombinant plasmid pCDF-lacY-cafF.

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

Example 3

Gene editing

This example uses CRISPR gene editing techniques to achieve wcaJ and lacZ gene knockouts and T7lac promoter knockins. The steps of gene editing are described in detail below.

1) Detection shows that the growth state of the original strain of the escherichia coli BL21 (DE 3) is normal, and detection of the knock-in position of target genes wcaJ and lacZ and the T7lac promoter and the sequence at the upstream and downstream thereof shows that the size of PCR amplified bands accords with the expectation, and the sequencing result accords with NCBI sequence;

2) According to the insertion position of the target gene sequence and the sequence characteristics around the target gene sequence, designing and preparing gRNA, 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 (DE 3) electrotransformation competence, transforming Cas9 plasmid into BL21 (DE 3) competence, picking spots to prepare BL21 (DE 3) -Cas9 electrotransformation competence, transforming Donor plasmid into BL21 (DE 3) -Cas9 electrotransformation competence, adding arabinose to induce and then coating a plate, and carrying out a gene editing strain screening experiment;

5) The size of a successful band of the wcaJ gene knocked out is 771bp, and the size of a non-knocked-out band is 1049bp after PCR amplification verification and editing; the successful band size of the lacZ gene knockout is 687bp, and the unsuccessful band size of the lacZ gene knockout is 983bp; the size of the band which is knocked in successfully by the T7lac promoter is 1076bp, and no band which is knocked in successfully is not formed; the electropherograms showed that monoclonals were successfully screened for simultaneous knockdown of wcaJ gene and lacZ gene and knockin of T7lac promoter.

In the embodiment, the CRISPR gene editing technology is adopted to realize the knockout of the escherichia coli BL21 (DE 3) gene wcaJ (the figure 1 is an electrophoresis diagram of PCR verification products of wcaJ gene knockout), so that the consumption of guanosine diphosphate fucose is reduced; the knockout of the lacZ gene of the escherichia coli BL21 (DE 3) is realized (FIG. 2 is an electrophoresis diagram of PCR verification products of lacZ gene knockout), and the hydrolysis of lactose in cells is reduced; the T7lac promoter was knocked in upstream of the gene manB of the E.coli genome (FIG. 3 is an electrophoretogram of PCR verification products knocked in by the T7lac promoter), the expression of the corresponding carbohydrase by the gene manB, manC, gmd, fcl was promoted, and the content of guanosine diphosphate fucose in the cell was increased. Successful implementation of the three aspects can effectively promote the synthesis of 3-fucosyl lactose by escherichia coli.

Example 4

Construction of recombinant E.coli for the Synthesis of 3-fucosyllactose

The CRISPR-edited escherichia coli of example 3 was subjected to a lysogenic treatment, and the metabolic pathway-constructing plasmid obtained in example 2 was transformed into a lysogenic bacterium to obtain a 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 recombinant E.coli to synthesize 3-fucosyllactose

The strain was inoculated 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. 1mL of the overnight cultured seed solution was transferred to 50mL of LB medium (kanamycin 50. Mu.g/mL, streptomycin 50. Mu.g/mL, glucose with a final concentration of 20 g/L) and cultured at 30℃and shaking table rotation speed of 250rpm for about 8 hours (bacterial liquid OD) ₆₀₀ About 0.8). Lactose at a final concentration of 12g/L and 0.3mM IPTG were added and the shaking table was rotated at 25℃and 250rpm to continue the induction fermentation for 48 hours. Centrifuging at 8000rpm for 10min, collecting supernatant, decocting at 100deg.C for 10min, centrifuging again, mixing 0.5mL supernatant with 0.5mL purified water, filtering with 0.22 μm filter membrane, and detecting by high performance liquid chromatography (FIG. 4 shows a chromatogram of the fermentation broth by ion chromatography, 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, thereby laying a foundation for further pilot scale up and even industrial production.

The foregoing embodiments are merely for illustrating the technical aspects of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical aspects described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof. Such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

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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 (3)

1. A recombinant escherichia coli for synthesizing 3-fucosyllactose, characterized by: the recombinant escherichia coli is obtained by knocking out a beta-galactosidase gene in a genome of an original escherichia coli strain, synthesizing a key enzyme gene of a colanic acid from guanosine diphosphate fucose, strengthening the expression of the lactose permease gene, the key enzyme gene of a guanosine diphosphate fucose head synthesis pathway, and expressing an exogenous fucosyltransferase gene;

the beta-galactosidase gene is knocked out by using a CRISPR gene editing technology, the key enzyme gene for synthesizing the lac from guanosine diphosphate fucose is knocked out by using the CRISPR gene editing technology;

when the CRISPR gene editing technology is utilized to knock out the undecadiene phosphate glucose phosphotransferase gene wcaJ, the nucleotide sequence of the target sequence of the gcaJ of the knocked-out gene wcaJ is shown as SEQ ID NO.1, the nucleotide sequence of the target sequence of the gcaJ 2 of the knocked-out gene wcaJ is shown as SEQ ID NO.2, the nucleotide sequence of the PCR identified upstream primer is shown as SEQ ID NO.3, and the nucleotide sequence of the PCR identified downstream primer is shown as SEQ ID NO. 4;

when the CRISPR gene editing technology is utilized to knock out the 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;

the key enzyme gene for the enhanced expression of lactose permease gene and guanosine diphosphate fucose synthesis pathway refers to that a T7lac promoter is knocked in upstream of a phosphomannose mutase gene manB of the guanosine diphosphate fucose synthesis pathway by using a CRISPR gene editing technology;

when guanosine diphosphate fucose is knocked in a T7lac promoter from the upstream of a mannosyl mutase gene manB of a head synthesis pathway by using a CRISPR gene editing technology, the nucleotide sequence of a knockin fragment is shown as SEQ ID NO.9, the nucleotide sequence of a gene at the upstream of a knockin position is shown as SEQ ID NO.10, the nucleotide sequence of a gene at the downstream of the knockin position 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;

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

the exogenous fucosyltransferase cafF gene is derived from Acremonium muciniphilum, and the codon-optimized cafF gene sequence is shown in SEQ ID No. 14.

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

3. A method of constructing recombinant escherichia coli for the synthesis of 3-fucosyllactose according to claim 1 or 2, comprising the steps of:

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

2) Detection shows that the growth state of the original strain of the escherichia coli BL21 (DE 3) is normal, and detection of the knock-in position of target genes wcaJ and lacZ and the T7lac promoter and the sequence at the upstream and downstream thereof shows that the size of PCR amplified bands accords with the expectation, and the sequencing result accords with NCBI sequence;

3) According to the insertion position of the target gene sequence and the sequence characteristics around the target gene sequence, designing and preparing gRNA, 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 (DE 3) electrotransformation competence, transforming Cas9 plasmid into BL21 (DE 3) competence, picking spots to prepare BL21 (DE 3) -Cas9 electrotransformation competence, transforming Donor plasmid into BL21 (DE 3) -Cas9 electrotransformation competence, adding arabinose to induce and then coating a plate, and carrying out a gene editing strain screening experiment;

5) The size of a successful band of the wcaJ gene knocked out is 771bp, and the size of a non-knocked-out band is 1049bp after PCR amplification verification and editing; the successful band size of the lacZ gene knockout is 687bp, and the unsuccessful band size of the lacZ gene knockout is 983bp; the size of the band which is knocked in successfully by the T7lac promoter is 1076bp, and no band which is knocked in successfully is not formed; the electropherogram shows that monoclonals in which the wcaJ gene and the lacZ gene are knocked out simultaneously and the T7lac promoter is knocked in are successfully screened;

6) And 5) carrying out a lysogeny treatment on the E.coli edited by CRISPR in the step 5), and then converting the metabolic pathway construction plasmid obtained in the step 1) into a lysogeny bacterium to obtain the recombinant E.coli capable of synthesizing 3-fucosyl lactose.