Alpha-1, 3-fucosyltransferase mutant as well as preparation method and application thereof

Abstract

The invention relates to an alpha-1, 3-fucosyltransferase mutant as well as a preparation method and application thereof. Compared with wild type alpha-1, 3-fucosyltransferase, the mutant has the advantages that mutation sites of the mutant comprise H129W and / or A188H, and any one of the mutation sites or accumulative mutation can obtain higher yield than that of the wild type in 3-FL synthesis. The obtained mutant obviously improves the yield of 3-FL, has important significance on biosynthesis of 3-FL, is suitable for the requirements of industrial production, and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an α-1,3-fucosyltransferase mutant, a preparation method thereof, and an application thereof. Background Art

[0002] Human milk oligosaccharides (HMOs) are the components with the largest proportion in human milk except lactose and lipids. HMOs have a complex composition, and currently more than 200 components are known, which play an important role in constructing the neonatal intestinal flora and regulating the neonatal intestinal function. Natural HMOs are difficult to mass-produce on a large scale. The current research direction is to artificially synthesize the main components of HMOs. In addition, some single products approved for application in infant formula foods in some countries and regions include 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-neotetraose, etc. Among them, the biosynthesis research of 2'-FL is the most thorough and has achieved large-scale production; while the research on 3-FL has made less progress at present because a high-performance α-1,3-fucosyltransferase has not been found.

[0003] Current research shows that: 3-FL has the same substrates as 2'-FL in biosynthesis: GDP-L-fucose and lactose. The difference is that 2'-FL is the α1,2 position of L-fucose linked to the galactose unit in lactose, and 3-FL is the α-1,3 position of L-fucose linked to the glucose unit in lactose. The structures of 2'-FL and 3-FL are shown as follows.

[0004]

[0005] There are various development strategies for industrial production of 3-FL. Compared with enzymatic catalysis or chemoenzymatic synthesis, the method of using genetically engineered microbial whole cells to synthesize 3-FL is obviously the best choice. Among the various factors that may affect the synthesis efficiency of 3-FL, the effective expression and catalytic activity of α-1,3-fucosyltransferase are very crucial for the biosynthesis of 3-FL. The de novo synthesis pathway of 3-FL must rely on this enzyme to synthesize 3-FL using GDP-L-fucose and lactose as substrates. In addition, in biosynthesis, since GDP-L-fucose can always only be maintained at a low level in cells, α-1,3-fucosyltransferase not only needs to be able to be effectively soluble expressed in industrial strains, but also has sufficient catalytic activity to adapt to the low level of GDP-L-fucose.

[0006] Three Ways of Industrial Production of 3-FL

[0007]

[0008]

[0009] α-1,3-fucosyltransferase is currently the key enzyme used in the biosynthesis of 3-FL. For the de novo synthesis pathway of 3-FL, this enzyme must use GDP-L-fucose with lactose as the substrate to synthesize 3-FL. Although multiple α-1,3-fucosyltransferases have been discovered, the results still show that the α-1,3-fucosyltransferase derived from Helicobacter pylori has the highest efficiency in 3-FL biosynthesis.

[0010] Wild-type FutA is derived from Helicobacter pylori (Helicobacter pylori strain 26695). Its heterologous expression in Escherichia coli results in relatively low protein solubility and reduced enzyme activity, which are undoubtedly disadvantages that need to be improved for industrial production of 3-FL. Therefore, in order to enhance its catalytic efficiency in Escherichia coli, the existing protein needs to be modified to further increase the yield of 3-FL. Summary of the Invention

[0011] Problems to be Solved by the Invention

[0012] In the biosynthesis of HMOs, compared with 2’-FL, the research on 3-FL is relatively less. According to the existing data, the participation of α-1,3-fucosyltransferase is an essential step in 3-FL synthesis, and its activity directly affects the yield of 3-FL. Existing improvement methods, including enhancing the expression of FutA, codon optimization, and seeking alternative enzymes from different sources, have not achieved satisfactory results and are difficult to be applied industrially. Therefore, the purpose of the present invention is to analyze and modify the structure of the existing futA to improve its soluble expression level and catalytic activity.

[0013] Solutions for Solving the Problems

[0014] The present invention provides an α-1,3-fucosyltransferase mutant, and the amino acid sequence of the mutant includes:

[0015] (1) Compared with the wild-type α-1,3-fucosyltransferase shown in the amino acid sequence SEQ ID NO.1, the mutation sites of the mutant include: H129W and / or A188H;

[0016] (2) Compared with the amino acid sequence described in (1), an amino acid sequence with one or more amino acid residue substitutions, deletions, additions, or any combination thereof, and the substitution is a conservative substitution; and

[0017] (3) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the amino acid sequence described in (1).

[0018] Preferably, the mutation site is H129W, or A188H, or H129W and A188H;

[0019] Preferably, the amino acid sequence of the α-1,3-fucosyltransferase mutant with the mutation site of H129W is as shown in SEQ ID NO.2;

[0020] Preferably, the amino acid sequence of the α-1,3-fucosyltransferase mutant with the mutation site of A188H is as shown in SEQ ID NO.3;

[0021] Preferably, the amino acid sequence of the α-1,3-fucosyltransferase mutant with the mutation sites of H129W and A188H is as shown in SEQ ID NO.4.

[0022] The present invention also provides a nucleic acid encoding the mutant.

[0023] The present invention also provides a vector comprising the nucleic acid.

[0024] The present invention also provides a recombinant engineered bacterium or recombinant engineered cell comprising the nucleic acid or the vector.

[0025] The present invention also provides a method for preparing the mutant, the method comprising: culturing the recombinant engineered bacterium or recombinant engineered cell to induce the expression of the mutant.

[0026] The present invention also provides a biological preparation, the biological preparation comprising: the mutant, or the nucleic acid, or the vector, or the recombinant engineered bacterium or recombinant engineered cell, or the product prepared according to the method.

[0027] The present invention also provides an application of the mutant, or the nucleic acid, or the vector, or the recombinant engineered bacterium or recombinant engineered cell, or the product prepared according to the method, or the biological preparation in the preparation of fucosylated oligosaccharides;

[0028] Preferably, the fucosylated oligosaccharides are selected from one or more of 2'-fucosyllactose, 3'-fucosyllactose, difucosyllactose, lacto-N-difuco-tetraose, lacto-N-fucopentaose I and lacto-N-difuco-hexaose I;

[0029] More preferably, the fucosylated oligosaccharide is 3'-fucosyllactose.

[0030] The present invention also provides a method for preparing fucosylated oligosaccharides by an in vitro enzymatic method, the method comprising:

[0031] (1) providing the α-1,3-fucosyltransferase mutant as described above;

[0032] (2) providing a donor substrate and an acceptor substrate, culturing a host cell under suitable nutritional conditions allowing for the production of the fucosylated oligosaccharide and conditions allowing for the expression of the α-1,3-fucosyltransferase mutant, such that the α-1,3-fucosyltransferase mutant in step (1) contacts the donor substrate and the acceptor substrate to prepare the fucosylated oligosaccharide;

[0033] Preferably, the fucosylated oligosaccharide is selected from 2'-fucosyllactose, 3'-fucosyllactose, difucosyllactose, lacto-N-difuco-tetraose, lacto-N-fucopentaose I, and lacto-N-difucohexaose I;

[0034] More preferably, the fucosylated oligosaccharide is 3'-fucosyllactose.

[0035] The present invention also provides the use of the mutant as described above, or the nucleic acid as described above, or the vector as described above, or the recombinant engineering bacterium or recombinant engineering cell as described above, or the product prepared according to the method as described above, or the biological preparation in the preparation of food or health products.

[0036] Effects of the Invention

[0037] Through enzyme engineering analysis, the present invention screened two beneficial mutations in the futA gene sequence, and any one or cumulative mutations at these mutation sites can obtain a higher yield than the wild type in the synthesis of 3-FL. The present invention uses enzyme engineering technology to modify futA derived from Helicobacter pylori strain 26695, thereby significantly improving its soluble expression level and catalytic activity. The obtained mutant significantly increases the yield of 3-FL, which is of great significance for the biosynthesis of 3-FL, meets the needs of industrial production, and has broad application prospects. Description of the Drawings

[0038] Figure 1 are the kinetic parameters of the interaction between futA and GDP-L-fucose.

[0039] Figure 2 is the transient conformation of futA-GDP-fuc.

[0040] Figure 3For the RMSF analysis of the instantaneous conformation of futA-GDP-fuc.

[0041] Figure 4 For the plasmid map of futA.pET.

[0042] Figure 5 For the plasmid map of BCGF-pRDA.

[0043] Figure 6 For the HPLC detection standard curve of the strain.

[0044] Figure 7 For the 3-FL production of the wild-type FA.BLD strain.

[0045] Figure 8 For the 3-FL production of the mutant H129W.BLD strain.

[0046] Figure 9 For the 3-FL production of the mutant A188H.BLD strain.

[0047] Figure 10 For the 3-FL production of the mutant H129W-A188H.BLD strain. Specific implementation manners

[0048] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following will be described in detail by listing specific embodiments. Among them, the drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0049] In the embodiments of the present invention, the experimental methods without specific conditions are usually carried out according to conventional conditions, or according to the conditions recommended by the raw material or commodity manufacturers. The reagents without specific sources are conventional reagents purchased from the market.

[0050] The three-letter codes and single-letter codes of amino acids used in the present invention are as described in J.biol.Chem, 1968, 243, 3558.

[0051] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the biological activity of a protein / polypeptide containing an amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue with another amino acid residue having a similar side chain, e.g., substitutions with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having a similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to substitute the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0052] As used herein, the term "identity" is used to refer to the sequence match between two polypeptides or two nucleic acids. When the same base or amino acid monomer subunit occupies a position in both of the two sequences being compared (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 of a total of 6 positions match). Generally, comparison is made when the two sequences are aligned to yield maximum identity. Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443 - 453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11 - 17 (1988)), incorporated into the ALIGN program (version 2.0), can also be used, with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the algorithm of Needleman and Wunsch (J Mol Biol. 48:444 - 453 (1970)) incorporated into the GAP program in the GCG software package (available at www.gcg.com) can be used, with a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences.

[0053] The terms related to the present invention are defined above, and those skilled in the art can also understand the above terms in combination with the prior art. The following further describes based on the content of the present invention and the definitions of the terms.

[0054] The present invention provides an α - 1,3 - fucosyltransferase mutant, and the amino acid sequence of the mutant comprises:

[0055] (1) Compared with the wild - type α - 1,3 - fucosyltransferase shown in the amino acid sequence SEQ ID NO.1, the mutation sites of the mutant include: H129W and / or A188H;

[0056] (2) An amino acid sequence having one or more amino acid residue substitutions, deletions, additions, or any combination thereof as compared to the amino acid sequence described in (1), wherein the substitutions are conservative substitutions; and

[0057] (3) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity as compared to the amino acid sequence described in (1).

[0058] In certain embodiments, the amino acid sequence having one or more amino acid residue substitutions, deletions, additions, or any combination thereof in (2) has the same or similar function as the amino acid sequence described in (1).

[0059] In certain embodiments, the amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity as compared to the amino acid sequence described in (1) in (3).

[0060] In certain embodiments, the amino acid sequence obtained in (3) has the same or similar function as the amino acid sequence described in (1).

[0061] In certain embodiments, the α-1,3-fucosyltransferase is FutA.

[0062] In certain embodiments, the α-1,3-fucosyltransferase is derived from the strain Helicobacter pylori strain 26695.

[0063] In certain embodiments, the amino acid sequence of the wild-type α-1,3-fucosyltransferase is as shown in SEQ ID NO.1:

[0064] MFQPLLDAFIESASIEKMASKSPPPPLKIAVANWWGDEEIKEFKKSVLYFILSQRYAITLHQNPNEFSDLVFSNPLGAARKILSYQNTKRVFYTGENESPNFNLFDYAIGFDELDFNDRYLRMPLYYAHLHYKAELVNDTTAPYKLKDNSLYALKKPSHHFKENHPNLCAVVNDESDLLKRGFASFVASNANAPMRNAFYDALNSIEPVTGGGSVRNTLGYKVGNKSEFLSQYKFNLCFENSQGYGYVTEKILDAYFSHTIPIYWGSPSVAKDFNPKSFVNVHDFNNFDEAIDYIKYLHTHPNAYLDMLYENPLNTLDGKAYFYQDLSFKKILDFFKTILENDTIYHKFSTSFMWEYDLHKPLVSIDDLRVNYDDLRVNYDRLLQNASPLLELSQNTTFKIYRKAYQKSLPLLRAVRKLVKKLGL*

[0065] In some embodiments, the mutation site is H129W, or A188H, or H129W and A188H.

[0066] In some embodiments, the mutation site is H129W.

[0067] In some embodiments, the amino acid sequence of the α-1,3-fucosyltransferase mutant with the mutation site of H129W is as shown in SEQ ID NO:2:

[0068] MFQPLLDAFIESASIEKMASKSPPPPLKIAVANWWGDEEIKEFKKSVLYFILSQRYAITLHQNPNEFSDLVFSNPLGAARKILSYQNTKRVFYTGENESPNFNLFDYAIGFDELDFNDRYLRMPLYYAWLHYKAELVNDTTAPYKLKDNSLYALKKPSHHFKENHPNLCAVVNDESDLLKRGFASFVASNANAPMRNAFYDALNSIEPVTGGGSVRNTLGYKVGNKSEFLSQYKFNLCFENSQGYGYVTEKILDAYFSHTIPIYWGSPSVAKDFNPKSFVNVHDFNNFDEAIDYIKYLHTHPNAYLDMLYENPLNTLDGKAYFYQDLSFKKILDFFKTILENDTIYHKFSTSFMWEYDLHKPLVSIDDLRVNYDDLRVNYDRLLQNASPLLELSQNTTFKIYRKAYQKSLPLLRAVRKLVKKLGL*

[0069] In some embodiments, the mutation site is A188H.

[0070] In some embodiments, the amino acid sequence of the α-1,3-fucosyltransferase mutant with the mutation site A188H is as shown in SEQ ID NO:3:

[0071] MFQPLLDAFIESASIEKMASKSPPPPLKIAVANWWGDEEIKEFKKSVLYFILSQRYAITLHQNPNEFSDLVFSNPLGAARKILSYQNTKRVFYTGENESPNFNLFDYAIGFDELDFNDRYLRMPLYYAHLHYKAELVNDTTAPYKLKDNSLYALKKPSHHFKENHPNLCAVVNDESDLLKRGFASFVHSNANAPMRNAFYDALNSIEPVTGGGSVRNTLGYKVGNKSEFLSQYKFNLCFENSQGYGYVTEKILDAYFSHTIPIYWGSPSVAKDFNPKSFVNVHDFNNFDEAIDYIKYLHTHPNAYLDMLYENPLNTLDGKAYFYQDLSFKKILDFFKTILENDTIYHKFSTSFMWEYDLHKPLVSIDDLRVNYDDLRVNYDRLLQNASPLLELSQNTTFKIYRKAYQKSLPLLRAVRKLVKKLGL*

[0072] In some embodiments, the mutation sites are H129W and A188H.

[0073] In some embodiments, the amino acid sequence of the α-1,3-fucosyltransferase mutant with the mutation sites of H129W and A188H is as shown in SEQ ID NO:4:

[0074] MFQPLLDAFIESASIEKMASKSPPPPLKIAVANWWGDEEIKEFKKSVLYFILSQRYAITLHQNPNEFSDLVFSNPLGAARKILSYQNTKRVFYTGENESPNFNLFDYAIGFDELDFNDRYLRMPLYYAWLHYKAELVNDTTAPYKLKDNSLYALKKPSHHFKENHPNLCAVVNDESDLLKRGFASFVHSNANAPMRNAFYDALNSIEPVTGGGSVRNTLGYKVGNKSEFLSQYKFNLCFENSQGYGYVTEKILDAYFSHTIPIYWGSPSVAKDFNPKSFVNVHDFNNFDEAIDYIKYLHTHPNAYLDMLYENPLNTLDGKAYFYQDLSFKKILDFFKTILENDTIYHKFSTSFMWEYDLHKPLVSIDDLRVNYDDLRVNYDRLLQNASPLLELSQNTTFKIYRKAYQKSLPLLRAVRKLVKKLGL*

[0075] The present invention also provides a nucleic acid encoding the mutant.

[0076] The present invention also provides a vector comprising the nucleic acid.

[0077] The present invention also provides a recombinant engineered bacterium or recombinant engineered cell comprising the nucleic acid or the vector.

[0078] The present invention also provides a method for preparing the mutant, the method comprising: culturing the recombinant engineered bacterium or recombinant engineered cell to induce the expression of the mutant.

[0079] The present invention also provides a biological preparation comprising: the mutant, or the nucleic acid, or the vector, or the recombinant engineered bacterium or recombinant engineered cell, or a product prepared according to the method.

[0080] The present invention also provides the use of the mutant, or the nucleic acid, or the vector, or the recombinant engineered bacterium or recombinant engineered cell, or a product prepared according to the method, or the biological preparation in the preparation of fucosylated oligosaccharides;

[0081] In certain embodiments, the fucosylated oligosaccharides are selected from one or more of 2'-fucosyllactose, 3'-fucosyllactose, difucosyllactose, lacto-N-difuco-tetraose, lacto-N-fucopentaose I, and lacto-N-difuco-hexaose I.

[0082] In some embodiments, the fucosylated oligosaccharide is 3'-fucosyllactose.

[0083] The present invention also provides a method for preparing fucosylated oligosaccharides by an in vitro enzymatic method, the method comprising:

[0084] (1) providing the α-1,3-fucosyltransferase mutant as described above;

[0085] (2) providing a donor substrate and an acceptor substrate, culturing a host cell under suitable nutrient conditions allowing production of the fucosylated oligosaccharide and conditions allowing expression of the α-1,3-fucosyltransferase mutant, so that the α-1,3-fucosyltransferase mutant in step (1) contacts the donor substrate and the acceptor substrate to prepare the fucosylated oligosaccharide;

[0086] In some embodiments, the fucosylated oligosaccharide is selected from 2'-fucosyllactose, 3'-fucosyllactose, difucosyllactose, lacto-N-difuco-tetraose, lacto-N-fucopentaose I and lacto-N-difuco-hexaose I;

[0087] In some embodiments, the fucosylated oligosaccharide is 3'-fucosyllactose.

[0088] The present invention also provides an application of the mutant as described above, or the nucleic acid as described above, or the vector as described above, or the recombinant engineering bacterium or recombinant engineering cell as described above, or the product prepared by the method as described above, or the biological preparation in the preparation of food or health products.

[0089] The preparation of the mutant as described in the present invention is further described below in conjunction with examples, but these examples do not limit the scope in the present disclosure.

[0090] Example 1: Screening for beneficial mutations in the futA gene sequence

[0091] In view of the current experimental fact that futA has poor affinity for GDP-L-fucose, kinetic parameters of the interaction between futA and GDP-L-fucose were calculated (1.2 ns), and the results are as Figure 1 shown. RMSD analysis was performed to obtain a relatively stable transient conformation (as Figure 2 shown), and the active pocket was analyzed based on this conformation. Amino acid residues that generate steric hindrance in the core region of the active pocket were mutated to amino acids with relatively small steric hindrance of the same type; in addition, based on RMSF analysis (as Figure 3As shown, amino acid residues with higher RMSF values (stronger flexibility) in the active pocket region and residues with higher RMSF values in the entire enzyme protein were identified, and mutations were made to them to adjust the overall rigidity (stability) of the protein. Finally, two beneficial mutations of futA were screened: H129W and A188H.

[0092] Example 2: Efficiency test of futA mutant protein

[0093] α-1,3-fucosyltransferase can be expressed through the pET28a(+) plasmid. Additionally, four genes, manB, manC, gmd, and fcl, of the GDP-L-fucose synthesis pathway are expressed using a high-copy plasmid pRSF Duet-1 and co-expressed in the BL21(DE3) strain with the lacZ gene knocked out, and 3-FL is obtained through fermentation. Therefore, this method can be used to test the efficiency of futA mutant protein.

[0094] 1. Entrust a gene synthesis company to biosynthesize the futA gene from Helicobacter pylori and install it on the pET28a(+) plasmid. The plasmid is named futA.pET, as Figure 4 shown.

[0095] 2. Replace the kanamycin resistance gene of the pRSF Duet-1 plasmid with the ampicillin resistance gene to obtain the plasmid pRDA.

[0096] (1) PCR amplify the ampicillin resistance DNA fragment from pUC19: 984bp

[0097]

[0098] PCR reaction system:

[0099]

[0100] The reaction conditions are pre-denaturation at 95°C for 30s, denaturation at 95°C for 15s, annealing at 56°C for 15s, extension for 40s, supplementary extension for 5min, and 30 cycles. After the reaction, agarose gel electrophoresis is performed, and the target fragment is recovered by cutting the gel.

[0101] (2) Amplify pRSF Duet-1 to obtain the pRSF Duet-1 linear plasmid: 2921bp

[0102]

[0103] PCR reaction system:

[0104]

[0105] (3) The two target fragments obtained above were ligated using Seamless Cloning master mix and then chemically transformed into DH5α competent cells. The cells were plated on LB agar plates with 100 μg / ml ampicillin resistance and cultured overnight at 37°C. Single colonies were selected for amplification and sequencing verification to obtain the pRDA plasmid.

[0106] 3. Construction of the BCGF-pRDA plasmid for synthesizing GDP-L-fucose:

[0107] (1) The manB gene fragment (1429 bp) was obtained by PCR from the genome of Escherichia coli strain BL21(DE3):

[0108]

[0109] PCR reaction system:

[0110]

[0111] The reaction conditions were pre-denaturation at 95°C for 30 s, denaturation at 95°C for 15 s, annealing at 48°C for 15 s, extension at 50 s, additional extension for 5 min, for 30 cycles. After the reaction, agarose gel electrophoresis was performed, and the target fragment was recovered by cutting the gel.

[0112] (2) The manC gene fragment (1479 bp) was obtained by PCR from the genome of Escherichia coli strain BL21(DE3):

[0113]

[0114] PCR reaction system:

[0115]

[0116] The reaction conditions were pre-denaturation at 95°C for 30 s, denaturation at 95°C for 15 s, annealing at 57°C for 15 s, extension at 50 s, additional extension for 5 min, for 30 cycles. After the reaction, agarose gel electrophoresis was performed, and the target fragment was recovered by cutting the gel.

[0117] (3) The gmd gene fragment (1122 bp) was obtained by PCR from the genome of Escherichia coli strain BL21(DE3):

[0118]

[0119] PCR reaction system:

[0120]

[0121] The reaction conditions were pre-denaturation at 95°C for 30 s, denaturation at 95°C for 15 s, annealing at 54°C for 15 s, extension at 50 s, supplementary extension at 5 min, for 30 cycles. After the reaction, agarose gel electrophoresis was performed, and the target fragment was recovered by gel cutting.

[0122] (4) The fcl gene fragment of 1009 bp was obtained by PCR from the genome of Escherichia coli strain BL21(DE3):

[0123]

[0124] PCR reaction system:

[0125]

[0126] The reaction conditions were pre-denaturation at 95°C for 30 s, denaturation at 95°C for 15 s, annealing at 50°C for 15 s, extension at 40 s, supplementary extension at 5 min, for 30 cycles. After the reaction, agarose gel electrophoresis was performed, and the target fragment was recovered by gel cutting.

[0127] (5) The linear fragment of 3546 bp was obtained by amplifying the pRDA plasmid:

[0128]

[0129] PCR reaction system:

[0130]

[0131] The reaction conditions were pre-denaturation at 95°C for 30 s, denaturation at 95°C for 15 s, annealing at 46°C for 15 s, extension at 3 min, supplementary extension at 5 min, for 30 cycles. After the reaction, agarose gel electrophoresis was performed, and the target fragment was recovered by gel cutting.

[0132] (6) The above five target fragments were ligated using Seamless Cloning master mix and then chemically transformed into DH5α competent cells. The cells were plated on a 50 μg / ml kanamycin-resistant LB agar plate and cultured overnight at 37°C. Single colonies were selected for expansion culture and then sequenced for verification to obtain the BCGF-pRDA plasmid, as Figure 5 shown.

[0133] 4. The gene lacZ that can metabolize lactose was knocked out from the BL21(DE3) strain, and then it was prepared into electrocompetent cells. The BCGF-pRDA plasmid was transferred into the cells. The cells were plated on a 100 μg / ml ampicillin-resistant LB agar plate and cultured overnight at 37°C. Single colonies were selected for culture to obtain the BCGF.BL21(DE3) strain.

[0134] Example 3: Comparative Test of Different Mutation Sites on the Effect of Producing 3-FL

[0135] 1. Construction of futA mutant plasmid:

[0136] (1) Design primers according to different mutation sites:

[0137]

[0138] (2) Use futA.pET plasmid as a template and amplify the mutant linear plasmid of about 6500bp with the above primers:

[0139] PCR reaction system:

[0140]

[0141] The reaction conditions are pre-denaturation at 95°C for 30s, denaturation at 95°C for 15s, annealing for 15s (annealing temperature is shown in the table), extension for 40s, supplementary extension for 5min, and 30 cycles. After the reaction, agarose gel electrophoresis is carried out, and the target fragment is recovered by cutting the gel.

[0142] (3) Use Seamless Cloning master mix to ligate the linear plasmid, and then chemically transform it into DH5α competent cells. Spread the cells on a 50ug / ml kanamycin-resistant LB agar plate and culture overnight at 37°C. Select monoclonal colonies for amplification and sequencing verification to obtain futA.pET plasmids with different mutations.

[0143] 2. Prepare electrocompetent cells from BCGF.BL21(DE3) strain and transfer the wild-type or mutant futA.pET plasmid into it respectively to obtain test strains:

[0144]

[0145] 3. To test the practicality of the test strains, use a fully synthetic artificial medium (hereinafter referred to as SE medium) with the following components:

[0146] Component Dosage / L Glucose 15.1g Potassium dihydrogen phosphate 5.0g Dipotassium hydrogen phosphate 10.3g Magnesium sulfate heptahydrate 0.8g Sodium chloride 0.8g Ammonium sulfate 7.0g Trace element solution 5ml

[0147] The components of the trace element solution are as follows:

[0148] Name g / L Ferric citrate hydrate 3.68 CaCl2 0.95 MnCl2·4H2O 0.52 CuCl2·2H2O 0.11 CoCl2·6H2O 0.15 ZnCl2 0.23 H3BO4 0.009 Na2EDTA·2H2O 0.12 Na2MoO4·2H2O 0.003 KI 0.05 Thiamine·HCl 0.3

[0149] 4. Induce protein expression to produce 3-FL:

[0150] (1) Inoculate each of the above strains into 20 ml of SE medium resistant to ampicillin (100 μg / ml) and kanamycin (50 ng / ml) (hereinafter referred to as SE(A / K) medium), and culture at 37°C and 220 rpm for 16 h.

[0151] (2) Measure the absorbance of the overnight culture at a wavelength of 600 nm, and then dilute the bacterial solution by a multiple of OD600 / 1.0. After dilution, inoculate the bacterial solution into 100 ml of SE(A / K) medium at a ratio of 1:10, and culture at 37°C and 220 rpm.

[0152] (3) When the OD600 of the strain culture reaches 0.4, add IPTG to a final concentration of 0.2 mM and lactose to a final concentration of 8.5 g / L, and continue to culture for 72 h.

[0153] (4) After the culture is completed, take 2.0 ml of the culture solution, centrifuge at 8000 xg for 10 min, and take the supernatant for HPLC detection of the 3-FL yield.

[0154] 5. Detection of 3-FL product: Analyze the sample by a high-performance liquid chromatography (HPLC) system (Shimadzu LC-20AT) and a Carbohydrate Analysis (Rezex ROA-organic acid H+(8%)) chromatographic column. Mobile phase: 8 mmol / L H2SO4; flow rate: 0.6 mL / min; column temperature: 50°C; injection volume: 10 μL. The detection standard curve is as Figure 6 shown.

[0155] The 3-FL yield of the wild-type FA.BLD strain is as Figure 7 shown, the 3-FL yield of the mutant H129W.BLD strain is as Figure 8 shown, the 3-FL yield of the mutant A188H.BLD strain is as Figure 9 shown, and the comparison of the 3-FL yields of different strains is as follows.

[0156] Strain 3-FL production (g / L) Wild type FA.BLD 0.113 Mutant H129W.BLD 0.218 Mutant A188H.BLD 0.233

[0157] Example 4: Test of the effect of multiple mutation combinations on the production of 3-FL

[0158] 1. Construction of the H129W-A188H plasmid:

[0159] (1) Using the H129W plasmid as a template, further PCR amplification is carried out to obtain the H129W-A188H mutant linear plasmid (6487 bp)

[0160] PCR reaction system:

[0161]

[0162] The reaction conditions were pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 15 s, annealing at 49 °C for 15 s, extension at 40 s, supplementary extension at 5 min, for 30 cycles. After the reaction, agarose gel electrophoresis was performed, and the target fragment was recovered by cutting the gel.

[0163] (2) The linear plasmid was ligated with the fragment using Seamless Cloning master mix and then chemically transformed into DH5α competent cells. The cells were plated on a 50 μg / ml kanamycin-resistant LB agar plate and cultured overnight at 37 °C. Single colonies were selected for expansion culture and sequencing verification to obtain H129W-A188H plasmids with different mutations.

[0164] 2. Prepare electrocompetent cells from BCGF.BL21(DE3) strain and transfer the H129W-A188H plasmid into it to obtain the test strain:

[0165] Original strain Transferred plasmid Test strain BCGF.BL21(DE3) H129W-A188H H129W-A188H.BLD

[0166] 3. Protein induction expression and 3-FL detection:

[0167] (1) Inoculate each of the above strains into 20 ml of SE medium resistant to ampicillin (100 μg / ml) and kanamycin (50 ng / ml), and culture at 37 °C and 220 rpm for 16 h.

[0168] (2) Measure the absorbance of the overnight culture at a wavelength of 600 nm, and then dilute the bacterial solution according to the multiple of OD600 / 1.0. After dilution, inoculate the bacterial solution into 100 ml of SE(A / K) medium at a ratio of 1:10 and culture at 37 °C and 220 rpm.

[0169] (3) When the OD600 of the bacterial strain culture reaches 0.4, add IPTG to a final concentration of 0.2 mM and lactose to a final concentration of 8.5 g / L, and continue to culture for 72 h.

[0170] (4) After the culture is completed, take 2.0 ml of the culture solution, centrifuge at 8000 xg for 10 min, and take the supernatant for HPLC detection of the 3-FL yield.

[0171] After the culture is completed, take 2.0 ml of the culture solution, centrifuge at 5000 xg for 10 min, and take the supernatant for HPLC detection of the 3-FL yield. The results are as Figure 10 shown. The 3-FL yield of the mutant H129W-A188H.BLD strain is 0.353 g / L, which is higher than that of the single mutants H129W-futA or A188H-futA.

[0172] It should be understood that the above embodiments are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various deformations and changes can also be made on the basis of the above embodiments. Similarly, the technical features of the above embodiments can also be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. An α-1,3-fucosyltransferase mutant, characterized in that, The amino acid sequence of the mutant includes: (1) Compared with the wild-type α-1,3-fucosyltransferase shown in the amino acid sequence SEQ ID NO.1, the mutation sites of the mutant include: H129W and / or A188H; (2) An amino acid sequence having one or more amino acid residue substitutions, deletions, additions, or any combination thereof compared with the amino acid sequence described in (1), and the substitution is a conservative substitution; and (3) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the amino acid sequence described in (1).

2. The mutant according to claim 1, wherein The mutation site is H129W, or A188H, or H129W and A188H; Preferably, the amino acid sequence of the α-1,3-fucosyltransferase mutant with the mutation site of H129W is as shown in SEQ ID NO.2; Preferably, the amino acid sequence of the α-1,3-fucosyltransferase mutant with the mutation site of A188H is as shown in SEQ ID NO.3; Preferably, the amino acid sequence of the α-1,3-fucosyltransferase mutant with the mutation sites of H129W and A188H is as shown in SEQ ID NO.

4.

3. A nucleic acid, characterized in that, The nucleic acid encodes the mutant according to any one of claims 1-2.

4. A carrier, characterized in that, The vector contains the nucleic acid according to claim 3.

5. A recombinant engineering bacterium or recombinant engineering cell comprising the nucleic acid according to claim 3, or the vector according to claim 4.

6. A method for preparing the mutant according to any one of claims 1-2, characterized in that, The method includes: culturing the recombinant engineering bacterium or recombinant engineering cell according to claim 5 to induce the expression of the mutant.

7. A biological preparation, characterized in that, The biological preparation includes: the mutant according to any one of claims 1-2, or the nucleic acid according to claim 3, or the vector according to claim 4, or the recombinant engineering bacterium or recombinant engineering cell according to claim 5, or the product prepared by the method according to claim 6.

8. Use of the mutant according to any one of claims 1-2, or the nucleic acid according to claim 3, or the vector according to claim 4, or the recombinant engineering bacterium or recombinant engineering cell according to claim 5, or the product prepared by the method according to claim 6, or the biological preparation according to claim 7 in the preparation of fucosylated oligosaccharides; Preferably, the fucosylated oligosaccharides are selected from one or more of 2'-fucosyllactose, 3'-fucosyllactose, difucosyllactose, lacto-N-difuco-tetraose, lacto-N-fucopentaose I, and lacto-N-difucohexaose I; More preferably, the fucosylated oligosaccharide is 3'-fucosyllactose.

9. A method for preparing fucosylated oligosaccharides by an in vitro enzymatic method, characterized in that, The method includes: (1) Providing the α-1,3-fucosyltransferase mutant according to any one of claims 1-2; (2) Provide a donor substrate and an acceptor substrate, and culture a host cell under suitable nutritional conditions allowing for the production of the fucosylated oligosaccharide and conditions allowing for the expression of the α-1,3-fucosyltransferase mutant, so that the α-1,3-fucosyltransferase mutant in step (1) contacts the donor substrate and the acceptor substrate to prepare the fucosylated oligosaccharide; Preferably, the fucosylated oligosaccharide is selected from 2'-fucosyllactose, 3'-fucosyllactose, difucosyllactose, lacto-N-difuco-tetraose, lacto-N-fucopentaose I, and lacto-N-difucohexaose I; More preferably, the fucosylated oligosaccharide is 3'-fucosyllactose.

10. Use of the mutant according to any one of claims 1-2, or the nucleic acid according to claim 3, or the vector according to claim 4, or the recombinant engineered bacterium or recombinant engineered cell according to claim 5, or the product prepared by the method according to claim 6, or the biological preparation according to claim 7 in the preparation of food or health products.

Images