CN113186142A - Escherichia coli engineering strain for efficiently producing 2' -fucosyllactose - Google Patents

Abstract

The invention provides an escherichia coli engineering strain for efficiently producing 2' -fucosyllactose, belonging to the technical field of synthetic biology and microbial metabolic engineering. The invention takes glycerol as a carbon source, lactose and L-fucose as substrates, and the enzyme complex is assembled and combined by introducing the key enzyme Fkp of the self-assembly short peptide mediated pathway and FutC, thereby reducing the loss of metabolic intermediate products, improving the production efficiency of the products and improving the production yield and the yield of the products 2' -FL. In addition, a series of genes related to substrate degradation and intermediate product shunting in escherichia coli are knocked out through a CRISPR/Cas9 gene editing system, and meanwhile, the cyclic regeneration of cofactors in a metabolic pathway is enhanced, so that the yield of 2' -FL is further improved. The obtained engineered Escherichia coli strain was fed in batch for 64 hours in a 3L fermenter to accumulate 30.5g/L of 2 ' -FL with molar yields of 0.661mol2 ' -FL/mol fucose and 0.495mol2 ' -FL/mol lactose. Has important significance for industrial production of 2' -FL.

Description

Escherichia coli engineering strain for efficiently producing 2' -fucosyllactose

Technical Field

The invention relates to an escherichia coli engineering strain for efficiently producing 2' -fucosyllactose, belonging to the technical field of synthetic biology and microbial metabolic engineering.

Background

Human Milk Oligosaccharides (HMOs) are the third most abundant solid component in breast milk, playing a very important role in infant growth and development. Research shows that HMO has prebiotic effect, and can promote the proliferation of intestinal probiotics, such as bifidobacterium, inhibit the growth of harmful bacteria, and regulate intestinal flora. Furthermore, HMOs are able to bind pathogenic bacteria and affect the barrier function of the intestine, thereby modulating local and systemic immunity. In HMOs, the proportion of fucosylated oligosaccharides is about 50%. Among them, 2' -FL is one of the most abundant and common fucosylated HMOs. 2' -FL can be used as a potent inhibitor of bacterial or viral adhesion to epithelial cells. In addition, 2' -FL can also be used as a prebiotic and plays an important role in gut development and immune maturation in infants and young children. In view of the potential value of 2 '-FL in nutraceutical and pharmaceutical applications, efficient and economical production of 2' -FL has become a research hotspot.

The chemical synthesis of 2' -FL has its limitations including difficulty in precise control of glycosidic linkages, cumbersome activation and (de) protection steps and the use of toxic reagents. More studies by researchers have utilized engineered microorganisms to synthesize 2' -FL via metabolic engineering and synthetic biological strategies. The metabolic pathway engineering for producing fucosyllactose using microbial cell factories mainly comprises the following parts: (1) absorption and metabolic modification of a substrate lactose; (2) supply of intracellular GDP-L-fucose; (3) expression and enzyme engineering of fucosyltransferase; (4) sugar transporters transport intracellular products to the outside of the cell. Among them, efficient supply of intracellular GDP-L-fucose and introduction of fucosyltransferase having excellent properties are essential for 2' -FL synthesis. Therefore, how to increase the accumulation of intracellular GDP-L-fucose and improve the utilization efficiency of this nucleotide precursor, while the discovery and engineering of superior fucosyltransferases is the key to efficient production of 2' -FL. GDP-L-fucose is available in two metabolic pathways as a key glycosyl donor for the production of fucosylated HMOs: de novo synthetic pathways and salvage pathways. The GDP-L-fucose is finally synthesized by starting from cheap renewable carbon sources such as glucose or glycerol through multi-step enzymatic reaction in a head synthesis way. The use of glucose as a carbon source in E.coli requires 7 steps of reaction to produce GDP-L-fucose. The remedy approach is to directly convert the externally provided L-fucose into GDP-L-fucose through two-step reaction by introducing bifunctional enzyme fucokinase/fucose-1-phosphate guanylyltransferase (Fkp) derived from Bacteroides fragilis 9343. Although the use of glucose or glycerol as substrate is less costly, the final yield is relatively low due to the loss of metabolic flux and intermediates from the long catalytic steps of the de novo synthetic pathway.

To date, various microbial cell metabolism factories have been developed for biosynthesis of 2' -FL. One recent study generated 2' -FL by constructing the GDP-L-fucose salvage pathway in recombinant Bacillus subtilis and introducing a Helicobacter pyrori-derived alpha-1, 2-fucosyltransferases (FutC) to transfer fucose residues from GDP-L-fucose donors to exogenously added lactose acceptors. In addition, in view of the absorption of substrates and the supply of cofactors, researchers have intensified the transport proteins of the respective substrates and genes related to cofactor regeneration pathways. Finally, fed-batch fermentation through a 3L fermenter achieved a 2' -FL yield of 5 g/L. The conversion of fucose and lactose was 0.85mol/mol and 0.27mol/mol, respectively. Recently, it has been found that microbial metabolic production of 2' -FL is achieved by constructing a de novo pathway for GDP-L-fucose within Saccharomyces cerevisiae and introducing the Escherichia coli-derived fucosyltransferase WbgL. In addition, by replacing the carbon source supply with xylose from glucose, the supply of cofactors necessary for the synthesis of 2 '-FL, such as NADPH and ATP, in the yeast cell is more sufficient, so that the 2' -FL production is significantly improved.

However, the introduction of enzymes from different organisms into target host strains faces a number of challenges, including low or even inactivation of enzyme activity for heterologous expression and an increased metabolic burden on the host leading to an imbalance in metabolic flux. In addition, the target pathway may interfere with the complex intracellular environment metabolites. These factors all affect the improvement of 2' -FL yield. Therefore, how to successfully and heterologously express a target enzyme and make the target enzyme play a role, and meanwhile, solving the interference between a target pathway and a host is still a problem which needs to be solved at present.

One effective strategy to address these problems is to recruit and assemble pathway enzymes into a multi-enzyme complex with the aid of an assembly tool. To date, researchers have developed a variety of protein assembly strategies including the construction of synthetic scaffolds based on proteins or nucleic acids, recruitment of pathway enzymes into pre-constructed cell microchambers, multi-enzyme self-assembly based on protein-protein or protein-peptide interactions, and the like. These strategies have been developed for the construction of complex multi-enzyme complexes for cascade biocatalysis. The construction of multiple enzyme complexes in metabolic pathways facilitates the formation of substrate channels, reduces diffusion of intermediates within the cytoplasm and increases the concentration of localized enzymes and corresponding substrates to achieve efficient conversion of substrates.

Disclosure of Invention

In order to solve the problems of low enzyme activity of heterologous expression target enzyme and low yield of 2 '-FL caused by influencing host metabolism, the invention improves the metabolic pathway of L-fucose → 2' -FL by knocking out endogenous genes of escherichia coli related to substrates L-fucose, lactose and key intermediate GDP-L-fucose catabolism, enhances the gene related to 2 '-FL synthesis and improves the combination efficiency and stability of an intracellular enzyme compound through short peptide, the remediation pathway is shorter, and the bifunctional enzyme Fkp can realize soluble expression in the escherichia coli, thereby being capable of more effectively supplying a GDP-L-fucose precursor and realizing the improvement of the yield of the 2' -FL.

The invention provides an engineering bacterium of escherichia coli for producing 2' -fucosyllactose, which is characterized by silencing and expressing genes related to substrates L-fucose, lactose and key intermediate GDP-L-fucose catabolism endogenous to escherichia coli, heterologously expressing GDP-L-fucose salvage pathway to synthesize key enzyme and 1,2-fucosyltransferase, and enhancing expression of one or more of guanosine-inosine kinase, guanylic acid kinase and nucleotide diphosphate kinase.

In one embodiment, the GDP-L-fucose salvage pathway synthesis key enzyme Fkp is derived from Bacteroides fragiliss 9343; the 1,2-fucosyltransferase FutC is derived from Helicobacter pylori.

In one embodiment, the nucleotide sequence of the gene encoding the key enzyme in the synthesis of the GDP-L-fucose salvage pathway is shown in SEQ ID NO. 1.

In one embodiment, the nucleotide sequence of the gene encoding the 1,2-fucosyltransferase is shown as SEQ ID NO. 2.

In one embodiment, the genes endogenous to E.coli involved in the catabolism of substrates L-fucose and lactose and the key intermediate GDP-L-fucose include the gene coding for beta-galactosidase lacZ, the gene coding for L-fucose isomerase and L-fucose kinase fucoik, the gene coding for D-arabinose isomerase araA, the gene coding for L-rhamnose isomerase rhaA and the gene coding for UDP-glucose lipid carrier transferase wcaJ.

In one embodiment, short peptides are used to modify the C-or N-terminus of the key enzyme in the synthesis of the GDP-L-fucose salvage pathway of the protein of interest and the 1, 2-fucosyltransferase; the short peptide is RIDD and RIAD.

In one embodiment, the nucleotide sequence of the RIDD is shown in SEQ ID NO. 6.

In one embodiment, the nucleotide sequence of the RIAD is as set forth in SEQ ID NO. 7.

In one embodiment, the C-terminus of FutC is modified with RIDD and the C-terminus or N of Fkp is modified with RIAD.

In one embodiment, the C-terminus of Fkp is modified with RIAD; or the C-terminal and the N-terminal of Fkp are modified by two RIADs; or Fkp was modified at the N-terminus with two RIADs.

In one embodiment, the short peptide is linked to the protein of interest using a linker peptide.

In one embodiment, the short peptide is (GGGGS)₃The nucleotide sequence for coding the polypeptide is shown as SEQ ID NAnd O.10.

In one embodiment, the guanosine-inosine kinase, guanylate kinase, and nucleotide diphosphate kinase are expressed using the vector pACM 4.

The invention provides a method for improving the production capacity of 2' -fucosyllactose of escherichia coli, which heterologously expresses GDP-L-fucose salvage way to synthesize key enzyme and 1,2-fucosyltransferase in the escherichia coli, and utilizes short peptides RIDD and RIAD to modify the GDP-L-fucose salvage way to synthesize the key enzyme and the 1, 2-fucosyltransferase.

In one embodiment, the C-terminus of FutC is modified with RIDD and the C-terminus or N of Fkp is modified with RIAD.

The invention provides a method for producing 2 ' -fucosyllactose, which takes escherichia coli engineering bacteria as fermentation microorganisms, takes glycerol as a carbon source, and takes L-fucose and lactose as substrates to synthesize 2 ' -fucosyllactose, and the 2 ' -fucosyllactose is produced by fermentation.

The invention provides application of the strain in preparation of 2' -fucosyllactose and derivatives thereof.

The invention has the beneficial effects that:

coli endogenous genes related to metabolic degradation of lactose and fucose serving as substrates and downstream metabolic shunt genes of GDP-L-fucose serving as key intermediate products are knocked out, self-assembled short peptides RIDD and RIAD are introduced to modify pathway enzymes Fkp and FutC, the spatial structure and the stoichiometric proportion of a formed Fkp-FutC enzyme compound are optimized, and a synthetic pathway of an auxiliary factor Guanosine Triphosphate (GTP) related to a metabolic pathway is strengthened, so that the loss of metabolic intermediate products is reduced, the production efficiency of the products is improved, and the yield of 2' -FL are obviously improved. 2.63 g/L2 '-FL with a molar yield of 0.654mol 2' -FL/mol fucose can be accumulated in 72 hours of shake flask fermentation with an initial addition of 2g/L fucose and 5g/L lactose. The results of fermentation experiments in the 3L fermentor showed that 30.5g/L of 2 ' -FL could be accumulated in 64 hours of fed-batch with a molar yield of 0.661mol2 ' -FL/mol fucose and 0.495mol2 ' -FL/mol lactose. Among them, the product conversion rate of fucose is obviously superior to the highest fucose conversion rate (0.52mol2 '-FL/mol fucose) reported so far in Escherichia coli for metabolic production of 2' -FL by using salvage pathway.

Drawings

FIG. 1 is a diagram of the metabolic pathway for the salvage pathway synthesis of 2' -FL;

FIG. 2 is a flow chart of recombinant plasmid construction: wherein (1): pET-fkp-futC; (2): pET-fkp^RD-futC^RA；(3)：pET-^RDfkp-^RAfutC；(4)：pACM4-gsk-gmk-ndk；(5)pET-fkp^RD-^RAfutC；

FIG. 3 is a flow chart of the CRISPR/Cas9 gene editing system knocking out E.coli genome gene;

FIG. 4 shows a gel electrophoresis of a knockout-related nucleic acid (taking knockout gene lacZ as an example);

FIG. 5 shows the protein expression in the relevant host under different short peptide modifications;

FIG. 6 is LC/MS spectra of product 2' -FL standard and product sample: wherein (1) a 2' -FL standard; (2) 2' -FL fermentation liquid treated product;

FIG. 7 is a conceptual diagram of a RIDD-RIAD mediated intracellular enzyme complex;

FIG. 8 shows the fermentation yield of 2' -FL under different conditions: wherein (1): production of 2' -FL by metabolic pathways in different hosts; (2): the yield of 2' -FL of metabolic pathway enzyme under the C-terminal modification of RIDD and RIAD short peptide; (3): the yield of 2' -FL of metabolic pathway enzyme under the modification of the C-terminal or the N-terminal of RIDD and RIAD short peptide; (4): the effect of adjusting the chemical dose ratio of the Fkp-FutC enzyme complex composition on 2' -FL production; (5): the yield of 2' -FL after enhancing the cofactor regeneration pathway; (6): effect of fed-batch fermentation on 2' -FL yield.

Detailed Description

The invention is further illustrated by the following specific examples.

LB liquid medium: 10g/L peptone, 5g/L yeast extract powder and 10g/L sodium chloride.

LB solid medium: 10g/L peptone, 5g/L yeast extract powder, 10g/L sodium chloride and 15g/L agar powder.

Fermentation medium (Defined medium, DM): 20g/L of glycerin, 13.5g/L of potassium dihydrogen phosphate, 4.0g/L of diammonium hydrogen phosphate, 1.7g/L of citric acid, 1.4g/L of magnesium sulfate heptahydrate and 10ml/L of trace metal elements, and adjusting the pH value to 6.8 by using sodium hydroxide.

The trace metal elements include: 10g/L ferrous sulfate, 2.25g/L zinc sulfate heptahydrate, 1.0g/L anhydrous copper sulfate, 0.35g/L manganese sulfate monohydrate, 0.23g/L sodium borate decahydrate, 0.11g/L ammonium molybdate and 2.0g/L calcium chloride dihydrate are dissolved in 5M hydrochloric acid.

Fed-batch fermentation feed liquid:

(1) and (3) carbon source supplement: 600g/L of glycerol, 20g/L of magnesium sulfate heptahydrate and 0.2g/L of thiamine.

(2) pH regulation and control: 14% aqueous ammonia (v/v).

Antibiotic concentration: ampicillin 100 mg.L^-1(liquid Medium), ampicillin 200 mg. multidot.L^-1(solid Medium), kanamycin 50 mg. L^-1Streptomycin 50 mg. L^-1Chloramphenicol 34 mg. L^-1Spectinomycin 50 mg. L^-1。

Inducer concentration: isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added at a concentration of 0.5mM in the flask, 0.2mM in the fed-batch fermentation, and 1mM in the gene knock-out where the pTargetT plasmid was removed. In the gene knockout experiment, the final concentration of L-arabinose addition was 20 mM.

Protein expression detection:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to compare the expression of the short peptide-modified enzyme in different hosts producing 2' -FL. The overnight cultured seed solution was inoculated into a 250mL Erlenmeyer flask containing 25mL DM medium, cultured at 37 ℃ and 200rpm, when OD₆₀₀Between 0.6 and 0.8, 1mL of the culture was taken 24 hours after induction with 1mM IPTG, and the cells were collected by centrifugation at 8,000 rpm. The cell pellet was resuspended in 40. mu.L of sterile water and 10. mu.L of 5 Xprotein electrophoresis loading buffer was added, followed by boiling for 10 minutes to rupture it. The supernatant was then collected by centrifugation at 12,000rpm for 10 minutes. For different host bacteriaSupernatant samples of the strains were analyzed by SDS-PAGE. The gel was run at 80V until the sample completely passed through the concentrate. Then, the voltage was maintained at 120V. After electrophoresis, the gel was stained with Coomassie Brilliant blue for 30 minutes and then destained with destaining solution. The gels were photographed using a Tanon gel imager and protein expression was analyzed.

Strain culture and fermentation:

colonies of each strain were inoculated into 4mL of LB medium from the plate. After overnight incubation, 0.5mL of the culture was inoculated into 25mL of DM medium containing the antibiotic of the corresponding plasmid. The culture was carried out in 250mL shake flasks at 200rpm and 37 ℃. When the cell OD₆₀₀When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.5mM and the substrate lactose was added to a final concentration of 5g/L and L-fucose was added to a final concentration of 2 g/L. Culturing at 25 deg.C, collecting fermentation liquor after 72 hr, and detecting 2' -FL yield.

Fed-batch fermentations were carried out in 3L fermentors containing 1LDM medium. The seed liquid was inoculated at 5% (v/v) and the initial temperature was maintained at 37 ℃. After complete depletion of 20g/L glycerol in the initial medium, the supply of a carbon source feed was started to meet cell growth. When OD is reached₆₀₀When about 20 ℃ was reached, 0.1mM IPTG was added to induce gene expression, and the temperature was lowered to 25 ℃. Lactose at a final concentration of 20g/L and L-fucose at a final concentration of 10g/L are added simultaneously, and the substrate is subsequently supplemented according to the actual conditions. By adding 14% ammonia water (ammonia water: H)₂O is 1: 1, v/v) the pH was kept at 6.8 and the foam was controlled by adding an antifoam. The dissolved oxygen is maintained at 20-30% by adjusting the stirring speed (200-1000 rpm) and the aeration rate (0.5-2 vvm).

Production amount detection of 2' -FL:

to measure the yield of 2' -FL in the fermentation broth after fermentation, 1mL of the broth supernatant was collected by centrifugation (8,000rpm, 10 minutes), and the supernatant was subjected to HPLC analysis by a High Performance Liquid Chromatography (HPLC) system (Waterse2695) equipped with a differential refraction Detector (Waters2414 RI Detector) and an Organic Acid chromatography column (Rezex ROA-Organic Acid H + column). The mobile phase is 5mM sulfuric acid aqueous solution, the flow rate is 0.6mL/min, and the column temperature is 60 ℃.

Identification of the Synthesis product of 2' -FL

The 2 '-FL standard and an equivalent amount of the 2' -FL fermentation broth treated sample were analyzed by WATERS MALDI SYNAPT Q-TOF MS equipped with a WATERS ACQUITY UPLC BEH AMIDE (2.1X 100mm 1.7 μm). The mobile phase is as follows: a: 80% acetonitrile and 20% ammonia (0.1%); b: 30% acetonitrile and 70% ammonia (0.1%) were subjected to gradient elution at 45 ℃ and a flow rate of 0.3 mL/min.

TABLE 1 primer sequences

Note: the underlined part is the cleavage site.

TABLE 2 PCR System

PCR conditions were as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1kb/min, 32 cycles, storage at 4 ℃ and gel recovery of PCR products.

TABLE 3 enzyme digestion System

Enzyme cutting conditions are as follows: the enzyme digestion reaction was carried out at 37 ℃ for 40 min.

TABLE 4 enzyme-linked reaction System

Enzyme-linked conditions: 22 ℃ for 3-5 hours, or 16 ℃ overnight.

TABLE 5 one-step cloning reaction System

One-step cloning reaction conditions: 50 ℃ for 5 minutes.

And calculating the vector dosage and the insert dosage according to a formula by X/Y:

the optimum amount of the insertion vector used was (0.02 × number of base pairs of cloning vector) ng (0.03 pmol);

the optimum amount of the insert used was (0.04X number of base pairs of the insert) ng (0.06 pmol).

TABLE 6 knockout of related primer sequence Listing

Note: the boxed sequence represents the N20 sequence used for gene deletion.

Example 1: construction of recombinant E.coli with lacZ knockout

(1) Carrying out PCR amplification on the existing pTargetF plasmid by using a primer N20-delta lacZ-F/R to obtain a pTargetF plasmid with a target lacZ gene;

(2) respectively amplifying upstream and downstream homologous fragments of lacZ gene by PCR by using primers delta lacZ-UH-F/R and delta lacZ-DH-F/R (the sequence of PCR primers is shown in Table 6);

(2) the two homologous fragments are obtained by

II one-step cloning kit was ligated to the linearized pTargetF-. DELTA.lacZN 20 vector (the vector was linearized by amplification with primers pTargetT-F/R) to construct plasmid pTargetT-. DELTA.lacZN 20;

(3) transformation of the pCas plasmid into e.coli bl21(DE3) by electroporation and addition of 20mM arabinose to induce expression of the λ -Red e.coli gene recombination system;

(4) the plasmid pTargetT-. DELTA.lacZN 20 was electroporated into the above E.coli BL21(DE3), spread on LB plates containing kanamycin and spectinomycin, cultured at 30 ℃ for 12 to 20 hours, and the colonies on the plates were verified for knockout (as shown in FIG. 4);

(5) after successful verification, a colony with the target gene knocked out is inoculated to 2mL of LB culture medium, 1mM IPTG is added to induce the expression of sgRNA-pMB1 to cut the pTargetT plasmid so as to achieve the aim of removing the pTargetT plasmid;

(6) selecting bacteria without pTargetT plasmid, inoculating to non-resistant LB culture medium at 42 deg.C, culturing overnight at 200rpm, taking appropriate amount of bacterial liquid, coating on non-resistant LB solid plate, after bacterial colony grows out, verifying whether to remove pCas plasmid, selecting the strain without pCas plasmid, and sequencing to verify to obtain E.coli BL21(DE 3). DELTA.lacZ.

Example 2: construction of E.coli for heterologous expression of futC and fkp

Genes futC (nucleotide sequence shown in SEQ ID NO. 2) and fkp (nucleotide sequence shown in SEQ ID NO. 1) were synthesized by Shanghai Biotechnology engineering Co., Ltd. Genes futC and fkp were amplified by primers futC-F/R (NcoI) and fkp-F/R (NdeI), respectively, and ligated by restriction enzymes to insert into the NcoI cleavage site of the first multiple cloning site and the NdeI cleavage site of the second multiple cloning site of pETDuet-1, to construct plasmid pET-futC-fkp (see Table 1 for primer sequences, FIG. 2(1) for plasmid construction scheme).

The plasmid pET-futC-fkp was transformed into e.coli bl21(DE3) Δ lacZ, and a strain containing the plasmid pET-futC-fkp was obtained after sequencing verification and named WLS 01.

Example 3: construction of recombinant Escherichia coli with fucoK, araA, rhaA, and wcaJ genes knocked out

Knocking out the fucoik gene in the strain WLS01 according to the method of example 1 to construct a strain WLS 02;

knocking out araA gene in a strain WLS02 according to the method of example 1 to construct a strain WLS 03;

knocking out rhaA gene in a strain WLS02 according to the method of example 1, and constructing a strain WLS 04;

knocking out rhaA gene in a strain WLS03 according to the method of example 1, and constructing a strain WLS 05;

the wcaJ gene in strain WLS05 was knocked out according to the method of example 1 to construct strain WLS 06.

Example 4: construction of short peptide fusion target protein plasmid and recombinant bacterium

(1) Construction of recombinant Escherichia coli with lacZ, fucoK, araA, rhaA, and wcaJ genes knocked out

The fucoik, araA, rhaA, wcaJ genes were deleted in the strain e.colibl21(DE3) Δ lacZ obtained in example 1 according to the method of example 1, to obtain strain WLS 06S.

(2) Facilitating expression of futC and fkp using tags

Plasmids pET-RD-RA (C-terminal modification, modification between two T7 promoters of pETDuet-1, sequence of the modified part is shown in SEQ ID NO. 8) and pET-NRD-NRA (N-terminal modification, modification between two T7 promoters of pETDuet-1, sequence of the modified part is shown in SEQ ID NO. 9) with labels of RIDD and RIAD are synthesized by a company, and common enzyme cutting sites are reserved for standby. Use of primer fkp^RD-F/R (Nco I/BamH I) and futC^RAAnd (4) amplifying fkp and futC respectively by F/R (BglII), and obtaining related target gene fragments by PCR amplification and DNA fragment gel recovery. Firstly, amplified fkp fragment and pET-RD-RA plasmid are subjected to Nco I/BamH I double enzyme digestion enzyme ligation (enzyme digestion system is shown in Table 3, enzyme ligation system is shown in Table 4), and plasmid pET-fkp is constructed^RD-RA. Then, the amplified fragment futC and plasmid pET-fkp^RDThe plasmid pET-fkp was constructed by enzymatic ligation of BglII single enzyme in RA^RD-futC^RA(the primer sequences are shown in Table 1, and the plasmid construction flow chart is shown in FIG. 2 (2)). Plasmid pET-futC^RD-fkp^RAThe construction method is similar.

Use of primers^RDfkp-F/R (BamHI/EcoRI) and^RAfkp and futC are respectively amplified by futC-F/R (Kpn I), and related target gene fragments are obtained by PCR amplification and DNA fragment gel recovery. First amplified fkp fragment and pETthe-RD-RA plasmid is subjected to BamH I/EcoR I double enzyme digestion enzyme ligation (the enzyme digestion system is shown in Table 3, and the enzyme ligation system is shown in Table 4) to construct a plasmid pET-^RDfkp-NRA. Then, the amplified fragment futC and the plasmid pET-^RDfkp-NRA for Kpn I single enzyme digestion to construct plasmid pET-^RDfkp-^RAfutC (primer sequences are shown in Table 1, and plasmid construction flow chart is shown in FIG. 2 (3)). Plasmid pET-^RDfutC-^RAfkp the construction method is similar.

Plasmid pET-fkp was constructed as described above^RD-^RAfutC、pET-^RDfutC-fkp^RA、pET-^RDfkp-futC^RA、pET-futC^RD-^RAfkp、pET-futC^RD-fkp^RARA、pET-futC^RD-^RAfkp^RA、pET-futC^RD-^RARAfkp。

Plasmid pET-fkp^RD-futC^RATransformed into WLS06S, and constructed into strain WLS 07.

Plasmid pET-futC^RD-fkp^RATransformed into WLS06S, and constructed into strain WLS 08.

Hosts WLS07 and WLS08 were obtained. The shake flask fermentation results showed that 2 '-FL yields of WLS07 and WLS08 were 1.39g/L and 1.82g/L, respectively, and were 65.5% and 117% higher, respectively, than the yield of WLS06 in a host expressing free enzyme (see FIGS. 8(2) and Table 7 for 2' -FL yield for each engineered strain).

(3) Optimizing spatial location of self-assembled short peptides

The modification positions of the short peptides on Fkp and FutC were adjusted at the same time (RIDD or RIAD was added to the N-terminus of Fkp or FutC), and a series of related plasmids were constructed as described in step 1. These plasmids were transferred to host WLS06S to obtain host WLS 9-14 (see Table 7 for details of hosts).

The results of the 2 '-FL production assay showed that when the short peptide RIDD was added to the N-terminus of Fkp, the production of 2' -FL dropped dramatically, and therefore this spatial position adjustment was disadvantageous. When RIDD or RIAD was added to the N-terminus of FutC, the strain had almost lost the ability to produce 2' -FL, and we speculated that N-terminal modifications to FutC could severely impair the original enzymatic activity of FutC. When the C-terminal modification of FutC by RIDD was maintained while RIAD was added to the N-terminus of Fkp, the 2 '-FL production was significantly improved on the original basis (see fig. 8(3) and table 7 for 2' -FL production for each engineered strain).

(4) Adjusting the chemical dose of pathway enzymes by optimizing the amount of self-assembled short peptides

Three plasmids pET-futC were constructed according to the procedure of step 1^RD-fkp^RARA(RIDD was added to the C-terminus of FutC, while two RIAD short peptide tags were added to Fkp), pET-futC^RD-^RAfkp^RA(RIDD was added to the C-terminus of FutC, while two RIAD short peptide tags were added to Fkp), and pET-futC^RD-^RARAfkp (RIDD was added to the C-terminus of FutC, while two RIAD short peptide tags were added to Fkp). The experimental results showed that the addition of two RIAD tags at Fkp N-terminal and C-terminal, respectively, was most effective, and the 2 '-FL yield reached 2.15g/L (see FIG. 8(4) and Table 7 for the 2' -FL yield of each engineered strain).

Example 5: construction of recombinant E.coli for enhancing GTP recycling

Enhanced expression of gsk, gmk and ndk in E.coli:

genes gsk (nucleotide sequence is shown as SEQ ID NO. 3), gmk (nucleotide sequence is shown as SEQ ID NO. 4) and ndk (nucleotide sequence is shown as SEQ ID NO. 5) in the Escherichia coli MG1655 are subjected to PCR amplification and DNA fragment glue recovery through primers gsk-F/R (NdeI/Kpn I), gmk-F/R (NdeI/Kpn I) and ndk-F/R (NdeI/Xho I) (primer sequences are shown in Table 1) to obtain related target gene fragments. The gsk, gmk and ndk are cloned to the multiple cloning site of pACM4 plasmid (Addgene is coded as plasmid #49797) by double enzyme digestion respectively to obtain plasmids pACM4-gsk, pACM4-gmk and pACM 4-ndk. Plasmids pACM4-gmk and pACM4-ndk were then cut with an AvrII/SalI quick cut, while plasmid pACM4-gsk was cut with a speI/SalI quick cut. The cleaved product was recovered by DNA gel and enzymatically ligated with T4 DNA ligase. Finally, plasmids pACM4-gsk-gmk and pACM4-gsk-gmk were obtained. The construction method of pACM4-gsk-gmk-ndk is similar (the plasmid construction flow chart is shown in FIG. 2 (4)).

And respectively transferring the pACM4-gsk, pACM4-gsk-gmk, pACM4-gsk-ndk and pACM4-gsk-gmk-ndk into WLS16 to obtain recombinant bacteria WLS 18-21.

The experimental results show that the obtained 4 strains are respectively expressed as WLS 18-21, and the yield is respectively 2.28g/L, 2.24g/L, 2.63g/L and 2.02 g/L. Wherein, the accumulation amount of host 2 '-FL of simultaneously over-expressing genes gsk and ndk reaches the highest and is 2.63g/L (see FIG. 8(5) and Table 7 for the yield of 2' -FL of each engineering strain).

Example 6: fed-batch fermentation of 3L fermenter to increase 2' -FL yield

And selecting the optimized optimal strain WLS20 to perform fed-batch fermentation experiments of a 3L fermentation tank. The fermentation experiments were performed as described for the strain culture and fermentation in the examples. The final fed-batch fermentation results are shown in FIG. 8 (6). The yield of 2' -FL after 64 hours of fermentation reaches 30.5g/L and the maximum OD₆₀₀Was 58.6. In addition, the conversion rates of the substrates fucose and lactose were 0.661mol2 '-FL/mol fucose and 0.495mol 2' -FL/mol lactose, respectively (see FIG. 6 for the fermentation yield of the engineered bacterium WLS 20).

TABLE 7 details of the respective engineering bacteria and 2' -FL yields

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

<110> university of south of the Yangtze river

<120> an engineered Escherichia coli strain for efficiently producing 2' -fucosyllactose

<130> BAA210285A

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 2847

<212> DNA

<213> Bacteroides fragilis

<400> 1

atgcaaaaac tactatcttt accgtccaat ctggttcagt cttttcatga actggagagg 60

gtgaatcgta ccgattggtt ttgtacttcc gacccggtag gtaagaaact tggttccggt 120

ggtggaacat cctggctgct tgaagaatgt tataatgaat attcagatgg tgctactttt 180

ggagagtggc ttgaaaaaga aaaaagaatt cttcttcatg cgggtgggca aagccgtcgt 240

ttacccggct atgcaccttc tggaaagatt ctcactccgg ttcctgtgtt ccggtgggag 300

agagggcaac atctgggaca aaatctgctt tctctgcaac ttcccctata tgaaaaaatc 360

atgtctttgg ctccggataa actccataca ctgattgcga gtggtgatgt ctatattcgt 420

tcggagaaac ctttgcagag tattcccgaa gcggatgtgg tttgttatgg actgtgggta 480

gatccgtctc tggctaccca tcatggcgtg tttgcttccg atcgcaaaca tcccgaacaa 540

ctcgacttta tgcttcagaa gccttcgttg gcagaattgg aatctttatc gaagacccat 600

ttgttcctga tggacatcgg tatatggctt ttgagtgacc gtgccgtaga aatcttgatg 660

aaacgttctc ataaagaaag ctctgaagaa ctaaagtatt atgatcttta ttccgatttt 720

ggattagctt tgggaactca tccccgtatt gaagacgaag aggtcaatac gctatccgtt 780

gctattctgc ctttgccggg aggagagttc tatcattacg ggaccagtaa agaactgatt 840

tcttcaactc tttccgtaca gaataaggtt tacgatcagc gtcgtatcat gcaccgtaaa 900

gtaaagccca atccggctat gtttgtccaa aatgctgtcg tgcggatacc tctttgtgcc 960

gagaatgctg atttatggat cgagaacagt catatcggac caaagtggaa gattgcttca 1020

cgacatatta ttaccggggt tccggaaaat gactggtcat tggctgtgcc tgccggagtg 1080

tgtgtagatg tggttccgat gggtgataag ggctttgttg cccgtccata cggtctggac 1140

gatgttttca aaggagattt gagagattcc aaaacaaccc tgacgggtat tccttttggt 1200

gaatggatgt ccaaacgcgg tttgtcatat acagatttga aaggacgtac ggacgattta 1260

caggcagttt ccgtattccc tatggttaat tctgtagaag agttgggatt ggtgttgagg 1320

tggatgttgt ccgaacccga actggaggaa ggaaagaata tctggttacg ttccgaacat 1380

ttttctgcgg acgaaatttc ggcaggtgcc aatctgaagc gtttgtatgc acaacgtgaa 1440

gagttcagaa aaggaaactg gaaagcattg gccgttaatc atgaaaaaag tgttttttat 1500

caacttgatt tggccgatgc agctgaagat tttgtacgtc ttggtttgga tatgcctgaa 1560

ttattgcctg aggatgctct gcagatgtca cgcatccata accggatgtt gcgtgcgcgt 1620

attttgaaat tagacgggaa agattatcgt ccggaagaac aggctgcttt tgatttgctt 1680

cgtgacggct tgctggacgg gatcagtaat cgtaagagta ccccaaaatt ggatgtatat 1740

tccgatcaga ttgtttgggg acgtagcccc gtgcgcatcg atatggcagg tggatggacc 1800

gatactcctc cttattcact ttattcggga ggaaatgtgg tgaatctagc cattgagttg 1860

aacggacaac ctcccttaca ggtctatgtg aagccgtgta aagacttcca tatcgtcctg 1920

cgttctatcg atatgggtgc tatggaaata gtatctacgt ttgatgaatt gcaagattat 1980

aagaagatcg gttcaccttt ctctattccg aaagccgctc tgtcattggc aggctttgca 2040

cctgcgtttt ctgctgtatc ttatgcttca ttagaggaac agcttaaaga tttcggtgca 2100

ggtattgaag tgactttatt ggctgctatt cctgccggtt ccggtttggg caccagttcc 2160

attctggctt ctaccgtact tggtgccatt aacgatttct gtggtttagc ctgggataaa 2220

aatgagattt gtcaacgtac tcttgttctt gaacaattgc tgactaccgg aggtggatgg 2280

caggatcagt atggaggtgt gttgcagggt gtgaagcttc ttcagaccga ggccggcttt 2340

gctcaaagtc cattggtgcg ttggctaccc gatcatttat ttacgcatcc tgaatacaaa 2400

gactgtcact tgctttatta taccggtata actcgtacgg caaaagggat cttggcagaa 2460

atagtcagtt ccatgttcct caattcatcg ttgcatctca atttactttc ggaaatgaag 2520

gcgcatgcat tggatatgaa tgaagctata cagcgtggaa gttttgttga gtttggccgt 2580

ttggtaggaa aaacctggga acaaaacaaa gcattggata gcggaacaaa tcctccggct 2640

gtggaggcaa ttatcgatct gataaaagat tataccttgg gatataaatt gccgggagcc 2700

ggtggtggcg ggtacttata tatggtagcg aaagatccgc aagctgctgt tcgtattcgt 2760

aagatactga cagaaaacgc tccgaatccg cgggcacgtt ttgtcgaaat gacgttatct 2820

gataagggat tccaagtatc acgatca 2847

<210> 2

<211> 900

<212> DNA

<213> Helicobacter pylori

<400> 2

atggctttta aagtggtgca aatttgtggg gggcttggga atcaaatgtt tcaatacgct 60

ttcgctaaaa gtttgcaaaa acaccttaat acgcccgtgc tattagacac tacttctttt 120

gattggagca ataggaaaat gcaattagag cttttcccta ttgatttgcc ctatgcgaat 180

gcaaaagaaa tcgctatagc taaaatgcaa catctcccca agttagtaag agatgcactc 240

aaatacatag gatttgatag ggtgagtcaa gaaatcgttt ttgaatacga gcctaaattg 300

ttaaagccaa gccgtttgac ttattttttt ggctatttcc aagatccacg atattttgat 360

gctatatcct ctttaatcaa gcaaaccttc actctacccc ccccccccga aaataataaa 420

aataataata aaaaagagga agaataccag cgcaagcttt ctttgatttt agccgctaaa 480

aacagcgtat ttgtgcatat aagaagaggg gattatgtgg ggattggctg tcagcttggt 540

attgattatc aaaaaaaggc gcttgagtat atggcaaagc gcgtgccaaa catggagctt 600

tttgtgtttt gcgaagactt aaaattcacg caaaatcttg atcttggcta ccctttcacg 660

gacatgacca ctagggataa agaagaagag gcgtattggg atatgctgct catgcaatct 720

tgcaagcatg gcattatcgc taatagcact tatagctggt gggcggctta tttgatggaa 780

aatccagaaa aaatcattat tggccccaaa cactggcttt ttgggcatga aaatattctt 840

tgtaaggaat gggtgaaaat agaatcccat tttgaggtaa aatcccaaaa atataacgct 900

<210> 3

<211> 1302

<212> DNA

<213> Escherichia coli

<400> 3

atgaaatttc ccggtaaacg taaatccaaa cattacttcc ctgtaaatgc acgcgatccg 60

ctgcttcagc agttccagcc agaaaacgaa accagcgccg cctgggtagt gggtatcgat 120

caaacgctgg tcgatattga agcgaaagtg gatgacgaat tcattgagcg ttatggatta 180

agcgccgggc attcactggt gattgaggat gacgtagccg aagcgcttta tcaggaacta 240

aaacagaaaa acctgattac ccatcagttt gcgggtggca ctattggtaa caccatgcac 300

aactactcgg tgctcgcgga cgaccgttcg gtgctgctgg gcgtcatgtg cagcaatatt 360

gaaattggca gctatgccta tcgttacctg tgtaacacct ccagccgtac cgatcttaac 420

tatctacaag gcgtggatgg tccgattggt cgttgcttta cgctgattgg cgagtccggg 480

gaacgtacct ttgctatcag ccctggccac atgaaccagc tgcgggctga aagtattccg 540

gaagatgtga ttgccggagc ctcggcactg gttctcacct cttatctggt gcgttgcaag 600

ccgggtgaac ccatgccgga agcaaccatg aaagccattg agtacgcgaa gaaatataac 660

gtaccggtgg tgctgacgct gggaactaag tttgtcattg ccgagaatcc gcagtggtgg 720

cagcaattcc tcaaagacca cgtctctatc cttgcgatga acgaagatga agccgaagcg 780

ttgaccggag aaagcgatcc gttgttggca tctgacaagg cgctggactg ggtagatctg 840

gtgctgtgca ccgccgggcc aatcggcttg tatatggcgg gctttaccga agacgaagcg 900

aaacgtaaaa cccagcatcc gctgctgccg ggcgctatag cggaattcaa ccagtatgag 960

tttagccgcg ccatgcgcca caaggattgc cagaatccgc tgcgtgtata ttcgcacatt 1020

gcgccgtaca tgggcgggcc ggaaaaaatc atgaacacta atggagcggg ggatggcgca 1080

ttggcagcgt tgctgcatga cattaccgcc aacagctacc atcgtagcaa cgtaccaaac 1140

tccagcaaac ataaattcac ctggttaact tattcatcgt tagcgcaggt gtgtaaatat 1200

gctaaccgtg tgagctatca ggtactgaac cagcattcac ctcgtttaac gcgcggcttg 1260

ccggagcgtg aagacagcct ggaagagtct tactgggatc gt 1302

<210> 4

<211> 621

<212> DNA

<213> Escherichia coli

<400> 4

atggctcaag gcacgcttta tattgtttct gcccccagtg gcgcgggtaa atccagcctg 60

attcaggctt tattaaaaac ccaaccgttg tatgacaccc aggtttctgt ttcacacacc 120

acacgccaac cgcgtcctgg tgaagtccac ggtgaacatt atttctttgt taatcatgat 180

gaatttaaag aaatgattag cagagatgcg ttcctcgaac acgcagaagt ttttggtaat 240

tactatggca cttcgcgtga ggccattgag caagtactgg cgaccggtgt cgatgttttt 300

ctcgatatcg actggcaggg cgcgcagcaa attcgccaga agatgccgca cgcgcggagt 360

atctttattt taccgccgtc caaaattgaa ctggaccgcc gtctacgcgg tcgcggtcag 420

gacagcgaag aggtcattgc aaagcgtatg gcgcaagctg ttgcagaaat gagccattac 480

gccgaatatg attatctgat tgtgaatgat gacttcgata ccgcgttgac cgatttgaag 540

accattattc gcgccgaacg tctgcgcatg agccgccaaa agcagcgtca tgacgcttta 600

atcagcaaat tgttggcaga c 621

<210> 5

<211> 429

<212> DNA

<213> Escherichia coli

<400> 5

atggctattg aacgtacttt ttccatcatc aaaccgaacg cggtagcaaa aaacgtcatt 60

ggtaatatct ttgcgcgctt tgaagctgca gggttcaaaa ttgttggcac caaaatgctg 120

cacctgaccg ttgaacaggc acgtggcttt tatgctgaac acgatggaaa accgttcttt 180

gatggtctgg ttgaattcat gacctctggc ccgatcgtgg tttccgtgct ggaaggtgaa 240

aacgccgttc agcgtcaccg cgatctgctg ggcgcgacca atccggcaaa cgcactggct 300

ggtactctgc gcgctgatta cgctgacagc ctgaccgaaa acggtaccca cggttctgat 360

tccgtcgaat ctgccgctcg cgaaatcgct tatttctttg gcgaaggcga agtgtgcccg 420

cgcacccgt 429

<210> 6

<211> 198

<212> DNA

<213> Artificial sequence

<400> 6

ggtggtggtg gttcaggtgg tggtggttca ggtggtggtg gttgtggtag cctgcgtgaa 60

tgtgaactgt atgttcagaa acataatatt caggccctgc tgaaagatag cattgttcag 120

ctgtgtaccg cacgtccgga acgtccgatg gcatttctgc gcgaatattt tgaacgtctg 180

gaaaaagaag aagccaaa 198

<210> 7

<211> 108

<212> DNA

<213> Artificial sequence

<400> 7

ggtggtggtg gttcaggtgg tggtggttca ggtggtggtg gttgtggtct ggaacagtat 60

gcaaatcagc tggcagatca gattatcaaa gaagcaaccg aaggttgc 108

<210> 8

<211> 415

<212> DNA

<213> Artificial sequence

<400> 8

taatacgact cactataggg gaattgtgag cggataacaa ttccccatct tagtatatta 60

gttaagtata agaaggagat atacatatgg cagatctggt ggtggtggtt caggtggtgg 120

tggttcaggt ggtggtggtt caggtggtgg tggttcaggt ggtggtggtt caggtggtgg 180

tggttgtggt ctggaacagt atgcaaatca gctggcagat cagattatca aagaagcaac 240

cgaaggttgc taaggtaccc tcgagtctgg taaagaaacc gctgctgcga aatttgaacg 300

ccagcacatg gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga 360

gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttg 415

<210> 9

<211> 386

<212> DNA

<213> Artificial sequence

<400> 9

taatacgact cactataggg gaattgtgag cggataacaa ttccccatct tagtatatta 60

gttaagtata agaaggagat atacatatgc tggaacagta tgcaaatcag ctggcagatc 120

agattatcaa agaagcaacc gaaggttgcg gtggtggtgg ttcaggtggt ggtggttcag 180

gtggtggtgg ttcaggtggt ggtggttcag gtggtggtgg ttcaggtacc ctcgagtctg 240

gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag 300

cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct 360

ctaaacgggt cttgaggggt tttttg 386

<210> 10

<211> 45

<212> DNA

<213> Artificial sequence

<400> 10

ggtggtggtg gttcaggtgg tggtggttca ggtggtggtg gttca 45

Claims (10)

1. An engineered escherichia coli strain for producing 2' -fucosyllactose is characterized in that genes related to catabolism of substrates L-fucose and lactose and key intermediate GDP-L-fucose endogenous to escherichia coli are silenced and expressed, key enzymes and 1,2-fucosyltransferase are synthesized by heterologous expression of GDP-L-fucose salvage pathway, and one or more of guanosine-inosine kinase, guanylate kinase and nucleotide diphosphate kinase are enhanced and expressed.

2. The engineered escherichia coli strain as claimed in claim 1, wherein the key enzyme for synthesis of GDP-L-fucose salvage pathway is derived from Bacteroides fragiliss 9343; the 1,2-fucosyltransferase is derived from Helicobacter pylori.

3. The engineered escherichia coli strain of claim 2, wherein the genes endogenous to escherichia coli and involved in catabolism of substrates L-fucose and lactose and a key intermediate GDP-L-fucose comprise genes encoding β -galactosidase, L-fucose isomerase, L-fucose kinase, D-arabinose isomerase, L-rhamnose isomerase, and UDP-glucose lipid carrier transferase.

4. The engineered Escherichia coli as claimed in claim 3, wherein C-terminal or N-terminal of GDP-L-fucose salvage pathway synthesis key enzyme and 1,2-fucosyltransferase of the target protein is modified with short peptide; the short peptide is RIDD and RIAD.

5. The engineered Escherichia coli as claimed in claim 4, wherein the short peptide is linked to the target protein by a linker peptide.

6. The engineered Escherichia coli as claimed in claim 5, wherein the guanosine-inosine kinase, guanylate kinase and nucleotide diphosphate kinase are expressed using a vector pACM 4.

7. A method for improving the production capacity of 2' -fucosyllactose of Escherichia coli is characterized in that GDP-L-fucose salvage pathway synthesis key enzyme and 1,2-fucosyltransferase are expressed in Escherichia coli in a heterologous mode, and short peptides RIDD and RIAD are used for modifying the GDP-L-fucose salvage pathway synthesis key enzyme and 1, 2-fucosyltransferase.

8. The method of claim 7, wherein the C-terminus of the 1,2-fucosyltransferase is modified by RIDD and the C-terminus or N of the key enzyme in the synthesis of GDP-L-fucose salvage pathway is modified by RIAD.

9. A method for producing 2 ' -fucosyllactose is characterized in that Escherichia coli engineering bacteria as claimed in claim 2 are used as fermentation microorganisms, glycerol is used as a carbon source, L-fucose and lactose are used as substrates to synthesize 2 ' -fucosyllactose, and fermentation is carried out to produce 2 ' -fucosyllactose.

10. Use of the strain according to claims 1-4 for the preparation of 2' -fucosyllactose and derivatives thereof.

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