High-throughput screening method of alpha-1, 2-fucosyltransferase and mutant obtained by screening

Abstract

The invention relates to a high-throughput screening method of alpha-1, 2-fucosyltransferase and a mutant obtained by screening, and belongs to the technical field of enzyme engineering. The invention provides a high-throughput screening method of alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase). The method comprises the following steps: constructing an indicator strain of 2 '-fucosyllactose, enabling the content of the 2'-fucosyllactose to be positively correlated with the fluorescence intensity, then expressing an L-fucokinase and alpha-1, 2-fucosyltransferase mutant library in a chassis strain, carrying out water-in-oil droplet embedding on the chassis strain, and carrying out fusion co-culture on the chassis strain and the indicator strain to obtain the 2 '-fucosyllactose. A chassis strain containing the alpha-1, 2-fucosyltransferase forward mutant is obtained through droplet microfluidic sorting according to a fluorescence detection result, the enzyme activity of the mutant FutCV93I is 2.31 times that of an original enzyme, and the enzyme activity is greatly improved. By using the method provided by the invention, the alpha-1, 2-fucosyltransferase can be subjected to high-throughput screening, and industrial production of the 2 '-fucosyllactose and large-scale application and popularization of the 2'-fucosyllactose in the fields of foods, medicines and the like are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to a high-throughput screening method for alpha-1,2-fucosyltransferase and mutants obtained by screening. Background Art

[0002] Fucosyltransferases (FucTs) play important roles in organisms and are involved in a variety of physiological and pathological processes, including inflammation, bacterial and viral infections, tumor metastasis, and genetic disorders. These enzymes catalyze the transfer of L-fucose from donor substrates to various sugar acceptor substrates, including oligosaccharides, glycoproteins, and glycolipids, thereby affecting the functions of these biomolecules. Fucosyltransferases also show their importance in disease diagnosis and drug development. For example, in some cancer patients, fucosyltransferase activity is high, and the activity of this enzyme can be detected to assist in tumor diagnosis and efficacy monitoring. Among them, α-1,2 fucosyltransferase (FutC) occupies an important position because it can catalyze the synthesis of 2'-fucosyllactose (2'-FL), a compound with multiple physiological functions, including inhibiting pathogen infection, regulating intestinal flora, and enhancing immunity. 2'-FL has been approved as a nutrient in infant formula and special needs foods. α-1,2-fucosyltransferase from Helicobacter pylori is currently the most widely used enzyme in 2'-FL biosynthesis, but its catalytic activity is low and heterologous expression is poor in microbial expression systems, which limits the synthesis efficiency of 2'-FL.

[0003] In order to improve the catalytic performance and expression of α-1,2-fucosyltransferase, directed evolution has become an effective strategy. Directed evolution simulates the Darwinian evolution process, creates a large number of mutations artificially through random mutation and recombination, and screens out proteins with desired characteristics by applying selection pressure according to specific needs and purposes. For example, the catalytic activity and thermal stability of Helicobacter pylori α-1,2-fucosyltransferase can be improved through semi-rational design of site saturation mutations. This strategy is of great significance for solving the technical bottleneck of FutC and the industrial application of 2'-FL.

[0004] In the process of directed evolution, it is indispensable to construct a high-throughput screening method. At present, researchers have developed a variety of photochemical analysis techniques based on the ultraviolet or fluorescent properties of substrates, products or their corresponding derivative compounds. These methods have been widely used in enzyme engineering, metabolic engineering and other fields. However, there is currently no reasonable and efficient high-throughput screening method for screening highly active α-1,2 fucosyltransferases, which limits the development of 2'-fucosyllactose biosynthesis. Therefore, the development of new high-throughput screening methods is crucial for the discovery and improvement of α-1,2 fucosyltransferases, which will help improve the production efficiency and reduce the cost of 2'-FL, and further promote its application in the food and pharmaceutical fields. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a high-throughput screening method for α-1,2-fucosyltransferase, comprising constructing an indicator strain of 2'-fucosyllactose so that the fluorescence intensity is positively correlated with 2'-fucosyllactose, expressing β-galactosidase, L-fucokinase and an α-1,2-fucosyltransferase mutant library in a chassis strain, embedding the chassis strain in oil-in-water droplets and co-culturing it with the indicator strain, and obtaining positively mutant α-1,2-fucosyltransferase mutants by droplet microfluidic sorting.

[0006] The first object of the present invention is to provide a high-throughput screening method for α-1,2-fucosyltransferase, comprising the following steps:

[0007] Step S1, constructing an indicator strain of 2'-fucosyllactose, wherein the indicator strain comprises a first expression vector and a second expression vector, wherein the first expression vector comprises an α-L-fucosidase encoding gene, and the second expression vector comprises a lactose operon and a fluorescent reporter gene, wherein the α-L-fucosidase degrades 2'-fucosyllactose into lactose, and combines with the lactose operon to regulate the expression of the fluorescent reporter gene, so that the 2'-fucosyllactose content is coupled to the fluorescence intensity;

[0008] Step S2, constructing a chassis strain, wherein the chassis strain expresses an L-fucokinase encoding gene and an α-1,2-fucosyltransferase mutant library;

[0009] Step S3, performing single-cell droplet embedding and droplet culture on the chassis strain to obtain a droplet microfluidic chip, wherein the droplet culture is performed in a culture medium containing fucose and lactose;

[0010] Step S4, fusing the indicator strain suspension with the droplets one-to-one, and after co-culturing, performing droplet microfluidic sorting according to the fluorescence intensity in the fused droplets to obtain a chassis strain containing the α-1,2-fucosyltransferase forward mutant.

[0011] Furthermore, in step S1, the first expression vector further comprises a gene encoding a signal peptide MalE.

[0012] Furthermore, in step S2, the chassis strain also includes a temperature-controlled expression vector, and the temperature-controlled expression vector regulates the expression of the β-galactosidase encoding gene.

[0013] Furthermore, in step S3, the droplets are first cultured at a first temperature to allow the chassis strain to convert fucose and lactose into 2'-fucosyllactose, and then the droplets are cultured at a second temperature until the chassis strain completely degrades the lactose.

[0014] Furthermore, the temperature-controlled expression vector is pBV220.

[0015] Furthermore, in step S2, the chassis strain knocks out the UDP-glucose lipid carrier transferase gene wcaJ.

[0016] Furthermore, in step S3, the droplets are water-in-oil droplets.

[0017] Furthermore, the oil phase was 2% Pico-Surf in Novec 7500.

[0018] Furthermore, in step S4, the absorbance of the indicator strain suspension at 600 nm is 10-12.

[0019] In one embodiment of the present invention, the absorbance value of the indicator strain suspension at 600nm is 10.

[0020] The second object of the present invention is to provide an α-1,2-fucosyltransferase mutant, wherein the amino acid sequence of the starting sequence shown in SEQ ID NO.X is modified in any of the following ways:

[0021] (1) Valine at position 93 mutated to isoleucine;

[0022] (2) glutamic acid at position 50 mutated to glycine;

[0023] (3) Lysine 81 mutated to methionine, and valine 173 mutated to isoleucine;

[0024] (4) Lysine at position 102 was mutated to glutamic acid, and tryptophan at position 284 was mutated to cysteine.

[0025] The third object of the present invention is to provide a gene encoding the above-mentioned α-1,2-fucosyltransferase mutant.

[0026] The fourth object of the present invention is to provide an expression vector comprising the above gene.

[0027] The fifth object of the present invention is to provide a host cell comprising the above α-1,2-fucosyltransferase mutant, the above gene or the above expression vector.

[0028] Furthermore, the host cell is a bacterium or a fungus.

[0029] Furthermore, the host cell is Escherichia coli.

[0030] In one embodiment of the present invention, the host cell is Escherichia coli K12 MG1655.

[0031] The sixth object of the present invention is to provide the use of the above-mentioned α-1,2-fucosyltransferase mutant, the above-mentioned gene, the above-mentioned expression vector or the above-mentioned host cell in the production of 2'-fucosyllactose.

[0032] The seventh object of the present invention is to provide an Escherichia coli for producing 2'-fucosyllactose, wherein the Escherichia coli has knocked out the β-galactosidase encoding gene and the UDP-glucose lipid carrier transferase encoding gene, and heterologously expresses the L-fucokinase encoding gene and the α-1,2-fucosyltransferase mutant described in claim 9.

[0033] The eighth object of the present invention is to provide a microbial agent comprising the above-mentioned Escherichia coli.

[0034] The ninth object of the present invention is to provide a method for producing 2'-fucosyllactose, comprising the step of fermenting the Escherichia coli according to claim 14 or the microbial agent according to claim 15.

[0035] Beneficial effects of the present invention:

[0036] The present invention provides a method for high-throughput screening of α-1,2-fucosyltransferase mutants. By constructing a 2'-fucosyllactose indicator strain and a chassis strain, and using droplet microfluidic screening technology, rapid screening of α-1,2-fucosyltransferase mutants is achieved, thereby improving screening efficiency. Through the positive correlation between the fluorescence intensity of the fluorescent reporter gene and the 2'-fucosyllactose content, the activity of α-1,2-fucosyltransferase can be evaluated intuitively and quickly, thereby screening out chassis strains with high production of 2'-fucosyllactose. Through the temperature-controlled expression vector, the transcription of the β-galactosidase encoding gene can be controlled at different temperatures, which solves the noise interference that may be caused by the substrate lactose and further optimizes the screening process. The mutant FutC obtained by screening using the method provided by the present invention V93IThe enzyme activity is 2.31 times that of FutC, which improves the screening efficiency of α-1,2-fucosyltransferase and the production efficiency of 2'-fucosyllactose. It has important industrial application prospects and is conducive to the industrial production of 2'-fucosyllactose and its large-scale application and promotion in food and medicine and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0038] Figure 1 This is the plasmid map of pJ23110-MalE-AFCA in Example 1 of the present invention;

[0039] Figure 2 This is the plasmid map of pACYC-T7lac-sfGFP in Example 1 of the present invention;

[0040] Figure 3 is the fluorescence response curve of the 2'-fucosyllactose indicator strain BL01 in Example 1 of the present invention;

[0041] Figure 4 This is a schematic diagram of the salvage synthesis pathway of 2'-fucosyllactose in Example 2 of the present invention;

[0042] Figure 5 This is the plasmid map of pRSF-trc-futC-fkp in Example 2 of the present invention;

[0043] Figure 6 This is the fermentation verification of the α-1,2-fucosyltransferase screening chassis strain in Example 2 of the present invention;

[0044] Figure 7 This is a schematic diagram of the screening process of the high-throughput screening platform for α-1,2-fucosyltransferase in Example 3 of the present invention;

[0045] Figure 8 The pET28a-gst-futc plasmid map used for protein expression in Example 4 of the present invention;

[0046] Fig. 9 This is a graph showing the gel electrophoresis results of the FutC protein and its mutants in Example 4 of the present invention;

[0047] Fig.10 This is a diagram showing the enzyme activity and enzyme amount detection results of the α-1,2-fucosyltransferase machinery mutant in Example 4 of the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0049] The temperature-controlled plasmid pBV220 used in the example is disclosed in the literature λpL / pR-cI 857 Modification of temperature control system and its effect on the preparation of Escherichia coli exuviae[J]. Chinese Journal of Biotechnology, 2012, 28(12): 1423-1430.

[0050] The culture medium and mother solution involved in the embodiment are as follows:

[0051] LB liquid medium: yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L;

[0052] TB solid medium: peptone 12 g / L, yeast extract 24 g / L, glycerol 4 mL / L, potassium dihydrogen phosphate 2.31 g / L, potassium dihydrogen phosphate trihydrate 16.43 g / L;

[0053] Fucose mother solution: fucose 150g / L;

[0054] Lactose mother liquor: lactose 200g / L.

[0055] The α-1,2-fucosyltransferase activity and 2'-fucosyllactose detection methods involved in the examples are as follows:

[0056] (1) The activity of wild-type FutC and its mutants was determined by measuring the titer of 2'-FL. 300 μL of the reaction mixture (containing 50 mM acetate buffer pH = 6.0, 2 mM GDP-L-fucose, 5 mM lactose, 2 mM divalent magnesium ions and 200 μg of pure enzyme) was incubated at 37°C for 20 minutes. The reaction was terminated by boiling the reaction solution for 10 minutes. Under standard experimental conditions, the enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol 2'-FL per minute;

[0057] (2) Take 1 mL of the fermentation broth or the above reaction solution, centrifuge at 10,000 rpm for 10 min, and take the supernatant for HPLC determination of 2'-FL content.

[0058] HPLC detection conditions: by high performance liquid chromatography (HPLC) system (Waters e2695); chromatographic column: RezexROA-Organic acid H + (8%); detector: Waters 2414RIDetector differential detector; mobile phase: 5mM H2SO4; flow rate: 0.6mL / min; column temperature: 60℃; injection volume: 10μL.

[0059] The strains involved in the embodiment are as follows:

[0060] Table 1 Strains and genotypes

[0061]

[0062] The primer sequences involved in the embodiment are as follows:

[0063] Table 2 Primers and sequences

[0064]

[0065]

[0066] Example 1: 2'-fucosyllactose indicator strain

[0067] (1) Obtaining plasmid pJ23110-MalE-AFCA

[0068] Using P J23110 The combined promoter (nucleotide sequence as shown in SEQ ID NO.1) expresses the signal peptide MalE (amino acid sequence as shown in SEQ ID NO.2) and the AfcA protein encoding gene (nucleotide sequence as shown in SEQ ID NO.3), wherein α-L-fucosidase AfcA can specifically degrade 2'-fucosyllactose to obtain lactose, and the signal peptide MalE can efficiently transport the AfcA protein to the periplasmic space, and the specific steps are as follows:

[0069] The gene sequence of afcA from Bifidobacterium bifidum was searched through NCBI, and the DNA sequence after codon optimization was sent to Suzhou Jinweizhi for synthesis. Then, the synthetic gene afcA and plasmid p15A-PJ23110 were PCR amplified using primers AfcA-F / AfcA-R and PJ23110-F / PJ23110-R, respectively. The PCR products were connected using a seamless cloning kit, and the resulting plasmid was named pJ23110-AFCA. The plasmid pJ23110-AFCA was subjected to full plasmid PCR using primers male-F / male-R with homology arms, and the PCR products were purified and transferred into E.coli JM109 competent cells. The correct positive clone was verified to be pJ23110-MalE-AFCA (map as shown in Figure 1 The plasmid was extracted using a plasmid extraction kit.

[0070] (2) Obtaining plasmid pACYC-T7lac-sfGFP

[0071] Using pACYCDuet-1 plasmid as the backbone, use P T7lac The inducible promoter (nucleotide sequence is shown in SEQ ID NO.4) expresses the fluorescent reporter gene sfgfp (nucleotide sequence is shown in SEQ ID NO.5), and the specific steps are as follows:

[0072] The green fluorescent protein gene sfgfp and pACYCDuet-1 were amplified by PCR using primers GFP-F / GFP-R and PACY-F / PACY-R, respectively. The PCR products were connected using a seamless cloning kit to obtain the plasmid pACYC-T7lac-sfGFP (see Figure 1). Figure 2 The clone was transformed into E. coli JM109 competent cells, and the correct positive clone was verified to be pACYC-T7lac-sfGFP, and the plasmid was extracted using a plasmid extraction kit.

[0073] (3) Construction and identification of 2'-fucosyllactose indicator strain

[0074] The plasmids pJ23110-MalE-AFCA and pACYC-T7lac-sfGFP constructed in step (1) and step (2) are co-transformed into competent cells of Escherichia coli BL21 (DE3), and plated into the corresponding double-resistance LB medium (streptomycin and chloramphenicol resistance). The normally growing transformant is the 2'-fucosyllactose indicator strain BL01. The lacI gene provided by the pACYC-T7lac-sfGFP plasmid expresses the LacI repressor protein, which binds to the lacO fragment to inhibit the transcription of the fluorescent reporter gene sfgfp. When the LacI repressor protein binds to the lactose obtained by the degradation of 2'-fucosyllactose provided by the pJ23110-MalE-AFCA plasmid, the transcription inhibition of the fluorescent reporter gene is released. Therefore, the indicator bacteria can be used to sense the concentration of 2'-fucosyllactose to linearly regulate the transcription intensity of the fluorescent reporter gene.

[0075] The LB liquid medium was dispensed into a 96-well microplate, 200 μL of the medium was added to each well, and then 2 μL of the BL01 seed solution containing 2'-fucosyllactose indicator strain was inoculated. The culture was continued on a 96-well microplate constant temperature shaking platform at 37°C and 900 rpm until the OD 600 =0.6, add different concentrations of 2'-FL (0, 62.5, 125, 250, 500, 1000, 2000, 4000 mg / L), continue to culture on a well plate shaker for 6 hours, and detect cell density and fluorescence value. The fluorescence intensity of the culture was detected by an ELISA reader, with a gain value of 60, an excitation wavelength of 480 nm, and an emission wavelength of 530 nm. The OD 600 The fluorescence level of a single cell is expressed as fluorescence value / OD600 To characterize.

[0076] The fluorescence response curve of 2'-fucosyllactose indicator strain BL01 is shown in Figure 3 When the concentration of 2'-FL is 62.5-4000 mg / L, the output fluorescence signal value is positively correlated with the concentration of 2'-FL. When the concentration of 2'-FL is 62.5 mg / L, the Fl / OD value of the sensor is 600 The value is 687±17. When the concentration of 2'-FL is 4000mg / L, the Fl / OD 600 The value was 69457±4000, which was 101.02 times that of 62.5 mg / L. The data were fitted nonlinearly using the logistic model, and the fitting equation was obtained: y=13885-13376 / (1+(x / 530)1.18)(R 2 =0.999). Therefore, the working range of the 2'-fucosyllactose indicator strain is 62.5-4000 mg / L.

[0077] Afterwards, in order to verify the accuracy and credibility of the indicator strain, 6 different concentrations of 2'-FL samples were prepared, and the indicator strain and HPLC were used for analysis and comparison. The results are shown in Table 3. The corresponding 2'-FL concentration was obtained by converting the obtained fluorescence ratio using the above fitting equation, and the error between the obtained fluorescence ratio and the standard value (HPLC) was no more than 5%, indicating that the 2'-FL indicator strain constructed in this example has a high credibility.

[0078] Table 3 Accuracy evaluation of 2'-FL indicator strains

[0079]

[0080]

[0081] Example 2: Construction of α-1,2-fucosyltransferase screening chassis strain

[0082] (1) Obtaining E. coli MG1655ΔlacZΔwcaJ strain

[0083] Using Escherichia coli K12 MG1655 as the starting strain, the β-galactosidase gene lacZ (nucleotide sequence as shown in Gene ID: 945006) and UDP-glucose lipid carrier transferase gene wcaJ (nucleotide sequence as shown in Gene ID: 946583) of the starting strain were knocked out. For details on the gene knockout method, please refer to the patent publication number CN110804577A, "A method for constructing a recombinant bacterium that efficiently produces 2'-fucosyllactose and its application", to obtain the recombinant strain E. coli MG1655ΔlacZΔwcaJ, named MGZ1. lacZ and wcaJ are both bypass pathway genes of the 2'-fucosyllactose salvage synthesis pathway. Knocking out these two genes can increase the utilization rate of lactose and GDP-L-fucose in the 2'-FL metabolic pathway, further directing the metabolic flow to the target metabolite, thereby further increasing the production of 2'-FL. The process of the 2'-fucosyllactose salvage synthesis pathway is as follows Figure 4 shown.

[0084] (2) Obtaining plasmid pRSF-trc-futC-fkp

[0085] Using the Escherichia coli genome as a template, primers fkp-F / fkp-R were used for PCR amplification to obtain the fkp gene (nucleotide sequence as shown in SEQ ID NO.6) for expressing L-fucokinase FKP, which can convert fucose into GDP-L-fucose, and then primers RSF-F / PSF-R were used to PCR amplify the plasmid pRSFDuet-1. The amino acid sequence of FutC from Helicobacter pylori (ATCC NO.26695) was searched through NCBI, and the DNA sequence after codon optimization was sent to Suzhou Jinweizhi for synthesis. Primers FutC-F / FutC-R were used with FutC (nucleotide sequence as shown in SEQ ID NO.7, amino acid sequence as shown in SEQ ID NO.8, used to express α-1,2-fucosyltransferase to convert GDP-L-fucose and lactose into 2'-fucosyllactose) as a template, and the QuickMutation TM FutC was mutated using a random gene mutation kit to construct a mutant library. The PCR products were connected using a seamless cloning kit and transferred into E. coli JM109 competent cells after purification. The correct positive clone was verified to be pRSF-trc-futC-fkp (plasmid map as shown in Figure 5 As shown), and the plasmid was extracted by a plasmid extraction kit, and the obtained plasmid was the mutant library plasmid.

[0086] (3) Obtaining plasmid pbv220-TB-lacZ

[0087] Using the Escherichia coli genome as a template, primers lacZ-F / lacZ-R were used for PCR amplification to obtain the lacZ gene (nucleotide sequence as shown in Gene ID: 945006), and then primers pbv220-F / pbv220-R were used to amplify the temperature-controlled plasmid pbv220 by PCR. The PCR products were connected using a seamless cloning kit and transferred into E. coli JM109 competent cells after purification. The correct positive clone was verified to be pbv220-TB-lacZ, and the plasmid was extracted using a plasmid extraction kit. The resulting plasmid consisted of P L Promoter-regulated expression of CI 857 Gene (nucleotide sequence as shown in SEQ ID NO.9), by P R The promoter regulates the expression of lacZ gene, making CI 857 The repressor protein of gene expression is in contact with P at 37℃ R The promoter binds to inhibit the transcription of the lacZ gene and releases the transcriptional inhibition of the lacZ gene at 42°C.

[0088] (4) Verification of α-1,2-fucosyltransferase screening chassis strain

[0089] The plasmids obtained in steps (2) and (3) were co-transformed into E. coli MG1655ΔlacZΔwcaJ competent cells to obtain the engineered strain MGZ2. MGZ2 carrying the original protein was inoculated into LB liquid medium and cultured overnight at 37°C, 200 rpm for 12 h to obtain seed solution. 500 μL of the seed solution was inoculated into 25 mL of TB medium and cultured at 37°C, 200 rpm until OD 600 The final concentration of IPTG was 0.6-0.8, and 3 g / L lactose and 2 g / L fucose were added at the final concentration. The induction culture was continued at 37°C and 200 rpm for 72 h, and then the culture temperature was adjusted to 42°C for 12 h. 1 mL of the fermentation broth was taken and centrifuged at 10,000 rpm for 10 min. The supernatant was taken and used for HPLC to determine the production of 2'-fucosyllactose and lactose. The results are as follows: Figure 6 As shown. The final concentration of 2'-fucosyllactose was 0.11 g / L and the final concentration of lactose was 0, indicating that MGZ2 could inhibit the transcription of lacZ gene at 37°C to generate 2'-fucosyllactose, and could also release the transcription inhibition of lacZ gene at 42°C to completely degrade lactose, thus solving the noise interference that could be caused by the substrate lactose and ensuring that all lactose in the screening process was obtained by the degradation of 2'-fucosyllactose by the AfcA protein in the indicator strain, thus meeting the screening requirements.

[0090] Example 3: Construction of recombinant strains Droplet microfluidic screening

[0091] (1) Optimization of droplet generation conditions

[0092] Wash the α-1,2-fucosyltransferase screening chassis strain MGZ2 three times with LB medium and resuspend it. 600 The value was adjusted to 0.05, and single-cell droplets were prepared using a droplet generation chip. The aqueous phase was a TB culture medium suspension containing MGZ2 bacteria; the oil phase was 2% Pico-Surf in FC40, 2% Pico-Surf in Novec 7500 or Bio-Rad droplet generation oil. The oil phase and the aqueous phase were respectively aspirated into a 1 mL sterile syringe, which was installed in a microinjection pump and connected to the chip with a catheter. The oil phase flow rate was set to 5 μL / min and the water phase flow rate was set to 2 μL / min. Single-cell droplets with a diameter of about 30 μm were generated and collected with a 1 mL sterile syringe. The syringe containing the single-cell droplets was placed in a 37°C incubator for incubation, and the droplets were sampled and observed at different times to evaluate the droplet stability. The most stable oil phase droplet was selected for subsequent experiments.

[0093] The most stable phase obtained in the above experiment was 2% Pico-Surf in Novec 7500. The MGZ2 strain was used to generate droplets according to the above conditions (the resuspension medium was TB medium, and 0.3 mM IPTG, 3 g / L lactose and 2 g / L fucose were added at final concentrations). The syringe containing the single-cell droplets was placed in an incubator at 37°C for 72 h and 42°C for 12 h, respectively, to eliminate the interference of the substrate lactose and carry out subsequent experiments.

[0094] (2) Optimization of droplet injection conditions

[0095] Sterile water was used as the aqueous phase and 2% Pico-Surf in Novec 7500 was used as the oil phase to generate oil-in-water single emulsion droplets. The droplets were collected with a 1 mL sterile syringe and re-injected into the microfluidic chip. Under the action of the electric field force, the sensor bacterial suspension merged with the passing droplets one-to-one at a rate of 0.8 μL / min. The fused droplets were collected and cultured in a 37°C droplet microfluidic incubator. After 24 hours, the samples were taken out and the injection of the strains in the droplets was observed under the bright and dark fields of a fluorescence microscope. The reporter strain concentration in which each droplet expressed green fluorescence and the droplet size was stable was selected as the optimal concentration. At this time, the OD of the indicator strain was 600 =10.

[0096] (3) Droplet screening

[0097] The collected single-cell droplets were placed in a 37°C incubator for culture. The culture medium was TB culture medium. Samples were taken at different times to observe the fluorescence changes in the droplets, and the appropriate culture time (12 hours) was selected for droplet microfluidic screening. During screening, the syringe containing the oil phase and droplets was installed in the microinjection pump, and the syringe outlet was connected to the corresponding inlet of the microfluidic droplet sorting chip with a catheter. The oil phase flow rate was set to 10μL / min and the droplet flow rate was set to 0.5μL / min. At this time, the droplet flow rate was about 500-1000 per second. Aim the blue laser (488nm) at the detection point of the screening chip, and collect the green fluorescence signal (520nm) of each droplet. The droplets with the highest fluorescence signal of about 3‰ were powered on, and the voltage was set to 300V or 500V, so that the target droplets flowed to the collection outlet of the screening chip under the action of electrostatics, and the remaining droplets flowed out from the waste liquid outlet. The collected droplets were spread on the LB plate. The screening process is as follows Figure 3 As shown, the strains obtained by screening were sequenced to obtain the mutant FutC V93I 、FutC K81M / V173I 、FutC E50G and FutC K102E / W284C .

[0098] Example 4: Enzyme activity assay

[0099] (1) Recombinant protein expression

[0100] The recombinant wild-type FutC and its mutant proteins obtained in Example 3 were overexpressed in Escherichia coli BL21 (DE3), respectively.

[0101] The GST protein tag was amplified by PCR using primers gst-f / gst-r, the recombinant wild-type FutC and its mutants were amplified by PCR using primers gst-futc-f / gst-futc-r, and the plasmid vector pET28a was amplified by PCR using primers pet28a-f / pet28a-r. The PCR products were connected by seamless cloning kit to obtain plasmid pET28a-gst-futc (plasmid map as shown in Figure 8 As shown). Transform into E.coli JM109 competent cells, verify that the correct positive clone is pET28a-gst-futc and extract the plasmid using a plasmid extraction kit. Transform the constructed plasmid pET28a-gst-futc into E.coli BL21 (DE3) competent cells, plate them in LB medium, and the normally growing transformants are futC overexpression strains BLM0-BLM4.

[0102] The cells were first grown in LB medium at 37°C with shaking (220 rpm) overnight. 4 mL of fresh cells grown in LB were transferred to fresh TB medium (200 mL) in a 500 mL non-baffled flask and incubated until the OD 600 IPTG with a final concentration of 0.3 mM was added to the culture medium to induce the expression of the target protein. After incubation at 20°C for 20 h, the recombinant cells were collected by centrifugation at 8000 g for 10 min.

[0103] (2) Protein purification and concentration detection

[0104] The recombinant FutC protein and its mutants were expressed by Ni 2+ Affinity chromatography separation and purification. The cell pellet was resuspended in lysis buffer and crushed on ice with an ultrasonic machine, turned on for 1s, turned off for 2s, and continued for 20min. The crushed mixture was centrifuged at 8000g for 10min and filtered with a 0.45μm filter to obtain a crude enzyme solution. The collected supernatant was uploaded to GST agarose purification resin (Sangong Bio, Shanghai) and balanced with 2 column volumes of protein purification A solution (20mM Tris-HCI buffer, pH = 8.0, adjusted to pH 6.5 with concentrated hydrochloric acid, made up to 2L with ultrapure water, and filtered with a 0.45μM filter). Then, 10% protein purification B solution (500mM imidazole, 20mM Tris-HCI buffer, pH = 8.0), adjusted to pH 6.5 with concentrated hydrochloric acid, made up to 2L with ultrapure water, and filtered with a 0.45μM filter to elute unbound proteins. Finally, the target protein was eluted with 100% protein purification solution B at pH 6.5. The concentration of the purified enzyme was determined using a BCA protein assay kit (Beyotime, Shanghai, China). The results are shown in Table 4.

[0105] Table 4 Protein concentrations corresponding to enzymes and mutants

[0106] Enzymes and mutants Protein concentration (mg / mL) FutC 0.55 FutCV93I 0.59 FutCK81M / V173I 0.61 FutCE50G 0.55 FutCK102E / W284C 0.54

[0107] (3) SDS-PAGE analysis

[0108] Protein sample acquisition and processing: break the bacterial cell wall, take 1-2mL of bacterial liquid, centrifuge at 4000g, 4℃ for 4min, pour out the supernatant and use lysis buffer (20mM Tris-HCI buffer pH value is 8.0, use concentrated hydrochloric acid to adjust the pH value to 6.5, and use ultrapure water to make up to 2L) to gently blow the cells, and repeat the above operation twice. Place the washed sample on the MPFastprep-24 5G fast sample preparation instrument for 40s. The treated liquid is centrifuged at 12000rpm, 4℃ for 2min. The supernatant is the protein sample. Sample processing: The protein sample obtained above is mixed with the Thermo Fisher NuPAGE TM Mix well with 4×LDS sample buffer, then denature in a metal bath at 98°C for 10 minutes and cool to room temperature before use. Gel electrophoresis operation: Fix the protein precast gel in the protein electrophoresis tank, add SDS-PAGE buffer to cover the protein gel wells, pipette an appropriate amount of processed protein sample and Protein Maker into the protein gel wells, set the voltage to 120V, and 120min. Protein gel staining and decolorization operation: Place the protein gel in a gel box with an appropriate amount of Coomassie Brilliant Blue dye, stain for 30 minutes, pour the dye solution back into the bottle after staining, add clean water or decolorization solution to decolorize, and when the protein bands are clearly visible, place it in the gel imaging system for observation. The protein gel results are as shown below. Fig. 9 shown.

[0109] (4) Enzyme activity detection

[0110] The activity of wild-type FutC and its mutants was determined by detecting the 2'-FL titer. The reaction solution was 300 μL, consisting of 20 mM Tris-HCl buffer (pH = 6.5), 2 mM GDP-L-fucose, 5 mM lactose, 2 mM Mn 2+ and 200 μg of pure enzyme, incubated at 37°C for 20 min. The reaction was terminated by boiling for 10 min. Under standard experimental conditions, the enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol 2'-FL per minute. The concentrations of various carbohydrates (including lactose, GDP-L-fucose and 2'-fucosyllactose) in the reaction solution were analyzed by high performance liquid chromatography. The results are shown in Fig.10 shown.

[0111] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A high-throughput screening method for α-1,2-fucosyltransferase, characterized in that: The following steps are involved: Step S1, constructing an indicator strain of 2'-fucosyllactose, wherein the indicator strain comprises a first expression vector and a second expression vector, wherein the first expression vector comprises an α-L-fucosidase encoding gene, and the second expression vector comprises a lactose operon and a fluorescent reporter gene, wherein the α-L-fucosidase degrades 2'-fucosyllactose into lactose, and combines with the lactose operon to regulate the expression of the fluorescent reporter gene, so that the 2'-fucosyllactose content is coupled to the fluorescence intensity; Step S2, constructing a chassis strain, wherein the chassis strain expresses an L-fucokinase encoding gene and an α-1,2-fucosyltransferase mutant library; Step S3, performing single-cell droplet embedding and droplet culture on the chassis strain to obtain a droplet microfluidic chip, wherein the droplet culture is performed in a culture medium containing fucose and lactose; Step S4, fusing the indicator strain suspension with the droplets one-to-one, and after co-culturing, performing droplet microfluidic sorting according to the fluorescence intensity in the fused droplets to obtain a chassis strain containing the α-1,2-fucosyltransferase forward mutant.

2. The high-throughput screening method according to claim 1, characterized in that: In step S1, the first expression vector further comprises a gene encoding a signal peptide MalE.

3. The high-throughput screening method according to claim 1, characterized in that: In step S2, the chassis strain also includes a temperature-controlled expression vector, and the temperature-controlled expression vector regulates the expression of the β-galactosidase encoding gene.

4. The high-throughput screening method according to claim 3, characterized in that: In step S3, the droplets are first cultured at a first temperature to allow the chassis strain to convert fucose and lactose into 2'-fucosyllactose, and then the droplets are cultured at a second temperature until the chassis strain completely degrades the lactose.

5. The high-throughput screening method according to claim 3, characterized in that: The temperature-controlled expression vector is pBV220.

6. The high-throughput screening method according to claim 1, characterized in that: In step S2, the chassis strain knocks out the UDP-glucose lipid carrier transferase gene wcaJ.

7. The high-throughput screening method according to claim 1, characterized in that: In step S3, the droplets are water-in-oil droplets, and the oil phase is 2% Pico-Surf in Novec 7500.

8. The high-throughput screening method according to claim 1, characterized in that: In step S4, the absorbance of the indicator strain suspension at 600 nm is 10-12.

9. An α-1,2-fucosyltransferase mutant, characterized in that: The amino acid sequence of SEQ ID NO.8 is modified by any of the following methods: (1) Valine at position 93 mutated to isoleucine; (2) glutamic acid at position 50 mutated to glycine; (3) Lysine 81 mutated to methionine, and valine 173 mutated to isoleucine; (4) Lysine at position 102 was mutated to glutamic acid, and tryptophan at position 284 was mutated to cysteine. 10 . A gene encoding the α-1,2-fucosyltransferase mutant according to claim 9 .

11. An expression vector comprising the gene according to claim 10. 12 . A host cell comprising the α-1,2-fucosyltransferase mutant of claim 9 , the gene of claim 10 , or the expression vector of claim 11 .

13. Use of the α-1,2-fucosyltransferase mutant of claim 9, the gene of claim 10, the expression vector of claim 11 or the host cell of claim 12 in producing 2'-fucosyllactose.

14. An Escherichia coli for producing 2'-fucosyllactose, characterized in that: The Escherichia coli knocks out the β-galactosidase encoding gene and the UDP-glucose lipid carrier transferase encoding gene, and heterologously expresses the L-fucokinase encoding gene and the α-1,2-fucosyltransferase mutant according to claim 9.

15. A microbial agent comprising the Escherichia coli according to claim 14.

16. A method for producing 2'-fucosyllactose, characterized in that: The method comprises the steps of using the Escherichia coli described in claim 14 or the microbial agent described in claim 15 for fermentation production.

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