CN114645007A - Recombinant strain, engineering bacterium for high yield of 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, an engineering bacterium for high yield of 2' -fucosyllactose based on the recombinant strain and application of the engineering bacterium. The invention provides a recombinant strain and an engineering bacterium for high yield of 2 ' -fucosyllactose based on the recombinant strain, wherein a fbp mutant enzyme gene is inserted into a maltodextrin glucosidase gene (malZ) gene locus of an escherichia coli JM109(DE3) strain in a chromosome integration mode, and simultaneously, double plasmids pETDuet-CBGF and pCDFDuet-TAB are designed to co-express genes ManC, ManB, Gmd, Fcl, FucT, RcsA and RcsB in the escherichia coli, so that the yield of 2 ' -fucosyllactose (2 ' -FL) is greatly improved.

Description

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

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 are concept defects, constitution differences and the like. Therefore, the development of the milk powder additive with similar efficacy to breast milk has great application value and social influence.

2 ' -fucosyllactose (2 ' -fucosyllactose, abbreviated herein as 2 ' -FL) is the most abundant oligosaccharide in human milk, and it has the following effects: (1) after 2' -FL is taken by people, the structure is stable and cannot be damaged by gastric acid and digestive enzyme, so that the probiotics can directly reach the large intestine to promote the growth of bifidobacteria and lactic acid bacteria in the intestinal tract; (2) the pathogenic bacteria adhesion is resisted, the first step after the pathogenic bacteria of the intestinal tract invade the human body is usually colonization on epithelial cells, then the infected tissue begins, the structure of 2' -FL is just similar to the partial structure of glycoprotein and sugar chain on the epithelial cells of the intestinal tract, so that the pathogenic bacteria can be tricked to be combined with the pathogenic bacteria and not be combined with the epithelial cells of the intestinal tract, and the colonization process is blocked; (3) the 2' -FL can be directly involved in the secretion regulation of cytokines to influence the immune system according to reports, for example, the concentration of inflammatory cytokines (IL-1ra, IL-1 alpha, IL-1 beta, IL-6, tumor necrosis factor-alpha) can be reduced in the body of an infant, which is basically consistent with the breast feeding result; while the normal milk powder feeding group shows a remarkable increase of inflammatory cytokines. The research results prove that the 2' -FL is the 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 all biological methods and are obtained in large quantities through microbial fermentation, and the process is green and environment-friendly and has high efficiency. The biological method can be further divided into a remedial way and a de novo way, wherein the remedial way is realized by adding L-fucose and lactose from external sources and using glycerol as a carbon source to complete fermentation; the de novo route can synthesize 2' -FL by directly using glycerol or sucrose as a carbon source and only adding lactose exogenously without using expensive L-fucose. Clearly, the de novo route is more cost effective, and glycerol has proven to be the most efficient carbon source. However, as can be seen from the research on the synthetic pathway of 2 '-FL, glycerol needs to be metabolized in multiple steps to be converted into 2' -FL intermediate fructose-6-phosphate, which has the defect of low utilization efficiency, so that the yield of 2 '-FL is difficult to be greatly improved, and the practical application of 2' -FL is limited.

Disclosure of Invention

In order to solve the problems, the invention provides a recombinant strain, an engineering bacterium for high yield of 2' -fucosyllactose based on the recombinant strain and application of the engineering bacterium. The engineering bacteria for high yield of 2 '-fucosyllactose provided by the invention can improve the efficiency of synthesizing 2' -FL by glycerol through overexpression of fbp enzyme mutant enzyme, and can greatly improve the yield of 2 '-FL produced by glycerol fermentation through plasmid overexpression of 2' -FL synthesis pathway genes ManC, ManB, Gmd, Fcl and 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 a gene encoding an fbp mutant enzyme into the chromosome of JM109(DE3) strain.

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

Preferably, the transcription of the fbp mutant enzyme gene is controlled by the promoter Ptrc.

Preferably, the JM109(DE3) strain has a chromosomal integration site at which the maltodextrin glucosidase gene is located.

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

The invention provides an engineering bacterium for high-yield 2' -fucosyllactose based on the recombinant strain, which is characterized in that the engineering bacterium is obtained by cotransforming a recombinant plasmid pETDuet-CBGF and a 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 to obtain the gene.

Preferably, the recombinant plasmid pCDFDuet-TAB is: the FucT gene, the RcsA gene and the RcsB gene are cloned on pCDFDuet-1 plasmid to obtain the recombinant plasmid.

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

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

In the prior art, the recombinant JM109(DE3) strain can synthesize 2 '-FL by using glycerol as a carbon source, but the path activity for converting glycerol into a key intermediate fructose-6-phosphate is low, so that the yield of 2' -FL is difficult to increase. The invention effectively improves the capacity of the strain for converting the glycerol into the intermediate fructose-6-phosphate by integrating the high-activity fructose-1, 6-diphosphatase (fbp enzyme) mutant enzyme in the chromosome, thereby greatly improving the yield of the 2' -FL and laying a foundation for the industrial application of the technology.

The invention provides a recombinant strain and an engineering bacterium for high yield of 2 '-fucosyllactose based on the same, wherein a fbp mutant enzyme gene (lysine residue (K) at the 104 th position of an amino acid sequence is mutated into glutamine (Q), arginine residue (R) at the 132 th position is mutated into isoleucine (I), tyrosine residue (Y) at the 210 th position is mutated into phenylalanine (F), lysine residue (K) at the 218 th position is mutated into glutamine (Q)) is inserted into a maltodextrin glucosidase gene (malZ) locus of an escherichia coli JM109(DE3) strain in a chromosome integration mode, and double plasmids pETDuet-GF and pCDFDuet-TAB are simultaneously 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 yield of 2 ' -FL of the engineering bacteria provided by the invention is 5.4g/L, and the yield of 2 ' -FL of the control strain is 2.1g/L, which shows that the strain provided by the invention can greatly improve the yield of 2 ' -FL.

Drawings

FIG. 1 shows the integration of the fbp mutant gene into the chromosome of JM109(DE3) strain.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not 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 purchased from conventional reagent manufacturers. In particular, the components referred to in the following additives are all conventional commercially available 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) from Escherichia coli E.coli K12 strain are obtained by gene total synthesis. The above genes were also subjected to codon-biased sequence optimization and synthesized by general biosystems (Anhui) Inc. The T7 promoter and RBS sequence on pETDuet-1 vector were amplified by PCR (polymerase chain reaction), and the ManC, ManB, Gmd, Fcl genes obtained by entrusted synthesis were 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 the fragments and a pET-Duet-1 vector into a recombinant plasmid A through an Overhang sequence by using a Golden gate connection method, wherein the clone carrying the recombinant plasmid with correct sequencing is positive clone; and extracting a recombinant plasmid from the positive clone, namely the pETDuet-CBGF recombinant plasmid. The golden gate connection method comprises the following experimental steps:

(1) obtaining gene fragments of ManC, ManB, Gmd and Fcl by PCR, obtaining a DNA fragment of 'T7 promoter and RBS sequence' on a pETDuet-1 vector by PCR, and obtaining a vector fragment of pETDuet-1 by PCR;

(2) the DNA fragment obtained above was purified by Gel cutting purification, and FastPure Gel DNAextractionMini Kit (cat # DC301-01) from Nanjing Novowed was selected;

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

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

(5) liquid in the PCR tube is transformed into escherichia coli DH5 alpha competent cells, and the cells are cultured overnight at 37 ℃;

(6) picking the grown monoclonal antibody into an LB liquid culture medium, culturing at 37 ℃ until the culture medium is turbid, sucking a small amount of bacterial liquid, sending the bacterial liquid to a general biological system (Anhui) Limited company for sequencing, and obtaining a positive clone if the result is correct;

(7) and culturing the positive clone to extract a recombinant plasmid, namely pETDuet-CBGF recombinant plasmid.

Example 2

Construction of recombinant plasmid pCDFDuet-TAB

1. FucT gene (shown as SEQ ID NO. 21), RcsA gene (shown as SEQ ID NO. 22) and RcsB gene (shown as SEQ ID NO. 23) derived from Escherichia coli E.coli K12 strain are obtained by gene total synthesis, and the genes are optimized by codon preference sequence and consigned to the synthesis of general biological system (Anhui) limited company. The T7 promoter and RBS sequence on pCDFDuet-1 vector were amplified by PCR (polymerase chain reaction), and FucT, RcsA, and RcsB genes obtained by synthesis were amplified, and the primers are shown in Table 2.

TABLE 2 primer sequence Listing

2. Purifying the amplified DNA fragments by using a gel cutting recovery method by using the method in the same example 1, connecting each fragment and the pCDFDuet-1 vector into recombinant plasmids by an Overhang sequence by using a golden gate connection method, wherein the clone carrying the recombinant plasmids with correct sequencing is a positive clone; extracting recombinant plasmid from positive clone, namely 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(DE3) strain to obtain JM109(DE3) -Cas strain by the following method:

(1) preparing LB agar plate containing 25mg/L kanamycin (10 g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, 20g/L agar);

(2) a1.5 ml centrifuge tube was taken, 100. mu.L of Escherichia coli JM109(DE3) competent cell suspension (Beijing Huayue biology, cat # NRR00980) was added, and placed on ice; adding 1 μ LpCas plasmid (100 ng/. mu.L), mixing by pipette gently, and standing on ice for 20 min;

(3) carrying out heat shock in a water bath at 42 ℃ for 90 seconds, then rapidly putting on ice for 3-5 min, and not oscillating bacterial liquid in the whole process;

(4) adding 1mLLB liquid culture medium (without antibiotics), mixing uniformly, performing shaking culture (100rpm) at 37 ℃ for 1 hour to restore the bacteria to a normal growth state, and expressing an antibiotic resistance gene coded by a plasmid to obtain a JM109(DE3) -Cas strain;

(5) 100 μ L of the bacterial solution was applied to LB agar plate containing 25mg/L kanamycin and spread evenly.

(6) After the bacterial liquid is absorbed by the culture medium, culturing for 12-16 hours at 37 ℃, picking out single bacteria and dropping the single bacteria into an LB liquid culture medium containing 25mg/L kanamycin to culture at 37 ℃ until the single bacteria are turbid, sucking 500 mu L of bacterial liquid to a sterilized EP tube, adding 500 mu L of 40% (w/w) concentration glycerol, uniformly mixing, and preserving at-80 ℃ for later use.

TABLE 3 primer sequence Listing

2. The N20 sequence (the nucleic acid sequence is shown as SEQ ID NO. 36) for breaking the malZ gene is inserted into a ptargetF Plasmid (Addgene number: Plasmid #62226) through inverse PCR to obtain a ptargetF-N20 recombinant Plasmid, and the specific method is as follows:

carrying out PCR amplification by using ptargetF plasmid as a template (the sequence of the used primer is shown in Table 3), transforming a PCR product into escherichia coli DH5 alpha competent cells, coating an LB agar plate containing 50mg/L spectinomycin, culturing at 37 ℃ for 12-16 hours, screening positive monoclonal, and extracting the plasmid to obtain ptargetF-N20 recombinant plasmid.

3. The fbp mutant enzyme gene (the sequence of the fbp mutant enzyme-encoding gene is shown in SEQ ID NO. 43) was amplified by PCR and cloned between XbaI and HindIII sites (see Table 3 for primer fbp-S, fbp-A) of ptrc99a vector (Beijing Wayuseyo Bion, cat # VECT5460), E.coli DH 5. alpha. competent cells were transformed using Clon ExpressII One Step Cloning Kit (cat # C112-01) of Nanjing Hooka, spread on LB plate containing 50mg/L ampicillin, and cultured at 37 ℃ for 12 to 16 hours. Positive clones were PCR screened with primers PTrc99C-F and PBV220-R in Table 3. And (3) overnight culturing the positive clone by using an LB liquid culture medium containing 50mg/L ampicillin, and extracting the plasmid to obtain the ptrc99a-fbp recombinant plasmid.

(fbp mutant enzyme has an amino acid sequence shown as SEQ ID NO.44, and fbp wild type enzyme has an amino acid sequence shown as SEQ ID NO. 45)

4. The ptarget F-N20 plasmid prepared in step 2 above and the homology arm DNA fragment "UP", "DOWN" for gene replacement and FBP mutant enzyme expression cassette DNA fragment "FBP" were ligated by Goldengate ligation to obtain a recombinant plasmid ptarget F-N20-UP-FBP-DOWN.

The method comprises the following steps: obtaining ptargetF-N20 fragment by PCR using primers "P-N20-F" and "P-N20-R"; obtaining an "UP" fragment by PCR by using primers "UP-F" and "UP-R"; PCR was carried out using primers "FBP-F" and "FBP-R" and a Ptrc99a-FBP recombinant plasmid as a template to obtain an "FBP" expression cassette fragment (containing the Ptrc promoter); the "DOWN" fragment was obtained by PCR using the primers "DOWN-F", "DOWN-R". Each DNA fragment was purified using a gel cutting recovery kit, added to a PCR tube in the same molar ratio, and BsaI enzyme, T4 DNA ligase, and buffer were added. And (3) incubating the PCR tube in a PCR instrument to finish enzyme digestion and connection, wherein the program is set to 37 ℃ for 1h and 60 ℃ for 5 min. Coli DH 5. alpha. competent cells were liquid transformed in the PCR tube and cultured overnight at 37 ℃. Picking the grown single clone into an LB liquid culture medium containing 50mg/L spectinomycin, culturing at 37 ℃ until the culture medium is turbid, sucking a small amount of bacterial liquid and sending the bacterial liquid to a general biological system (Anhui) Limited company for sequencing, wherein the correct result is positive clone; and extracting a recombinant plasmid from the positive clone, namely the ptargetF-N20-UP-FBP-DOWN recombinant plasmid.

The primers required above are shown in Table 4.

TABLE 4 primer sequence Listing

5. JM109(DE3) -Cas strain was prepared in a competent form by:

the strain was cultured overnight in LB liquid medium at 37 ℃ and sterilized at high temperature in a 500mL centrifuge flask for shaking the flask the next day and sterile water (about 1.5L) for resuspending the cells the next day. Transferring 0.2-1 mL of overnight strain liquid to a 1-2L shake flask filled with 500mL of LB liquid medium, performing shake culture at 37 ℃ for 2-6 hours, and regularly monitoring OD₆₀₀Values (measured every half hour after 1 hour of incubation). When OD is reached₆₀₀When the value reaches 0.5-1.0, taking out the shake flask from the shaking table, and placing on ice for cooling for at least 15 minutes; the cells were centrifuged at 5000g for 15 minutes at 4 ℃ and the supernatant was discarded; resuspend cells in sterile ice water, resuspend cells in a small volume (a few milliliters) with a vortex apparatus, then dilute to 2/3 volumes in the centrifuge tube with water; centrifuging repeatedly according to the above steps, carefully discarding the supernatant; resuspending the cells with sterilized ice water as above, centrifuging and discarding the supernatant; resuspending the cells with 20mL of sterilized ice-cold 10% glycerol, centrifuging at 5000g at 4 ℃ for 15 minutes, carefully discarding the supernatant, and resuspending the cells with 10% glycerol to a final volume of 2-3 mL; cells were loaded in 100 μ L aliquots into microcentrifuge tubes and stored in a-80 ℃ freezer.

5. Transformation method

During electrotransformation, adding 80ng of the prepared competent cells into the recombinant plasmid ptarget F-N20-UP-FBP-DOWN prepared in the step (9), mixing gently, adding the mixture into a precooled 1mm electrotransfer cup, placing the cup into an electrotransfer instrument (Bio-Rad micro Pulser) for electrotransfer under the condition of 1.8kV, quickly adding 1mL of LB culture medium (room temperature) after electrotransfer, culturing at 30 ℃ and 180rpm for 1h for resuscitation, then coating the cell on an LB double-resistance plate containing 50mg/L kanamycin and 50mg/L spectinomycin, and culturing at 30 ℃ overnight; transformants were tested by PCR.

6. The recombinant strain claimed by the invention is obtained by completely eliminating pCas plasmid and recombinant plasmid ptargetF-N20-UP-FBP-DOWN in JM109(DE3) malZ.

The elimination method comprises the following steps: the two-plasmid-containing JM109(DE3) malZ:: the fbp strain was inoculated with LB liquid medium (containing 50mg/L of kanamycin and induced by addition of IPTG at a final concentration of 0.5 mM) at 30 ℃ overnight and spread on a plate containing 50mg/L of kanamycin for elimination of pTargetF recombinant plasmid. When a single colony grows on the plate, the single colony is sequentially selected and cultured in an LB liquid culture medium containing 50mg/L spectinomycin at 30 ℃ overnight, and the strain without growth is the strain with successfully eliminated pTargetF plasmid. Then the bacterium is selected to be cultured in an LB liquid culture medium at 37 ℃ overnight, and then pCas plasmid can be eliminated; JM109(DE3) malZ:: fbp strain which completed plasmid elimination was ready for use.

Example 4

Construction of strains for fermentation

The recombinant plasmid pETDuet-CBGF prepared in examples 1 and 2 and the recombinant plasmid pCDFDuet-TAB were co-transformed into JM109(DE3) malZ prepared in example 3 by a plasmid transformation method, i.e., a JM109(DE3) V1 strain for fermentation (i.e., the engineered bacterium producing 2' -fucosyllactose as claimed in the present invention) was obtained from the fbp strain. The method comprises the following specific steps:

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

(2) a1.5 ml centrifuge tube was filled with 100. mu.L of the competent cell suspension of JM109(DE3) malZ:: fbp prepared in example 3, and placed on ice; adding 1 μ L of recombinant plasmid pETDuet-CBGF (concentration of 100 ng/. mu.L) and 1 μ L of recombinant plasmid pCDFDuet-TAB (concentration of 100 ng/. mu.L), gently mixing by using a pipette, and standing on ice for 20 min;

(3) carrying out heat shock in a water bath at 42 ℃ for 90 seconds, then rapidly putting on ice for 3-5 min, and not oscillating bacterial liquid in the whole process;

(4) adding 1mL LB liquid culture medium (without antibiotic), mixing uniformly, shaking culturing at 37 deg.C (100rpm) for 1 hr to make bacteria restore normal growth state, and expressing antibiotic resistance gene coded by plasmid;

(5) taking 100 mu L of bacterial liquid to 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, carrying out inverted culture at 37 ℃ for 12-16 hours, picking out JM109(DE3) V1 single bacterial colony after single bacterial colony appears, culturing the single bacterial colony in LB liquid culture medium containing 50mg/L ampicillin and 25mg/L streptomycin until the single bacterial colony is turbid at 37 ℃, sucking 500 mu L of 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, 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(DE3) competent cells, to obtain JM109(DE3) V0 strain which was used as a control experiment.

Example 5

Fermentation synthesis of 2' -fucosyllactose

Using a triangular flask fermentation of 500mL size and a 100mL liquid content, JM109(DE3) V0 (control) and JM109(DE3) V1 (test) provided in example 4 were inoculated with 1% (V/V) inoculum of each of LB medium (15 g/L glycerol, 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, streptomycin 25mg/L, ampicillin 50mg/L), glycerol in each of the flasks, cultured at 37 ℃ and 220rpm to OD 1.0, cooled to 25 ℃, added with 0.2mM IPTG and 5g/L lactose at final concentrations, and cultured at 25 ℃ and 220rpm for 60 hours.

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

The measured result shows that: the 2 '-FL yield of JM109(DE3) V1 strain was 5.4g/L, while the 2' -FL yield of JM109(DE3) V0 strain was 2.1g/L, i.e.V 1 strain was 2.57 times that of V0 strain.

The results show that the yield of 2' -fucosyllactose produced by fermenting the recombinant JM109(DE3) strain under the condition of taking glycerol as a unique carbon source can be greatly improved after the fbp mutant enzyme is introduced.

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

Sequence listing

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<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 (10)

1. A recombinant strain characterized in that it is a strain integrating the fbp mutant enzyme-encoding gene into the chromosome of JM109(DE3) strain.

2. The recombinant strain of claim 1, wherein the fbp mutant enzyme encoding gene is: and (2) mutating the 104 th lysine residue of the amino acid sequence of the wild type fbp enzyme coding gene into glutamine, the 132 th arginine residue into isoleucine, the 210 th tyrosine residue into phenylalanine and the 218 th lysine residue into glutamine.

3. The recombinant strain of claim 1, wherein transcription of the fbp mutant enzyme gene is controlled by a promoter Ptrc.

4. The recombinant strain according to claim 1, wherein the site of chromosomal integration of the JM109(DE3) strain is at the position of the maltodextrin glucosidase gene.

5. The application of the recombinant strain of any one of claims 1 to 4 in preparing engineering bacteria for producing 2' -fucosyllactose with high yield.

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

7. The engineered bacterium of claim 6, wherein 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 to obtain the gene.

8. The engineered bacterium of claim 6, wherein the recombinant plasmid pCDFDuet-TAB is: the FucT gene, the RcsA gene and the RcsB gene are cloned on pCDFDuet-1 plasmid to obtain the recombinant plasmid.

9. A method of producing 2' -fucosyllactose, and characterized in that the method is: the engineering bacteria of claim 6 is used as a fermentation strain to produce 2' -fucosyllactose in a fermentation system with glycerol as a substrate.

10. The use of the engineered bacteria of claim 6 for high production of 2 '-fucosyllactose and for production of products containing 2' -fucosyllactose.

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