A hyaluronan synthase mutant synthesizing hyaluronan with different molecular weights

Abstract

The present invention relates to a hyaluronic acid synthase mutant for synthesizing hyaluronic acid of different molecular weights, belonging to the field of bioenzyme engineering technology. The hyaluronic acid synthase mutant of the present invention is a point mutation or a compound mutation of the hyaluronic acid synthase of the amino acid sequence as shown in SEQ ID NO.1, and the hyaluronic acid synthase mutant screened out on this basis can synthesize hyaluronic acid of different molecular weights. The hyaluronic acid synthase after mutation of the present invention can synthesize hyaluronic acid of different molecular weights compared with the hyaluronic acid synthase before mutation, and the hyaluronic acid molecular weight range obtained is 300KDa-40000KDa, and the output of hyaluronic acid increases at the same time, with very high industrial application value.

Description

Technical Field

[0001] The invention relates to the technical field of bioenzyme engineering, in particular to a hyaluronan synthase mutant for synthesizing hyaluronan with different molecular weights. Background Art

[0002] Hyaluronic acid is a linear, high molecular weight, non-sulfated glycosaminoglycan, a polymer formed by repeated alternation of N-acetylglucosamine and D-glucuronic acid through β-1,4- and β-1,3-glycosidic bonds. The molecular weight of natural hyaluronic acid is widely distributed, ranging from thousands of Daltons to millions or even tens of millions of Daltons. The properties of hyaluronic acid are directly related to its molecular weight. Ultra-high molecular weight hyaluronic acid (molecular weight greater than 3000KDa) has an inhibitory effect on cell migration, proliferation, differentiation and phagocytosis and can be used as a solid filler; high molecular weight (molecular weight between 1000KDa and 3000KDa) hyaluronic acid has good moisturizing and anti-inflammatory functions and can be used as a viscoelastic agent and intra-articular injection therapy in ophthalmic surgery; medium molecular weight (molecular weight between 100KDa and 1000KDa) hyaluronic acid has good moisturizing and lubricating effects and is widely used in the cosmetics field; low molecular weight (molecular weight less than 100KDa) hyaluronic acid and its oligosaccharides have anti-tumor, wound healing, angiogenesis, immune regulation and other effects, and have good medical application prospects.

[0003] At present, the main methods for regulating the molecular weight of hyaluronic acid are physical and chemical degradation and enzymatic depolymerization. The physical and chemical method mainly degrades hyaluronic acid by treatment methods such as thermal degradation, ultrasound, strong acid and strong alkali. The molecular weight distribution of the product obtained by this method is uneven, the efficiency is low, and it will destroy the molecular structure of hyaluronic acid and reduce the product quality. Compared with the physical and chemical method, the enzymatic depolymerization of hyaluronic acid has mild and controllable reaction conditions and good repeatability, but the cost is high, and the performance of the degradation enzyme also limits the further development of this method. Direct synthesis using microorganisms is an ideal method for producing hyaluronic acids of different molecular weights. At present, researchers have optimized and regulated the molecular weight of hyaluronic acid by modifying enzyme molecules and optimizing the fermentation process to achieve the synthesis of hyaluronic acids of different molecular weights. However, the molecular weight range of hyaluronic acid produced by microbial fermentation in the prior art is 1KDa-3000KDa, and ultra-high molecular weight hyaluronic acid (molecular weight greater than 3000KDa) cannot be synthesized, and the yield of hyaluronic acid in the prior art is low. Summary of the invention

[0004] To this end, the technical problem to be solved by the present invention is to overcome the problem in the prior art that the controllable synthesis of hyaluronic acid molecular weight in the range of 300KDa-4000KDa cannot be achieved and the yield of the synthesized hyaluronic acid is low.

[0005] To solve the above-mentioned technical problems, the present invention uses a hyaluronan synthase mutant, which is screened out by point mutation or compound mutation of hyaluronan synthase to synthesize hyaluronan synthase of different molecular weights, and the mutant can synthesize hyaluronan of medium and high molecular weight, and can also synthesize hyaluronan of ultra-high molecular weight, and the molecular weight range of the obtained hyaluronan is 300KDa-40000KDa. At the same time, the screened hyaluronan synthase mutant is introduced into recombinant bacteria, and the hyaluronan output produced is significantly improved.

[0006] The first object of the present invention is to provide a hyaluronan synthase mutant for synthesizing hyaluronic acids of different molecular weights, characterized in that the hyaluronan synthase mutant is a hyaluronan synthase with an amino acid sequence as shown in SEQ ID NO.1 mutated:

[0007] (a) The arginine at position 2 is mutated to proline, the threonine at position 3 is mutated to isoleucine, and the leucine at position 4 is mutated to phenylalanine (R2P / T3I / L4F);

[0008] (b) mutating asparagine at position 6 to lysine (N6K);

[0009] (c) the leucine at position 7 is mutated to threonine, the isoleucine at position 8 is mutated to leucine, the threonine at position 9 is mutated to isoleucine, the valine at position 11 is mutated to leucine, the alanine at position 12 is mutated to serine, the serine at position 14 is mutated to isoleucine, the isoleucine at position 15 is mutated to phenylalanine, and the phenylalanine at position 16 is mutated to leucine (L7T / I8L / T9I / V11L / A12S / S14I / I15F / F16L);

[0010] (d) mutating the tryptophan at position 17 to isoleucine (W17I);

[0011] (e) The valine at position 18 was mutated to serine, the leucine at position 19 was mutated to isoleucine, the valine at position 23 was mutated to leucine, and the valine at position 25 was mutated to methionine (V18S / L19I / V23L / V25M).

[0012] Specifically, the sequence of SEQ ID NO.1 is: MRTLKNLITVVAFSIFWVLLIYVNV YLFGAKGSLSIYGFLLIAYLLVKMSLSFFYKPFKGRAGQYKVAAIIPSYNEDAESLLETLKSVQQQTYPLAEIYVVDDGSADETGIKRIEDYVRDTGDLSSNVIVHRSEKNQGKRHAQAWAFERSDADVFLTVDSDTYIYPDALEELLKTFNDPTVFAATGHLNVRNRQTNLLTRLTDIRYDNAFGVERAAQSVTG NILVCSGPLSVYRREVVVPNIDRYINQTFLGIPVSIGDDRCLTNYATDLGKTVYQSTAKCITDVPDKMSTYLKQQNRWNKSFFRESIISVKKIMNNPF VALWTILEVSMFMMLVYSVVDFFVGNVREFDWLRVLAFLVIIFIVALCRNIHYMLKHPLSFLLSPFYGVLHLFVLQPLKLYSLFTIRNADWGTRKKLL

[0013] Furthermore, compared with the amino acid sequence shown in SEQ ID NO: 1, the hyaluronan synthase mutant has any of the following mutations:

[0014] (1) N6K, V18S, L19I, V23L and V25M;

[0015] (2) R2P, T3I, L4F and W17I;

[0016] (3) W17I, V18S, L19I, V23L and V25M;

[0017] (4) V18S, L19I, V23L and V25M;

[0018] (5)N6K;

[0019] (6) R2P, T3I, L4F, N6K and W17I;

[0020] (7) R2P, T3I, L4F, L7T, I8L, T9I, V11L, A12S, S14I, I15F, F16L and W17I;

[0021] (8) R2P, T3I, L4F, V18S, L19I, V23L and V25M;

[0022] (9)W17I;

[0023] (10) R2P, T3I, L4F, L7T, I8L, T9I, V11L, A12S, S14I, I15F and F16L;

[0024] (11) R2P, T3I and L4F;

[0025] (12) L7T, I8L, T9I, V11L, A12S, S14I, I15F and F16L;

[0026] (13) L7T, I8L, T9I, V11L, A12S, S14I, I15F, F16L and W17I;

[0027] (14) R2P, T3I, L4F, L7T, I8L, T9I, V11L, A12S, S14I, I15F, F16L, V18S, L19I, V23L, and V25M;

[0028] (15) N6K and W17I;

[0029] (16) R2P, T3I, L4F, N6K, V18S, L19I, V23L and V25M;

[0030] (17) N6K, L7T, I8L, T9I, V11L, A12S, S14I, I15F and F16L;

[0031] (18) R2P, T3I, L4F, N6K, L7T, I8L, T9I, V11L, A12S, S14I, I15F and F16L;

[0032] (19) R2P, T3I, L4F, W17I, V18S, L19I, V23L and V25M;

[0033] (20) R2P, T3I, L4F and N6K.

[0034] The second object of the present invention is to provide a gene encoding the above hyaluronan synthase mutant.

[0035] The third object of the present invention is to provide a recombinant plasmid carrying the above gene.

[0036] The fourth object of the present invention is to provide a host cell expressing the above hyaluronan synthase mutant.

[0037] Furthermore, the host cell is a bacterium or a fungus. Preferably, the host cell is Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli, Lactococcus lactis, Pichia pastoris,

[0038] (Pichia pastoris), Saccharomyces cerevisiae, Streptococcus zooepidemicus

[0039] (Streptococcus zooepidemicus), Streptococcus thermophilus, Corynebacterium pekinense and other strains.

[0040] The fifth object of the present invention is to provide an application of the hyaluronan synthase mutant, the gene, the recombinant plasmid or the host cell in the preparation of hyaluronic acid.

[0041] Furthermore, the application is to add the hyaluronan synthase mutant or the expression system containing the hyaluronan synthase mutant into the reaction system to generate hyaluronic acid.

[0042] The sixth object of the present invention is to provide a method for synthesizing hyaluronic acid of different molecular weights, using the hyaluronic acid synthase mutant to be introduced into a plasmid to form a recombinant plasmid, and the recombinant plasmid is introduced into a host cell to form a recombinant bacterium, and the recombinant bacterium is fermented and cultured to synthesize hyaluronic acid, and the molecular weight of the synthesized hyaluronic acid ranges from 300KDa to 4000KDa.

[0043] Furthermore, the recombinant plasmid also includes glutamine-fructose-6-phosphate aminotransferase

[0044] The gene sequences of glmS, glmM and UDP-glucose dehydrogenase (ugd) are shown in Figure 2. The gene sequence of glmS is shown in SEQ ID NO.24, the gene sequence of glmM is shown in SEQ ID NO.25, and the gene sequence of ugd is shown in SEQ ID NO.26. Hyaluronic acid is mainly synthesized through the UDP-N-acetylglucosamine pathway and the UDP-glucuronic acid pathway. The enzymes expressed by the ugd, glmS and glmM genes are the key enzymes in these two pathways. Enhancing the synthesis of these three enzymes can effectively increase the yield of hyaluronic acid.

[0045] Specifically, the gene sequence of SEQ ID NO.24 is:

[0046]

[0047] The gene sequence of SEQ ID NO.25 is:

[0048]

[0049] The gene sequence of SEQ ID NO.26 is:

[0050]

[0051] Furthermore, the fermentation culture contains a carbon source, a nitrogen source, an inorganic salt and a metal ion. Preferably, the carbon source and the nitrogen source in the fermentation culture are provided by glucose, corn syrup powder and (NH4)2SO4, and the inorganic salt and the metal ion are provided by KH2PO4, K2HPO4, MgSO4, 3-morpholinepropanesulfonic acid (MOPS).

[0052] Beneficial effects of the present invention:

[0053] The hyaluronan synthase mutant of the present invention is obtained by performing point mutation or compound mutation on the hyaluronan synthase with an amino acid sequence as shown in SEQ ID NO: 1. The hyaluronan synthase mutant can synthesize hyaluronic acid of different molecular weights, and can synthesize hyaluronic acid of medium and high molecular weight as well as ultra-high molecular weight. The molecular weight of hyaluronic acid obtained by using the hyaluronan synthase mutant ranges from 300KDa to 40000KDa, and the yield of the hyaluronic acid synthesized by the present invention is higher, and the hyaluronic acid has high industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0055] Figure 1 It is a schematic diagram of the structure of type I hyaluronan synthase;

[0056] Figure 2 is the molecular weight and yield of hyaluronic acid produced by recombinant Corynebacterium glutamicum CG(01-20)-ugd-glmS-glmM;

[0057] Figure 3 is the hyaluronic acid yield of recombinant Corynebacterium glutamicum CG01-ugd-glmS-glmM in a 5L fermenter;

[0058] Figure 4 It is the hyaluronic acid production of recombinant Corynebacterium glutamicum CG20-ugd-glmS-glmM in a 5L fermenter. DETAILED DESCRIPTION

[0059] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0060] Strain: Corynebacterium glutamicum ATCC 13032.

[0061] Plasmids: pXMJ19, pEC-XK99E.

[0062] LB medium: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L.

[0063] BHI: Brain Heart Infusion 37g / L, Sorbitol 91g / L.

[0064] Fermentation medium: glucose 40 g / L, corn steep liquor powder 20 g / L, (NH4)2SO4 30 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4 25 g / L, 3-morpholinepropanesulfonic acid (MOPS) 42 g / L.

[0065] Determination of hyaluronic acid production:

[0066] Related reagents: borax sulfuric acid solution (weigh 4.77g sodium tetraborate, dissolve in 500mL concentrated sulfuric acid); carbazole solution (weigh 1.25g carbazole, dissolve in 500mL anhydrous ethanol); glucuronic acid solution 1g / L.

[0067] Sample purification: Take the fermentation liquid, centrifuge at 12000rpm for 2min. Take the supernatant, add 4 times the volume of anhydrous ethanol, place in a -20℃ refrigerator, precipitate for 12h, centrifuge at 12000rpm for 5min, and discard the supernatant. After the ethanol evaporates, add water to resuspend, add to the original volume after fully dissolving, centrifuge at 12000rpm for 5min, collect the supernatant, and repeat the above operation. After the alcohol precipitation is completed, collect the supernatant and the sample purification is completed.

[0068] Borax sulfuric acid-carbazole method: dilute 1g / L glucuronic acid solution to 0, 10, 20, 30, 40, 50mg / L, take 1mL borax sulfuric acid + 200μL glucuronic acid solution, mix in a glass colorimetric tube, and boil in water for 15min. Add 50μL carbazole solution to the colorimetric tube and boil in water for 10min. Transfer the reaction system to a 96-well plate and use an enzyme reader to measure the absorbance of the sample at a wavelength of 530nm. With the absorbance as the horizontal axis and the glucuronic acid concentration (mg / L) as the vertical axis, draw a curve showing the relationship between the absorbance and the glucuronic acid concentration, and obtain the standard curve equation: y=125.7x-11.590, R 2 =0.999; similarly, the purified sample was subjected to a borax-sulfuric acid-carbazole colorimetric reaction, the absorbance of the sample at a wavelength of 530 nm was measured, the absorbance obtained was substituted into the above curve equation, and the hyaluronic acid content in the sample was calculated according to the following formula: hyaluronic acid content (g / L) = (concentration measured by the standard curve * dilution factor * 2.067) / 1000.

[0069] Determination of Molecular Weight of Hyaluronic Acid:

[0070] The sample was purified by repeated alcohol precipitation according to the above sample purification method, using an 18-angle laser light scattering gel permeation chromatography system, mobile phase: ultrapure water (0.02% sodium azide), pH = 6.0; column temperature: 25°C; flow rate: 1.0 mL / min; chromatographic column: Shodex OHpak SB-806HQ connected in series with SB-804HQ.

[0071] Example 1: Construction of hyaluronan synthase mutants

[0072] (1) Construction of hyaluronan synthase mutant library

[0073] In vivo, HA is synthesized from two precursors, UDP-G1cA and UDP-G1cNAc, by hyaluronan synthase HAS. Hyaluronan synthase is a membrane-bound glycosyltransferase that has multiple functions in synthesizing HA, including binding to two precursors, UDP-G1cA and UDP-G1cNAc, glycosidic bond transport, continuous catalysis of the polymerization of the two precursors, and transfer of the synthesized HA chain to the extracellular space. Therefore, hyaluronan synthase itself plays an important role in the elongation and molecular weight regulation of HA chains (e.g. Figure 1 As shown). Therefore, based on the amino acid sequence of hyaluronan synthase shown in SEQ ID NO.1, primers HasA-F / HasA-R were designed to amplify the fragment SeHasA, and the appropriate restriction site of plasmid pXMJ19 was selected to obtain the linear vector plasmid pXMJ through restriction reaction. pXMJ and SeHasA fragments were connected by Taiji assembly reaction, transformed into Escherichia coli Top 10 by heat shock method, and cultured at 37°C for 24h. Transformants were selected for plasmid extraction and sequencing to obtain plasmid pXMJ19-SeHasA. On this basis, primers HasA were designed. 2-4 -F / HasA 2-4 -R, HasA 6 -F / HasA 6 -R, HasA 7-16 -F / HasA 7-16 -R, HasA 17 -F / HasA 17 -R and HasA 18-25 -F / HasA 18-25 -R (as shown in Table 1). By circular PCR amplification of plasmid pXMJ19-SeHasA, amino acid mutations at positions 2-4, 6, 7-16, 17, and 18-25 were achieved, respectively, to obtain hyaluronan synthase mutants 01-20 (as shown in Table 2).

[0074] Table 1 Primers used for construction of hyaluronan synthase mutant library

[0075]

[0076] (2) Expression of hyaluronan synthase mutants

[0077] Using an electroporator, set the instantaneous voltage to 1.5KV, the voltage duration to 5ms, the number of electric shocks to 2 times, and use a 1mm electric rotating cup to transform the above hyaluronan synthase mutant 01-20 into Corynebacterium glutamicum. Incubate at 46°C for 6min, culture at 30°C for 1.5h, apply to a BHI plate (containing chloramphenicol 15g / L), and culture at 30°C for 48h to obtain recombinant Corynebacterium glutamicum CG01-CG20. Pick the transformant into 5mL BHI liquid culture medium, culture at 30°C, 220rpm for 14h, and transfer 2% inoculum to 25mL fermentation medium. After 30°C, 220rpm culture for 4h, add a final concentration of 0.25mM IPTG to induce the expression of hyaluronan synthase mutants, and continue to culture for 48h. Collect the fermentation broth and determine the average molecular weight of hyaluronic acid in the fermentation broth sample. The hyaluronan synthase mutant sequence and the average molecular weight of the synthesized hyaluronic acid are shown in Table 2.

[0078] Table 2 Hyaluronan synthase mutant library (only the first 25 mutant amino acid sequences are indicated)

[0079]

[0080] (3) Construction of recombinant Corynebacterium glutamicum

[0081] Take out E. coli Nissle 1917 from the -80℃ refrigerator, streak it on the LB plate to revive, culture it at 37℃ for 24h, pick a single colony and inoculate it into 5mL LB medium, culture it at 220rpm, 37℃ for 12h, and use the cell genome extraction kit to extract genomic DNA. Using the genomic DNA of E. coli Nissle 1917 as a template, design primers ugd-F / ugd-R, glmS-F / glmS-R, glmM-F / glmM-R, and amplify the ugd, glmS, and glmM genes through the PCR amplification system and procedure. UP-F / UP-R and Down-F / Down-R using the Corynebacterium glutamicum genome as templates were selected, and the upstream and downstream homologous arms UP and Down of the genomic integration site were amplified through a PCR amplification system and program. Suitable restriction sites of plasmid pK18mobsacB were selected, and a linear vector plasmid pK18mobsacB was obtained through restriction digestion reaction. The fragments ugd, glmS, glmM, Up and Down were respectively connected to the vector pK18mobsacB through Tai Chi assembly reaction to obtain the recombinant plasmid pK18-ugd / glmS / glmM, which was transformed into Corynebacterium glutamicum ATCC 13032 by electroporation, spread on a BHI plate (containing 25 g / L kanamycin), and cultured at 30°C for 48 hours.

[0082] Pick the transformants and inoculate them into 5mL BHI, and culture them overnight at 30℃ and 220rpm. Dilute and spread the bacterial solution according to Table 4. If the number of colonies on the BHI plate containing 10% sucrose + 25g / L kanamycin is far less than that on the BHI plate containing 25g / L kanamycin, pick 100 transformants on the corresponding BHI plate (containing 10% sucrose) and inoculate them onto the BHI plate containing 25g / L kanamycin and the BHI plate without resistance at the same time. Pick the colonies that grow normally under the condition of no resistance but do not grow on the kanamycin plate, inoculate them into 5mL BHI medium, and culture them at 30℃ and 220rpm for 24h. Use the cell genome extraction kit to extract genomic DNA, perform genome sequencing, and obtain the recombinant strain CG-ugd-glmS-glmM. The hyaluronan synthase mutant in step (2) was transformed into CG-ugd-glmS-glmM by electroporation to obtain a recombinant strain producing hyaluronan of a specific molecular weight, namely CG(01-20)-ugd-glmS-glmM.

[0083] Table 3 Primers used for the construction of recombinant plasmid pK18-ugd / glmS / glmM

[0084]

[0085]

[0086] Table 4 Strain dilution coating method

[0087]

[0088] Example 2: Production of Specific Molecular Weight Hyaluronic Acid by 250 mL Shake Flask Fermentation of Recombinant Corynebacterium glutamicum

[0089] The recombinant Corynebacterium glutamicum CG(01-20)-ugd-glmS-glmM constructed in step (3) of Example 1 was inoculated into a shaking tube filled with 5 mL of BHI and cultured overnight at 30°C and 220 rpm. 600 =0.2 was transferred to a baffled conical flask containing 25 mL of fermentation medium. After culturing at 220 rpm and 30°C for 3.5 h, IPTG with a final concentration of 0.25 mM was added to induce gene expression. The fermentation cycle was 48 h. During the fermentation period of 20 h and 24 h, 2 M NaOH was added to adjust the pH of the fermentation liquid to 6.5-7. The fermentation liquid was collected and centrifuged at 10,000 rpm for 5 min. The supernatant was taken and repeatedly precipitated with alcohol twice. The hyaluronic acid content and molecular weight in the fermentation liquid were determined by the borax sulfuric acid-carbazole method and gel permeation chromatography. Figure 2It can be seen that the molecular weight of hyaluronic acid produced by CG(01-20)-ugd-glmS-glmM shake flask fermentation ranges from 300KDa to 4000 KDa, and the yield of CG01-ugd-glmS-glmM is the highest, which is 10g / L. The yield of hyaluronic acid decreases with the increase of hyaluronic acid molecular weight.

[0090] Example 3: Production of hyaluronic acid of different molecular weights by fermentation of recombinant Corynebacterium glutamicum in a 5L fermenter

[0091] The recombinant Corynebacterium glutamicum CG01-ugd-glmS-glmM and CG20-ugd-glmS-glmM constructed in step (3) of Example 1 were inoculated into 5 mL of BHI medium and cultured overnight at 220 rpm and 30°C. 600 =0.1 inoculum was transferred to a baffled conical flask of 25mL fermentation medium, and after culturing at 220rpm and 30℃ for 10h, it was inoculated into a 5L fermenter with an inoculum of 10%. The initial temperature was set to 30℃, the speed was 300r / min, and after fermentation for 3.5h, IPTG with a final concentration of 0.25mM was added to induce gene expression. During the fermentation process, 14% ammonia water was used to control the pH of the fermentation liquid at about 7, and glucose was added to maintain the glucose content in the fermenter at about 10g / L. The fermentation cycle was 72h. The results showed that the average molecular weight of hyaluronic acid produced by strain CG01-ugd-glmS-glmM was 300KDa, and the yield was 45g / L. The average molecular weight of hyaluronic acid produced by strain CG20-ugd-glmS-glmM was 3980KDa, and the yield of hyaluronic acid was 10g / L.

[0092] Comparative Example

[0093] The plasmid pXMJ19-SeHasA without mutation in step (1) of Example 1 was transformed into Corynebacterium glutamicum ATCC13032, and the recombinant strain was transformed according to the same strategy as step (3) of Example 1, and pXMJ19-SeHasA-ugd-glmS-glmM was fermented in a 5L fermenter to produce hyaluronic acid according to the method in Example 3. After 72h fermentation, the hyaluronic acid output was 5g / L, and the average molecular weight was 1800KDa. Therefore, it can be proved that the recombinant strain without mutation synthesized hyaluronic acid with a single molecular weight and low yield.

[0094] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A hyaluronan synthase mutant for synthesizing hyaluronic acids of different molecular weights, characterized in that: The hyaluronan synthase mutant is a hyaluronan synthase with an amino acid sequence as shown in SEQ ID NO.1, subjected to the following modifications: Asparagine at position 6 was mutated to lysine, valine at position 18 was mutated to serine, leucine at position 19 was mutated to isoleucine, valine at position 23 was mutated to leucine, and valine at position 25 was mutated to methionine.

2. A gene encoding the hyaluronan synthase mutant according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2.

4. A host cell expressing the hyaluronan synthase mutant according to claim 1.

5. The host cell according to claim 4, characterized in that The host cell is a bacterium or a fungus.

6. Use of the hyaluronan synthase mutant according to claim 1, the gene according to claim 2, the recombinant plasmid according to claim 3 or the host cell according to claim 4 or 5 in the preparation of hyaluronic acid.

7. The use according to claim 6, characterized in that: The application is to add the hyaluronan synthase mutant or the expression system containing the hyaluronan synthase mutant into the reaction system to generate hyaluronic acid.

8. A method for synthesizing hyaluronic acid of different molecular weights, characterized in that: The hyaluronan synthase mutant of claim 1 is introduced into a plasmid to form a recombinant plasmid, and the recombinant plasmid is introduced into a host cell to form a recombinant bacterium. The recombinant bacterium is fermented and cultured to synthesize hyaluronic acid. The molecular weight of the synthesized hyaluronic acid ranges from 300KDa to 4000KDa.

9. The method according to claim 8, characterized in that The recombinant plasmid also includes gene sequences of glutamine-fructose-6-phosphate aminotransferase, phosphoglucomutase and uridine diphosphate-glucose dehydrogenase.

10. The method according to claim 8, characterized in that The fermentation culture contains carbon source, nitrogen source, inorganic salt and metal ions.

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