In vitro two-step enzymatic process for efficient synthesis of GDP-L-fucose and its application

Abstract

The present invention relates to the field of biotechnology, and in particular to an in vitro two-step enzymatic process for efficiently synthesizing GDP-L-fucose and its application. The present invention utilizes a bifunctional enzyme (Uniprot: Q58T34) having the amino acid sequence shown in SEQ ID NO: 1 as a template for site-directed mutagenesis to obtain variants M07 with high L-Fucose kinase activity and M10 with high GDP-L-Fucose pyrophosphorylase activity. The variants are then used to prepare GDP-L-Fucose using an ATP regeneration system in the presence of FKP and PPA enzymes using a two-step enzymatic process. The resulting GDP-L-Fucose has high yield and purity, low production cost and energy consumption, is suitable for large-scale production, and has broad application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an in vitro two-step enzymatic process for efficiently synthesizing GDP-L-fucose and its application. Background Art

[0002] GDP-L-fucose (guanosine 5'-diphosphate (GDP)-L-Fucose), a nucleotide sugar, is an important substrate in the synthesis of fucosyl compounds, especially an essential intermediate in the biosynthesis of fucosylated human milk oligosaccharides (HMOs).

[0003] The existing process for the in vitro enzymatic preparation of GDP-L-fucose is to use de novo synthesis or direct salvage methods; the de novo synthesis is to obtain GDP-L-fucose through a multi-enzyme cascade reaction catalyzed by: using cheap guanosine diphosphate and sucrose as substrates, GDP-L-fucose is catalyzed by sucrose synthase, CDP-tyvetose 2-epimerase, GDP-mannose dehydratase and isomeroreductase. The direct salvage is to use the bifunctional enzyme FKP enzyme to catalyze the one-pot synthesis of GDP-L-fucose using the exogenous substrate L-fucose, ATP and GTP, as shown in the reaction formula: Figure 3 shown.

[0004] De novo synthesis using a multi-enzyme cascade is cost-effective, but this approach requires a long preparation route, multiple enzyme types, and high enzyme dosages. Direct salvage synthesis methods, due to the discrepancies in the enzymatic properties of the two active domains of the bifunctional FKP enzyme, present difficulties in designing reaction conditions. Current technologies often utilize a single reaction (simultaneous feeding, single pH conditions) to achieve milligram-scale reactions. This results in a small reaction system, long reaction times, and low conversion rates of 10% to 30%. Low reaction temperatures lead to incomplete conversion, while high reaction temperatures can cause GDP-L-fucose to decompose unstably. This directly leads to a time-consuming preparation process, low overall yields, and difficulties in isolating and purifying the final product. Therefore, there is an urgent need to develop scalable converting enzymes and catalytic methods that can rapidly and stably obtain GDP-L-fucose. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an in vitro two-step enzymatic process for efficiently synthesizing GDP-L-fucose and its application.

[0006] The present invention provides L-fucokinase / GDP-L-fucose pyrophosphorylase variants, which include L-fucokinase variants and / or GDP-L-fucose pyrophosphorylase variants;

[0007] The L-fucokinase variant includes an L-fucokinase / GDP-L-fucose pyrophosphorylase with an amino acid sequence as shown in SEQ ID NO: 1 having the following mutations:

[0008] Glutamine Q at position 623 is mutated to serine S; and / or

[0009] Glutamine Q at position 627 is mutated to glycine G; and / or

[0010] Tyrosine Y at position 629 is mutated to Serine S; and / or

[0011] Lysine K at position 631 is mutated to arginine R; and / or

[0012] Aspartic acid D at position 644 was mutated to alanine A;

[0013] The GDP-L-fucose pyrophosphorylase variant includes an L-fucokinase / GDP-L-fucose pyrophosphorylase with an amino acid sequence as shown in SEQ ID NO: 1 having the following mutations:

[0014] Arginine R at position 592 is mutated to lysine K; and / or

[0015] Proline P at position 711 is mutated to alanine A; and / or

[0016] Glycine G at position 713 is mutated to alanine A; and / or

[0017] Serine S at position 714 is mutated to Threonine T; and / or

[0018] The leucine L at position 722 was mutated to alanine A.

[0019] Furthermore, in the L-fucokinase / GDP-L-fucose pyrophosphorylase variant of the present invention,

[0020] The L-fucokinase variants include:

[0021] The L-fucokinase / GDP-L-fucose pyrophosphorylase of SEQ ID NO: 1 wherein the glutamine Q at position 623 is mutated to serine S and the glutamine Q at position 627 is mutated to glycine G;

[0022] and / or the L-fucokinase / GDP-L-fucose pyrophosphorylase described in SEQ ID NO: 1, wherein the tyrosine at position 629 is mutated to serine S and the lysine at position 631 is mutated to arginine R;

[0023] and / or the L-fucokinase / GDP-L-fucose pyrophosphorylase as described in SEQ ID NO: 1, wherein the tyrosine at position 629 is mutated to serine S, the lysine at position 631 is mutated to arginine R, and the aspartic acid at position 644 is mutated to alanine A;

[0024] and / or the L-fucokinase / GDP-L-fucose pyrophosphorylase as described in SEQ ID NO: 1, wherein glutamine Q at position 623 is mutated to serine S, glutamine Q at position 627 is mutated to glycine G, tyrosine Y at position 629 is mutated to serine S, and lysine K at position 631 is mutated to arginine R;

[0025] The GDP-L-fucose pyrophosphorylase variants include:

[0026] and / or the L-fucokinase / GDP-L-fucose pyrophosphorylase described in SEQ ID NO: 1 wherein the arginine R at position 592 is mutated to lysine K and the proline P at position 711 is mutated to alanine A;

[0027] and / or the L-fucokinase / GDP-L-fucose pyrophosphorylase described in SEQ ID NO: 1, wherein proline P at position 711 is mutated to alanine A, glycine G at position 713 is mutated to alanine A, and serine S at position 714 is mutated to threonine T;

[0028] And / or the L-fucokinase / GDP-L-fucose pyrophosphorylase described in SEQ ID NO: 1, wherein the arginine R at position 592 is mutated to lysine K, the proline P at position 711 is mutated to alanine A, the serine S at position 714 is mutated to threonine T, and / or the leucine L at position 722 is mutated to alanine A.

[0029] In the present invention, a bifunctional enzyme BFFKP (Uniprot: Q5LC59) derived from Bacteroides fragilis is mutated and used for the synthesis of GDP-L-fucose; the bifunctional enzyme BFFKP has both L-fucokinase and GDP-L-fucose pyrophosphorylase activities, and its amino acid sequence is shown in SEQ ID NO: 1.

[0030] The present invention constructs variants M01 to M10 using the bifunctional enzyme BFFKP as a template through truncation and point mutation. Variants M04 to M07 have high L-fucokinase activity, and M07 to M10 have high GDP-L-fucose pyrophosphorylase activity. Test results show that among the variants M04 to M07 with L-fucokinase activity, the activity of M07 is significantly higher than that of the other variants; and among the variants M07 to M10 with GDP-L-fucose pyrophosphorylase activity, the activity of M10 is significantly better than that of the other variants.

[0031] In a specific embodiment of the present invention, the mutation sites corresponding to the variants M04 to M10 are shown in Table 1.

[0032] The present invention provides a composition comprising composition 1 and / or composition 2:

[0033] The composition 1 comprises the L-fucokinase variant and polyphosphate kinase (PPK enzyme) in the L-fucokinase / GDP-L-fucose pyrophosphorylase variant of the present invention;

[0034] The composition 2 comprises the GDP-L-fucose pyrophosphorylase variant and pyrophosphatase (PPA enzyme) among the L-fucokinase / GDP-L-fucose pyrophosphorylase variants of the present invention.

[0035] The present invention provides a biomaterial comprising at least one of the following A) to D):

[0036] A), a nucleic acid encoding the L-fucokinase / GDP-L-fucose pyrophosphorylase variant of the present invention or the composition of the present invention;

[0037] B), a recombinant vector containing the nucleic acid described in A);

[0038] C), transforming or transfecting the host cell with the recombinant vector described in B);

[0039] D) Cultivate the mixture obtained by culturing the host cells described in C).

[0040] Further,

[0041] The backbone of the recombinant vector is pET28a.

[0042] The host cell is Escherichia coli.

[0043] The nucleic acid described in the present invention 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 forms include cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The 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). The nucleic acid can be linear or circular in topology. The nucleic acid 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 recombinant, enzymatic or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0044] In the present invention, the nucleic acid may be codon-optimized or non-codon-optimized, and these optimizations include but are not limited to: codon usage bias, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, mRNA secondary structure, 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.

[0045] The present invention also provides a transcription unit comprising the nucleic acid, which refers to a DNA sequence starting with a promoter and ending with a terminator. The promoter and terminator may also be flanked or interposed with regulatory segments, which may include a promoter, enhancer, transcription termination signal, polyadenylation sequence, replication origin, nucleic acid restriction site, and homologous recombination site operably linked to the nucleic acid sequence, such as a promoter enhancer and poly(A) signal.

[0046] The present invention provides a recombinant vector comprising a vector backbone and the nucleic acid of the present invention.

[0047] Furthermore, the vector backbones described in the present invention may be derived from plants, animals, bacteria, fungi, phages, or viruses, but are not limited thereto. Bacterial vectors include, but are not limited to, pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, and pColdII. Fungal vectors include, but are not limited to, pYES2, pYES3, pYES6, and pAUR23.

[0048] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences or elements necessary for the expression of the operably linked coding gene in a specific host organism. Prokaryotes are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably, as plasmids are the most commonly used vector form. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to plasmids, phage particles, viral vectors, and / or simply potential genomic inserts. In a specific embodiment, the vector of the present invention is pET28a.

[0049] In the present invention, the host cell includes but is not limited to plants, animals, bacteria, fungi, phages or viruses, and the present invention is not limited to this; specifically, it is a bacterial cell, more specifically Escherichia coli. In a specific embodiment of the present invention, the cell is E. coli (BL21).

[0050] The present invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells, so that the transformed host cells have the ability to replicate protein-encoding vectors or express desired proteins.

[0051] In the present invention, the transformation methods include chemical transformation and electroporation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection methods include adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0052] The present invention provides the use of at least one of the following i) to iii) in the synthesis of GDP-L-Fucose:

[0053] i), the L-fucokinase / GDP-L-fucose pyrophosphorylase variant of the present invention;

[0054] ii), the composition of the present invention;

[0055] iii) The biomaterial of the present invention.

[0056] The present invention provides a method for preparing GDP-L-Fucose, which comprises synthesizing GDP-L-Fucose using at least one of the following methods I) to IV):

[0057] I), the L-fucokinase / GDP-L-fucose pyrophosphorylase variant of the present invention;

[0058] II), the composition of the present invention;

[0059] III), the biomaterial of the present invention.

[0060] Furthermore, the preparation method of the present invention comprises the following steps:

[0061] Step 1, L-fucose kinase variant catalyzes L-fucose to generate Fuc-1-P in the presence of auxiliary 1;

[0062] Step 2, the GDP-L-fucose pyrophosphorylase variant catalyzes Fuc-1-P to generate GDP-L-fucose in the presence of auxiliary substance 2;

[0063] The auxiliary material 1 includes an auxiliary material 1a and / or an auxiliary material 1b;

[0064] The auxiliary substance 1a includes Tris-HCl, ATP, manganese chloride and polyphosphate kinase (PPK enzyme);

[0065] The auxiliary substance 1b includes Tris-HCl, ATP, and manganese chloride;

[0066] The auxiliary 2 includes GTP, magnesium chloride and pyrophosphatase (PPA enzyme).

[0067] Furthermore,

[0068] In step 1, the pH value for catalyzing L-fucose to generate Fuc-1-P is 8.5-9.0, and the reaction time is 1.5-3 h, specifically 2 h;

[0069] In step 2, the pH for catalyzing Fuc-1-P to generate GDP-L-fucose is 6.5-7.0, and the reaction time is 3-5 h.

[0070] The present invention optimizes a method for preparing GDP-L-fucose using the variant. In a specific embodiment of the present invention, the preparation method includes preparing GDP-L-Fucose by a one-pot enzymatic method, preparing GDP-L-Fucose by an enzyme-catalyzed cascade reaction using a two-step enzymatic method and PPA, and preparing GDP-L-Fucose by using an ATP regeneration system in the presence of FKP enzyme and PPA enzyme using the two-step enzymatic method. Experimental results of the present invention indicate that the GDP-L-Fucose prepared by using an ATP regeneration system in the presence of FKP enzyme and PPA enzyme using the two-step enzymatic method has a higher yield and purity.

[0071] Specifically, in some embodiments of the present invention, when the auxiliary substance 1 is auxiliary substance 1a, the preparation method is the preparation of GDP-L-Fucose using an ATP regeneration system in the presence of FKP enzyme and PPA enzyme by means of a two-step enzymatic method, which comprises the following steps:

[0072] Step 1: L-fucose kinase variant catalyzes L-fucose to generate Fuc-1-P in the presence of auxiliary 1a;

[0073] Step 2, the GDP-L-fucose pyrophosphorylase variant catalyzes Fuc-1-P to generate GDP-L-fucose in the presence of auxiliary substance 2;

[0074] At this time, the concentration of the bacterial cells containing the L-fucokinase variant is 0.01 g / mL~0.025 g / mL (10 g / L~25 g / L), the concentration of Tris-HCl is 40 mM~60 mM, the concentration of ATP is 5 mM~6 mM, the concentration of manganese chloride is 40 mM~60 mM, and the concentration of polyphosphate kinase (PPK) is 1500 U / L~2500 U / L; the concentration of the bacterial cells containing the GDP-L-fucose pyrophosphorylase variant is 0.008 g / mL~0.009 g / mL (8 g / L~9 g / L), the concentration of GTP is 150 mM~250 mM, the concentration of magnesium chloride is 40 mM~60 mM, and the concentration of pyrophosphatase (PPA) is 1500 U / L~2500 U / L;

[0075] Specifically, as in Example 5, the amount of the bacterial cells containing the L-fucokinase variant was 10 g / L, the concentration of Tris-HCl was 50 mM, the concentration of ATP was 5.4 mM, the concentration of manganese chloride was 50 mM, and the concentration of polyphosphate kinase (PPK) was 2000 U / L; the amount of the bacterial cells containing the GDP-L-fucose pyrophosphorylase variant was 9 g / L, the concentration of GTP was 200 mM, the concentration of magnesium chloride was 50 mM, and the concentration of pyrophosphatase (PPA) was 2000 U / L;

[0076] Specifically, in other embodiments of the present invention, when the auxiliary substance 1 is auxiliary substance 1b, the preparation method is a two-step enzymatic method and PPA enzyme-catalyzed cascade reaction to prepare GDP-L-Fucose, which comprises the following steps:

[0077] Step 1: L-fucose kinase variant catalyzes L-fucose to generate Fuc-1-P in the presence of auxiliary 1b;

[0078] Step 2, the GDP-L-fucose pyrophosphorylase variant catalyzes Fuc-1-P to generate GDP-L-fucose in the presence of auxiliary substance 2;

[0079] At this time, when the auxiliary substance 1 is auxiliary substance 1b, the concentration of the bacterial cells containing the L-fucokinase variant is 0.01 g / mL~0.025 g / mL (10 g / L~25 g / L), the concentration of Tris-HCl is 40 mM~60 mM, the concentration of ATP is 150 mM~250 mM, and the concentration of manganese chloride is 40 mM~60 mM. The concentration of the bacterial cells containing the GDP-L-fucose pyrophosphorylase variant is 0.05 g / mL~1.5 g / mL, the concentration of GTP is 150 mM~250 mM, the concentration of magnesium chloride is 40 mM~60 mM, and the concentration of pyrophosphatase is 1500 U / L~2500 U / L;

[0080] Specifically, as in Example 4, when the auxiliary substance 1 is auxiliary substance 1b, in the 10 mL system in step 1, the concentration of the bacteria containing the L-fucokinase variant is 0.009 g / mL (9 g / L), the concentration of Tris-HCl is 50 mM, the concentration of ATP is 160 mM, and the concentration of manganese chloride is 50 mM; in step 2, the reaction substances are further added to the system in step 1, and the total reaction system is 11.5 mL, wherein the concentration of the bacteria containing the GDP-L-fucose pyrophosphorylase variant is 0.0082 g / mL (8.2 g / L), the concentration of GTP is 181 mM, the concentration of magnesium chloride is 43.4 mM, and the concentration of pyrophosphatase is 1739 U / L;

[0081] In the present invention, when the auxiliary substance is auxiliary substance 1a or auxiliary substance 1b, the L-fucokinase variant and the GDP-L-fucose pyrophosphorylase variant need to be prepared as pure enzymes and / or crude enzyme solutions when used. In a specific embodiment of the present invention, the crude enzyme solution is obtained by mixing Escherichia coli (BL21) cells containing the enzyme variant with a buffer at a mass ratio of 1:10 and then crushing the cells. The buffer is a Tris-HCl buffer, specifically 50 mM Tris-HCl (pH 7.5). In step 1, the volume of the crude enzyme solution of the L-fucokinase variant is 10% to 25% of the total reaction system volume, corresponding to the amount of cells used being 0.01 g / mL to 0.025 g / mL. In step 2, the volume of the crude enzyme solution of the GDP-L-fucose pyrophosphorylase variant is 8% to 10% of the total volume of the first reaction system, corresponding to the amount of cells used being 0.008 g / mL to 0.009 g / mL.

[0082] More specifically, in the preparation method of the present invention, the combination of enzymes in the preparation method is optimized; in a specific embodiment of the present invention, the L-fucokinase variant in step 1 may be any one of M04 to M07, and the GDP-L-fucose pyrophosphorylase in step 2 may be at least one of M08 to M10; or the L-fucokinase variant in step 1 may be M07, and the GDP-L-fucose pyrophosphorylase in step 2 may be M07; or the L-fucokinase variant in step 1 may be M10, and the GDP-L-fucose pyrophosphorylase in step 2 may be M10;

[0083] The test results of the present invention show that the yield and purity of GDP-L-fucose are the best when the L-fucokinase variant in step 1 is M07 and the GDP-L-fucose pyrophosphorylase in step 2 is M10.

[0084] The present invention designs a two-step method to carry out reactions at different pH values; considering that the L-fucokinase of FKP is inhibited by GTP and ADP, a step-by-step method is adopted, in which GTP is not introduced first and only ATP is added. In addition, the use of an ATP cycle system can also alleviate the inhibition of the kinase. After the phosphate-activated Fuc-1P is generated, GTP is added to generate the target product. Because it is a step-by-step reaction, it is also possible to realize the two-step enzyme reaction using the optimal metal ions: Mn 2+ and Mg 2+ Therefore, in the present invention, the enzymes, raw materials and reaction conditions used cooperate with each other and work synergistically to achieve high yield and high purity of GDP-L-fucose.

[0085] The present invention utilizes a bifunctional enzyme (Uniprot: Q58T34) with the amino acid sequence shown in SEQ ID NO: 1 as a template for site-directed mutagenesis, resulting in variants M07 and M10, respectively, with high L-Fucose kinase activity and GDP-L-Fucose pyrophosphorylase activity. These variants are then used to prepare GDP-L-Fucose using an ATP regeneration system in the presence of FKP and PPA enzymes in a two-step enzymatic process. This method achieves high GDP-L-Fucose yield and purity, with low production costs and energy consumption, making it suitable for large-scale production and possessing broad application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Shown is the comparison of L-Fucose kinase reaction activities of mutants (WT enzyme activity is 100%, and the activities of other mutants are relative activity values ​​%);

[0087] Figure 2Comparison of GDP-Fucose pyrophosphorylase activity is shown (WT activity is 100%, and the activities of other mutants are relative activity values ​​%);

[0088] Figure 3 The reaction formula for the one-pot enzymatic preparation of GDP-L-Fucose by the natural enzyme BFFKP is shown;

[0089] Figure 4 The results of one-pot enzymatic preparation of GDP-L-Fucose by natural enzyme BFFKP are shown;

[0090] Figure 5 The reaction formula for preparing GDP-L-Fucose by enzyme-catalyzed cascade reaction using a two-step enzymatic method (WT+WT, M07+M07, M10+M10, M07+M10) is shown;

[0091] Figure 6 Comparison of the yield of GDP-L-Fucose prepared by a two-step reaction using a single enzyme (WT, M07, or M10) and a dual enzyme (M07+M10) reaction;

[0092] Figure 7 It shows that the FKP mutant M07 catalyzes the "fucose + ATP" reaction for 2 hours;

[0093] Figure 8 The FKP mutants M07+M10 catalyzed the synthesis of GDP-L-Fucose for 2+4 hours;

[0094] Figure 9 The reaction formula for preparing GDP-L-Fucose using a 1L system, FKP enzyme (M07+M10) and a step-by-step enzymatic method, utilizing an ATP regeneration system and PPA to hydrolyze pyrophosphate is shown.

[0095] Figure 10 Liquid phase detection after the reaction is completed;

[0096] Figure 11 shows the HPLC spectrum of GDP-fucose after purification;

[0097] Figure 12 Shown is the mass spectrum of the product GDP-fucose. DETAILED DESCRIPTION

[0098] The present invention provides an in vitro two-step enzymatic process and application for the efficient synthesis of GDP-L-fucose. Those skilled in the art can refer to the contents herein and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications will be apparent to those skilled in the art and are considered to be encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel will clearly be able to modify or appropriately alter and combine the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0099] Enzyme related information:

[0100] In this patent: L-fucokinase / GDP-L-fucose pyrophosphorylase is a bifunctional enzyme derived from Bacteroides fragilis (Uniprot: Q5LC59), numbered BFFKP in this patent; in order to obtain a highly active monomer capable of performing a two-step reaction, BFFKP was used as a template for modification. The specific mutation site information is shown in Table 1.

[0101] Polyphosphate kinase (PPK), first expressed for enzyme catalysis: Polyphosphate kinase from a salmon bacterium: Renibacterium salmoninarum, Uniprot ID: A9WPD3, numbered PPK in this invention.

[0102] Pyrophosphatase (PPA): Inorganic pyrophosphatase derived from Escherichia coli (E Coli K12), Uniprot: P0A7A9, numbered as PPA in the present invention.

[0103] Amino acid sequence of BFFKP: MQKLLSLPSNLVQSFHELERVNRTDWFCTSDPVGKKLGSGGGTSWLLEECYNEYSDGATFGEWLEKEKRILLHAGGQSRRLPGYAPSGKILTPVPVFRWERGQHLGQNLLSLQLPLYEKIMSLAPDKLHTLIASGDVYIRSEKPLQSIPEADVVCYGLWVDPSLATHHGVFASDRKHPEQLDFMLQKPSLAELESLSKTHLFLMDIGIWLLSDRAVEILMKRSHKESSEELKYYDLYSDFGLALGTHPRIEDEEVNTLSVAILPLPGGEFYHYGTSKELISSTLSVQNKVYDQRRIMHRKVKPNPAMFVQNAVVRIPLCAENADLWIENSHIGPKWKIASRHIITGVPENDWSLAVPAGVCVDVVPMGDKGFVARPYGLDDVFKGDLRDSKTTLTGIPFGEWMSKRGLSYTDLKGRTDDLQAVSVFPMVNSVEELGLVLRWMLSEPELEEGKNIWLRSEHFSADEISAGANLKRLYAQREEFRKGNWKALAVNHEKSVFYQLDLADAAEDFVRLGLDMPELLPEDALQMSRIHNRMLRARILKLDGKDYRPEEQAAFDLLRDGLLDGISNRKSTPKLDVYSDQIVWGRSPVRIDMAGGWTDTPPYSLYSGGNVVNLAIELNGQPPLQVYVKPCKDFHIVLRSIDMGAMEIVSTFDELQDYKKIGSPFSIPKAALSLAGFAPAFSAVSYASLEEQLKDFGAGIEVTLLAAIPAGSGLGTSSILASTVLGAINDFCGLAWDKNEICQRTLVLEQLLTTGGGWQDQYGGVLQGVKLLQTEAGFAQSPLVRWLPDHLFTHPEYKDCHLLYYTGITRTAKGILAEIVSSMFLNSSLHLNLLSEMKAHALDMNEAIQRGSFVEFGRLVGKTWEQNKALDSGTNPPAVEAIIDLIKDYTLGYKLPGAGGGGYLYMVAKDPQAAVRIRKILTENAPNPRARFVEMTLSDKGFQVSRS (SEQ ID NO:1);

[0104] Amino acid sequence of PPK: MSLGRPAMASKHGFKKSPAELLVVGPEFNLDQVDTSGAPGFSGKKRDAEQVLAEGERKLSVLQEQLFAQSKFGGKHSVLLVLQAMDTAGKGGIVSHVVGSVDPQGMQLHAFKAPTDEEKQHDFLWRIEKGVPKAGMLGVFDRSHYEDVLIHRVHGWADETELERRYQAINDFEEQLTSNGTKIVKVMLDISPETQKERLTARLAEPTKHWKYSPTDVTEREFWPQYMEAYQLAFEKTSTKIAPWHVVPADKKWYARLAVQELLLDAFESLDLSWPEGDYDLALERKRVAAS (SEQ ID NO:2);

[0105] Amino acid sequence of PPA: MGSSHHHHHHSSGLVPRGSHMSLLNVPAGKDLPEDIYVVIEIPANADPIKYEIDKESGALFVDRFMSTAMFYPCNYGYINHTLSLDGDPVDVLVPTPYPLQPGSVIRCRPVGVLKMTDEAGEDAKLVAVPHSKLSKEYDHIKDVNDLPELLKAQIAHFFEHYKDLEKGKWVKVEGWENAEAAKAEIVASFERAKNK (SEQ ID NO:3);

[0106] Amino acid sequence of BFFKP-M07: MQKLLSLPSNLVQSFHELERVNRTDWFCTSDPVGKKLGSGGGTSWLLEECYNEYSDGATFGEWLEKEKRILLHAGGQSRRLPGYAPSGKILTPVPVFRWERGQHLGQNLLSLQLPLYEKIMSLAPDKLHTLIASGDVYIRSEKPLQSIPEADVVCYGLWVDPSLATHHGVFASDRKHPEQLDFMLQKPSLAELESLSKTHLFLMDIGIWLLSDRAVEILMKRSHKESSEELKYYDLYSDFGLALGTHPRIEDEEVNTLSVAILPLPGGEFYHYGTSKELISSTLSVQNKVYDQRRIMHRKVKPNPAMFVQNAVVRIPLCAENADLWIENSHIGPKWKIASRHIITGVPENDWSLAVPAGVCVDVVPMGDKGFVARPYGLDDVFKGDLRDSKTTLTGIPFGEWMSKRGLSYTDLKGRTDDLQAVSVFPMVNSVEELGLVLRWMLSEPELEEGKNIWLRSEHFSADEISAGANLKRLYAQREEFRKGNWKALAVNHEKSVFYQLDLADAAEDFVRLGLDMPELLPEDALQMSRIHNRMLRARILKLDGKDYRPEEQAAFDLLRDGLLDGISNRKSTPKLDVYSDQIVWGRSPVRIDMAGGWTDTPPYSLYSGGNVVNLAIELNGSPPLGVSVRPCKDFHIVLRSIDMGAMEIVSTFDELQDYKKIGSPFSIPKAALSLAGFAPAFSAVSYASLEEQLKDFGAGIEVTLLAAIPAGSGLGTSSILASTVLGAINDFCGLAWDKNEICQRTLVLEQLLTTGGGWQDQYGGVLQGVKLLQTEAGFAQSPLVRWLPDHLFTHPEYKDCHLLYYTGITRTAKGILAEIVSSMFLNSSLHLNLLSEMKAHALDMNEAIQRGSFVEFGRLVGKTWEQNKALDSGTNPPAVEAIIDLIKDYTLGYKLPGAGGGGYLYMVAKDPQAAVRIRKILTENAPNPRARFVEMTLSDKGFQVSRS (SEQ ID NO: 4);

[0107] Amino acid sequence of BFFKP - M10: MQKLLSLPSNLVQSFHELERVNRTDWFCTSDPVGKKLGSGGGTSWLLEECYNEYSDGATFGEWLEKEKRILLHAGGQSRRLPGYAPSGKILTPVPVFRWERGQHLGQNLLSLQLPLYEKIMSLAPDKLHTLIASGDVYIRSEKPLQSIPEADVVCYGLWVDPSLATHHGVFASDRKHPEQLDFMLQKPSLAELESLSKTHLFLMDIGIWLLSDRAVEILMKRSHKESSEELKYYDLYSDFGLALGTHPRIEDEEVNTLSVAILPLPGGEFYHYGTSKELISSTLSVQNKVYDQRRIMHRKVKPNPAMFVQNAVVRIPLCAENADLWIENSHIGPKWKIASRHIITGVPENDWSLAVPAGVCVDVVPMGDKGFVARPYGLDDVFKGDLRDSKTTLTGIPFGEWMSKRGLSYTDLKGRTDDLQAVSVFPMVNSVEELGLVLRWMLSEPELEEGKNIWLRSEHFSADEISAGANLKRLYAQREEFRKGNWKALAVNHEKSVFYQLDLADAAEDFVRLGLDMPELLPEDALQMSRIHNRMLRARILKLDGKDYRPEEQAAFDLLRDGLLDGISNRKSTPKLDVYSDQIVWGRSPVKIDMAGGWTDTPPYSLYSGGNVVNLAIELNGQPPLQVYVKPCKDFHIVLRSIDMGAMEIVSTFDELQDYKKIGSPFSIPKAALSLAGFAPAFSAVSYASLEEQLKDFGAGIEVTLLAAIAAGTGLGTSSIAASTVLGAINDFCGLAWDKNEICQRTLVLEQLLTTGGGWQDQYGGVLQGVKLLQTEAGFAQSPLVRWLPDHLFTHPEYKDCHLLYYTGITRTAKGILAEIVSSMFLNSSLHLNLLSEMKAHALDMNEAIQRGSFVEFGRLVGKTWEQNKALDSGTNPPAVEAIIDLIKDYTLGYKLPGAGGGGYLYMVAKDPQAAVRIRKILTENAPNPRARFVEMTLSDKGFQVSRS (SEQ ID NO:5);

[0108]

[0109] Nucleotide sequence of PPK: atgagcctgggccgtccggcaatggccagtaaacatggctttaaaaaaagtccggcagaactgctggtggttggcccggaatttaatctggatcaggttgataccagtggtgcacctggttttagcggcaaaaaacgtgatgcagaacaggttctggccgaaggcgaacgtaaactgagcgttctgcaggaacagctgtttgcccagagtaaatttggtggcaaacatagtgtgctgctggtgctgcaggcaatggataccgccggcaaaggcggtattgtgagccatgtggtgggcagtgttgatccgcagggtatgcagctgcatgcatttaaagcacctaccgatgaagaaaaacagcatgattttctgtggcgcattgaaaaaggcgttccgaaagcaggtatgctgggcgtttttgatcgtagccattatgaagatgtgctgattcatcgtgttcatggttgggcagatgaaaccgaactggaacgtcgttatcaggccattaatgattttgaagaacagctgaccagcaatggtaccaaaattgtgaaagtgatgctggatattagcccggaaacccagaaagaacgtctgaccgcccgcctggcagaaccgaccaaacattggaaatatagtccgaccgatgtgaccgaacgcgaattttggccgcagtatatggaagcctatcagctggcctttgaaaaaaccagcaccaaaattgcaccgtggcatgttgttccggccgataaaaaatggtatgcccgcctggcggttcaggaactgctgctggatgcctttgaaagcctggatctgagttggccggaaggtgattatgatctggcactggaacgtaaacgcgttgcagcaagttaa (SEQ ID NO:7);

[0110] Nucleotide sequence of PPA: (SEQ ID NO: 8);

[0111] In the present invention, the preparation of the crude enzyme solution in Examples 3 to 5 is the same as that in Example 2. The bacterial cells and 50 mM Tris-HCl (pH 7.5) solution are uniformly mixed at a mass ratio of 1:10, and the cells are broken by homogenization to prepare the crude enzyme solution.

[0112] 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 with reference to the following examples:

[0113] Example 1 Construction of mutants

[0114] 1. Enzyme mutation construction

[0115] In order to obtain a monofunctional enzyme, the present invention modified the enzyme by truncation and point mutation, and obtained the mutants shown in Table 1 after modification:

[0116] Table 1. Mutants

[0117]

[0118] 1.1 Construction of mutants

[0119] The target gene BFFKP was synthesized at a gene synthesis company. The amino acid sequence of BFFKP is shown in SEQ ID NO: 1. Other mutants were introduced with corresponding point mutations by PCR using BFFKP as a template. The template fragments or point mutation fragments were then digested with NdeI and XhoI restriction endonucleases and ligated into the pET28a expression vector to form recombinant products pET28a-BFFKP, pET28a-M01, pET28a-M02, pET28a-M03, pET28a-M04, pET28a-M05, pET28a-M06, pET28a-M07, pET28a-M08, pET28a-M09, and pET28a-M10.

[0120] Transfer 4 μL of the recombinant product into 30 μL of DH5α competent cells. Incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, and incubate on ice for 2-3 minutes. Add 500 μL of SOC and incubate on a shaker for 1.5 hours. Centrifuge the entire plate and incubate overnight at 37°C for 12-16 hours. Pick a single clone, confirm the correct construction of the vector by sequencing, and store it for future use.

[0121] 1.2 Enzyme fermentation production

[0122] The plasmid successfully constructed in 1.1 was transferred into E. coli (BL21) competent cells for plate culture and single clone screening, as well as small-scale liquid culture of single clones. The bacteria with correct protein expression were finally amplified in liquid culture step by step.

[0123] The specific steps are as follows: a single colony was inoculated into 5 mL of LB culture medium containing 50 μM kanamycin (37 oC) Culture, when the cells grow to the logarithmic phase, inoculate into 250mL LB culture medium containing the same antibiotics, and when they grow to the logarithmic phase, transfer them into a 5L culture fermenter for culture and final protein expression. In the 5L fermenter, when the cell OD reaches 20, add 0.3mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 25 o Protein expression was induced by C for 6 hours, and finally, cells were collected by high-speed centrifugation (4000 rpm, 20 minutes) to obtain 25g-50g of enzyme-overexpressing wet cells. A small amount of cells was first mixed with Tris-HCl buffer (50mM, pH 8.0) on ice, and then the cells were disrupted by freeze-thaw and centrifuged at high speed to obtain a supernatant. The supernatant was subjected to SDS-PAGE gel electrophoresis, confirming the successful expression of BFFKP and its variant proteins.

[0124] The LB culture medium consists of: 1 wt% tryptone, 0.5 wt% yeast powder, 1 wt% NaCl, 1 wt% dipotassium hydrogen phosphate, 1 wt% dipotassium hydrogen phosphate and 5 wt% glycerol.

[0125] 2. Screening of mutants with L-Fucose kinase activity and GDP-Fucose pyrophosphorylase activity

[0126] The host cells containing the above mutation vector were lysed, and the lysate was as shown in Table 2.

[0127] Table 2. Lysis buffer

[0128]

[0129] The cracking operation is as follows:

[0130] The cultured deep-well plate containing only bacterial cells was taken out from the -20°C refrigerator, 100 μL of lysis buffer was added to each well, and the suspended bacterial cells were thoroughly pipetted and disrupted at 25°C, 800 rpm, for 2 h to obtain a crude enzyme solution containing the BFFKP and its variants M01 to M10;

[0131] 2.1 Screening of L-Fucose kinase activity

[0132] Prepare 20 mL of 2× reaction solution (pH = 8.5-9.0, temperature 38°C) as shown in Table 3.

[0133] Table 3. L-Fucose kinase screening reaction solution

[0134]

[0135] The above reagents were weighed and completely dissolved in 15 mL of water. 4 M NaOH was added to adjust the pH to 9.0, and the volume was then adjusted to 20 mL.

[0136] The steps for screening L-Fucose kinase activity are as follows (deep-well plate reaction):

[0137] Add 200 μL of 2× reaction solution and 100 μL of H2O to each well, mix well by pipetting, then add 100 μL of the wild-type BFFKP (WT) or mutant M01~M10 crude enzyme solution of FKP, place on a shaker at 38°C, 800 rpm, and react for 2 h.

[0138] Pipette 100 μL of supernatant into each well of a deep-well plate. Quench the reaction by adding 900 μL of 60% acetonitrile (diluted 10-fold). Incubate at 37°C, shake at 1000 rpm for 10 minutes, and centrifuge at 4000 rpm for 10 minutes. Filter the supernatant through a 0.45 μm filter before performing liquid chromatography analysis.

[0139] After comparing the results (such as Figure 1 As shown in the figure), after truncation, the C-terminal mutant M03 (497~949aa) has L-Fucose kinase activity, but the enzyme activity is relatively low. Among the mutants, M07 has the highest L-Fucose kinase reaction activity.

[0140] 2.2 Screening of GDP-Fucose pyrophosphorylase activity

[0141] First, prepare fucose-1-phosphate (Fuc-1-P) reaction solution 1 using M07. The preparation process of reaction solution 1 is as follows:

[0142] Dissolve 0.3283g of L-Fucose, 1.1023g of ATP-2Na, 0.2517g of MnCl2, and 0.2423g of Tris in 15mL of water. Add 4M NaOH to adjust the pH to 9.0, and then dilute to 20mL. Add 10mL of water, mix thoroughly, and add 10mL of the M07 bacterial suspension. Place in a shaking water bath at 38°C and incubate for 4 hours.

[0143] Then adjust to acidic pH = 6.5~7.0 to obtain reaction solution 1, which serves as the starting substrate for the second step reaction.

[0144] Take 20 mL of reaction solution 1 (containing Fuc-1-P at a concentration of approximately 50 mM) and adjust the pH to 6.5-7.0 with HCl. Then, add 0.952 g of anhydrous magnesium chloride (50 mM) and 0.68 g of GTP-2Na (Fuc-1-P and GTP-2Na are added at a ratio of 1:1.2, 60 mM). Then, adjust the pH to 7.0 with NaOH to obtain step 2 reaction solution 2.

[0145] Take a 96-well plate, add 300 μL of reaction solution 2 to each well, add 100 μL of FKP wild type (WT) or mutant M01~M10 bacterial solution, mix well by pipetting, and place on a shaker at 38°C, 800 rpm, and react for 4 hours.

[0146] Pipette 100 μL of supernatant into each well of a deep-well plate. Quench the reaction by adding 900 μL of 60% acetonitrile (diluted 10-fold). Incubate at 37°C, shake at 1000 rpm for 10 minutes, and centrifuge at 4000 rpm for 10 minutes. Filter the supernatant through a 0.45 μm filter before performing liquid chromatography analysis.

[0147] Comparison results of GDP-Fucose pyrophosphorylase activity are as follows Figure 2 As shown in the figure, after truncation, the N-terminal mutant M01 (1-496 aa) still has GDP-Fucose pyrophosphorylase activity, but it is reduced. The activity of M02 (1-564 aa) is improved, and the mutant M10 has the highest pyrophosphorylase activity.

[0148] Example 2 One-pot enzymatic preparation of GDP-L-Fucose using the natural enzyme BFFKP

[0149] GDP-L-Fucose was prepared by a one-pot enzymatic method using the natural enzyme BFFKP. The reaction formula is as follows: Figure 3 As shown;

[0150] 1. Reagent preparation

[0151] Substrate solution: Accurately weigh 1662 mg of ATP-2Na (molecular weight 551.145 g / mol), 490 mg of L-fucose (molecular weight 164.16 g / mol), and 2041 mg of GTP-2Na (molecular weight 567.2 g / mol), dissolve in 10 mL of deionized water, adjust the pH to 7.5, and finally make up to 15 mL; final concentrations: 200 mM ATP, 200 mM L-fucose, 240 mM GTP).

[0152] Tris-HCl solution (1 M, molecular weight 121.1 g / mol, pH 7.5): Weigh 12.11 g of Tris into a 250 mL beaker, add approximately 80 mL of deionized water, stir thoroughly to dissolve, add concentrated hydrochloric acid to adjust the pH to 7.5, and dilute the solution to 100 mL.

[0153] MgCl2 solution (1 M, molecular weight 95.211 g / mol): Accurately weigh 0.952 g of anhydrous magnesium chloride, dissolve it in deionized water and dilute to 10 mL.

[0154] 2. Preparation of crude enzyme solution

[0155] Dilute 1M Tris-HCl (pH 7.5) to 50mM. Mix BFFKP cells and 50mM Tris-HCl (pH 7.5) at a mass ratio of 1:8. Disrupt the cells in a cell disruptor (homogenizer) at 40% power for 15 minutes to dissolve intracellular proteins in the buffer. Centrifuge and remove the supernatant to obtain a crude BFFKP enzyme solution. Mix PPA cells and 50mM Tris-HCl (pH 7.5) at a mass ratio of 1:10. Disrupt the cells in a cell disruptor at 40% power for 15 minutes to dissolve intracellular proteins in the buffer. Centrifuge and remove the supernatant to obtain a crude PPA enzyme solution.

[0156] 3. Reaction solution

[0157] Take 800 μL of substrate solution, add 50 μL of Tris-HCl solution (1M, pH 7.5), 50 μL of MgCl2 solution (1M) and 100 μL of BFFKP crude enzyme solution in sequence, and finally add 2 U of PPA crude enzyme solution. Mix well and react at 38°C for 6 hours. After the reaction, take 100 μL of supernatant into a test tube, add 900 μL of 60% acetonitrile to quench the reaction according to the dilution of 10 times, shake at 37°C, 1000 rpm for 10 minutes, and centrifuge at 4000 rpm for 10 minutes. Take the supernatant and filter it with a 0.45 μm filter head. Send the sample for HPLC analysis. The results are as follows Figure 4 As shown, there is a large amount of ATP and GTP remaining. Calculated yield: 8.5 g / L of GDP-L-Fucose produced.

[0158] Example 3 A two-step enzymatic method (WT+WT, M07+M07, M10+M10, M07+M10) was used to prepare GDP-L-Fucose by enzyme-catalyzed cascade reaction.

[0159] A two-step enzymatic method (WT+WT, M07+M07, M10+M10, M07+M10) was used to perform an enzyme-catalyzed cascade reaction to prepare GDP-L-Fucose. The reaction formula is as follows: Figure 5 As shown;

[0160] 1. Solvent preparation

[0161] Tris-HCl solution (1 M, pH 8.5): Weigh 12.11 g of Tris into a 250 mL beaker. Add approximately 80 mL of deionized water and stir thoroughly to dissolve. Adjust the pH to 8.5 and bring the solution to 100 mL.

[0162] Substrate solution 1: Accurately weigh 1662 mg of ATP-2Na and 490 mg of L-fucose, dissolve in 10 mL of deionized water, adjust the pH to 8.5-9.0, and finally make up to 15 mL; final concentrations: 200 mM ATP, 200 mM L-fucose.

[0163] Substrate solution 2: MnCl2 solution (1 M, molecular weight 125.844 g / mol): Accurately weigh 1.258 g of anhydrous manganese chloride, dissolve it in deionized water and dilute to 10 mL;

[0164] Substrate Solution 3: MgCl2 solution (1 M, molecular weight 95.211 g / mol): Accurately weigh 0.952 g of anhydrous magnesium chloride, dissolve it in deionized water, and dilute to 10 mL.

[0165] 2. Reaction steps

[0166] Step 1: Take 800 μL of substrate solution 1, add 50 μL of Tris-HCl solution (1 M, pH 8.5), 50 μL of substrate solution 2 (1 M MnCl2 solution) and 100 μL of crude enzyme solution (WT, M07, M10, M07) in sequence, mix well and react at 38°C for 2 hours.

[0167] Step 2: After the first step, add 50 μL of substrate solution 3 (1 M MgCl2 solution), slowly add 0.136 g of GTP-2Na (240 mM) and adjust the pH to 6.5-7.0 with ammonia water, add 2 U of PPA crude enzyme solution, and finally add 100 μL of crude enzyme solution (WT, M07, M10 or M10 crude enzyme solution), mix well and react at 38°C for 4 h.

[0168] For each reaction, aspirate 100 μL of the reaction solution and dilute 10-fold by adding 900 μL of 60% acetonitrile to quench the reaction. Incubate at 37°C, shake at 1000 rpm for 10 minutes, and centrifuge at 4000 rpm for 10 minutes. Filter the supernatant through a 0.45 μm filter and analyze by liquid chromatography. Calculate the GDP-fucose yield (g / L).

[0169] The results of the two-step enzymatic method (WT+WT, M07+M07, M10+M10, M07+M10) for the preparation of GDP-L-Fucose by enzyme-catalyzed cascade reaction are shown in the figure. Figure 6As shown in Table 4, the GDP-L-Fucose yields of WT+WT, M07+M07, M10+M10, and M07+M10 prepared via a two-step enzymatic cascade reaction were 10.8 g / L, 40.3 g / L, 30.5 g / L, and 64.1 g / L, respectively. The GDP-L-Fucose yield of M07+M10 prepared via a two-step enzymatic cascade reaction was significantly higher than that of the other three groups. The table also compares the GDP-L-Fucose yields of the one-pot method used in Example 1 with the two-step method of this example. The results show that the two-step method produces a higher GDP-L-Fucose yield.

[0170] Table 4. Single enzyme (WT, M07 or M10) and dual enzyme (M07+M10)

[0171] Two-step reaction results

[0172]

[0173] Example 4: Preparation of GDP-L-Fucose in a 10 mL system using two FKP enzyme mutants (M07 + M10) and a two-step enzymatic method and PPA (pyrophosphohydrolase) enzyme-catalyzed cascade reaction

[0174] Step 1: Take 8 mL of substrate solution 1 (200 mM ATP and 200 mM L-Fucose), add 500 μL Tris-HCl solution (1 M, pH 8.5), 500 μL of substrate solution 2 (1 M MnCl2 solution) and 1 mL of M07 crude enzyme solution in sequence, mix well and react at 38°C for 2 h. The L-fucose reaction was complete as detected by HPLC. The conversion rate of substrate L-fucose to the product Fuc-1-P obtained after the first step reaction was 80% (see Figure 7 ).

[0175] Step 2: After the reaction, add 500 μL of substrate solution 3 (1 M MgCl2 solution), slowly add 1.36 g of GTP-2Na, and adjust the pH to 6.5-7.0 with ammonia water. Add 20 U of PPA enzyme, and finally add 1 mL of M10 crude enzyme solution. Mix well and react at 38°C for 4 hours. HPLC liquid chromatography detection reaction showed that the product of the first step was not completely converted into GDP-L-fucose (such as Figure 8 As shown), the GDP-L-fucose yield was 136 mM, and the conversion rate of Fuc-1-P to GDP-L-fucose was 85%.

[0176] Example 5 1L system - using FKP enzyme (M07 + M10) - step-by-step enzymatic method, using ATP regeneration system and PPA to hydrolyze pyrophosphate to prepare GDP-L-Fucose

[0177] 1L system - using FKP enzyme (M07 + M10) - step-by-step enzymatic method - using ATP regeneration system and PPA to hydrolyze pyrophosphate to prepare GDP-L-Fucose. Figure 9 As shown;

[0178] Step 1: Add 32.8g L-fucose (200mM), 3.0g adenosine triphosphate monosodium salt (ATP-2Na, 5.4mM), and 6.3g anhydrous manganese chloride (50mM) to 1L 50mM Tris-HCl solution (pH 8.5). Adjust the pH value of the solution to 8.5-9.0, then add 2000U of PPK crude enzyme solution and 100mL of M07 crude enzyme solution (the amount added is 10% of the total reaction system in step 1). Incubate at 38 o The reaction mixture was stirred gently at C and the pH of the reaction system was maintained between 8.5 and 9.0. After 2 hours, HPLC detected that the L-fucose reaction was basically complete, and the conversion rate of the substrate L-fucose to the Fuc-1-P product obtained after the first step reaction was nearly 100%.

[0179] Step 2: The pH of the reaction solution was adjusted to 7.0 with HCl, and 113 g of guanosine triphosphate monosodium salt (GTP, 200 mM) and 4.8 g of anhydrous magnesium chloride (50 mM) were added. Then, 100 mL of M10 crude enzyme solution (the addition amount was 9% to 11% of the reaction system, which was 9.09% at this time) and 2000 U of PPA enzyme were added. o C and gently stirred while maintaining the pH of the reaction system between 6.5 and 7.0. After 4 hours, GDP-L-fucose was detected to be almost completely generated by HPLC (such as Figure 10 As shown), the yield of GDP-L-fucose was 170 mM, and the conversion rate of the product after the first step reaction to GDP-L-fucose was 85%.

[0180] The reaction solution containing GDP-L-Fucose was adjusted to pH 2.0, the enzyme in the reaction system was denatured and precipitated, and the protein solid was removed by centrifugation. The crude product was obtained by two steps of isopropanol purification: first, at -20 oUnder C conditions, add 40% (v / v) by volume and let it stand for 0.5h. Centrifuge to separate the supernatant and precipitate. Under -20 degrees, add isopropanol to the supernatant again to 80% (v / v), let it stand for 0.5h, and centrifuge to separate the precipitate. Continue to purify, dissolve the solid in water, and directly load it onto a D201 anion exchange resin purification column to remove impurities such as adenosine diphosphate and free phosphate. Finally, the product is desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol: water, 2:1, v:v) to obtain 60.1 grams of white powder. The product was confirmed to be GDP-L-fucose by mass spectrometry. The HPLC purity was 95.6% (see spectrum Figure 11 ), mass spectrometry results are shown in Figure 12 , GDP-L-fucose, molecular weight: 587.334 g / mol.

[0181] 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. L-fucokinase variant, characterized in that Its amino acid sequence is based on that shown in SEQ ID NO: 1, with the glutamine at position 623 mutated to serine, the glutamine at position 627 mutated to glycine, the tyrosine at position 629 mutated to serine, and the lysine at position 631 mutated to arginine.

2. A GDP-L-fucose pyrophosphorylase variant, characterized in that Its amino acid sequence is based on that shown in SEQ ID NO: 1, with the arginine at position 592 mutated to lysine, the proline at position 711 mutated to alanine, the serine at position 714 mutated to threonine, and the leucine at position 722 mutated to alanine.

3. A composition, characterized in that Comprising Composition 1 and / or Composition 2: The composition 1 comprises the L-fucokinase variant and polyphosphate kinase according to claim 1; The composition 2 comprises the GDP-L-fucose pyrophosphorylase variant according to claim 2 and pyrophosphatase.

4. Biomaterial, characterized in that Including at least one of the following A) to D): A), a nucleic acid encoding the L-fucokinase variant of claim 1, the GDP-L-fucose pyrophosphorylase variant of claim 2, or the composition of claim 3; B) a recombinant vector containing the nucleic acid described in A); C), transforming or transfecting the host cell with the recombinant vector described in B); D) Culturing the mixture obtained by culturing the host cells described in C).

5. The biomaterial according to claim 4, characterized in that The backbone of the recombinant vector is pET28a.

6. The biomaterial according to claim 4, characterized in that The host cell is Escherichia coli.

7. Application of at least one of the following i) to iv) in the synthesis of GDP-L-Fucose: i) the L-fucokinase variant according to claim 1; ii), the GDP-L-fucose pyrophosphorylase variant according to claim 2; iii) the composition according to claim 3; iv) The biomaterial according to any one of claims 4 to 6.

8. A method for preparing GDP-L-Fucose, characterized in that: The method comprises synthesizing GDP-L-Fucose by using at least one of the following methods I) to IV): 1), the L-fucokinase variant according to claim 1; II), the GDP-L-fucose pyrophosphorylase variant according to claim 2; III), the composition according to claim 3; IV) The biomaterial according to any one of claims 4 to 6.

9. The preparation method according to claim 8, characterized in that The steps include: Step 1, L-fucose kinase variant catalyzes L-fucose to generate Fuc-1-P in the presence of auxiliary 1; Step 2, the GDP-L-fucose pyrophosphorylase variant catalyzes Fuc-1-P to generate GDP-L-fucose in the presence of auxiliary substance 2; The auxiliary material 1 includes an auxiliary material 1a and / or an auxiliary material 1b; The auxiliary substance 1a includes Tris-HCl, ATP, manganese chloride and polyphosphate kinase; The auxiliary substance 1b includes Tris-HCl, ATP, and manganese chloride; The auxiliary substance 2 includes GTP, magnesium chloride and pyrophosphatase.

10. The preparation method according to claim 9, characterized in that In step 1, the pH for catalyzing L-fucose to generate Fuc-1-P is 8.5-9.0, and the reaction time is 1.5-3 h; In step 2, the pH for catalyzing Fuc-1-P to generate GDP-L-fucose is 6.5-7.0, and the reaction time is 3-5 h.

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