Method for producing hyaluronic acid through cell-free catalysis

Abstract

The invention relates to a method for producing hyaluronic acid through cell-free catalysis, and belongs to the technical field of biological catalysis. According to the invention, a system capable of being applied to cell-free catalytic production of hyaluronic acid is constructed, and genetically engineered bacteria are used for expressing N-acetyl hexosamine 1-kinase, glucosamine-1-phosphate acetyl transferase, glucuronide kinase, GlcA-1-P uridinetransferase, hyaluronic acid synthase, polyphosphate kinase, uridine monophosphate kinase and inorganic pyrophosphatase; according to the present invention, N-acetylglucosamine GlcNAc, glucuronic acid GlcA, ATP and UMP are adopted as substrates to perform catalytic production, UDP and ADP produced during the reaction process are converted into ATP and UTP, such that nucleotide circulation is achieved, raw material consumption is reduced, the final hyaluronic acid yield reaches 1.28 g / L, and an economic and efficient brand new path is provided for hyaluronic acid industrial production.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, and in particular to a cell-free catalytic method for producing hyaluronic acid. Background Technology

[0002] Hyaluronic acid (HA) is a linear polysaccharide composed of GlcA and GlcNAc disaccharide repeating units. Due to its advantages of water retention, biocompatibility, and non-immunogenicity, it has been widely used in food, pharmaceuticals, and cosmetics. Microbial fermentation is currently the main method for producing hyaluronic acid. This method utilizes specific microorganisms as bioreactors. In a fermenter, by controlling temperature, pH, and dissolved oxygen conditions, the microorganisms metabolize carbon sources (such as glucose) in the culture medium into hyaluronic acid. This process typically lasts 48-72 hours, covering the cell proliferation phase and the HA synthesis phase. Its core relies on the catalytic reaction of endogenous hyaluronic acid synthase within the microbial cells. However, this method has significant bottlenecks: on the one hand, the lengthy fermentation cycle and complex downstream processing severely restrict efficiency. After fermentation is terminated, it requires high-temperature inactivation, centrifugation, proteolysis, nucleic acid degradation, activated carbon adsorption of pigments, and multi-step ethanol precipitation or chromatography purification, resulting in excessively high yield losses. On the other hand, endotoxins (lipopolysaccharides) remaining in the microbial cell walls are difficult to completely remove, potentially triggering fever or inflammatory reactions in humans, thus limiting the application of the product in highly sensitive scenarios such as pharmaceutical injections and ophthalmic surgery. These inherent defects have prompted the industry to seek more efficient, safe, and controllable alternatives.

[0003] In recent years, with the advancement of molecular biology techniques, the technology of synthesizing hyaluronic acid using isolated and purified hyaluronic acid synthase (HAS) has been developed. However, the enzymatic synthesis of hyaluronic acid requires the use of large amounts of glyconucleotides as substrates, and the high price of glyconucleotides has limited the development of research on enzymatic HA synthesis. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a cell-free catalytic method for producing hyaluronic acid. The method involves using a host bacterium to express N-acetylglucosamine 1-kinase, glucosamine-1-phosphate acetyltransferase, glucuronide kinase, GlcA-1-P uridine transferase, hyaluronic acid synthase, polyphosphate kinase, uridine kinase, and inorganic pyrophosphatase. After purification, these enzymes are added to a reaction system using N-acetylglucosamine GlcNAc, glucuronide GlcA, ATP, and UMP as substrates.

[0005] The first objective of this invention is to provide a cell-free method for catalyzing hyaluronic acid production, comprising the following steps:

[0006] Step S1: Recombinant expression of N-acetylglucosamine 1-kinase NahK, glucosamine-1-phosphoacetyltransferase GlmU, glucuronide kinase GlcAK, GlcA-1-P uridine transferase USP, hyaluronic acid synthase HAS, polyphosphate kinase PPK3, uridine kinase URA6, and inorganic pyrophosphatase PpA was performed using host bacteria.

[0007] Step S2: Add NahK, GlmU, GlcAK, USP, PPK3, URA6 and PpA obtained in step S1 to the reaction system, wherein the reaction system includes N-acetylglucosamine GlcNAc, glucuronic acid GlcA, adenosine triphosphate ATP and uridine monophosphate UMP.

[0008] Step S3: After the reaction has proceeded for 0.5-24 hours, the HAS obtained in step S1 is added to continue the reaction. After the reaction is completed, the product is purified to obtain hyaluronic acid.

[0009] Further, in step S1, the amino acid sequence of hyaluronic acid synthase HAS as shown in SEQ ID NO.1 is truncated, and the truncation includes any of the following:

[0010] (1) Remove amino acids from positions 2 to 45;

[0011] (2) Remove amino acids from positions 2 to 95;

[0012] (3) Remove amino acids from positions 710 to 972;

[0013] (4) Remove amino acids from position 800 to 972.

[0014] Furthermore, in step S2, the ratio of NahK, GlmU, GlcAK and USP is 1-3:1-3:1-3:1-3.

[0015] Furthermore, in step S2, the ratio of GlcNAc, GlcA, ATP and UMP is 1-2:1-2:1-2:1-2.

[0016] Furthermore, in step S2, the reaction system also includes Mg. 2+ .

[0017] A second object of the present invention is to provide an enzyme composition for cell-free catalytic production of hyaluronic acid, the enzyme composition comprising N-acetylglucosamine 1-kinase NahK, glucosamine-1-phosphate acetyltransferase GlmU, glucuronide kinase GlcAK, GlcA-1-P uridine transferase USP, and hyaluronic acid synthase HAS.

[0018] Furthermore, the amino acid sequence of hyaluronic acid synthase HAS, as shown in SEQ ID NO.1, was truncated, and the truncation included any of the following:

[0019] (1) Remove amino acids from positions 2 to 45;

[0020] (2) Remove amino acids from positions 2 to 95;

[0021] (3) Remove amino acids from positions 710 to 972;

[0022] (4) Remove amino acids from position 800 to 972.

[0023] Furthermore, the enzyme composition also includes polyphosphate kinase PPK3, uridine kinase URA6, and inorganic pyrophosphatase PpA.

[0024] A third objective of this invention is to provide genetically engineered bacteria that express the above-described enzyme composition.

[0025] A fourth object of the present invention is to provide the application of the above-described enzyme composition or the above-described genetically engineered bacteria in the production of hyaluronic acid.

[0026] The beneficial effects of this invention are:

[0027] This invention addresses the problems of long fermentation cycles and microbial endotoxins in the production of hyaluronic acid via microbial fermentation. It utilizes genetically engineered bacteria to express N-acetylglucosamine 1-kinase, glucosamine-1-phosphate acetyltransferase, glucuronide kinase, GlcA-1-P uridine transferase, hyaluronic acid synthase, polyphosphate kinase, uridine kinase, and inorganic pyrophosphatase, using N-acetylglucosamine GlcNAc, glucuronide GlcA, ATP, and UMP as substrates for catalytic production. To further improve the soluble expression of hyaluronic acid synthase (HAS), it was truncated. Combining multi-enzyme stepwise cascade and nucleotide regeneration technology, this invention achieves, for the first time, highly efficient synthesis of hyaluronic acid using inexpensive monosaccharides (GlcNAc / GlcA) and UMP as substrates, with a yield of 1.28 g / L. The product has a wide molecular weight range and no endotoxin residue, providing an economical, efficient, high-purity product with a molecular weight distribution of 1.28 × 10⁻⁶ for industrial hyaluronic acid production. 4 Up to 1.02×10 6 Da's entirely new path. Attached Figure Description

[0028] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0029] Figure 1 This is a schematic diagram of the cell-free catalytic synthesis pathway for hyaluronic acid (HA) according to the present invention.

[0030] Figure 2 The product characterization of hyaluronic acid in Example 1 of the present invention is shown in the figure, where a is the predicted truncated soluble expression of pmHAS and b is the SDS-PAGE verification of pmHASΔ710-972.

[0031] Figure 3 This invention provides an optimization of the synthesis of UDP-GlcNAc and UDP-GlcA in Example 1 of this invention. In this figure, a represents the UDP-GlcNAc and UDP-GlcA separated and identified by LC-MS; b represents the SDS-PAGE results; lanes 1-4 represent the four pathway enzymes NahK, GlmU, GlcAK, and USP, respectively. c and d represent the conversion rates of UDP-GlcNAc and UDP-GlcA at different substrate concentrations through cascade catalysis; e and f represent the optimized enzyme loading results.

[0032] Figure 4 Infrared spectra of the HA product and standard in Example 1 of this invention;

[0033] Figure 5 This refers to the LC-MS determination of the HA product after degradation into HA disaccharide in Example 1 of the present invention.

[0034] Figure 6 This is the construction and verification of the nucleotide cycling system in Example 2 of the present invention, where a and b are schematic diagrams of the relevant pathways, c is the SDS-PAGE verification of PPK3, PpA and URA6, lane M is the marker, lanes 1-3 are PPK3, PpA and URA6 respectively, and d is the HPLC detection of the ATP and UTP cycling system.

[0035] Figure 7 This refers to the establishment of the nucleotide sugar cycle system and the cell-free enzyme cascade for de novo HA synthesis in Example 2 of the present invention, where a and b are the nucleotide conversion rates before and after the establishment of the nucleotide cycle, c is the conversion rate of the precursor nucleotide sugar during the enzyme cascade, and d is the HA yield detection. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0037] Some of the experimental methods involved in the embodiments are shown below:

[0038] (1) Qualitative and molecular weight determination of HA

[0039] Purified HA was prepared into a 2 g / L solution in a 50 mM, pH 7.0 sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH₂PO₄-Na₂HPO₄) buffer solution. 0.9 mL of the HA solution was reacted with 0.1 mL of hyaluronic acid lyase at 37 °C for 12 h. The reaction was terminated by heating in a boiling water bath for 10 min, followed by centrifugation to remove the precipitate. Degradation products were detected using liquid chromatography-mass spectrometry (LC-MS).

[0040] The molecular weight of the final product was accurately determined using negative ion electrospray mass spectrometry and gel permeation chromatography. Mass spectrometry acquisition range: 50-2000 m / z. ESI-MS analysis was performed under the following conditions:

[0041] Capillary: 3.0 kV, Cone: 20 / 50 V, Source block temperature: 100 °C, Desolvation temperature: 400 °C, Desolvation gas flow rate: 700 L / h, Cone gas flow rate: 50 L / h, Collision energy: 6 eV, Detector: 1800 V. The chromatographic column was an Ohpak SB-806HQ2.0300, using a differential detector (RI), with 0.1 M NaNO3 as the mobile phase, a flow rate of 0.5 mL / min, a column temperature of 25 °C, an injection concentration of 5-10 mg / mL, and an injection volume of 200 μL.

[0042] (2) Determination of UDP-GlcNAc and UDP-GlcA

[0043] To quantitatively analyze the content of hyaluronic acid precursors (UDP-GlcA and UDP-GlcNAc), an Agilent 1100 series high-performance liquid chromatography (HPLC) system equipped with an autosampler was used for separation on an Aminex HPX-87H cation exchange column (300 × 7.8 mm, Bio-Rad). After sample injection, linear gradient elution was performed at a flow rate of 0.6 mL / min, with mobile phases A (methanol) and B (methanol:water:acetic acid:tetrabutylammonium hydroxide = 3:97:1:0.6), and a total run time of 50 minutes. The eluent was quantitatively analyzed using calibration curves of UDP-GlcA and UDP-GlcNAc standards.

[0044] Example 1: Cell-free biosynthesis of HA

[0045] (1) Using Escherichia coli BL21(DE3) and Rosseta as hosts to efficiently express exogenous proteins

[0046] Using pET32a plasmid as a backbone, a recombinant plasmid containing hyaluronic acid synthase pmHAS (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) from Pasteurella multocida was constructed. Using E. coli Rosseta as the host bacterium, the recombinant strain R-pmHAS was obtained.

[0047] Using pET32a plasmid as a backbone, recombinant plasmids containing NahK (nucleotide sequence as shown in SEQ ID NO.3) from Bifidobacterium longum, GlmU (nucleotide sequence as shown in SEQ ID NO.4) from Escherichia coli, GlAcK (nucleotide sequence as shown in SEQ ID NO.5) from Arabidopsis thaliana, and USP (nucleotide sequence as shown in SEQ ID NO.6) were constructed. Using E. coli BL21(DE3) as the host bacterium, recombinant strains B-NahK, B-GlmU, B-GlAcK, and B-USP were obtained.

[0048] To increase the soluble expression of pmHAS, the amino acid sequence of pmHAS, as shown in SEQ ID NO.1, was truncated. The truncation strategies were as follows: truncating amino acids 2-45, 2-75, 2-95, 710-972, 800-972, and 900-972. The solubility of pmHAS obtained with different truncation strategies was predicted, and the prediction results are shown below. Figure 2 As shown in Figure a, pmHASΔ710-972 exhibits significantly improved solubility compared to wild-type pmHAS and pmHAS obtained through other truncation strategies. Therefore, pmHASΔ710-972 was selected for subsequent experiments. The SDS-PAG validation results of pmHASΔ710-972 are shown below. Figure 2 As shown in b.

[0049] Linearized primers with the corresponding truncated amino acid sequence Δ710-972 were designed, and homologous sequences were introduced at both ends of the primers. The linear plasmid fragment was recovered by digestion with the SwiftCut DpnⅠ restriction endonuclease. The DpnⅠ digestion product was aspirated and added to E. coli JM109 competent cells in a clean bench for subsequent transformation. The transformed cells were used to verify whether the construction was successful. Then, using E. coli Rosseta as the host, the recombinant strain R-pmHASΔ710-972 was obtained.

[0050] Recombinant strains R-pmHASΔ710-972, B-NahK, B-GlmU, B-GlAcK, and B-USP were inoculated into 50 mL of LB medium and cultured at 37°C and 220 rpm until OD500. 600 The expression level was increased to 0.8-1.0. Then, the culture was induced with 0.5 mM IPTG, and the expression was further induced at 16-25℃ for 14-30 h depending on the expression of different enzymes.

[0051] The bacterial cells were centrifuged at 8000g for 10 min, washed twice with PBS buffer and resuspended. The collected cells were lysed by sonication and centrifuged at 6000 rpm for 10 min at 4°C to remove cell debris. The supernatant was purified by nickel affinity chromatography, using a buffer with a high concentration of imidazole to replace and elute the target proteins binding to Ni, yielding five enzymes: pmHAS, NahK, GlmU, GluAcK, and USP. The purified proteins were evaluated by SDS-PAGE and protein concentration was determined using the Bradford assay. The results are shown below. Figure 3 As shown in b.

[0052] Following the steps described above, recombinant strains R-pmHASΔ710-972, B-NahK, B-GlmU, B-GlAcK, and B-USP were fermented, and the bacterial cells were collected and purified to obtain five enzymes: pmHASΔ710-972, NahK, GlmU, GlAcK, and USP.

[0053] Four enzymes, NahK, GlmU, GluAcK, and USP, were added to a reaction system containing 50 mM PBS buffer (pH 7.5), 10 mM MgCl2, GlcNAc, GlcA, UTP, and ATP. The reaction was carried out at 30-37°C, and the products were the HA precursors UDP-GlcNAc and UDP-GlcA. The ratio of the four enzymes was 1:1.5:1.5:2. The ratio of GlcNAc, GlcA, UTP, and ATP was adjusted, and the conversion rates of the precursors UDP-GlcNAc and UDP-GlcA were obtained using LC-MS. The results are shown below. Figure 3 c and Figure 3 As shown in Figure d, the highest conversion rates of the precursor were observed when the ratio of GlcNAc, ATP, and UTP was 1:1:1 and when the ratio of GlcA, ATP, and UTP was 2:1:1. The LC-MS identification results of the generated UDP-GlcNAc and UDP-GlcA are shown in Figure d. Figure 3 As shown in a. Further adjustments were made to the proportions of the four enzymes, and the results are as follows. Figure 3 e and Figure 3 As shown in f, Figure 3 The ratio of the four enzymes added for e is 1:2:1:2. Figure 3The ratio of the four enzymes added is 1:1.5:1.5:2. When the ratio of the four enzymes is 1:1.5:1.5:2, the synthesis of the two precursors is equivalent, which makes it easier to determine the timing of adding pmHASΔ710-972.

[0054] When the nucleotide sugar synthesis reaction proceeded to the point where the amounts of precursors UDP-GlcNAc and UDP-GlcA were equal, pmHASΔ710-972 was added, and the mixture was incubated at 30°C to synthesize HA. After 24 hours of HA synthesis, the protein was denatured by boiling in a water bath for 10 minutes. The supernatant was collected by centrifugation at 8000 rpm for 10 minutes, and the product was extracted using 4 times the volume of the supernatant. The supernatant was then purified using a dialysis bag with a molecular weight cutoff of 3.5 kDa and lyophilized to obtain the HA sample. The HA was qualitatively analyzed and its molecular weight was determined according to the above method. The infrared spectrum is shown below. Figure 4 As shown, the LC-MS results after the HA product was degraded into HA disaccharide are as follows: Figure 5 As shown, the yield of hyaluronic acid (HA) was 1.43 g / L, with a molecular weight range of 1.28 × 10⁻⁶. 4 Up to 1.02×10 6 Da.

[0055] Example 2: Cell-free biosynthesis of HA using a nucleotide cycling system

[0056] Recombinant strains R-pmHASΔ710-972, B-NahK, B-GlmU, B-GlAcK, and B-USP were constructed according to the method in Example 1, and then expressed by fermentation to obtain purified proteins pmHASΔ710-972, NahK, GlmU, GlAcK, and USP.

[0057] Using pET32a plasmid as a backbone, recombinant strains B-PPK3, B-URA6, and B-PpA were constructed, comprising PPK3 from Ruegeria pomeroyi (nucleotide sequence as shown in SEQ ID NO. 7), URA6 from Arabidopsis thaliana (nucleotide sequence as shown in SEQ ID NO. 8), and PpA from Pasteurella multocida (nucleotide sequence as shown in SEQ ID NO. 9). E. coli BL21(DE3) was used as the host bacterium to obtain recombinant strains B-PPK3, B-URA6, and B-PpA. Purified PPK3, URA6, and PpA were obtained according to the method in Example 1. The purified proteins were evaluated by SDS-PAGE, and the protein concentration was determined using the Bradford assay. The results are shown below. Figure 6 As shown in c.

[0058] In a reaction system containing 50 mM pH 7.5P BS buffer, 10 mM MgCl2, 10 mM GlcNAc, 10 mM GlcA, 10 mM MUMP, and 10 mM ATP, seven enzymes (NahK, GlmU, GluAcK, USP, PPK3, URA6, and PpA) were added. The ratio of NahK, GlmU, GluAcK, and USP was 1:1.5:1.5:2 (the remaining enzymes were added in a 1:1:1 ratio). After reacting at 30-37℃ for 8 hours, the yields of UDP-GlcNAc and UDP-GlcA exceeded 60% within 8 hours, and ATP and UTP were continuously regenerated. The conversion rates of the precursors UDP-GlcNAc and UDP-GlcA during the reaction were as follows: Figure 7 As shown in c. When the nucleotide sugar synthesis reaction proceeded to a point where the amounts of the two precursors were roughly equal, pmHASΔ710-972 was added, and the mixture was incubated at 30°C to synthesize HA. This combined the nucleotide sugar cycle with HA biosynthesis, and after 12 hours, the HA yield reached 1.28 g / L. The reactions involved in the nucleotide cycle system are as follows: Figure 6 a and Figure 6 As shown in b, the HPLC detection results of the circulating system are as follows: Figure 6 As shown in d.

[0059] The nucleotide conversion rates in the cell-free catalytic production of hyaluronic acid (HA) were statistically analyzed and compared. The nucleotide conversion rates before the establishment of the nucleic acid recycling system (in Example 1) were as follows: Figure 7 As shown in Figure a, the nucleotide conversion rate after the nucleic acid recycling system is established (in this embodiment) is as follows: Figure 7 As shown in b.

[0060] After 24 hours of HA synthesis, the protein was denatured by boiling in a water bath for 10 minutes. The supernatant was collected by centrifugation at 8000 rpm for 10 minutes. The product was extracted using 4 times its volume of water, centrifuged at 6000 rpm for 10 minutes, and the supernatant was purified using a dialysis bag with a molecular weight cutoff of 3.5 kDa and then lyophilized to obtain the HA sample. The results are as follows: Figure 7 As shown in d.

[0061] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A cell-free method for catalyzing hyaluronic acid production, characterized in that, Includes the following steps: Step S1: Recombinant expression of N-acetylglucosamine 1-kinase NahK, glucosamine-1-phosphoacetyltransferase GlmU, glucuronide kinase GlcAK, GlcA-1-P uridine transferase USP, hyaluronic acid synthase HAS, polyphosphate kinase PPK3, uridine kinase URA6, and inorganic pyrophosphatase PpA was performed using host bacteria. Step S2: Add NahK, GlmU, GlcAK, USP, PPK3, URA6 and PpA obtained in step S1 to the reaction system, wherein the reaction system includes N-acetylglucosamine GlcNAc, glucuronic acid GlcA, adenosine triphosphate ATP and uridine monophosphate UMP. Step S3: After the reaction has proceeded for 0.5-24 hours, the HAS obtained in step S1 is added to continue the reaction. After the reaction is completed, the product is purified to obtain hyaluronic acid.

2. The method according to claim 1, characterized in that, In step S1, the amino acid sequence of hyaluronic acid synthase HAS, as shown in SEQ ID NO.1, is truncated, and the truncation includes any of the following: (1) Remove amino acids from positions 2 to 45; (2) Remove amino acids from positions 2 to 95; (3) Remove amino acids from positions 710 to 972; (4) Remove amino acids from position 800 to 972.

3. The method according to claim 1, characterized in that: In step S2, the ratio of NahK, GlmU, GlcAK and USP is 1-3:1-3:1-3:1-3.

4. The method according to claim 1, characterized in that: In step S2, the ratio of GlcNAc, GlcA, ATP and UMP is 1-2:1-2:1-2:1-2.

5. The method according to claim 1, characterized in that: In step S2, the reaction system also includes Mg. 2+ .

6. An enzyme composition for cell-free catalytic production of hyaluronic acid, characterized in that: The enzyme composition includes N-acetylglucosamine 1-kinase NahK, glucosamine-1-phosphate acetyltransferase GlmU, glucuronide kinase GlcAK, GlcA-1-P uridine transferase USP, and hyaluronic acid synthase HAS.

7. The enzyme composition according to claim 6, characterized in that, In step S1, the amino acid sequence of hyaluronic acid synthase HAS, as shown in SEQ ID NO. 1, is truncated, and the truncation includes any of the following: (1) Remove amino acids from positions 2 to 45; (2) Remove amino acids from positions 2 to 95; (3) Remove amino acids from positions 710 to 972; (4) Remove amino acids from position 800 to 972.

8. The enzyme composition according to claim 6, characterized in that: The enzyme composition also includes polyphosphate kinase PPK3, uridine kinase URA6, and inorganic pyrophosphatase PpA.

9. Genetically engineered bacteria expressing the enzyme composition of any one of claims 6-8.

10. The use of any of the enzyme compositions of claims 6-8 or the genetically engineered bacteria of claim 9 in the production of hyaluronic acid.

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