CN114645007B - Recombinant strain, engineering bacterium for high-yield 2' -fucosyllactose based on recombinant strain and application of engineering bacterium - Google Patents

Abstract

The invention relates to the technical field of bioengineering, in particular to a recombinant strain, engineering bacteria for high-yield 2' -fucosyllactose based on the recombinant strain and application of the engineering bacteria. The invention provides a recombinant strain and engineering bacteria for high-yield 2' -fucosyllactose based on the recombinant strain, wherein an fbp mutant enzyme gene is inserted into a maltodextrin glucosidase gene (malZ) gene locus of an escherichia coli JM109 (DE 3) strain in a chromosome integration mode, and simultaneously, double plasmids pETDuet-CBGF and pCDFDuet-TAB are designed to co-express ManC, manB, gmd, fcl, fucT, rcsA and rcsB genes in the escherichia coli, so that the yield of the 2' -fucosyllactose (2 ' -FL) is greatly improved.

Description

Recombinant strain, engineering bacterium for high-yield 2' -fucosyllactose based on recombinant strain and application of engineering bacterium

Technical Field

The invention relates to the technical field of bioengineering, in particular to a recombinant strain, engineering bacteria for high-yield 2' -fucosyllactose based on the recombinant strain and application of the engineering bacteria.

Background

The concept of breast milk is always the development direction of formula milk powder, and the current breast feeding rate is low, and the main reasons of the concept defect, the physique difference and the like are the same. Therefore, the development of the milk powder additive with the breast milk approximate effect has great application value and social influence.

2'-fucosyllactose (2' -FL), which is an oligosaccharide most abundant in human milk, has the following effects: (1) Promoting the reproduction of intestinal probiotics and inhibiting the growth of harmful bacteria, and after the human intakes 2' -FL, the structure of the intestinal probiotics is stable and can not be damaged by gastric acid and digestive enzymes, so that the intestinal probiotics can directly reach the large intestine part and the growth of bifidobacteria and lactobacillus in the intestinal tract can be promoted; (2) Anti-pathogen adhesion, the first step after invasion of intestinal pathogens into the human body is usually colonization of epithelial cells, and then tissue infection begins, and the structure of 2' -FL is exactly similar to that of glycoprotein and sugar chain part on intestinal epithelial cells, so that the pathogenic bacteria can be decoy to bind with the intestinal epithelial cells but not to the intestinal epithelial cells, and thus the colonization process of the intestinal epithelial cells is blocked; (3) Modulation of the immune system to reduce inflammatory response, 2' -FL has been reported to be directly involved in cytokine secretion modulation to affect the immune system, e.g., inflammatory cytokine (IL-1 ra, IL-1 alpha, IL-1 beta, IL-6, tumor necrosis factor-alpha) concentrations can be reduced in infants, which is substantially consistent with breast feeding results; whereas the common milk powder feeding group had a significant rise in inflammatory cytokines. The research results prove that 2' -FL is a breast milk concept formula milk powder additive with great application value, and the research on the preparation method is very valuable.

The existing 2' -FL production methods are biological methods, are obtained in large quantity through microbial fermentation, and the process is green and has high environmental protection efficiency. Biological processes can be further divided into two types, salvage pathway and de novo pathway, wherein the salvage pathway completes fermentation by exogenously adding L-fucose and lactose, taking glycerol as a carbon source; the de novo approach takes glycerol or sucrose as a carbon source directly, and can synthesize 2' -FL by adding lactose only exogenously without using expensive L-fucose. Clearly, the de novo approach is more cost advantageous, while glycerol has proven to be the most effective carbon source. However, as is known from studies on the synthetic route of 2'-FL, glycerol is required to be metabolized in multiple steps to be converted into fructose-6-phosphate as an intermediate of 2' -FL, and the utilization efficiency is low, so that the yield of 2'-FL is difficult to be greatly improved, and practical application of 2' -FL is limited.

Disclosure of Invention

In order to solve the problems, the invention provides a recombinant strain, engineering bacteria for high-yield 2' -fucosyllactose based on the recombinant strain and application of the engineering bacteria. The engineering bacteria for high-yield 2'-fucosyllactose provided by the invention can improve the efficiency of synthesizing 2' -FL by over-expressing the mutant enzyme of fbp enzyme, and can greatly improve the yield of 2'-FL produced by glycerol fermentation by over-expressing 2' -FL synthesis pathway gene ManC, manB, gmd, fcl, fucT and forward transcription factors rcsA and rcsB.

The invention provides a recombinant strain, which is characterized in that the recombinant strain is a strain integrating an fbp mutant enzyme coding gene into a JM109 (DE 3) strain chromosome.

Preferably, the fbp mutant enzyme coding gene is: the lysine residue at position 104 of the amino acid sequence of the wild-type fbp enzyme coding gene is mutated to glutamine, the arginine residue at position 132 is mutated to isoleucine, the tyrosine residue at position 210 is mutated to phenylalanine, and the lysine residue at position 218 is mutated to glutamine.

Preferably, the promoter Ptrc is used to control transcription of the fbp mutant enzyme gene.

Preferably, the JM109 (DE 3) strain has the chromosome integrated at the location of the maltodextrin glucosidase gene.

The invention also provides application of the recombinant strain in preparing engineering bacteria for high-yield 2' -fucosyllactose.

The invention provides engineering bacteria for high-yield 2' -fucosyllactose based on the recombinant strain, which is characterized in that the engineering bacteria are obtained by co-transforming recombinant plasmid pETDuet-CBGF and recombinant plasmid pCDFDuet-TAB into the recombinant strain.

Preferably, the recombinant plasmid pETDuet-CBGF is: the ManC gene, the ManB gene, the Gmd gene and the Fcl gene are cloned on a pETDuet-1 plasmid.

Preferably, the recombinant plasmid pCDFDuet-TAB is: the FucT gene, the rcsA gene and the rcsB gene were cloned into the pCDFDuet-1 plasmid.

The invention also provides a method for producing 2' -fucosyllactose, and is characterized in that the method comprises the following steps: the engineering bacteria are used as fermentation strains, and 2' -fucosyllactose is produced in a fermentation system with glycerol as a substrate.

The invention also provides application of the engineering bacteria in high-yield 2'-fucosyllactose and production of products containing 2' -fucosyllactose.

Recombinant JM109 (DE 3) strain in the prior art can synthesize 2'-FL by using glycerol as a carbon source, but the path activity of the glycerol to convert into key intermediate fructose-6-phosphate is low, so that the yield of 2' -FL is difficult to improve. According to the invention, the high-activity fructose-1, 6-bisphosphatase (fbp enzyme) mutant enzyme is integrated in the chromosome, so that the capability of converting the glycerol into the intermediate fructose-6-phosphate by the strain is effectively improved, the yield of 2' -FL is greatly improved, and a foundation is laid for the industrial application of the technology.

The invention provides a recombinant strain and engineering bacteria for high-yield 2'-fucosyllactose based on the recombinant strain, wherein an fbp mutant enzyme gene (lysine residue (K) at 104 th site of an amino acid sequence is mutated into glutamine (Q), arginine residue (R) at 132 th site is mutated into isoleucine (I), tyrosine residue (Y) at 210 th site is mutated into phenylalanine (F), lysine residue (K) at 218 th site is mutated into glutamine (Q)) is inserted into a maltodextrin glucosidase gene (malZ) gene locus of an escherichia coli JM109 (DE 3) strain in a chromosome integration mode, and simultaneously, double plasmids pETDuet-CBGF and pCDFDuet-TAB are designed to co-express ManC, manB, gmd, fcl, fucT, rcsA and rcsB genes in the escherichia coli, so that the yield of 2' -FL is greatly improved.

The results of the examples show that under the same conditions, the 2' -FL yield of the engineering bacteria provided by the invention is 5.4g/L, and the 2' -FL yield of the control strain is 2.1g/L, which indicates that the strain provided by the invention can greatly improve the 2' -FL yield.

Drawings

FIG. 1 is a schematic representation of the integration of the fbp mutant gene into the JM109 (DE 3) strain chromosome.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and examples. It should be understood that the detailed description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the invention. The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were all purchased from conventional reagent manufacturers. In particular, the components involved in the following additives are all conventional commercial products.

Example 1

Construction of recombinant plasmid pETDuet-CBGF

1. The ManC gene (the nucleic acid sequence is shown as SEQ ID NO. 1), the ManB gene (the nucleic acid sequence is shown as SEQ ID NO. 2), the Gmd gene (the nucleic acid sequence is shown as SEQ ID NO. 3) and the Fcl gene (the nucleic acid sequence is shown as SEQ ID NO. 4) derived from the E.coli K12 strain are obtained through total gene synthesis. The genes are also subjected to codon preference sequence optimization and are consigned to be synthesized by general biological systems (Anhui) limited company. The T7 promoter and RBS sequence on pETDuet-1 vector were amplified by PCR (polymerase chain reaction), and the ManC, manB, gmd, fcl gene obtained by the entrusted synthesis was amplified, and the primer sequences are shown in Table 1.

TABLE 1 primer sequence listing

2. Purifying the amplified DNA fragments by using a gel cutting recovery method, connecting each fragment and the pET-Duet-1 vector by using a Golden gate connecting method to form a recombinant plasmid A through an Overhang sequence, and obtaining positive clones carrying recombinant plasmids with correct sequencing; and extracting recombinant plasmid from the positive clone to obtain pETDuet-CBGF recombinant plasmid. The Goldengate connection method comprises the following experimental steps:

(1) Obtaining ManC, manB, gmd, fcl gene fragments by PCR, obtaining DNA fragments of a T7 promoter and a RBS sequence on a pETDuet-1 vector by PCR, and obtaining vector fragments of pETDuet-1 by PCR;

(2) Purifying the obtained DNA fragment by gel cutting purification method, and selecting FastPure Gel DNAExtractionMini Kit (product number DC 301-01) of Nanjinouzan company;

(3) The fragments were mixed in a PCR tube at an equimolar ratio, and BsaI enzyme (NEB Co., cat# R3733S), T4 DNA ligase (NEB Co., cat# M0202S) and Buffer were added;

(4) Incubating the PCR tube in a PCR instrument to finish enzyme digestion and connection, wherein the procedure is set to 37 ℃ for 1h and 60 ℃ for 5min;

(5) Transforming the liquid in the PCR tube into E.coli DH5 alpha competent cells, and culturing at 37 ℃ overnight;

(6) Selecting the grown monoclonal into LB liquid culture medium, culturing at 37 ℃ until the culture medium is turbid, sucking a small amount of bacterial liquid, and sending the bacterial liquid to general biological systems (Anhui) limited company for sequencing, wherein the result is correct, namely positive clone;

(7) And (3) culturing positive clones to extract recombinant plasmids, namely pETDuet-CBGF recombinant plasmids.

Example 2

Construction of recombinant plasmid pCDFDuet-TAB

1. The FucT gene (the nucleic acid sequence is shown as SEQ ID NO. 21), the RcsA gene (the nucleic acid sequence is shown as SEQ ID NO. 22) and the RcsB gene (the nucleic acid sequence is shown as SEQ ID NO. 23) which are derived from E.coli K12 strain are obtained through total synthesis of genes, and the genes are subjected to codon preference sequence optimization and are entrusted to synthesis by general biological systems (Anhui) limited company. The T7 promoter and RBS sequence on the pCDFDuet-1 vector were amplified by PCR (polymerase chain reaction), and the FucT, rcsA, rcsB gene obtained by the synthesis was amplified, and the primers are shown in Table 2.

TABLE 2 primer sequence listing

2. Using the same method as in example 1, using a cut gel recovery method to purify amplified DNA fragments, connecting each fragment and pCDFDuet-1 vector through a Goldengate connection method to form recombinant plasmids through Overhang sequences, and obtaining positive clones as clones carrying the recombinant plasmids with correct sequencing; and extracting recombinant plasmid from the positive clone to obtain pCDFDuet-TAB recombinant plasmid.

Example 3

Chromosome integrated fbp mutant enzyme coding gene

1. The pCas Plasmid (Addgene number: plasmid # 62225) was transformed into JM109 (DE 3) strain to obtain JM109 (DE 3) -Cas strain by:

(1) LB agar plates (formula 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, 20g/L agar) containing 25mg/L kanamycin were prepared;

(2) A1.5 ml centrifuge tube was taken, 100. Mu.L of E.coli JM109 (DE 3) competent cell suspension (Beijing Hua Vietnam organism, cat# NRR 00980) was added, and the mixture was placed on ice; 1 mu LpCas plasmid (100 ng/. Mu.L concentration) was added, gently mixed with a pipette, and left on ice for 20min;

(3) Heat shock is carried out for 90 seconds in a water bath at the temperature of 42 ℃, then ice is quickly placed for 3-5 minutes, and bacterial liquid is not required to be oscillated in the whole process;

(4) Adding 1mLLB liquid culture medium (without antibiotics), shaking culturing at 37deg.C (100 rpm) for 1 hr to recover bacteria to normal growth state, and expressing plasmid-encoded antibiotic resistance gene to obtain JM109 (DE 3) -Cas strain;

(5) mu.L of the bacterial liquid was taken out and spread evenly on LB agar plates containing 25mg/L kanamycin.

(6) After the bacterial liquid is absorbed by the culture medium, culturing for 12-16 hours at 37 ℃, after single colony appears, picking up the single colony, culturing to be turbid in LB liquid culture medium containing 25mg/L kanamycin at 37 ℃, sucking 500 mu L bacterial liquid to a sterilized EP tube, adding 500 mu L of 40% (w/w) glycerol, uniformly mixing, and preserving at-80 ℃ for later use.

TABLE 3 primer sequence listing

2. The N20 sequence for disrupting the malZ gene (nucleic acid sequence shown in SEQ ID NO. 36) was inserted into the ptargetF Plasmid (Addgene number: plasmid # 62226) by inverse PCR to obtain the ptargetF-N20 recombinant Plasmid, as follows:

PCR amplification was performed using the ptargetF plasmid as a template (the primer sequences used are shown in Table 3), the PCR product was transformed into E.coli DH 5. Alpha. Competent cells, and then the competent cells were plated on LB agar plates containing 50mg/L spectinomycin, and cultured at 37℃for 12-16 hours, and positive monoclonal was selected, followed by plasmid extraction to obtain ptargetF-N20 recombinant plasmid.

3. The fbp mutant enzyme gene (the sequence of the fbp mutant enzyme encoding gene is shown as SEQ ID NO. 43) was amplified by PCR and cloned between XbaI and HindIII sites of the ptrc99a vector (Beijing Wash Vietnam, cat# VECT 5460) (primer fbp-S, fbp-A see Table 3), E.coli DH 5. Alpha. Competent cells were transformed with the use of Nanjinovirzan ClonExpressII One Step Cloning Kit (cat# C112-01), spread on LB plates containing 50mg/L ampicillin, and cultured at 37℃for 12 to 16 hours. Positive monoclonal was PCR screened, primers PTrc99C-F and PBV220-R in Table 3. Positive clones were cultured overnight with LB liquid medium containing 50mg/L ampicillin, and plasmids were extracted to obtain ptrc99a-fbp recombinant plasmids.

(the amino acid sequence of the fbp mutant enzyme is shown as SEQ ID NO.44, and the amino acid sequence of the fbp wild-type enzyme is shown as SEQ ID NO. 45)

4. And (3) connecting the ptargetF-N20 plasmid prepared in the step (2) with homologous arm DNA fragments "UP", "DOWN" and FBP mutant enzyme expression frame DNA fragment "FBP" for gene replacement by a Goldengate connection method to obtain a recombinant plasmid ptargetF-N20-UP-FBP-DOWN.

The method comprises the following steps: the ptargetF-N20 fragment was obtained by PCR using primers "P-N20-F", "P-N20-R"; the primer "UP-F" and the primer "UP-R" are used for obtaining a "UP" fragment through PCR; PCR (polymerase chain reaction) is carried out by using primers of 'FBP-F', 'FBP-R' and Ptrc99a-FBP recombinant plasmid as a template to obtain 'FBP' expression frame fragments (comprising Ptrc promoter); the "DOWN" fragment was obtained by PCR using the primers "DOWN-F", "DOWN-R". The DNA fragments were purified using a gel cutting recovery kit, added to a PCR tube at the same molar ratio, and BsaI enzyme, T4 DNA ligase, buffer were added. Incubating the PCR tube in a PCR instrument, completing enzyme digestion and connection, and setting the procedure at 37 ℃ for 1h and 60 ℃ for 5min. The liquid in the PCR tube was transformed into E.coli DH 5. Alpha. Competent cells, and cultured overnight at 37 ℃. Selecting the grown monoclonal into LB liquid medium containing 50mg/L spectinomycin, culturing at 37 ℃ until the medium is turbid, sucking a small amount of bacterial liquid, and sending the bacterial liquid to general biological systems (Anhui) limited company for sequencing, wherein the result is positive clone; and extracting recombinant plasmid from the positive clone, namely the ptargetF-N20-UP-FBP-DOWN recombinant plasmid.

The above-mentioned desired primers are shown in Table 4.

TABLE 4 primer sequence listing

5. JM109 (DE 3) -Cas strain was prepared in competent form by:

the strain was cultured overnight in LB liquid medium at 37℃and autoclaved in 500mL centrifuge bottles for the next day shake flasks and sterilized water (about 1.5L) for the next day re-suspension of cells. Transferring 0.2-1 mL of overnight bacterial liquid to a 1-2L shaking flask filled with 500mL of LB liquid medium, culturing at 37 ℃ for 2-6 hours in an oscillating way, and monitoring OD at regular time ₆₀₀ Values (measured every half hour after 1 hour of incubation). When OD is ₆₀₀ When the value reaches 0.5-1.0, taking out the shake flask from the shake table, and placing the shake flask on ice for cooling for at least 15 minutes; cells were centrifuged at 5000g for 15 min at 4℃and the supernatant was discarded; re-suspending the cells with sterilized ice water, re-suspending the cells in a small volume (a few milliliters) with a vortex meter, and then diluting with water to 2/3 of the volume of the centrifuge tube; repeating the above stepsHeart, carefully discard supernatant; re-suspending the cells with sterilized ice water according to the above procedure, centrifuging and discarding the supernatant; re-suspending the cells with 20mL sterilized, ice-cold 10% glycerol, centrifuging for 15 minutes at 5000g at 4 ℃, carefully discarding the supernatant, re-suspending the cells with 10% glycerol to a final volume of 2-3 mL; the cells were loaded into microcentrifuge tubes in 100. Mu.L aliquots and stored in a-80℃freezer.

5. Conversion process

In the process of electrotransformation, 80ng of the competent cells obtained by preparation are added into a recombinant plasmid ptargetF-N20-UP-FBP-DOWN prepared in the step (9), after being gently mixed, the competent cells are added into a precooled 1mm electrorotating cup, are put into an electrorotating instrument (Bio-Rad micro Pulser) under the condition of 1.8kV for electrotransformation, after the electrotransformation is finished, 1mL of LB culture medium (room temperature) is rapidly added, and the culture is carried out at 30 ℃ and 180rpm for 1h for resuscitation, and then the resuscitated cells are coated on an LB double-resistance flat plate containing 50mg/L kanamycin and 50mg/L spectinomycin, and are cultured at 30 ℃ overnight; transformants were detected by PCR.

6. The pCas plasmid and recombinant plasmid ptargetF-N20-UP-FBP-DOWN in JM109 (DE 3) malZ:: FBP strain are all eliminated to obtain the recombinant strain claimed in the invention.

The elimination method comprises the following steps: the two-plasmid-containing JM109 (DE 3) malZ:: fbp strain was inoculated with LB liquid medium (containing 50mg/L kanamycin and induced by addition of IPTG at a final concentration of 0.5 mM) and cultured overnight at 30℃and plated on a plate containing 50mg/L kanamycin for elimination of pTargetF recombinant plasmid. When single bacterial colony grows on the flat plate, sequentially picking single bacterial colony, and culturing in LB liquid medium containing 50mg/L spectinomycin at 30 ℃ overnight, wherein the strain is the strain with pTargetF plasmid successfully eliminated. Then picking the strain and culturing the strain in LB liquid medium at 37 ℃ overnight, so as to eliminate pCas plasmid; JM109 (DE 3) malZ:: fbp strain was used after completion of plasmid elimination.

Example 4

Construction of strains for fermentation

The recombinant plasmids pETDuet-CBGF and pCDFDuet-TAB obtained in the examples 1 and 2 are co-transformed into JM109 (DE 3) malZ:: fbp strain obtained in the example 3 by a plasmid transformation method, namely JM109 (DE 3) V1 strain (namely engineering bacterium of the invention for producing high yield 2' -fucosyllactose) for fermentation is obtained. The method comprises the following specific steps:

(1) LB agar plates (formula 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, 20g/L agar) containing 50mg/L ampicillin and 25mg/L streptomycin were prepared;

(2) A1.5 ml centrifuge tube was taken, 100. Mu.L of the competent cell suspension of JM109 (DE 3) malZ:: fbp prepared in example 3 was added, and the mixture was placed on ice; 1. Mu.L of recombinant plasmid pETDuet-CBGF (100 ng/. Mu.L concentration) and 1. Mu.L of recombinant plasmid pCDFDuet-TAB (100 ng/. Mu.L concentration) were added, gently mixed with a pipette, and left on ice for 20min;

(3) Heat shock is carried out for 90 seconds in a water bath at the temperature of 42 ℃, then ice is quickly placed for 3-5 minutes, and bacterial liquid is not required to be oscillated in the whole process;

(4) Adding 1mL of LB liquid medium (without antibiotics), mixing uniformly, carrying out shaking culture (100 rpm) for 1 hour at 37 ℃ to enable bacteria to recover to a normal growth state, and expressing an antibiotic resistance gene coded by a plasmid;

(5) Taking 100 mu L of bacterial liquid onto an LB agar plate containing 50mg/L ampicillin and 25mg/L streptomycin, and uniformly coating;

(6) After the bacterial liquid is absorbed by the culture medium, culturing for 12-16 hours at 37 ℃ in an inversion way, after single colony appears, picking JM109 (DE 3) V1 single colony, culturing in LB liquid culture medium containing 50mg/L ampicillin and 25mg/L streptomycin at 37 ℃ until turbidity occurs, sucking 500 mu L of bacterial liquid to a sterilized EP tube, adding 500 mu L of 40% (w/w) glycerol, uniformly mixing, preserving at-80 ℃ for later use, and marking as an experimental group;

(7) In the same manner, the recombinant plasmid pETDuet-CBGF and the recombinant plasmid pCDFDuet-TAB were co-transformed into JM109 (DE 3) competent cells to obtain JM109 (DE 3) V0 strain used as a control experiment.

Example 5

Synthesis of 2' -fucosyllactose by fermentation

JM109 (DE 3) V0 (control) and JM109 (DE 3) V1 (experimental) provided in example 4 were inoculated into shake flasks at 1% (V/V) of each of the inoculum sizes, respectively, using a flask for fermentation, 500mL of shake flask size and 100mL of liquid loading, and LB medium (formulation 15g/L glycerol, 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, 25mg/L streptomycin, 50mg/L ampicillin) containing glycerol was added thereto, and the mixture was cultured at 37℃and 220rpm until OD=1.0, cooled to 25℃and then subjected to culture fermentation at 220rpm for 60 hours at 25℃with the final concentrations of 0.2mM IPTG and 5g/L lactose.

The 2' -FL concentration in the fermentation broth was determined by High Performance Liquid Chromatography (HPLC) as follows: liquid phase device: agilent 1260InfinityII; differential detector detects, model: G7162A-1260RId; liquid phase column model: sepax HP-Amino,4.6 x 250mm, 5 micron particle size (or equivalent size Amino columns); flow rate: 0.8ml/min, mobile phase: 80% pure acetonitrile: 20% water (v/v), system temperature: the temperature is 35 ℃, and the sample injection amount is 10-20 microlitres.

The measured results show that: the 2'-FL yield of JM109 (DE 3) V1 strain was 5.4g/L, while the 2' -FL yield of JM109 (DE 3) V0 strain was 2.1g/L, i.e., the V1 strain was 2.57 times that of the V0 strain.

The results show that after the fbp mutant enzyme is introduced, the yield of 2' -fucosyllactose produced by fermenting the recombinant JM109 (DE 3) strain under the condition of taking glycerol as the sole carbon source can be greatly improved.

While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.

Sequence listing

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gcactggccg tggatcgtac cgatggtatt agtatgacct ttgcagattg gcgctttaat 1260

ctgcgcacca gtaataccga accggtggtt cgtctgaatg tggaaagccg cggtgacgtg 1320

ccgctgatgg aagcccgtac ccgtaccctg ctgaccctgc tgaatgaata a 1371

<210> 3

<211> 1122

<212> DNA

<213> Artificial Sequence

<400> 3

atgagtaagg ttgccctgat taccggtgtg accggccagg atggcagcta tctggccgag 60

ttcctgctgg aaaaaggtta tgaagtgcat ggtattaaac gtcgtgccag cagcttcaat 120

accgaacgtg ttgatcatat ctatcaggac cctcatacct gcaatccgaa attccatctg 180

cattatggtg atctgagcga taccagcaat ctgacccgta ttctgcgtga agttcagccg 240

gatgaagtgt ataatctggg tgcaatgagt catgttgcag ttagcttcga aagtccggaa 300

tataccgcag atgtggatgc aatgggcacc ctgcgtctgc tggaagcaat tcgcttcctg 360

ggcctggaaa aaaaaaccag attctatcag gccagcacca gcgaactgta tggcctggtg 420

caggaaattc cgcagaaaga aaccaccccg ttctatccgc gtagtccgta tgcagttgcc 480

aaactgtatg cctattggat taccgttaat tatcgtgaaa gctatggtat gtatgcctgt 540

aatggtattc tgttcaatca tgaaagtccg cgtcgtggcg aaaccttcgt gacccgcaaa 600

attacccgtg ccattgccaa tattgcacag ggcctggaaa gttgtctgta tctgggtaat 660

atggatagcc tgcgcgattg gggtcatgcc aaagattatg ttaaaatgca gtggatgatg 720

ctgcagcagg aacagccgga agacttcgtt attgccaccg gtgttcagta tagcgtgcgt 780

cagttcgtgg aaatggccgc agcccagctg ggcattaaac tgcgcttcga aggcaccggt 840

gtggaagaaa aaggcattgt ggtgagtgtt accggccatg atgcaccggg cgtgaaaccg 900

ggcgatgtga ttattgccgt ggaccctcgc tacttccgcc cggctgaagt tgaaacctta 960

ctgggtgatc cgaccaaagc ccatgaaaaa ctgggctgga aaccggaaat taccctgcgt 1020

gaaatggtta gcgaaatggt tgcaaatgat ctggaagccg caaaaaaaca tagtctgctg 1080

aaaagccacg gttatgatgt ggcaattgcc ctggaaagct aa 1122

<210> 4

<211> 966

<212> DNA

<213> Artificial Sequence

<400> 4

atgtctaagc agcgcgtctt cattgctggc catcgtggca tggttggttc tgctattcgt 60

cgccagttgg aacaacgtgg cgatgttgaa ttggttcttc gcacccgtga tgaattgaat 120

cttcttgata gccgcgctgt tcatgacttc ttcgctagcg aacgtattga tcaagtctat 180

cttgctgccg ctaaagttgg tggcattgtt gctaataata cctatccggc cgacttcatc 240

tatcaaaata tgatgattga aagcaacatc atccatgctg cccatcaaaa tgatgttaat 300

aaattgttgt tcctgggtag cagctgtatc tatcctaaat tggctaaaca accgatggcc 360

gaatctgaat tgttgcaggg taccttggaa cctaccaatg aaccttatgc cattgccaaa 420

attgctggca ttaaattgtg tgaaagctat aatcgtcagt atggccgtga ttatcgcagc 480

gttatgccta ccaatctgta tggtcctcat gataacttcc atccttctaa ttctcatgtt 540

attccggcct tgttgcgtcg cttccatgaa gccaccgccc aaaatgctcc tgatgttgtt 600

gtctggggca gcggtacccc tatgcgcgag ttcttgcatg ttgatgatat ggccgctgct 660

tctattcatg ttatggaatt ggctcatgaa gtctggttgg aaaataccca gcctatgttg 720

agccatatta atgttggcac cggcgttgat tgcaccattc gtgaattggc ccaaaccatt 780

gctaaagttg ttggctataa aggtcgtgtt gtcttcgatg ccagcaaacc tgatggtacc 840

ccgcgtaaat tgttggatgt tacccgcttg catcagttgg gttggtatca tgaaattagc 900

ttggaagctg gtcttgcttc tacctatcag tggttcttgg aaaatcagga tcgcttccgt 960

ggctaa 966

<210> 5

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 5

ggctacggtc tcgtccgaat tcatggccca gagtaaactg 40

<210> 6

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 6

ggctacggtc tcttcgtatt attacacacg accataacg 39

<210> 7

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 7

ggctacggtc tctacgactc actatagggg aattg 35

<210> 8

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 8

ggctacggtc tctaggtcag cttcttcatg gtatatctcc ttcttaaagt taaacaaaat 60

tatttc 66

<210> 9

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 9

ggctacggtc tctacctgct ttaaagccta tg 32

<210> 10

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 10

ggctacggtc tctttcattc agcagggtca g 31

<210> 11

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 11

ggctacggtc tcttgaataa taatacgact cactataggg 40

<210> 12

<211> 49

<212> DNA

<213> Artificial Sequence

<400> 12

ggctacggtc tctccttact catggtatat ctccttctta aagttaaac 49

<210> 13

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 13

ggctacggtc tctaaggttg ccctgattac c 31

<210> 14

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 14

ggctacggtc tcgattatta gctttccagg gcaattg 37

<210> 15

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 15

ggctacggtc tcgtaatacg actcactata ggg 33

<210> 16

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 16

ggctacggtc tcaggtatat ctccttctta aagttaaac 39

<210> 17

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 17

ggctacggtc tcataccatg tctaagcagc gcgtc 35

<210> 18

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 18

ggctacggtc tctgaagcga tcctgatttt cc 32

<210> 19

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 19

ggctacggtc tctcttccgt ggctaactcg agtctggtaa agaaaccg 48

<210> 20

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 20

ggctacggtc tcgcggatcc tggctgtggt g 31

<210> 21

<211> 903

<212> DNA

<213> Artificial Sequence

<400> 21

atggcgttca aagttgttca gatctgcggt ggcttaggta accagatgtt ccagtacgcg 60

ttcgcgaaat ctctgcagaa acacagcaac accccggttc tgctggatat caccagcttc 120

gattggtctg atcgtaaaat gcagctggaa ctgttcccga tcgatctgcc gtacgcgtct 180

gcgaaagaaa ttgccatcgc gaaaatgcag cacctcccaa agctggtacg cgatgccctg 240

aagtgtatgg ggtttgatcg tgttagccag gaaatcgttt tcgaatacga accgaaactg 300

ctgaaaccgt cccgtctgac ctacttcttc ggctacttcc aggacccgcg ttacttcgat 360

gcgatcagcc cgctgatcaa acagaccttc accctgccgc cgccgccgga aaacaacaaa 420

aacaacaaca aaaaagaaga agaataccag tgcaaactga gcctgatcct ggcggcgaaa 480

aacagcgttt tcgttcacat ccgtcgtggc gattacgttg gtatcggttg ccagctgggt 540

atcgattacc agaaaaaagc gctggaatac atggcgaaac gtgttccgaa catggaactg 600

ttcgttttct gcgaagatct ggaatttacc cagaacctgg atctgggtta cccgttcatg 660

gatatgacca cccgtgataa agaagaagaa gcgtactggg atatgctgct gatgcagagc 720

tgccagcacg gcatcatcgc gaacagcacc tattcttggt gggcggcgta cctgatcgaa 780

aacccggaaa aaatcatcat cggtccgaaa cactggctgt tcggccacga aaacatcctg 840

tgcaaagaat gggttaaaat cgaaagccac ttcgaagtta aatctcagaa atacaacgcg 900

taa 903

<210> 22

<211> 624

<212> DNA

<213> Artificial Sequence

<400> 22

atgagcacca ttattatgga tctgtgttct tatacccgct tgggtcttac cggttatctt 60

cttagccgtg gcgttaaaaa acgtgaaatt aatgatatcg aaaccgttga tgatcttgct 120

attgcctgtg atagccaacg cccgagcgtt gtcttcatta atgaagattg cttcattcat 180

gatgcttcta attctcaacg cattaaattg attatcaacc agcatccgaa taccttgttc 240

attgtcttca tggctattgc taatgttcac ttcgatgaat atcttcttgt tcgtaaaaac 300

ttgttgatct cttctaaaag catcaaaccg gaaagccttg atgatattct tggtgatatt 360

cttaagaagg aaaccaccat taccagcttc ttgaatatgc cgaccttgtc tcttagccgc 420

accgaaagct ctatgttgcg tatgtggatg gctggccaag gcaccattca aattagcgat 480

cagatgaata ttaaggctaa aaccgttagc agccataaag gtaatattaa acgcaaaatc 540

aagacccata acaaacaggt tatctatcat gttgttcgtc ttaccgataa tgttaccaat 600

ggcatcttcg ttaatatgcg ctaa 624

<210> 23

<211> 651

<212> DNA

<213> Artificial Sequence

<400> 23

atgaacaata tgaacgtaat tattgccgat gaccatccga tagtcttgtt cggtattcgc 60

aaatcacttg agcaaattga gtgggtgaat gttgtcggcg aatttgaaga ctctacagca 120

ctgatcaaca acctgccgaa actggatgcg catgtgttga ttaccgatct ctccatgcct 180

ggcgataagt acggcgatgg cattacctta atcaagtaca tcaagcgcca tttcccaagc 240

ctgtcgatca ttgttctgac tatgaacaac aacccggcga ttcttagtgc ggtattggat 300

ctggatatcg aagggatcgt gctgaaacaa ggtgcaccga ccgatctgcc gaaagctctc 360

gccgcgctgc agaaagggaa gaaatttacc ccggaaagcg tttctcgcct gttggaaaaa 420

atcagtgctg gtggttacgg tgacaagcgt ctctcgccaa aagagagtga agttctgcgc 480

ctgtttgcgg aaggcttcct ggtgaccgag atcgctaaaa agctgaaccg cagtattaaa 540

accatcagta gccagaagaa atctgcgatg atgaagctgg gtgtcgagaa cgatatcgcc 600

ctgctgaatt atctctcttc agtgacctta agtccggcag ataaagacta a 651

<210> 24

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 24

ggctacggtc tcgaggtggt gcaaatttgc g 31

<210> 25

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 25

ggctacggtc tccagtcgta ttattaagcg ttatactttt gggatttc 48

<210> 26

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 26

ggctacggtc tccgactcac tataggggaa ttg 33

<210> 27

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 27

ggctacggtc tctccttctt aaagttaaac aaaattattt c 41

<210> 28

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 28

ggctacggtc tctaaggaga tataccatga gcaccattat tatgg 45

<210> 29

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 29

ggctacggtc tcgcgtatta ttagcgcata ttaacgaag 39

<210> 30

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 30

ggctacggtc tcgtacgact cactataggg g 31

<210> 31

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 31

ggctacggtc tctattgttc atggtatatc tccttcttaa agttaaac 48

<210> 32

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 32

ggctacggtc tctcaatatg aacgtaatta ttgcc 35

<210> 33

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 33

ggctacggtc tccgttagtc tttatctgcc gg 32

<210> 34

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 34

ggctacggtc tcctaactcg agtctggtaa agaaac 36

<210> 35

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 35

ggctacggtc tcgaccttaa aagccatgaa ttcggatcct ggctg 45

<210> 36

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 36

gcagcataat gcgctgcggt 20

<210> 37

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 37

gcagcataat gcgctgcggt gttttagagc tagaaatagc 40

<210> 38

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 38

accgcagcgc attatgctgc actagtatta tacctaggac 40

<210> 39

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 39

ggtacccggg gatcctctag aatgaaaacg ttaggtgaat ttattgtc 48

<210> 40

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 40

tccgccaaaa cagccaagct tttacgcgtc cgggaactca 40

<210> 41

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 41

ttgcgccgac atcataac 18

<210> 42

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 42

ctgcgttctg atttaatctg 20

<210> 43

<211> 999

<212> DNA

<213> Artificial Sequence

<400> 43

atgaaaacgt taggtgaatt tattgtcgaa aagcagcacg agttttctca tgctaccggt 60

gagctcactg ctttgctgtc ggcaataaaa ctgggcgcca agattatcca tcgcgatatc 120

aacaaagcag gactggttga tatcctgggt gccagcggtg ctgagaacgt gcagggcgag 180

gttcagcaga aactcgactt gttcgctaat gaaaaactga aagccgcact gaaagcacgc 240

gatatcgttg cgggcattgc ctctgaagaa gaagatgaga ttgtcgtctt tgaaggctgt 300

gaacacgcac agtacgtggt gctgatggac cccctggatg gctcgtccaa catcgatgtt 360

aacgtctctg tcggtaccat tttctccatc tacatccgcg ttacgcctgt tggcacgccg 420

gtaacggaag aagatttcct ccagcctggt aacaaacagg ttgcggcagg ttacgtggta 480

tacggctcct ctaccatgct ggtttacacc accggatgcg gtgttcacgc ctttacttac 540

gatccttcgc tcggcgtttt ctgcctgtgc caggaacgga tgcgcttccc ggagaaaggc 600

aaaacctact ccatcaacga aggaaacttt attaagtttc cgaacggggt gcagaagtac 660

attaaattct gccaggaaga agataaatcc accaaccgcc cttatacctc acgttatatc 720

ggttcactgg tcgcggattt ccaccgtaac ctgctgaaag gcggtattta tctctaccca 780

agcaccgcca gccacccgga cggcaaactg cgtttgctgt atgagtgcaa cccgatggca 840

ttcctggcgg aacaagcggg cggtaaagcg agcgatggca aagagcgtat tctggatatc 900

atcccggaaa ccctgcacca gcgccgttca ttctttgtcg gcaacgacca tatggttgaa 960

gatgtcgaac gctttatccg tgagttcccg gacgcgtaa 999

<210> 44

<211> 332

<212> PRT

<213> Artificial Sequence

<400> 44

Met Lys Thr Leu Gly Glu Phe Ile Val Glu Lys Gln His Glu Phe Ser

1 5 10 15

His Ala Thr Gly Glu Leu Thr Ala Leu Leu Ser Ala Ile Lys Leu Gly

20 25 30

Ala Lys Ile Ile His Arg Asp Ile Asn Lys Ala Gly Leu Val Asp Ile

35 40 45

Leu Gly Ala Ser Gly Ala Glu Asn Val Gln Gly Glu Val Gln Gln Lys

50 55 60

Leu Asp Leu Phe Ala Asn Glu Lys Leu Lys Ala Ala Leu Lys Ala Arg

65 70 75 80

Asp Ile Val Ala Gly Ile Ala Ser Glu Glu Glu Asp Glu Ile Val Val

85 90 95

Phe Glu Gly Cys Glu His Ala Gln Tyr Val Val Leu Met Asp Pro Leu

100 105 110

Asp Gly Ser Ser Asn Ile Asp Val Asn Val Ser Val Gly Thr Ile Phe

115 120 125

Ser Ile Tyr Ile Arg Val Thr Pro Val Gly Thr Pro Val Thr Glu Glu

130 135 140

Asp Phe Leu Gln Pro Gly Asn Lys Gln Val Ala Ala Gly Tyr Val Val

145 150 155 160

Tyr Gly Ser Ser Thr Met Leu Val Tyr Thr Thr Gly Cys Gly Val His

165 170 175

Ala Phe Thr Tyr Asp Pro Ser Leu Gly Val Phe Cys Leu Cys Gln Glu

180 185 190

Arg Met Arg Phe Pro Glu Lys Gly Lys Thr Tyr Ser Ile Asn Glu Gly

195 200 205

Asn Phe Ile Lys Phe Pro Asn Gly Val Gln Lys Tyr Ile Lys Phe Cys

210 215 220

Gln Glu Glu Asp Lys Ser Thr Asn Arg Pro Tyr Thr Ser Arg Tyr Ile

225 230 235 240

Gly Ser Leu Val Ala Asp Phe His Arg Asn Leu Leu Lys Gly Gly Ile

245 250 255

Tyr Leu Tyr Pro Ser Thr Ala Ser His Pro Asp Gly Lys Leu Arg Leu

260 265 270

Leu Tyr Glu Cys Asn Pro Met Ala Phe Leu Ala Glu Gln Ala Gly Gly

275 280 285

Lys Ala Ser Asp Gly Lys Glu Arg Ile Leu Asp Ile Ile Pro Glu Thr

290 295 300

Leu His Gln Arg Arg Ser Phe Phe Val Gly Asn Asp His Met Val Glu

305 310 315 320

Asp Val Glu Arg Phe Ile Arg Glu Phe Pro Asp Ala

325 330

<210> 45

<211> 332

<212> PRT

<213> Artificial Sequence

<400> 45

Met Lys Thr Leu Gly Glu Phe Ile Val Glu Lys Gln His Glu Phe Ser

1 5 10 15

His Ala Thr Gly Glu Leu Thr Ala Leu Leu Ser Ala Ile Lys Leu Gly

20 25 30

Ala Lys Ile Ile His Arg Asp Ile Asn Lys Ala Gly Leu Val Asp Ile

35 40 45

Leu Gly Ala Ser Gly Ala Glu Asn Val Gln Gly Glu Val Gln Gln Lys

50 55 60

Leu Asp Leu Phe Ala Asn Glu Lys Leu Lys Ala Ala Leu Lys Ala Arg

65 70 75 80

Asp Ile Val Ala Gly Ile Ala Ser Glu Glu Glu Asp Glu Ile Val Val

85 90 95

Phe Glu Gly Cys Glu His Ala Lys Tyr Val Val Leu Met Asp Pro Leu

100 105 110

Asp Gly Ser Ser Asn Ile Asp Val Asn Val Ser Val Gly Thr Ile Phe

115 120 125

Ser Ile Tyr Arg Arg Val Thr Pro Val Gly Thr Pro Val Thr Glu Glu

130 135 140

Asp Phe Leu Gln Pro Gly Asn Lys Gln Val Ala Ala Gly Tyr Val Val

145 150 155 160

Tyr Gly Ser Ser Thr Met Leu Val Tyr Thr Thr Gly Cys Gly Val His

165 170 175

Ala Phe Thr Tyr Asp Pro Ser Leu Gly Val Phe Cys Leu Cys Gln Glu

180 185 190

Arg Met Arg Phe Pro Glu Lys Gly Lys Thr Tyr Ser Ile Asn Glu Gly

195 200 205

Asn Tyr Ile Lys Phe Pro Asn Gly Val Lys Lys Tyr Ile Lys Phe Cys

210 215 220

Gln Glu Glu Asp Lys Ser Thr Asn Arg Pro Tyr Thr Ser Arg Tyr Ile

225 230 235 240

Gly Ser Leu Val Ala Asp Phe His Arg Asn Leu Leu Lys Gly Gly Ile

245 250 255

Tyr Leu Tyr Pro Ser Thr Ala Ser His Pro Asp Gly Lys Leu Arg Leu

260 265 270

Leu Tyr Glu Cys Asn Pro Met Ala Phe Leu Ala Glu Gln Ala Gly Gly

275 280 285

Lys Ala Ser Asp Gly Lys Glu Arg Ile Leu Asp Ile Ile Pro Glu Thr

290 295 300

Leu His Gln Arg Arg Ser Phe Phe Val Gly Asn Asp His Met Val Glu

305 310 315 320

Asp Val Glu Arg Phe Ile Arg Glu Phe Pro Asp Ala

325 330

<210> 46

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 46

ggctacggtc tctacctgca gaagcttaga tc 32

<210> 47

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 47

ggctacggtc tcaagaattc aaaaaaagca ccg 33

<210> 48

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 48

ggctacggtc tcattctctc gctgtatgtc ggtttc 36

<210> 49

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 49

ggctacggtc tcagctccga tatccggtac atcg 34

<210> 50

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 50

ggctacggtc tcagagctgt tgacaattaa tcatc 35

<210> 51

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 51

ggctacggtc tcttttgtcc tactcaggag ag 32

<210> 52

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 52

ggctacggtc tctcaaatcc gccgatgaag taggactgga tg 42

<210> 53

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 53

ggctacggtc tctaggtcga ctctagacgt tttatcaatc tgaacaatta c 51

Claims (9)

1. A recombinant strain, characterized in that the recombinant strain is a strain in which an fbp mutant enzyme encoding gene is integrated into the chromosome of JM109 (DE 3) strain;

the fbp mutant enzyme coding gene is as follows: mutating the lysine residue at the 104 th site of the amino acid sequence of the wild fbp enzyme coding gene into glutamine, mutating the arginine residue at the 132 th site into isoleucine, mutating the tyrosine residue at the 210 th site into phenylalanine, mutating the lysine residue at the 218 th site into glutamine;

the nucleotide sequence of the wild fbp enzyme coding gene is shown as SEQ ID NO. 45;

the nucleotide sequence of the fbp mutant enzyme coding gene is shown as SEQ ID NO. 44.

2. Recombinant strain according to claim 1, characterized in that the fbp mutant enzyme gene transcription is controlled using the promoter Ptrc.

3. The recombinant strain according to claim 1, wherein the site of integration of the chromosome of the JM109 (DE 3) strain is the location of the maltodextrin glucosidase gene.

4. Use of the recombinant strain according to any one of claims 1 to 3 for the preparation of engineering bacteria for the production of high yields of 2' -fucosyllactose.

5. An engineering bacterium for producing 2' -fucosyllactose with high yield based on the recombinant strain of claim 1, wherein the engineering bacterium is obtained by co-transforming the recombinant plasmid pETDuet-CBGF and the recombinant plasmid pCDFDuet-TAB into the recombinant strain of claim 1.

6. The engineering bacterium according to claim 5, wherein the recombinant plasmid petdouet-CBGF is: the ManC gene, the ManB gene, the Gmd gene and the Fcl gene are cloned on a pETDuet-1 plasmid.

7. The engineered bacterium of claim 5, wherein the recombinant plasmid pcdfduret-TAB is: the FucT gene, the rcsA gene and the rcsB gene were cloned into the pCDFDuet-1 plasmid.

8. A method for producing 2' -fucosyllactose, and characterized in that the method is: the engineering bacterium of claim 5 is used as a fermentation strain to produce 2' -fucosyllactose in a fermentation system using glycerol as a substrate.

9. The use of the engineering bacteria of claim 5 in high yield of 2'-fucosyllactose and in the production of products containing 2' -fucosyllactose.