Alpha-1, 3-fucosyltransferase, genetically engineered bacteria for producing 3-fl and construction and application thereof

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to an alpha-1, 3-fucosyltransferase, a genetically engineered bacterium for producing 3-FL and construction and application thereof. The present application provides a novel alpha-1, 3-fucosyltransferase, and provides a new route for the synthesis of 3-FL. The present application further provides a genetically engineered bacterium capable of encoding and expressing the novel alpha-1, 3-fucosyltransferase, and the yield of 3-FL can be improved.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to an α-1,3-fucosyltransferase, a genetically engineered bacterium for producing 3-FL, and their construction and application. Background Technology

[0002] 3-Fucose-based lactose (3-FL) is an important human milk oligosaccharide (HMO) abundant in breast milk and plays a vital role in infant health. 3-FL possesses various biological activities, including promoting the growth of beneficial bacteria, inhibiting harmful bacteria, and regulating the immune system. Studies have shown that 3-FL can reduce the risk of gut microbiota imbalance caused by harmful bacteria and selectively stimulate the growth of beneficial bifidobacteria, supporting overall gut health. Regarding safety, 3-FL has passed safety evaluations for acute oral toxicity, genotoxicity, and subchronic toxicity and has been approved as Generally Recognized As Safe (GRAS).

[0003] Currently, the commercial production of 3-FL mainly includes two methods: chemical synthesis and biosynthesis. Biosynthesis, requiring only inexpensive carbon sources and renewable intracellular donors, achieves high economic output with relatively low environmental costs, thus holding greater application potential. The key enzyme in the biosynthesis of 3-FL is α-1,3-fucosyltransferase. Only about 30 α-1,3-fucosyltransferases have been reported so far, most of which originate from Helicobacter pylori. Their generally low catalytic activity and poor solubility limit the yield of 3-FL. Therefore, discovering novel α-1,3-fucosyltransferases is an effective way to increase 3-FL production. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a novel α-1,3-fucosyltransferase for increasing 3-FL production.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a novel α-1,3-fucosyltransferase, the amino acid sequence of which is shown in (a) or (b):

[0007] (a) The amino acid sequence is shown in SEQ ID NO.1;

[0008] (b) A protein derived from (a) whose amino acid sequence in (a) has been substituted, deleted or added with one or more amino acids and has α-1,3-fucosyltransferase activity.

[0009] The nucleotide sequence of the novel α-1,3-fucosyltransferase is shown in SEQ ID NO.2.

[0010] Preferably, the novel α-1,3-fucosyltransferase is derived from a viral metagenomics.

[0011] Secondly, the present invention provides a genetically engineered bacterium for producing 3-fucosylated lactose, wherein the genetically engineered bacterium expresses the aforementioned novel α-1,3-fucosylated transferase.

[0012] Thirdly, the present invention provides a method for constructing the genetically engineered bacteria for producing 3-fucosylated lactose, comprising the following steps:

[0013] (1) The α-1,3-fucosyltransferase gene was cloned into the vector pETDuet-1 to obtain the VmFucT coding sequence expression cassette;

[0014] (2) Using Escherichia coli as the chassis strain, knock out the araA, wcaJ, and lacZ genes, insert the manC and manB genes at the rhaA site, and insert the gmd and wcaG genes at the ugd site to obtain strain A.

[0015] (3) The lacA-N32 sequence was cloned into the pSPIN plasmid to obtain the pSPIN-lacA(N32) plasmid;

[0016] (4) The VmFucT coding sequence expression cassette obtained in step (1) is cloned into the plasmid pSPIN-lacA(N32) obtained in step (3) to obtain the pSPIN-lacA-VmFucT plasmid.

[0017] (5) Transform the pSPIN-lacA-VmFucT plasmid into strain A obtained in step (2), screen for positive transformants, remove the plasmid to obtain the genetically engineered bacteria that produce 3-fucosylated lactose.

[0018] This invention involved the following steps in *E. coli*: araA gene knockout, which inhibits GDP-fucose degradation; wcaJ gene knockout, which prevents GDP-L-fucose from being metabolized into colacid, thus achieving GDP-L-fucose accumulation in the cytoplasm; lacZ gene knockout, which inhibits intracellular lactose hydrolysis and maintains intracellular lactose concentration; and insertion of manC and manB genes at the ugd site. Through these steps, strain A84 was obtained to enhance the synthesis efficiency of the precursor GDP-fucose. Finally, VmFucT was introduced to further increase the yield of 3-fucosyllactose.

[0019] Preferably, the Escherichia coli is BL21(DE3).

[0020] Preferably, the lacA-N32 sequence is as shown in SEQ ID NO.3.

[0021] The function of N32 is similar to that of N20. The transposon will precisely locate the bases about 48-50 bp after the N32 specific sequence and insert the gene into that position.

[0022] Fourthly, the present invention provides a method for producing 3-fucosyllactose by fermentation using the genetically engineered bacteria described above for producing 3-fucosyllactose.

[0023] Preferably, the fermentation conditions are: temperature 29.5℃-37℃, pH=6.8, and fermentation time 90h.

[0024] Fifthly, the present invention provides the application of the above-mentioned genetically engineered bacteria for producing 3-fucosylated lactose in the preparation of food and pharmaceutical products.

[0025] The beneficial effects of this invention are as follows:

[0026] Currently reported α-1,3-fucosyltransferases are few in number and have low activity. This invention provides a novel α-1,3-fucosyltransferase, VmFucT, offering a new pathway for 3-FL synthesis. This invention also provides a genetically engineered bacterium encoding and expressing the novel α-1,3-fucosyltransferase. After fermentation in a 5L tank, the 3-FL yield reached 25.01 g / L, demonstrating a significant improvement in 3-FL production and possessing high application value. Detailed Implementation

[0027] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0028] The control transferases FutM2-Q126A, FucTa-Y218K, m3FT, WP_042442472.1, and FucT7 used in this invention's experiments (codon optimization and synthesis were performed by the Guangzhou branch of Beijing Liuhe BGI Genomics Co., Ltd.) are as follows:

[0029] The source of the above-mentioned transferase is:

[0030] FutM2-Q126A comes from the literature Combinatorial Optimization Strategies for 3-Fucosyllactose Hyperproduction in Escherichia coli (Du Z, Zhu Y, et al. Journal of Agricultural and Food Chemistry);

[0031] FucTa-Y218K comes from the literature Rational Design of anα-1,3-Fucosyltransferase for the Biosynthesis of 3-Fucosyllactose in Bacillus subtilis ATCC 6051a via DeNovo GDP-l-Fucose Pathway (Xie Y, Wu X, et al. J Agric Food Chem.);

[0032] m3FT comes from the literature Enhanced bioproduction of fucosylated oligosaccharide3-fucosyllactose in engineered Escherichia coli with an improved de novopathway. (Ni Z, Wu J, et al. Biosci Biotechnol Biochem.);

[0033] WP_042442472.1 (Organism: Azospirillum lipoferum) is derived from patent WO2020127417A2;

[0034] FucT7 is derived from patent EP2439264A1.

[0035] Example 1:

[0036] Using uniprot, enzymes containing the two basic domains of α-1,3-fucosyltransferase, pfam18025 and pfam00852, were searched. The screened α-1,3-fucosyltransferases were subjected to molecular docking simulations with lactose and GDP-L-fucose, and enzymes with higher scores (ranked by the absolute value of the difference between the product and substrate Gibbs free energy) were selected. After optimization, a novel α-1,3-fucosyltransferase, named VmFucT, was obtained. Its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2.

[0037] VmFucT originates from the viral metagenome, contains 891 bases, and encodes a protein with 296 amino acids. It exhibits low sequence similarity to α-1,3-fucosyltransferases FutM2-Q126A, FucTa-Y218K, m3FT, WP_042442472.1, and FucT7, with homology rates of 21%, 29%, 29%, 22%, and 15%, respectively. It is a novel enzyme for the production of 3-FL.

[0038] Example 2: Construction of genetically engineered bacteria for producing 3-FL

[0039] 1. Plasmid construction:

[0040] (1.1) The α-1,3-fucosyltransferase VmFucT was synthesized by BGI and subcloned into the vector pETDuet-1 to construct the plasmid pET-VmFucT containing the complete coding sequence expression cassette of VmFucT.

[0041] (1.2) The pET-GW plasmid (containing the complete gmd-wcaG expression cassette) was constructed as follows:

[0042] The vector pETDuet-1 was linearized using NcoI and KpnI restriction endonucleases. PCR amplification was performed using primers GW-F and GW-R, with the *E. coli* Bl21(DE3) genome as a template, to obtain the target fragment GW. A seamless cloning kit was then used. DNAAssembly Mix Plus was used to perform homologous recombination ligation of the target fragment and the linearized vector, which was then transformed into E. coli DH5α to construct the pET-GW plasmid containing the complete gmd-wcaG expression cassette.

[0043] (1.3) pET-CB plasmid (containing the complete manC-manB expression cassette), the construction process is as follows:

[0044] The vector pETDuet-1 was linearized using NcoI and KpnI restriction endonucleases. PCR amplification was performed using primers CB-F and CB-R, with the *E. coli* Bl21(DE3) genome as a template, to obtain the target fragment CB. A seamless cloning kit was then used. DNA Assembly Mix Plus was used to perform homologous recombination ligation of the target fragment and the linearized vector, which was then transformed into E. coli DH5α to construct the pET-CB plasmid containing the complete manC-manB expression cassette.

[0045] pETDuet-1 was purchased from BGI Genomics.

[0046] 2. Using *Escherichia coli* Bl21(DE3) as the starting strain, the following gene modifications were performed using a modified CRISPR-Cas9 technology:

[0047] (2.1) The upstream and downstream homologous arms were amplified using araA-up-F, araA-up-R and araA-down-F, araA-down-R as primers, respectively. Then, the donor fragment was amplified by fusion PCR using the upstream and downstream homologous arms as templates. The araA was knocked out by targeted gene editing using pEcgRNA containing sgRNA sequence and N20 specific sequence. The araA was verified using araA-DF and araA-DR.

[0048] (2.2) The upstream and downstream homologous arms were amplified using wcaJ-up-F, wcaJ-up-R and wcaJ-down-F, wcaJ-down-R as primers, respectively. Then, the upstream and downstream homologous arms were used as templates to fuse PCR amplification of the donor fragment. The target gene editing of wcaJ was performed using pEcgRNA containing sgRNA sequence and N20 specific sequence to knock out wcaJ. The results were verified using wcaJ-DF and wcaJ-DR.

[0049] (2.3) Using lacZ-up-F, lacZ-up-R and lacZ-down-F, lacZ-down-R as primers, the upstream and downstream homologous arms were amplified respectively. Then, using primers lacZ-up-F and lacZ-down-R as primers, the upstream and downstream homologous arms were used as templates to fuse PCR amplification of the donor fragment. lacZ was knocked out by targeted gene editing using pEcgRNA containing sgRNA sequence and N20 specific sequence. lacZ-DF and lacZ-DR were used for verification.

[0050] Through the above steps (2.1)-(2.3), strain A30 was obtained, with the genotype BL21(DE3)ΔaraAΔwcaJΔlacZ.

[0051] 3. Utilizing modified CRISPR-mediated transposon technology:

[0052] (3.1) Insert the gmd and wcaG genes at the rhaA site.

[0053] The rhaA-N32 specific sequence was cloned into the pSPIN plasmid to obtain the pSPIN-rhaA(N32) plasmid. Using pET-GW as a template, the complete gmd-wcaG expression cassette obtained in step (1) was cloned into pSPIN-rhaA(N32) using primers T7-F and T7-R to obtain the pSPIN-rhaA-GW plasmid. The transformants were verified using primers pSPIN-DF and pSPIN-DR, and positive transformants were screened and transformed into strain A30. The transformants were cultured on resistant plates, and the transformants were verified using primers rhaA-DF and rhaA-DR, and positive transformants were screened. After removing the plasmid from the positive transformants, strain A61 was obtained with the genotype BL21(DE3)ΔaraA ΔwcaJΔlacZ,rhaA::GW, thus realizing the insertion of the gmd and wcaG genes at the rhaA site.

[0054] (3.2) Insert the manC and manB genes at the ugd site.

[0055] The ugd-N32 specific sequence was cloned into the pSPIN plasmid to obtain the pSPIN-ugd(N32) plasmid. Using pET-CB as a template, the complete manC-manB expression cassette obtained in step (1) was cloned into pSPIN-ugd(N32) using primers T7-F and T7-R. The transformants were verified using primers pSPIN-DF and pSPIN-DR, and positive transformants were screened to obtain the pSPIN-ugd-CB plasmid. The transformant was transformed into strain A61 and cultured on a resistant plate. The transformants were verified using primers ugd-DF and ugd-DR, and positive transformants were screened. After removing the plasmid from the positive transformants, the chassis strain A84 was obtained with the genotype BL21(DE3)ΔaraA ΔwcaJΔlacZ,rhaA::GW,ugd::CB, thus achieving the insertion of manC and manB genes at the ugd site.

[0056] The above construction process uses pEcgRNA containing sgRNA and N20 specific sequences for targeted gene editing; the gene knockout technology uses pEcCas vector containing Cas9 and λ-Red recombinase; targeted gene editing is performed using pSPIN containing N32 specific sequences; and the gene transposition technology uses pSPIN vector containing N32 specific sequences.

[0057] 4. The lacA-N32 specific sequence was cloned into the pSPIN plasmid to obtain the pSPIN-lacA(N32) plasmid. Using pET-VmFucT as a template, the complete VmFucT expression cassette obtained in step (1) was cloned into pSPIN-lacA(N32) using primers T7-F and T7-R. The transformants were verified using primers pSPIN-DF and pSPIN-DR, and positive transformants were screened to obtain the pSPIN-lacA-VmFucT plasmid. The transformant was transformed into strain A84 and cultured on an resistant plate. The transformants were verified using primers lacA-DF and lacA-DR, and positive transformants were screened. After removing the plasmid from the positive transformants, strain A193 was obtained. Thus, the genetically engineered bacteria that produces 3-FL was obtained.

[0058] The specific sequences and primer sequences used in this embodiment are shown in Table 1.

[0059] Table 1

[0060]

[0061]

[0062] Comparative Examples 1-5:

[0063] Comparative Examples 1-5 provide genetically engineered bacteria that produce 3-FL. The only difference between the strain construction method and the construction method in Example 2 is that the coding sequence expression cassette imported in step 4 is different.

[0064] Comparative Examples 1-5 were introduced with expression cassettes encoding different α-1,3-fucosyltransferases, namely FutM2-Q126A, FucTa-Y218K, m3FT, WP_042442472.1, and FucT7, respectively, to obtain the corresponding genetically engineered bacteria that produce 3-FL.

[0065] Experimental Example 1: Fermentation Production of 3-FL

[0066] The genetically engineered bacteria for producing 3-FL obtained in Example 2 and Comparative Examples 1-5 were fed-batch fermented in a 5L tank under the following conditions:

[0067] (1) Liquid volume: 2L; Inoculum volume: 150mL; Fermentation time: 90h;

[0068] (2) Control parameters: pH 6.8; fermentation temperature 37℃, adjusted to 29.5℃ after feeding; IPTG and lactose were added 4 hours after feeding for induction;

[0069] (3) Ventilation rate: 2.5 L / min;

[0070] (4) Rotation speed: Starting speed 200 rpm;

[0071] (5) Dissolved oxygen control: Before feeding, DO is linked to the rotation speed, and DO is set to 30-40%. The rotation speed range is set to 200-1000 rpm.

[0072] (6) Carbon source is fed at a constant rate: the rate is 18g of feed liquid / h;

[0073] (7) Lactose: Add in batches, 70g of lactose each time.

[0074] 3-FL yield was determined by liquid chromatography under the following conditions: 75% acetonitrile, 25% water, v / v, flow rate 1 mL / min, column: Shim-pack Scepter Diol-HILIC-120 (4.6 × 250 mm, 5 μm), column temperature 40 °C, injection volume 10 μL. Isocratic elution was performed, and the detection time was 15 min.

[0075] Engineered bacteria obtained by introducing different transferases into the same chassis strain (A84) were fermented under the above fermentation conditions. A comparison was made: the engineered bacteria introduced with transferase VmFucT, fermented in a 5L tank, achieved a 3-FL yield of 25.01 g / L. In contrast, the control strains introduced with transferases FutM2-Q126A, FucTa-Y218K, m3FT, WP_042442472.1, and FucT7, fermented under the same conditions in a 5L tank, yielded 3-FL yields of 17.37 g / L, 0.29 g / L, 0 g / L, 19.3 g / L, and 20.03 g / L, respectively. VmFucT showed a 24.8% increase in 3-FL yield compared to the highest-yielding control strain, FucT7. Therefore, the genetically engineered bacteria provided by this invention have significant effects and high application value.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An α-1,3-fucosyltransferase, characterized in that, The amino acid sequence of the α-1,3-fucosyltransferase is shown in SEQ ID NO.

1.

2. The α-1,3-fucosyltransferase as described in claim 1, characterized in that, The nucleotide sequence of the α-1,3-fucosyltransferase is shown in SEQ ID NO.

2.

3. The α-1,3-fucosyltransferase as described in claim 1, characterized in that, The α-1,3-fucosyltransferase is derived from a viral metagenomics.

4. A genetically engineered bacterium for producing 3-fucosyllactose, characterized in that, The genetically engineered bacteria express the α-1,3-fucosyltransferase as described in any one of claims 1-3.

5. The method for constructing the genetically engineered bacteria for producing 3-fucosylated lactose as described in claim 4, characterized in that, Includes the following steps: (1) The α-1,3-fucosyltransferase gene was cloned into the vector pETDuet-1 to obtain the VmFucT coding sequence expression cassette; (2) Using Escherichia coli as the chassis strain, knock out the araA, wcaJ, and lacZ genes, insert the manC and manB genes at the rhaA site, and insert the gmd and wcaG genes at the ugd site to obtain strain A; (3) The lacA-N32 sequence was cloned into the pSPIN plasmid to obtain the pSPIN-lacA(N32) plasmid; (4) The VmFucT coding sequence expression cassette obtained in step (1) is cloned into the plasmid pSPIN-lacA(N32) obtained in step (3) to obtain the pSPIN-lacA-VmFucT plasmid. (5) Transform the pSPIN-lacA-VmFucT plasmid into strain A obtained in step (2), screen for positive transformants, remove the plasmid to obtain the genetically engineered bacteria that produce 3-fucosylated lactose.

6. The construction method as described in claim 5, characterized in that, The Escherichia coli was BL21(DE3).

7. The construction method as described in claim 5, characterized in that, The lacA-N32 sequence is shown in SEQ ID NO.

3.

8. A method for producing 3-fucosyllactose, characterized in that, 3-Fucose is produced by fermentation using the genetically engineered bacteria for producing 3-fucosylated lactose as described in claim 4.

9. The method for producing 3-fucosylated lactose as described in claim 8, characterized in that, The fermentation conditions were: temperature 29.5℃-37℃, pH=6.8, and fermentation time 90 h.

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