Heparin-modifying enzyme gene, high-efficiency expression engineering strain and application

Abstract

The present application relates to the field of biotechnology and the field of heparin biosynthesis, and discloses a heparin modification enzyme gene, a high-efficiency expression engineering strain and application. Specifically, the present application relates to a bifunctional N-deacetylase / N-sulfotransferase NDST gene, a glucuronate C5-epimerase C5epi gene, a sulfated heparan 2-sulfotransferase (2-OST) gene, a sulfated heparan 6-sulfotransferase 6-OST gene derived from a Limosa lapponica and a sulfated heparan 3-sulfotransferase 3-OST gene derived from a Hirundo rustica. The heparin modification enzyme genes obtained by mining are derived from various bird genomes that have not been annotated, and the high-efficiency expression and purification of the genes in Pichia pastoris or Escherichia coli are realized. Meanwhile, it is verified that the genes have biological catalytic activity and can be used for in vitro multi-enzyme catalytic synthesis of heparin. Therefore, the present application has potential application value.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and heparin biosynthesis, specifically to heparin-modifying enzyme genes, highly efficient expression engineered strains, and applications. Background Technology

[0002] Heparin is a highly sulfated, heterogeneous linear glycosaminoglycan produced by mast cells in animal connective tissue. It is the most effective and clinically used anticoagulant drug in the world. The global heparin market reached $10 billion in 2024. With the continued increase in the elderly population and the rising incidence of cardiovascular and venous thromboembolism in recent years, the market is projected to reach $14.9 billion by 2029. my country is a major supplier of heparin raw materials globally, exporting approximately 100-200 tons annually, accounting for about 80% of global production. Currently, the main production method for heparin is extraction from pig small intestine, which suffers from severe pollution during the production process, significant environmental pressure on enterprises, numerous byproducts, substantial clinical side effects, and high dependence on pig supply for raw materials, leading to an urgent need for industrial transformation and upgrading.

[0003] Enzymatic synthesis of heparin offers advantages such as fewer byproducts, independence from raw material supply, a green and environmentally friendly production process, and controllable molecular structure, making it the most promising method for heparin production. However, the enzymatic synthesis of bioactive heparin from proheparinogen requires at least 4-5 enzymatic steps, involving numerous heparin-modifying enzymes. Currently, the lack of heparin-modifying enzyme gene resources, low catalytic efficiency, and poor heterologous expression levels hinder the industrialization of heparin biosynthesis. Patent document CN 114763570B discloses the application of heterologous expression of heparin synthesis pathway-related genes in Pichia pastoris cells: bifunctional N-deacetyl / N-sulfate transferase (NDST), glucuronide C5-allostericase (C5epi), heparan sulfonate 2-sulfate transferase (2-OST), heparan sulfonate 6-sulfate transferase (6-OST), and heparan sulfonate 3-sulfate transferase (3-OST). Using the above series of heparin-modifying enzymes, functional heparin was synthesized by enzymatic modification of heparin precursors at a concentration of 0.1-0.2 g / L. This series of heparin-modifying enzymes is mainly derived from known functional enzymes from human or chicken sources.

[0004] Therefore, although the heparin-modifying enzymes currently revealed have multiple sources, their protein structures all show high similarity, limiting their catalytic mechanisms and capabilities. There is a need to discover more enzymes with different molecular structures, catalytic mechanisms, and capabilities to address the current issues of limited variety and generally low catalytic efficiency of heparin-modifying enzymes. Simultaneously, although heparin-modifying enzymes have achieved active expression in various chassis cell types, their expression levels and activities have not yet reached industrial-scale levels. Therefore, it is necessary to focus on chassis cell types, which have relatively greater potential, and to focus on elucidating the mechanisms of molecular tool adaptation during heterologous expression of heparin-modifying enzyme genes from different sources in chassis cells. Summary of the Invention

[0005] This invention utilizes bioinformatics techniques to analyze the NCBI genome and proteome databases. Through multiple homology alignment of sequences and structures, a series of previously unannotated heparin-modifying enzyme-encoding genes were discovered. Through heterologous expression and enzyme activity assays, these novel heparin-modifying enzyme-encoding genes with biocatalytic activity were identified. These include a bifunctional N-deacetyl / N-sulfate transferase (NDST) gene from the Common Bunting (Melospiza georgiana), a glucuronide C5-epi gene from the Common Cuckoo (Cuculus canorus), a heparin sulfonate 2-sulfate transferase (2-OST) gene from the Dryobates pubescens, a heparin sulfonate 6-sulfate transferase (6-OST) gene from the Barn Godwit (Limosa lapponica baueri), and a heparin sulfonate 3-sulfate transferase (3-OST) gene from the Barn Swallow (Hirundo rustica rustica). Furthermore, enzyme activity characterization of some genes demonstrated that their catalytic activity was significantly higher than that of the corresponding human modifying enzymes previously reported. This expands the source of heparin modifying enzyme genes, providing a wider and better selection of enzyme elements for heparin enzymatic synthesis.

[0006] This invention also systematically optimized the heterologous expression of the novel heparin-modifying enzyme gene (including host bacteria, expression vector, and fusion tag), discovering numerous molecular elements that can significantly enhance the expression level of the target enzyme. This significantly improved the heterologous expression level of the novel heparin-modifying enzyme gene in this invention, with enzyme production reaching up to 1.2 g / L or more after fermentation in a 5L fermenter. This helps to solve the technical bottleneck of high enzyme element costs in the enzymatic heparin synthesis process, thus bridging the last mile of the heparin enzymatic synthesis industrial process, and thus completing this invention.

[0007] This invention provides a heparin-modifying enzyme gene, which is a bifunctional N-deacetyl / N-sulfate transferase NDST gene derived from the Songbird (Melospiza georgiana), a glucuronide C5-allostericase C5epi gene derived from the Common Cuckoo (Cuculus canorus), a sulfoheparin 2-sulfate transferase 2-OST gene derived from the Dryobates pubescens, a sulfoheparin 6-sulfate transferase 6-OST gene derived from the Barn Godwit (Limosa lapponica baueri), or a sulfoheparin 3-sulfate transferase 3-OST gene derived from the Barn Swallow (Hirundo rustica rustica).

[0008] Preferably, its nucleotide sequence is obtained by codon optimization based on the expression preferences of Pichia pastoris or Escherichia coli.

[0009] More preferably, it is further fused with a fusion expression tag, specifically such as TrxA, UB, GB1, NusA, CaBP, SUMO(Smt3), FTN-H, or Skp.

[0010] The present invention also provides the protein encoded by the heparin-modifying enzyme gene.

[0011] The present invention contains an expression vector containing the heparin-modifying enzyme gene, which is a prokaryotic expression vector or a eukaryotic expression vector.

[0012] Preferably, it is an expression vector suitable for expression in Pichia pastoris or Escherichia coli.

[0013] The present invention also provides a recombinant host bacterium containing the heparin-modifying enzyme gene or the expression vector described herein.

[0014] Preferably, it is Pichia pastoris or Escherichia coli.

[0015] This invention provides the application of the heparin-modifying enzyme gene or its encoded protein in the in vitro multi-enzyme catalytic synthesis of heparin.

[0016] Specifically, the application involves catalytic reactions of deacetylation, C5 isomerization, or 2-,6-,3-sulfonation.

[0017] The present invention also provides a method for expressing the heparin-modifying enzyme gene or preparing the encoded protein, characterized in that the heparin-modifying enzyme is produced by culturing the recombinant host bacteria.

[0018] This invention is based on a series of novel heparin-modifying enzymes with high catalytic activity discovered for the first time. Combined with the construction of an efficient heterologous expression system, it can realize the subsequent catalytic synthesis of heparin from high-concentration heparin precursors with a concentration of 0.8-1.2 g / L, showing great potential for industrial application of enzymatic heparin synthesis.

[0019] This invention addresses the green development needs of the heparin industry by establishing a green biomanufacturing process for heparin, solving industrial application problems such as efficient catalysis of heparin synthesis-related enzymes, removing obstacles and difficulties in the industrialization process, promoting the demonstration of green biomanufacturing of heparin, ensuring human drug safety, and effectively consolidating and enhancing my country's leading international position in the heparin industry. Attached Figure Description

[0020] Figure 1 Phylogenetic analysis of novel heparin sulfate transferase sequences.

[0021] Figure 2 Different chaperone proteins in Pichia pastoris cells promote heterologous expression of C5-epi(1). Among them, M: Marker, 1 is pPICK empty vector, 2-3 are C5-epi(1) engineered bacteria, 4 is C5-epi(1) engineered bacteria: pPICZA-PDI, 5 is C5-epi(1) engineered bacteria: pPICZA-HAC1, 6 is C5-epi(1) engineered bacteria: pPICZαA-PDI, and 7 is C5-epi(1) engineered bacteria: pPICZαA-HAC1.

[0022] Figure 3 The effect of different fusion tags in Escherichia coli on promoting the soluble expression of 2-OST(1). Among them, M:Marker, 1:TrxA, 2:MBP, 3:UB, 4:GST, 5:ZZ-tag, 6:GB1, 7:DsbA, 8:NusA, 9:IF2, 10:CaBP, 11:Smt3, 12:FTN-H, 13:skp, 14:T7PK.

[0023] Figure 4 .2-OST enzyme activity comparison diagram.

[0024] Figure 5 The reaction route and catalytic activity diagram of a novel recombinant heparin-modifying enzyme catalyzing the synthesis of heparin from proheparin. Detailed Implementation

[0025] The present invention will be described below through specific embodiments in order to better understand the present invention, but these embodiments do not constitute a limitation thereof.

[0026] Example 1: Discovery of new genes for heparin-modifying enzymes

[0027] 1.1 Establishing a local database and conducting data analysis using bioinformatics techniques.

[0028] This embodiment establishes a standard analytical method and screening process for discovering new genes targeting heparin-modifying enzymes based on a bioinformatics analysis method using a hidden Markov model. First, based on reported heparin-modifying enzyme sequence information, multiple sequence alignment and iterative protein search were used to establish sequence conservation characteristics. Then, target species were identified, and genome and proteome data files were downloaded. A target protein database was established using the BLAST program to obtain species distribution information. A total of 324,693 similar sequences were obtained, the vast majority of which are distributed in eukaryotic cells. This task first selected the *Aves* species, which has few reported sequences but rich sequence information, for further sequence alignment analysis.

[0029] 1.2 Constructing an HMM dataset to mine new gene sequences

[0030] Further analysis was conducted on similar sequences in the Aves species. An HMM dataset was constructed using the training results of multiple sequence alignment, and this dataset was used to scan the target species (Aves) and protein databases to obtain the target sequences.

[0031] 1.3 Analysis and organization of new gene sequences of heparin-modifying enzymes

[0032] Thirty-five target-similar sequences from birds were selected for analysis, and a gene family phylogenetic tree was constructed using MEGA, as shown in the attached figure. Figure 1 As shown, several similar novel enzyme-coding genes were obtained through screening for 2-O-sulfate transferase, 6-O-sulfate transferase, and 3-O-sulfate transferase, respectively. It was also found that isozymes of 6-O-sulfate transferase and 3-O-sulfate transferase are at a certain distance on the phylogenetic tree, indicating certain sequence and structural differences. Simultaneously, two novel encoding genes for C5-epimerase and two bifunctional N-deacetyl / N-sulfate transferases (NDST) were obtained using the same method. Therefore, this embodiment has preliminarily established a novel enzyme resource database covering all key heparin-modifying enzymes. Specific sequence information is shown in Table 1 below.

[0033] Table 1. Sequences of different heparin-modifying enzymes and their gene sources in this invention

[0034]

[0035] Example 2: Construction of heterologous expression strains of heparin-modifying enzymes from different sources

[0036] 2.1 Construction of heterologous expression engineered strains of Pichia pastoris

[0037] Based on the codon bias of Pichia pastoris, analysis of the original lipase gene using online software revealed the presence of several low-frequency codons, which affected the expression of the original heparin-modifying enzyme gene in Pichia pastoris. After optimization, the GC content of the original heparin-modifying enzyme genes in Table 1 was reduced to approximately 50%, reducing AT and GC enrichment regions and thus facilitating the expression of each heparin-modifying enzyme gene in Pichia pastoris. The similarity between the optimized heparin-modifying enzyme genes and the original genes was 65.67-77.33%. Twelve chaperone protein encoding genes, namely PDI, EROI, KAR2, CPR6, FES1, HAC1, SSA1, STL1, VGB, RPL10, RPL38, and RPL43, were constructed on plasmids pPICZA and pPICZαA, respectively, resulting in 24 recombinant plasmids: pPICZA-PDI, pPICZA-EROI, pPICZA-KAR2, pPICZA-CPR6, pPICZαA-PDI, pPICZαA-EROI, pPICZαA-KAR2, and pPICZαA-CPR6. 6, pPICZA-RPL10, pPICZA-RPL38, pPICZA-RPL43, pPICZαA-RPL10, pPICZαA-RPL38, pPICZαA-RPL43, pPICZA-FES1, pPICZA-HAC1, pPICZA-SSA1, pPICZA-STL1, pPICZA-VGB, pPICZαA-FES1, pPICZαA-HAC1, pPICZαA-SSA1, pPICZαA-STL1, pPICZαA-VGB. The above 24 plasmids were transformed into Pichia pastoris engineered strains of each heparin-modifying enzyme listed in Table 1. The heparin-modifying enzyme genes in Table 1 were integrated into the Pichia pastoris genome using pPIC9k. A summary of the chaperone proteins that enhance the expression of the target enzymes is shown in Table 2. Taking C5-epi(1) expression as an example, ... Figure 2 As shown, pPICZαA-PDI and pPICZαA-HAC1 can significantly enhance the expression of C5-epi.

[0038] Table 2. Statistical analysis of the effect of Pichia pastoris expression system chaperone proteins on promoting extracellular expression of heparin-modifying enzymes

[0039] Gene chaperone protein <![CDATA[Promotion effect a > NDST(1) pPICZA-PDI, pPICZA-EROI 6 NDST(2) pPICZA-HAC1, pPICZαA-RPL10 2 C5-epi(1) pPICZαA-PDI, pPICZαA-HAC1 6 C5-epi(2) pPICZαA-PDI, pPICZαA-HAC1 6 2-OST(1) pPICZA-VGB 1 2-OST(2) pPICZA-VGB 1 6-OST-1 pPICZA-KAR2 3 6-OST-3 pPICZA-RPL10 3 3-OST-1 pPICZαA-HAC1, pPICZA-PDI 6 3-OST-5 pPICZαA-HAC1, pPICZA-RPL10 6

[0040] a The expression-promoting effects of each chaperone protein on the original engineered strains were rated as follows: 1: 10-20%; 2: 21-40%; 3: 41-60%; 4: 61-80%; 5: 81-100%; 6: from none to one (i.e., the strain could not express protein originally, but expression was achieved after the addition of the chaperone protein).

[0041] 2.2 Construction of heterologous expression engineered strains of Escherichia coli

[0042] For the newly discovered heparin-modifying enzyme genes in Table 1, codon optimization was performed on Escherichia coli. The plasmid pET32a was used as a heterologous expression vector and transformed into the expression host strain E. coil BL21(DE3) to construct heterologous expression engineered strains. In order to reduce the excessive proportion of inclusion bodies formed during the expression of target proteins in engineered strains, in addition to the commonly used TrxA fusion tag on vectors, this invention also selected UB, GB1, NusA, CaBP, SUMO(Smt3), FTN-H, Skp and other fusion expression tags. It was found that these tags can all promote the soluble expression of target genes. Among them, 2-OST(1) is the most significant in MBP crude fusion effect, which can achieve a high level of expression of specific target proteins. Figure 3 ).

[0043] 2.3 Detection of Heterologous Protein Expression Levels by SDS-PAGE

[0044] 2.3.1 Preparation of relevant solutions for SDS-PAGE protein electrophoresis

[0045] 1.5 mol / L pH 8.8 separating gel buffer: Weigh 18.15 g Tris powder, dissolve it thoroughly in sterile water, and bring the volume to 90 mL. Adjust the pH to 8.8 with 1 mol / L HCl, bring the volume to 100 mL with sterile water, and store at 4°C.

[0046] 1.0 mol / L pH 6.8 Stacking Gel Buffer: Weigh 12.10 g Tris powder, dissolve it completely in sterile water, bring the volume to 90 mL, then adjust the pH to 6.8 with 1 mol / L HCl, bring the volume to 100 mL with sterile water, and store at 4°C.

[0047] 5×Tris-glycine electrophoresis buffer: Weigh 5g SDS, 15g Tris, and 72g glycine, dissolve them thoroughly in 800mL of deionized water, and bring the volume to 1L.

[0048] 10% SDS solution (w / v): Weigh 10g of SDS powder, dissolve it thoroughly in deionized water, and bring the volume to 100mL. Store at room temperature.

[0049] 10% ammonium persulfate solution (w / v): Weigh 1g of ammonium persulfate solid, dissolve it completely in 10mL of deionized water, and store at 4℃.

[0050] 12% separating gel (25 mL): Using a pipette, pipette 6.6 mL of sterile water, 8.8 mL of 30% acrylamide stock solution, 5.0 mL of 1.5 M pH 8.8 separating gel buffer, 200 μL of 10% SDS, 200 μL of 10% ammonium persulfate, and 8 μL of LTEMED into a beaker and mix thoroughly.

[0051] Preparation of 5% stacking gel (6 mL): Using a pipette, pipette 4.1 mL of sterile water, 1.0 mL of 30% acrylamide stock solution (750 μL), 1.5 M pH 6.8 separating gel buffer, 60 μL of 10% SDS, 60 μL of 10% ammonium persulfate, and 6 μL of LTEMED into a beaker and mix thoroughly.

[0052] Coomassie Brilliant Blue Staining Solution: Weigh 1g of Coomassie Brilliant Blue R-250 and 50mL of isopropanol, dissolve thoroughly, then add 650mL of deionized water and 100mL of glacial acetic acid, and stir well. Filter through a membrane and store at room temperature for later use.

[0053] Protein gel decolorizing solution: Weigh 100 mL of glacial acetic acid and 50 mL of anhydrous ethanol, mix them, and then dilute to 1 L with distilled water. Store at room temperature for later use.

[0054] 0.4 mol / L trichloroacetic acid (TCA): Weigh 6.54 g of trichloroacetic acid, dilute to 100 mL with distilled water, and store at room temperature for later use.

[0055] 2.3.2 SDS-polyacrylamide gel electrophoresis

[0056] 1) Preparing the adhesive. Fix the mold, pour water in, and observe for any leakage. If the seal is intact, pour out the water. Prepare the separating adhesive by pouring it from the top to 3cm from the top edge, pressing it flat with anhydrous ethanol, and letting it stand for 30 minutes until the adhesive solidifies. Prepare the concentrating adhesive by pouring it from the top and inserting a comb, and letting it stand for 30 minutes until it solidifies.

[0057] 2) Sample preparation. Take 20 ml of fermentation supernatant, mix it with 6 μL of protein loading buffer, and boil in water for 10 min.

[0058] Collect all the sample solution from the centrifuge tube and spot 10-20 μL of the sample.

[0059] 3) Sample loading. Place the prepared gel into the electrophoresis tank, add buffer, remove the comb, spot the protein marker in the first well, and then load the remaining wells sequentially.

[0060] 4) Sample running. First, run the sample at a constant voltage of 120V until the sample is separated from the stacking gel. Then, run the sample at a constant voltage of 150V until the sample is separated from the gel.

[0061] 5) Staining. Remove the entire gel from the gel plate and stain with rapid staining solution for 15 minutes.

[0062] 6) Decolorization. Decolorize with water for 30 minutes until the bands are clear.

[0063] 7) Take photos. Use a gel imaging device to take photos and save them.

[0064] The yields of various heparin-modifying enzymes expressed by different engineered strains were detected by SDS-PAGE. Preliminary grayscale value calculations showed that the yields of each heparin-modifying enzyme expressed in *Pichia pastoris* ranged from 20 to 500 mg / L. In contrast, the expression levels of each heparin-modifying enzyme in the *E. coli* expression system reached 400 mg / L to >1.2 g / L. Figure 3 As shown, taking 2-OST as an example, the expression level of NusA-2-OST(1) fusion can reach >1.2g / L.

[0065] Example 3: Characterization of the catalytic activity of a novel recombinant heparin-modifying enzyme

[0066] 3.1 Method for detecting the activity of heparin-modifying enzymes

[0067] The concentration of the test protein was determined using a BCA kit, and the amount of each modified enzyme in the enzyme activity assay system was kept as uniform as possible. Enzyme activity assays were performed using a reaction system reported in the literature (reduced fivefold: 200 μl system). Continuous assays were performed at a constant temperature of 37°C using a microplate reader, 2 h, 2 min / time.

[0068] Reaction system (200 μl):

[0069] ① 0.4 mg deacetylated sulfonated heparin precursor — 0.2 mg 10 μl

[0070] ②50mM PNPS——20μl

[0071] ③ 5mM PAPS — 4μl

[0072] ④ Pure enzyme 1 (C5-epi): 1 mg / ml — 20 μl (0.02 mg)

[0073] ⑤ Pure enzyme 2 (2-OST-Y94A): 1 mg / ml — 20 μl (0.02 mg)

[0074] ⑥M-AST IV: 1mg / ml——20μl (0.02mg)

[0075] ⑦ Add 200 μl of 20 mM Tris-HCl to make up the difference.

[0076] ⑧ Enterokinase: 0.1-0.2 μl

[0077] The control group consisted of pure enzyme 1 and pure enzyme 2, which were deactivated by boiling in a water bath for 10 minutes.

[0078]

[0079] 2-OST, 6-OST and 3-OST enzyme activity assay (250 μL):

[0080] ①OST——0.1mg / 0.2mg

[0081] ②AST-Ⅳ——0.1mg

[0082] ③PNPS——0.5μM

[0083] ④PAPS—4nmol

[0084] ⑤ N-sulfonyptane decasaccharide – 25 μg

[0085] ⑥ Add MES buffer (pH 7.4) to a final volume of 250 μL.

[0086] The reaction time was 2 hours, and the detection interval was 2 minutes.

[0087] The control group consisted of OST that was deactivated by boiling water bath for 10 minutes, centrifuged, and then added in equal volume.

[0088] 3.2 Comparison of enzyme activities of recombinant heparin-modifying enzymes

[0089] The activity of the newly discovered C5 epimerase C5epi(2) from Cuculus canorus (0.063 U / L) is significantly higher than that of the previously reported D-C5epi enzyme (0.032 U / L), nearly twice that of D-C5epi. Furthermore, the activity of the newly discovered 2-OST enzyme from Dryobates pubescens is also 2.4 times that of the previously reported M-2-OST. Figure 4 As shown in Figure 3.3. Activity characterization of heparin synthesized via proheparinase modification.

[0090] This invention uses proheparin as a precursor and applies the newly discovered heparin-modifying enzymes listed in Table 1, combined with the engineered strain successfully constructed in Example 2, to characterize the stepwise catalytic reaction activity of the obtained pure heparin-modifying enzymes in synthesizing heparin. Specifically, [the process is described in the original text]. Figure 5 The assay was performed step by step as shown. The results are shown in Figure 6. PNPs were produced in each step of the modified enzyme catalytic reaction, which proves the effectiveness of the recombinant enzyme catalytic reaction in each step. The synthetic route from proheparin to heparin can be opened up by the newly discovered and heterologously expressed recombinant heparin modified enzyme of this invention.

Claims

1. A heparin-modifying enzyme gene, characterized in that, It originates from the song-bearing bunting ( Melospiza georgiana The bifunctional N-deacetyl / N-sulfate transferase NDST gene, derived from the rhododendron (Rhododendron molle) Cuculus canorus The glucuronide C5-allostericase C5 epi gene, derived from the downy woodpecker ( Dryobates pubescens The heparin sulfonate 2-sulfate transferase 2-OST gene, derived from the bar-tailed godwit ( Limosa lapponica baueri The heparin sulfonate 6-sulfate transferase 6-OST gene of ) may be derived from swallows ( Hirundo rustica rustica The heparan sulfonate 3-sulfate transferase 3-OST gene.

2. The heparin-modifying enzyme gene as described in claim 1, characterized in that, Its nucleotide sequence was obtained by codon optimization based on the expression preferences of Pichia pastoris or Escherichia coli.

3. The heparin-modifying enzyme gene as described in claim 1, characterized in that, It further merges with fusion expression tags, such as TrxA, UB, GB1, NusA, CaBP, SUMO (Smt3), FTN-H, or Skp.

4. The protein encoded by the heparin-modifying enzyme gene as described in any one of claims 1 to 3.

5. An expression vector containing the heparin-modifying enzyme gene as described in any one of claims 1 to 3, wherein the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

6. The expression vector as described in claim 5, characterized in that, It is an expression vector suitable for expression in Pichia pastoris or Escherichia coli.

7. A recombinant host bacterium containing the heparin-modifying enzyme gene as described in any one of claims 1 to 3, or the expression vector as described in claim 5 or 6.

8. The recombinant host bacterium as described in claim 7, characterized in that, It is either Pichia pastoris or Escherichia coli.

9. The use of a heparin-modifying enzyme gene as described in any one of claims 1 to 3, or the protein encoded thereon, in the in vitro multi-enzyme catalytic synthesis of heparin; specifically, the use relates to catalytic reactions involving deacetylation, C5 isomerization, or 2-, 6-, 3-sulfonation.

10. A method for expressing the heparin-modifying enzyme gene as described in any one of claims 1 to 3 or for preparing the protein encoded as described in claim 4, characterized in that, It is achieved by culturing the recombinant host bacteria as described in claim 7 or 8 to produce the heparin-modifying enzyme.

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