Method for improving soluble expression quantity of alpha-1, 3-fucosyltransferase

Abstract

The invention relates to the technical field of biology, in particular to a method for improving the soluble expression quantity of alpha-1, 3-fucosyltransferase. According to the invention, fucosyltransferase from helicobacter pylori is cut off, and a carrier RBS sequence is optimized, so that a fucosyltransferase high-yield strain is obtained, the expression quantity of fucosyltransferase is successfully increased to nearly 2.99 times of that of wild strain fermentation by the strain, the activity is increased by nearly 1.9 times under the same concentration, and the fucosyltransferase high-yield strain is obtained. The high-efficiency expression of fucosyltransferase in engineered escherichia coli is realized, and the fucosyltransferase has a huge application prospect in industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for increasing the soluble expression amount of alpha-1,3-fucosyltransferase. Background Art

[0002] α-1,3-fucosyltransferase (EC 2.4.1.152.) can catalyze the transfer of nucleotide-activated fucosyl to a galactosyl-terminated receptor to form an α-1,3-glycosidic bond. 3'-fucosyllactose synthesized by α-1,3-fucosyltransferase is one of the most abundant HMOs at the end of lactation. At the same time, 3'-fucosyllactose has a wide range of health benefits. Studies have shown that 3'-fucosyllactose can selectively stimulate the growth of beneficial bifidobacteria to support overall intestinal health. In addition, 3'-fucosyllactose has been shown to have certain immunomodulatory effects and antibacterial and anti-adhesion effects. Therefore, it also has broad application prospects in the pharmaceutical, health care, food, cosmetics and other industries.

[0003] Since the enzymatic synthesis of the substrate GDP-fucose is expensive, the use of genetically engineered strains to heterologously express α-1,3-fucosyltransferase is currently the mainstream means of biosynthesizing fucosylated products. However, the soluble expression level of the heterologously expressed α-1,3-fucosyltransferase gene in commercial strains such as Escherichia coli is extremely low, resulting in extremely poor enzyme catalytic efficiency; at the same time, the random mutation method to transform the substrate binding domain and catalytic domain of α-1,3-fucosyltransferase to improve enzyme activity has the uncertainty of random mutation, which is time-consuming and has low efficiency. None of the above technologies can be truly applied to industrial production, which greatly hinders the large-scale application of their products. Therefore, it is urgent to find a new method that can overcome the defects of low soluble expression level and easy formation of inclusion bodies and can significantly increase the soluble expression level of fucosyltransferase. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for increasing the soluble expression level of α-1,3-fucosyltransferase.

[0005] The present invention provides a fucosyltransferase variant, which includes a fucosyltransferase derived from Helicobacter pylori having at least one of the following mutations:

[0006] The amino acid at position 434 to position 478 is deleted; and / or

[0007] The amino acid at position 427 to 478 is deleted; and / or

[0008] The amino acid at position 399 to position 478 is deleted; and / or

[0009] The amino acids at positions 364 to 478 are missing.

[0010] Furthermore, the amino acid sequence of the fucosyltransferase derived from Helicobacter pylori is shown in SEQ ID NO:1.

[0011] In the present invention,

[0012] The amino acid sequence of the fucosyltransferase shown in SEQ ID NO:1, wherein the amino acid at positions 434 to 478 is deleted, is shown in SEQ ID NO:2;

[0013] The amino acid sequence of the fucosyltransferase shown in SEQ ID NO:1, wherein the amino acid at positions 427 to 478 is deleted, is shown in SEQ ID NO:3;

[0014] The amino acid sequence of the fucosyltransferase shown in SEQ ID NO:1, wherein the amino acid at positions 399 to 478 is deleted, is shown in SEQ ID NO:4;

[0015] The amino acid sequence of the fucosyltransferase shown in SEQ ID NO:1, wherein the amino acid at positions 364 to 478 is deleted, is shown in SEQ ID NO:5;

[0016] The present invention provides a nucleic acid encoding the fucosyltransferase variant of the present invention.

[0017] The present invention provides a transcription unit comprising an RBS and the nucleic acid of the present invention.

[0018] Furthermore, the RBS is selected from at least one or more of the nucleotide sequences shown in SEQ ID NO:6 to SEQ ID NO:8.

[0019] The present invention provides a recombinant vector, which comprises at least one of the nucleic acid of the present invention and / or the transcription unit of the present invention and a vector backbone.

[0020] Furthermore, the vector backbone includes pET22b.

[0021] The present invention provides a host cell which is transfected or transformed with the recombinant vector of the present invention.

[0022] The present invention truncated the fucosyltransferase derived from Helicobacter pylori and optimized RBS to obtain R1-Δ45, R1-Δ52, R1-Δ80, R1- Δ115, R2-Δ45, R2-Δ52, R2-Δ80, R2-Δ115, R3-Δ45, R3-Δ52, R3-Δ80, and R3-Δ115 mutants. The test results showed that the yield of fucosyltransferase after the Δ80 (SEQ ID NO: 4) truncation was combined with each RBS could reach the highest, followed by Δ52. Secondly, among the three RBSs, the R2-Δ80 strain obtained by combining R2 (SEQID NO: 7) with the Δ80 truncation had the highest expression amount of fucosyltransferase.

[0023] The existing strategy to increase the expression of enzyme protein is mainly to regulate gene expression elements, that is, to optimize from aspects such as promoter, transcription factor, RBS, N-terminal sequence and enzyme gene itself. It has been reported in previous articles that when expressing fucosyltransferase, the enzyme can be expressed smoothly by adding a special sequence at the N-terminus, but its expression level is very low. The present invention finds that the heptapeptide repeat sequence and the hydrophobic residue-rich sequence sequence at the C-terminus of fucosyltransferase can effectively enhance the soluble expression of fucosyltransferase and reduce the formation of its inclusion bodies. At the same time, the synergistic optimization of the RBS region proposed in this scheme can effectively improve the enzyme production level of engineered bacteria and further increase the expression of fucosyltransferase.

[0024] In the present invention, the nucleic acid can be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. Nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acids can be linear or circular in topology. Nucleic acids can be part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0025] In the present invention, the nucleic acid may be optimized or unoptimized; the optimization includes but is not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0026] The present invention also provides a transcription unit, and the expression unit refers to a DNA sequence starting from a promoter recognition region (RBS) and ending at a terminator. A regulatory segment may also be included on both sides or between the promoter and the terminator, and the regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably linked to the nucleic acid sequence, such as an enhancer of the promoter, a poly (A) signal, etc.

[0027] The present invention provides a recombinant vector, which comprises at least one of the nucleic acid or expression element described in the present invention and a vector backbone.

[0028] Further, the sources of the vector backbone include plants, animals, bacteria, fungi, phages, or viruses, which are not limited in the present invention. The viral vectors include: adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, or lentivirus vectors, etc. The phage vectors include phagemids and auxiliary vectors, and the phagemids include but are not limited to pBluescript II-KS (+), pcomb3XSS, pCANTAB5E or pKK233.3. The animals include mammals and non-mammals, and the mammalian expression vectors include but are not limited to pcDNA 3.1, pIRES, pTT3, pCEP4, pATX1, or pCHO1.0. The bacterial vector includes but is not limited to pET22b, pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, pColdII, etc. The fungal vector includes but is not limited to pYES2, pYES3, pYES6, pAUR23, etc.

[0029] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule, which comprises a desired coding sequence and an appropriate nucleic acid sequence or element that is essential for the expression of an operably connected coding gene in a specific host organism. The nucleic acid sequence or element necessary for the expression in prokaryotic cells includes a promoter, a ribosome binding site and possible other sequences. Known eukaryotic cells utilize promoters, enhancers and terminators. Once transformed into a suitable host, the vector can be replicated and play a role independently of the host genome, or, in some cases, integrate into the genome by itself. In this specification, "plasmid" and "vector" can sometimes be interchanged and used, because plasmid is the most commonly used vector form at present. However, the present invention is intended to include such other forms of expression vectors, which play an equivalent role, which is known or will become known in the art, including but not limited to: plasmid, phage particle, viral vector and / or only potential genomic insert. In a specific embodiment, the nucleic acid encoding the fusion protein provided by the present invention can be constructed in various prokaryotic expression vectors, such as pET22b.

[0030] The present invention provides a host cell transformed or transfected with the recombinant vector. The host cell is transformed or transfected with a vector constructed using recombinant DNA technology, so that the transformed host cell has the ability to replicate the protein-encoding vector or express the desired protein.

[0031] Furthermore, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0032] The host cells provided by the present invention may be derived from plants, animals, bacteria, fungi, bacteriophages or viruses, but the present invention is not limited thereto.

[0033] The present invention provides the use of at least one of the following I) to VI) in the preparation of a fucosylated product:

[0034] I), the fucosyltransferase variant of the present invention;

[0035] II), the nucleic acid of the present invention;

[0036] III), the transcription unit of the present invention;

[0037] IV), the recombinant vector of the present invention;

[0038] V), the host cell of the present invention;

[0039] VI), culturing the host cell of the present invention to obtain a culture.

[0040] The present invention provides a product for preparing a fucosylated product, characterized in that it comprises auxiliary materials and at least one of the following A) to F):

[0041] A), the fucosyltransferase variant of the present invention;

[0042] B), the nucleic acid of the present invention;

[0043] C), the transcription unit of the present invention;

[0044] D), the recombinant vector of the present invention;

[0045] E), the host cell of the present invention;

[0046] F), culturing the host cell of the present invention to obtain a culture.

[0047] Furthermore, the auxiliary materials include buffers, culture media, antibiotics, stabilizers, antioxidants and / or reducing agents used to maintain the activity of the product, assist the product to function or obtain the product, and the present invention is not limited to this.

[0048] The present invention provides a method for preparing a fucosylated product, characterized in that the method comprises obtaining the fucosylated product by using at least one of the following i) to vii):

[0049] i), the fucosyltransferase variant of the present invention;

[0050] ii), the nucleic acid of the present invention;

[0051] iii), the transcription unit of the present invention;

[0052] iv), the recombinant vector of the present invention;

[0053] v), the host cell of the present invention;

[0054] vi), culturing a culture obtained by culturing the host cell of the present invention;

[0055] vii), the product described in the present invention.

[0056] In the present invention, fucose is used as a substrate and the fucosyltransferase of the present invention is used for catalysis to obtain a fucosylated product.

[0057] The present invention obtains a high-yield strain of fucosyltransferase by truncating the fucosyltransferase from Helicobacter pylori and optimizing the vector RBS sequence. The strain successfully increases the expression amount of fucosyltransferase to nearly 2.99 times that of the wild-type strain fermentation, and the activity at the same concentration is increased by nearly 1.9 times, thereby achieving efficient expression of fucosyltransferase in engineered Escherichia coli, and has great application prospects in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The results of measuring the concentration of recombinant protein by Coomassie Brilliant Blue method are shown;

[0059] Figure 2 The SDS-PAGE results of the wild type and R2-futAΔ80 are shown, wherein A is the SDS-PAGE result of the wild type; B is the SDS-PAGE result of R2-futAΔ80;

[0060] Figure 3 The results of enzyme activity measurement by high performance liquid chromatography are shown;

[0061] Figure 4 The results of enzyme activity determination using a glycosyltransferase kit (R&D Systems) are shown. DETAILED DESCRIPTION

[0062] The present invention provides a method for increasing the soluble expression of α-1,3-fucosyltransferase. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0063] Original sequence of fucosyltransferase futA: MFQPLLDAYVESASIEKMASKSPPPLKIAVANWWGDEEIKEFKNSVLYFILSQRYTITLHQNPNEFSDLVFGNPLGSARKILSYQNAKRVFYTGENESPNFNLFDYAIGFDELDFNDRYLRMPLYYDRLHHKAESVNDTTAPYKLKDNSLYALKKPSHCFKEKHPNLCAVVNDESDPLKRGFASFVASNPNAPIRNAFYDALNSIEPVTGGGSVRNTLGYNVKNKNEFLSQYKFNLCFENTQGYGYVTEKIIDAYFSHTIPIYWGSPSVAKDFNPKSFVNVHDFKNFDEAIDYIKYLHTHKNAYLDMLYENPLNTLDGKAYFYQNLSFKKILAFFKTILENDTIYHDNPFIFCRDLNEPLVTIDDLRVNYDDLRVNYDDLRINYDDLRVNYDDLRINYDDLRVNYDDLRVNYDDLRINYDDLRVNYDDLRVNYERLLSKATPLLELSQNTTSKIYRKAYQKSLPLLRAIRRWVKKLGL(SEQ ID NO:1);

[0064] Amino acid sequence of fucosyltransferase variant futAΔ45: MFQPLLDAYVESASIEKMASKSPPPLKIAVANWWGDEEIKEFKNSVLYFILSQRYTITLHQNPNEFSDLVFGNPLGSARKILSYQNAKRVFYTGENESPNFNLFDYAIGFDELDFNDRYLRMPLYYDRLHHKAESVNDTTAPYKLKDNSLYALKKPSHCFKEKHPNLCAVVNDESDPLKRGFASFVASNPNAPIRNAFYDALNSIEPVTGGGSVRNTLGYNVKNKNEFLSQYKFNLCFENTQGYGYVTEKIIDAYFSHTIPIYWGSPSVAKDFNPKSFVNVHDFKNFDEAIDYIKYLHTHKNAYLDMLYENPLNTLDGKAYFYQNLSFKKILAFFKTILENDTIYHDNPFIFCRDLNEPLVTIDDLRVNYDDLRVNYDDLRINYDDLRVNYDDLRINYDDLRVNYDDLRVNYDDLRINYDDLRVNY (SEQ ID NO:2);

[0065] Amino acid sequence of fucosyltransferase variant futAΔ52: MFQPLLDAYVESASIEKMASKSPPPLKIAVANWWGDEEIKEFKNSVLYFILSQRYTITLHQNPNEFSDLVFGNPLGSARKILSYQNAKRVFYTGENESPNFNLFDYAIGFDELDFNDRYLRMPLYYDRLHHKAESVNDTTAPYKLKDNSLYALKKPSHCFKEKHPNLCAVVNDESDPLKRGFASFVASNPNAPIRNAFYDALNSIEPVTGGGSVRNTLGYNVKNKNEFLSQYKFNLCFENTQGYGYVTEKIIDAYFSHTIPIYWGSPSVAKDFNPKSFVNVHDFKNFDEAIDYIKYLHTHKNAYLDMLYENPLNTLDGKAYFYQNLSFKKILAFFKTILENDTIYHDNPFIFCRDLNEPLVTIDDLRVNYDDLRVNYDDLRINYDDLRVNYDDLRINYDDLRVNYDDLRVNYDDLRINYDDLRVNY (SEQ ID NO:3);

[0066] Amino acid sequence of fucosyltransferase variant futAΔ80: MFQPLLDAYVESASIEKMASKSPPPLKIAVANWWGDEEIKEFKNSVLYFILSQRYTITLHQNPNEFSDLVFGNPLGSARKILSYQNAKRVFYTGENESPNFNLFDYAIGFDELDFNDRYLRMPLYYDRLHHKAESVNDTTAPYKLKDNSLYALKKPSHCFKEKHPNLCAVVNDESDPLKRGFASFVASNPNAPIRNAFYDALNSIEPVTGGGSVRNTLGYNVKNKNEFLSQYKFNLCFENTQGYGYVTEKIIDAYFSHTIPIYWGSPSVAKDFNPKSFVNVHDFKNFDEAIDYIKYLHTHKNAYLDMLYENPLNTLDGKAYFYQNLSFKKILAFFKTILENDTIYHDNPFIFCRDLNEPLVTIDDLRVNYDDLRVNYDDLRINYDDLRVNYDDLRINY (SEQ ID NO:4);

[0067] Amino acid sequence of fucosyltransferase variant futAΔ115: MFQPLLDAYVESASIEKMASKSPPPLKIAVANWWGDEEIKEFKNSVLYFILSQRYTITLHQNPNEFSDLVFGNPLGSARKILSYQNAKRVFYTGENESPNFNLFDYAIGFDELDFNDRYLRMPLYYDRLHHKAESVNDTTAPYKLKDNSLYALKKPSHCFKEKHPNLCAVVNDESDPLKRGFASFVASNPNAPIRNAFYDALNSIEPVTGGGSVRNTLGYNVKNKNEFLSQYKFNLCFENTQGYGYVTEKIIDAYFSHTIPIYWGSPSVAKDFNPKSFVNVHDFKNFDEAIDYIKYLHTHKNAYLDMLYENPLNTLDGKAYFYQNLSFKKILAFFKTILENDTIYHDNPFIFCRDLNEPLVTI (SEQ ID NO:5);

[0068] Nucleotide sequence of R1: tttgtttaactttaagaaggaga (SEQ ID NO:6);

[0069] Nucleotide sequence of R2: taaatatactataaggaggttacttt (SEQ ID NO: 7);

[0070] The nucleotide sequence of R3: aagttttagactataaggaggtttttt (SEQ ID NO: 8);

[0071] At present, the mainstream means of producing fucosylated products is biological method, which uses genetically engineered strains for fermentation, and fucosyltransferase catalyzes the reaction in the strain to achieve the conversion and synthesis from substrate to product. However, the key step of this method is still the catalysis of fucosyltransferase. The expression level of the enzyme in the heterologous strain will greatly affect the efficiency of catalytic synthesis, which is further related to the concentration of the product. It is now widely reported that the yield of fucosylated products produced by whole-cell fermentation, such as 3'-fucosyllactose, is relatively low. Although the yield of this method has reached the commercial production level through multi-faceted regulation and optimization, the current targeted optimization gap of fucosyltransferase is still the key node that limits the further increase of yield. At present, this method still has the problems of low yield and poor substrate conversion efficiency.

[0072] In response to the above problems, this protocol proposes a method for increasing the expression of fucosyltransferase:

[0073] (1) The present invention first designs RBS sequences of different strengths to replace the original part on the vector, and systematically truncates the heptapeptide repeat sequence (DDLRVNY) and the hydrophobic residue-rich sequence at the C-terminus of the fucosyltransferase, and expresses it in an expression host, thereby successfully increasing the expression level of the fucosyltransferase to nearly 2.99 times that of the wild-type strain fermentation;

[0074] (2) After fermenting the recombinant Escherichia coli of the present invention for 6 hours, the apparent enzyme activity of the intracellular fucosyltransferase can be increased by 5.7 (2.99×1.9) times;

[0075] (3) The present invention achieves efficient expression of fucosyltransferase in engineered Escherichia coli and has great application prospects in industrial production.

[0076] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0077] Example 1 Fucosyltransferase mutants and their expression

[0078] 1. Heterologous expression of α-1,3-fucosyltransferase from Helicobacter pylori

[0079] The α-1, 3-fucosyltransferase gene futA sequence (AAB81031.1) of Helicobacter pylori (NCTC 11639) published on NCBI was synthesized. The futA gene and expression vector pET22b were amplified by PCR, and the template plasmid was digested with DpnI enzyme, then transformed into E. coli DH5α competent cells, and transformed into E. coli BL21 (DE3) competent cells after overnight culture. The cells were revived at 37℃ for 1h and coated on LB plates with a final concentration of 0.1mM ampicillin resistance, and cultured at 37℃ for 10-16h. Finally, single colonies on the ampicillin resistance plate were selected for colony PCR verification, and sequencing was used to confirm whether the α-1,3-fucosyltransferase FutA was successfully constructed. The plasmid with the correct sequencing was pET22b-futA.

[0080] 2. RBS sequence optimization design

[0081] The fucosyltransferase sequence was calculated by the RBS Calculator online server, and three RBS sequences with different strengths were obtained, namely R1, R2, and R3; primers were designed to replace the original RBS sequence on pET22b by point mutation PCR, and the RBS sequence optimized vectors pET22b-R1-futA, pET22b-R2-futA, and pET22b-R3-futA were obtained. The above RBS sequence replacement vectors were transformed into the expression host for expression. The specific operations are as follows: the recombination reaction system was transformed into Escherichia coli DH5α competent cells, and after overnight culture, it was transformed into Escherichia coli DH5α competent cells, revived at 37℃ for 1h, coated with ampicillin resistance LB plates with a final concentration of 0.1mM, and cultured at 37℃ for 10~16h. Finally, single colonies on the ampicillin resistance plate were selected for colony PCR verification, and sequencing was used to confirm whether the RBS sequence replacement vector was successfully constructed.

[0082] 3. Systematic truncation of the C-terminal sequence of fucosyltransferase

[0083] Using the recombinant plasmid pET22b-futA sequenced correctly in the above step (1) as a template, primers were designed to select appropriate restriction sites to truncate the heptapeptide repeat sequence (DDLRVNY) and the hydrophobic residue-rich sequence at the C-terminus of the fucosyltransferase, truncating 45, 52, 80, and 115 amino acids, respectively, to obtain truncated sequences Δ45, Δ52, Δ80, and Δ115 with restriction sites at both ends. The mutant was integrated into pET22b, and pET22b-futAΔ45, pET22b-futAΔ52, pET22b-futAΔ80, and pET22b-futAΔ115 were obtained and then transformed into an expression host for expression. The specific operation was as follows: the recombination reaction system was transformed into Escherichia coli DH5α competent cells, and after overnight culture, it was transformed into Escherichia coli DH5α competent cells, revived at 37°C for 1h, coated with an ampicillin resistance LB plate with a final concentration of 0.1mM, and cultured at 37°C for 10-16h. Finally, a single colony on the ampicillin resistance plate was selected for colony PCR verification, and sequencing was used to confirm whether the recombinant expression vector was successfully constructed.

[0084] 4. Construction of synergistically optimized fusion protein particles

[0085] The plasmid vectors and sequences constructed in "two" and "three" were digested with enzymes, and the linearized vector fragments and the truncated sequence fragments after digestion were purified and recovered, and then the two were recombined and connected using T4 ligase. The RBS sequence was connected to the N-terminus of the futA gene to construct a synergistically optimized fusion protein sequence to obtain the recombinant expression vectors pET22b-R1-futAΔ45, pET22b-R1-futAΔ52, pET22b-R1-futAΔ80, pET22b-R1-futAΔ115, pET22b-R2-futAΔ45, pET22b-R2-futAΔ52, pET22b-R2-futAΔ80, pET22b-R2-futAΔ115, pET22b-R3-futAΔ45, pET22b-R3-futAΔ52, pET22b-R3-futAΔ80, and pET22b-R3-futAΔ115.

[0086] The above recombinant expression vectors were transformed into expression hosts for expression. The specific operations were as follows: the recombinant reaction system was transformed into E. coli DH5α competent cells, and after overnight culture, it was transformed into E. coli DH5α competent cells, revived at 37°C for 1 hour, coated with LB plates with a final concentration of 0.1 mM ampicillin resistance, and cultured at 37°C for 10-16 hours. Finally, single colonies on the ampicillin resistance plates were selected for colony PCR verification, and sequencing was used to confirm whether the recombinant expression vector was successfully constructed.

[0087] 5. Shake flask fermentation to verify the enzyme production capacity of recombinant bacteria

[0088] The recombinant expression vectors sequenced correctly in the above examples were transferred into BL21 (DE3) competent cells, revived at 37°C for 1 hour, coated with LB plates with a final concentration of 0.1 mM ampicillin resistance, and cultured at 37°C for 10-16 hours to obtain fermentation recombinant bacteria containing different RBS and C-terminal sequence truncations. Single colonies were picked and cultured in LB medium (tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L) with a final concentration of 0.1 mM ampicillin for 8-10 hours as seed liquid for shake flask fermentation. Then, the seed solution was inoculated into a 250mL Erlenmeyer flask containing 20~25mL fermentation medium at a 1% inoculation rate, and ampicillin with a final concentration of 0.1mM was added. The formula of the fermentation medium was: 1.2% (W / V) tryptone, 2.4% (W / V) yeast extract, 0.4% (V / V) glycerol, phosphate buffer 17mM KH2PO4, 72mM K2HPO4. After that, the Erlenmeyer flask was placed at 37°C and 200r / min for 2~4h, and IPTG with a final concentration of 0.25mM was added to the shake flask, and cultured at 26°C and 150r / min for 2~10h.

[0089] After the fermentation broth was centrifuged at 12000rpm for 15-20min, the cells were collected, resuspended and ultrasonically broken, and low-temperature high-speed centrifugation was performed to obtain the cell-breaking supernatant. The target protein was purified using Ni-NTA using the histidine tag affinity purification principle. The cell-breaking supernatant was passed through the nickel column three times, then washed with a washing solution (Tris-HCl buffer system, 10mM imidazole concentration) to remove non-affinity adsorbed impurities, and finally eluted with an eluent (Tris-HCl buffer system, 500mM imidazole concentration) The target protein was eluted three times and collected separately. The imidazole in the eluent was removed by dialysis, and a relatively pure recombinant fusion protein was finally obtained.

[0090] 1. Bradford method to measure the concentration of recombinant protein

[0091] The concentration of the purified recombinant protein was measured by the Bradford method and the correct expression of the recombinant protein was confirmed by SDS-PAGE. The results of the recombinant protein concentration measurement by the Bradford method are as follows: Figure 1 As shown in Table 1. Under the same bacterial cell disruption-protein purification operation conditions, the strain BL21-pET22b-R2-futAΔ80 had the strongest enzyme production capacity, which was about 2.99 times that of the wild type ().

[0092] Table 1. Protein concentration

[0093]

[0094] 2. SDS-PAGE results

[0095] SDS-PAGE results are as follows Figure 2 As shown in the figure, Sup: cell disruption supernatant, E1: first elution, E2: second elution. WT is the FUTA protein without RBS replacement and C-terminal sequence truncation synergistic optimization, and RBS modification & C-terminal truncation is the recombinant fusion protein with the best expression after RBS replacement and C-terminal sequence truncation synergistic optimization (BL21- pET22b-R2-futAΔ80). Comparing the gel running results of the wild-type and optimized recombinant protein purification products, there are obvious bands at 44.3~66.4 kDa, which is consistent with the molecular weight of fucosyltransferase, and the expression level of the optimized recombinant protein is significantly improved.

[0096] 6. Enzyme activity measurement

[0097] The fermentation broth of the strain BL21-pET22b-R2-futAΔ80 with the best synergistic optimization expression in "Five" was centrifuged at 12000rpm for 15-20min, and the cells were collected, resuspended, and ultrasonically disrupted. The cell supernatant was obtained by low-temperature high-speed centrifugation. A relatively pure enzyme solution was obtained by histidine tag purification, and imidazole was removed by dialysis and the salt concentration of the buffer was adjusted. The final enzyme solution (R2-futAΔ80) was stored for later use.

[0098] 1. Determination of enzyme activity by high performance liquid chromatography

[0099] Add 100μL of 100μM GDP-L-fucose and 100μL of 5mM lactose, and then add 250μL of enzyme solution, incubate at 37℃ for 30~60min to achieve catalytic generation of 3'-FL. After the catalysis is completed, put it on ice immediately to stop the reaction. Then, after high-speed centrifugation at low temperature and ultrafiltration through a desalting column to remove impurities, a high-performance liquid chromatography (HPLC) is used to determine the 3'-FL yield in the catalytic system and the remaining amount of the substrate. The reaction solution obtained by catalysis is passed through a 0.22μm filter membrane for use. Prepare the mobile phase required for liquid chromatography detection, mobile phase A: acetonitrile, mobile phase B = 0.5% ammonium acetate aqueous solution. Select amide chromatographic column (150mm×2.1μm), the detection conditions are: mobile phase ratio 80:20, flow rate 0.2m / L, column temperature 35℃, detector using evaporative light scattering detector, evaporation temperature 70℃, carrier gas flow rate 2.0SLM. The product is quantified by external standard method. HPLC test results are as follows Figure 3 shown.

[0100] like Figure 3As shown, in the sample of the catalytic system, a new substance peak was generated, the product 3'-FL was detected, and the reduction and disappearance of the GDP-fucose substance peak were observed. The content of the substance was calculated using the peak area by the external standard method, and the product yield was 10.01μM 3'-FL / mM Lactose.

[0101] 2. Glycosyltransferase kit to determine enzyme activity

[0102] The enzyme activity was measured by comparison using a glycosyltransferase kit (R&D Systems). The purified non-optimized fucosyltransferase WT and the optimized recombinant protein R2-futAΔ80 were diluted to the same concentration and reacted according to the kit instructions, and the absorbance was measured using a microplate reader.

[0103] After measuring the absorbance with a microplate reader, the WT enzyme activity was calculated to be 1.65U, and the enzyme activity of R2-futAΔ80 was 3.12U. ​​After synergistic optimization, the enzyme activity increased by about 1.9 times.

[0104] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fucosyltransferase variant, characterized in that The fucosyltransferase derived from Helicobacter pylori has at least one of the following mutations: The amino acid at position 434 to position 478 is deleted; and / or The amino acid at position 427 to 478 is deleted; and / or The amino acid at position 399 to position 478 is deleted; and / or The amino acids at positions 364 to 478 are missing.

2. The fucosyltransferase variant according to claim 1, characterized in that The amino acid sequence of the fucosyltransferase derived from Helicobacter pylori is shown in SEQ ID NO:

1.

3. A nucleic acid, characterized in that Encoding the fucosyltransferase variant according to claim 1 or 2.

4. A transcription unit, characterized in that Comprising RBS and the nucleic acid as claimed in claim 3.

5. The transcription unit according to claim 4, characterized in that The RBS is selected from at least one or more of the nucleotide sequences shown in SEQ ID NO: 6 to SEQ ID NO:

8.

6. A recombinant vector, characterized in that It comprises at least one of the nucleic acid according to claim 3 and / or the transcription unit according to claim 4 and a vector backbone.

7. A host cell, characterized in that Transfect or transform the recombinant vector as described in claim 6.

8. Use of at least one of the following I) to VI) in the preparation of a fucosylated product: 1), the fucosyltransferase variant according to claim 1 or 2; II), the nucleic acid according to claim 3; III), the transcription unit according to claim 4; IV), the recombinant vector according to claim 6; V), the host cell according to claim 7; VI), culturing a culture obtained by culturing the host cell according to claim 7.

9. A method for preparing a fucosylated product, characterized in that Including auxiliary materials and at least one of the following A) to F): A), the fucosyltransferase variant according to claim 1 or 2; B), the nucleic acid according to claim 3; C), the transcription unit according to claim 4; D), the recombinant vector according to claim 6; E), the host cell according to claim 7; F), culturing a culture obtained by culturing the host cell according to claim 7.

10. A method for preparing a fucosylated product, characterized in that: Including using at least one of the following i) to vii): i), the fucosyltransferase variant according to claim 1 or 2; ii), the nucleic acid according to claim 3; iii), the transcription unit according to claim 4; iv) The recombinant vector according to claim 6; v), the host cell according to claim 7; vi), culturing a culture obtained by culturing the host cell according to claim 7; vii) The product according to claim 9.

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