A hyaluronate synthase mutant and use thereof

Abstract

The application provides a hyaluronan synthase mutant and application thereof, and relates to the technical field of biology.The hyaluronan synthase mutant is obtained by deleting N-terminal and C-terminal amino acids of the amino acid sequence such as SEQ ID NO.1 and performing point mutation on 280 and 540 sites and the like.The hyaluronan synthase mutant is introduced into a host bacterium and subjected to fermentation culture, and the hyaluronan yield is greatly improved compared with the unmodified hyaluronan synthase.In addition, under the premise of strengthening the expression of UDP-glucose dehydrogenase, the hyaluronan synthase and hyaluronan degradation enzyme are connected through a GS linker and subjected to heterologous expression, and the yield in a 5L fermenter is about 15g / L, and the generated hyaluronan is hyaluronan tetrasaccharide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a hyaluronan synthase mutant and its application. BACKGROUND

[0002] Hyaluronic acid (HA) is a high molecular weight polysaccharide widely exists in human skin, joints, eyes and connective tissue. Hyaluronic acid has excellent moisturizing properties and biocompatibility, can form a protective film between cells, maintain moisture, promote normal metabolism and repair of cells, its main functions include moisturizing, lubricating and anti-inflammatory effect. In the medical and cosmetic fields, hyaluronic acid is widely used. In medicine, hyaluronic acid is used for the treatment of arthritis, by injecting hyaluronic acid to supplement joint fluid, reduce pain and improve joint function; in ophthalmic surgery, hyaluronic acid can be used as a surgical auxiliary material to protect the cornea and maintain intraocular pressure. In the cosmetic field, hyaluronic acid is widely used in skin care products and injection fillers, which can deeply hydrate, improve skin elasticity and reduce wrinkles, and also shows good effect in wound repair and burn treatment. In summary, hyaluronic acid has important application value in many fields due to its unique physical and biological properties.

[0003] The function of hyaluronic acid is closely related to its molecular weight, and different molecular weight hyaluronic acid has different functions and applications. High molecular weight hyaluronic acid has excellent lubricating, moisturizing ability and gel forming ability, medium molecular weight hyaluronic acid has better permeability and moisturizing ability, and low molecular weight hyaluronic acid has better permeability and biological activity. However, the most widely used hyaluronic acid on the market is still high molecular weight and medium molecular weight, mainly due to the difficulty in preparing low molecular weight hyaluronic acid, resulting in high cost and poor molecular weight uniformity. Therefore, developing a low molecular weight hyaluronic acid biosynthesis technology with good uniformity is crucial to promote the wide application of low molecular weight hyaluronic acid and its derivative materials in more fields, and there is a lack of a biosynthesis method for high-yield low molecular weight hyaluronic acid in the prior art. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the problem that there is a lack of a biosynthesis method for high-yield low molecular weight hyaluronic acid in the prior art.

[0005] To solve the above technical problems, the present application provides a hyaluronan synthase mutant and its application. The hyaluronan synthase mutant of the present application is a hyaluronan synthase with the following modifications to the amino acid sequence shown in SEQ ID NO. 1: deletion of N-terminal and C-terminal amino acids, mutation of aspartic acid at position 280 to serine, and mutation of cysteine at position 540 to serine. The hyaluronan synthase mutant of the present application is heterologously expressed and subjected to fermentation culture, and the yield of hyaluronic acid is greatly increased compared to the unmutated hyaluronan synthase. In addition, under the premise of strengthening the expression of UDP-glucose dehydrogenase, the hyaluronan synthase and hyaluronan-degrading enzyme of the present application are heterologously expressed, and the yield in a 5L fermenter is about 15g / L, and the generated hyaluronic acid is tetrasaccharide hyaluronic acid with a molecular weight of about 1100Da. The present application provides a method for biosynthesis of high-yield low-molecular-weight hyaluronic acid, which is convenient for industrial production.

[0006] The first object of the present application is to provide a hyaluronan synthase mutant, which is one or more of the following modifications to the hyaluronan synthase with the amino acid sequence shown in SEQ ID NO. 1:

[0007] (1) deletion of N-terminal 2-30 amino acids;

[0008] (2) deletion of N-terminal 2-47 amino acids;

[0009] (3) deletion of N-terminal 2-64 amino acids;

[0010] (4) deletion of N-terminal 2-107 amino acids;

[0011] (5) deletion of C-terminal 920-972 amino acids;

[0012] (6) deletion of C-terminal 850-972 amino acids;

[0013] (7) deletion of C-terminal 790-972 amino acids;

[0014] (8) mutation of aspartic acid at position 280 to serine (D280S);

[0015] (9) mutation of cysteine at position 540 to serine (C540S).

[0016] Further, the sequence of SEQ ID NO. 1 is shown as follows:

[0017] MNTLSQAIKAYNSNDYQLALKLFEKSAEIYGRKIVEFQITKCKEKLSAHPSVNSAHLSVNKEEKVNVCDSPLDIATQLLLSNVKKLVLSDSEKNTLKNKWKLLTEKKSENAEVRAVALVPKDFPKDLVLAPLPDHVNDFTWYKKRKKRLGIKPEHQHVGLSIIVPTFNRPAILSITLACLVNQKTHYPFEVIVTDDGSQEDLSPIIRQYENKLDIRYVRQKDNGLQASAARNMGLRLAKYDFIGLLDCDMAPNPLWVHSYVAELLEDDDLTIIGPRKYIDTQHIDPKDFLNNASLLESLPEVKTNNSVAAKGEGTVSLDWRLEQFEKTENLRLSDSPFRFFAAGNVAFAKKWLNKSGFFDEEFNHWGGEDVEFGYRLFRYGSFFKTIDGIMAYHQEPPGKENETDREAGKNITLDIMREKVPYIYRKLLPIEDSHINRVPLVSIYIPAYNCANYIQRCVDSALNQTVVDLEVCICNDGSTDNTLEVINKLYGNNPRVRIMSKPNGGIASASNAAVSFAKGYYIGQLDSDDYLEPDAVELCLKEFLKDKTLACVYTTNRNVNPDGSLIANGYNWPEFSREKLTTAMIAHHFRMFTIRAWHLTDGFNEKIENAVDYDMFLKLSEVGKFKHLNKICYNRVLHGDNTSIKKLGIQKKNHFVVVNQSLNRQGITYYNYDEFDDLDESRKYIFNKTAEYQEEIDILKDIKIIQNKDAKIAVSIFYPNTLNGLVKKLNNIIEYNKNIFVIVLHVDKNHLTPDIKKEILAFYHKHQVNILLNNDISYYTSNRLIKTEAHLSNINKLSQLNLNCEYIIFDNHDSLFVKNDSYAYMKKYDVGMNFSALTHDWIEKINAHPPFKKLIKTYFNDNDLKSMNVKGASQGMFMTYALAHELLTIIKEVITSCQSIDSVPEYNTEDIWFQFALLILEKKTGHVFNKTSTLTYMPWERKLQWTNEQIESAKRGENIPVNKFIINSITL.

[0018] A second object of the present application is to provide a gene encoding the hyaluronan synthase mutant.

[0019] A third object of the present application is to provide a recombinant plasmid carrying the gene.

[0020] A fourth object of the present application is to provide a recombinant cell expressing the hyaluronan synthase mutant.

[0021] Further, the recombinant cell is a bacterium or a fungus.

[0022] A fifth object of the present application is to provide a use of the hyaluronan synthase mutant, the gene, the recombinant plasmid or the recombinant cell in the synthesis of hyaluronic acid.

[0023] Further, the use is to add the hyaluronan synthase mutant or an expression system containing the hyaluronan synthase mutant into a reaction system to generate hyaluronic acid.

[0024] A sixth object of the present application is to provide a method for synthesizing hyaluronic acid, which comprises introducing the hyaluronan synthase mutant into a plasmid to form a recombinant plasmid, introducing the recombinant plasmid into a host cell to form a recombinant bacterium, and culturing the recombinant bacterium to synthesize hyaluronic acid.

[0025] Further, the recombinant plasmid further comprises the gene sequences of a hyaluronan-degrading enzyme and a UDP-glucose dehydrogenase.

[0026] Further, the amino acid sequence of the hyaluronan-degrading enzyme is shown in SEQ ID NO. 2, and the sequence of SEQ ID NO. 2 is shown as follows.

[0027] MADRVIQRNDTAARWQSINPVLAQGELGIVSDGAKGYKIGDGVTAWNSLEYPANPASVVQELGNSETAVISQNAITNNFANTNSFLKNSIFITEIERTRLIIDGNSSNIELTSGYIKTDGNLGDIVNLTVNQSLNTRYAIIPCKEGDYFVISGNGGDLSRLFCFIDSSNRIILNAESRLVLSTYIKVPYNTSKLIVNFEQASDYIKKFNINNDSVRKYWVLNNSLNIYTNTFTPSPLYIQYDNNYIQSINPLDTTPITPSKNGFLVFNKKDSKYIFRLNSVDIDVINDIIVFVVRNNIFYNGDSLSYCLSYHVQNLVKYKTRNTYMLYSPNNDTSYTNNSVTTLGAWYLDNGTIHNHFILENGIVTPKTFVFTINSYLVYNKANYTARIVDSYKDIGDDDVIWLYYQDGKFVDGLFYSYYMASKMNSTSIESENSVETTIKKRAYQFATIPWVALKPIASTSSSTGIEIGNHVGLPYTSCMEVDKFVGYEVSLRTFMTCANNPYSLLYTEDLSRNISGYGFTYHNTGRGTIGGYMGIVCNIFGMNAISYKIPYDTGNWKFLRKKGYFKELQYQEAKYLNIGDIIVEPGHCNVITNIEKNDSGYSKIIYWGESVMDFPKINRYSEEQANNRIAERGGIIYRRDNLYKDSYYERSPFVAVEDEILDSSYVYNDDICTFAGDYAVFRENQKIVINYNLKNTNSDWNQIELYKNDELIGTYSLVDSHNYDLSSLNLKYGNYKARLKYNSTFSDYTYFQILDTEVSYTLNDSTIKIVFNSHNGEPLFIRLCKVDGGPICIVELTEDDKRKGFIEFDYNYYGNGQGYSIVNGGTYLKVYFESEYGRVTNEPILTTI.

[0028] Further, the amino acid sequence of the UDP-glucose dehydrogenase is shown as SEQ ID NO. 3, and the sequence of SEQ ID NO. 3 is shown as follows.

[0029] MRMTVIGTGYLGATHAACMAELSHEVLGVDVDEAKIASLKDSKVPFFEPGLPEVLERNLENGRLNFTTDYAEAAAFAQVHFLGVGTPQQKGTYAADLTYVRQVVEDLVPLLEGEHIIFGKSTVPVGTAEQLQELADSLVKPGSHVEIAWNPEFLREGYAVKDTITPDRIVVGVREGATAEAIAREVYATAIAADTPFLVTDLATAELVKVSANAFLATKISFINAVAEICEQTGADVVALADAIGHDDRIGRKFLGAGLGFGGGCLPKDIRAFMARAGELGADQALTFLREVDSINMRRRDRVVQLAKEMCGGSLLGKRVTVLGAAFKPNSDDVRDSPALSVAGSLSLQGAAVSVYDPEAMDNARRVFPTLSYASSTKEALIDAHLVVLATEWQEFRDLDPEVAGGVVEKRAIIDGRNVLDVAKWKAAGWEMEALGRNLGKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDVPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.

[0030] Further, the fermentation culture contains carbon source, nitrogen source, inorganic salt and metal ion.

[0031] Advantages of the present application:

[0032] The hyaluronan synthase mutant of the present application is a hyaluronan synthase with N-terminal and / or C-terminal amino acid deletion and modification of aspartic acid at position 280 to serine, cysteine at position 540 to serine, etc. The hyaluronan synthase mutant of the present application is introduced into a recombinant bacterium for fermentation culture, and the hyaluronan yield is greatly improved compared with the unmutated hyaluronan synthase. In addition, under the premise of strengthening the expression of UDP-glucose dehydrogenase, the hyaluronan synthase and hyaluronan degrading enzyme of the present application are expressed heterogeneously, and the yield in a 5L fermenter is about 15g / L, and the generated hyaluronan is hyaluronan tetrasaccharide with a molecular weight of 1100Da. The present application provides a convenient and feasible biosynthesis method for high-yield low-molecular-weight hyaluronan, which is convenient for industrial large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which

[0034] Figure 1 is a secondary structure diagram of hyaluronan synthase;

[0035] Figure 2 is a diagram showing the effect of N and C terminal truncation of hyaluronan synthase on hyaluronan yield; wherein, HasA represents untruncated hyaluronan synthase, HasA-N1 represents hyaluronan synthase with deletion of N-terminal 2-30 amino acids, HasA-N2 represents hyaluronan synthase with deletion of N-terminal 2-47 amino acids, HasA-N3 represents hyaluronan synthase with deletion of N-terminal 2-64 amino acids, HasA-N4 represents hyaluronan synthase with deletion of N-terminal 2-107 amino acids, HasA-C1 represents hyaluronan synthase with deletion of C-terminal 920-972 amino acids, HasA-C2 represents hyaluronan synthase with deletion of C-terminal 850-972 amino acids, HasA-C3 represents hyaluronan synthase with deletion of C-terminal 790-972 amino acids, HasA-N4C1 represents hyaluronan synthase with deletion of N-terminal 2-107 amino acids and deletion of C-terminal 920-972 amino acids, HasA-N4C2 represents hyaluronan synthase with deletion of N-terminal 2-107 amino acids and deletion of C-terminal 850-972 amino acids, and HasA-N4C3 represents hyaluronan synthase with deletion of N-terminal 2-107 amino acids and deletion of C-terminal 790-972 amino acids;

[0036] Figure 3Figure 1 is a diagram showing the effect of hyaluronic acid synthase mutation on hyaluronic acid production; wherein, HasA-N4C3-A represents a hyaluronic acid synthase with N-terminal 2-107 amino acid deletion and C-terminal 790-972 amino acid deletion and D at position 280 mutated to S, HasA-N4C3-B represents a hyaluronic acid synthase with N-terminal 2-107 amino acid deletion and C-terminal 790-972 amino acid deletion and C at position 540 mutated to S, and HasA-N4C3-C represents a hyaluronic acid synthase with N-terminal 2-107 amino acid deletion and C-terminal 790-972 amino acid deletion and D at position 280 mutated to S and C at position 540 mutated to S;

[0037] Figure 4 Figure 2 is a diagram showing the production of batch fed fermentation in a 5 L fermenter;

[0038] Figure 5 Figure 3 is a mass spectrum of hyaluronic acid. DETAILED DESCRIPTION

[0039] The present application will be further described with reference to the following figures and specific examples, so that those skilled in the art can better understand the present application and implement it, but the examples are not intended to limit the present application.

[0040] Strain: Streptococcus thermophilus CNRZ1066 (ATCC 53513) Streptococcus thermophilus CNRZ1066);

[0041] Plasmid: pNZ8148

[0042] MB medium: Tryptone: 10 g / L, Beef extract: 10 g / L, Yeast extract: 5 g / L, Glucose: 5 g / L

[0043] Fermentation medium: Tryptone: 10 g / L, Beef extract: 10 g / L, Yeast extract: 5 g / L, Glucose: 20 g / L, K2HPO4: 2.0 g / L, MgSO4·7H2O: 0.2 g / L, MnSO4·H2O: 0.05 g / L, FeSO4·7H2O: 0.05 g / L, CaCO3: 20 g / L, NaCl: 5 g / L, Trisodium citrate: 2.0 g / L

[0044] Preparation of Streptococcus thermophilus competence: Strain activation: Streptococcus thermophilus strain stored in -80°C refrigerator was streaked onto MB solid medium and cultured at 37°C for 24-48 hours until obvious colonies appeared. Seed culture: A single colony was picked from the plate and inoculated into 5 mL MB liquid medium, which was cultured at 37°C with 180 rpm shaking for 8-12 hours until OD 600To 0.4-0.6. Expansion culture: take 1 mL seed culture solution, add to 100 mL MB liquid medium, 37°C, 180 rpm shaking culture to OD 600 To 0.4-0.6. Centrifugal collection of bacterial cells: transfer the culture solution to a centrifuge tube, centrifuge at 4000 rpm for 10 minutes at 4°C, discard the supernatant. Washing: resuspend the bacterial cells with pre-cooled 10% glycerol solution, so that the final volume is about 1 / 10 of the original culture solution, gently mix and then centrifuge at 4000 rpm for 10 minutes at 4°C, discard the supernatant. Resuspension and aliquoting: resuspend the bacterial cells again with pre-cooled 10% glycerol solution, so that the final volume is about 1 / 100 of the original culture solution, gently mix and then aliquot into sterile 1.5 mL centrifuge tubes, about 100 μL per tube.

[0045] Purification and content determination of hyaluronic acid: To quantify the hyaluronic acid, the cells were removed by centrifugation at 16000 x g for 15 minutes. The supernatant was combined with four volumes of ice-cold ethanol and the sample was precipitated at -30°C for 8 hours. The insoluble fraction was then removed by centrifugation at 16000 x g for 10 minutes and the supernatant was freeze-dried. The fraction was dissolved in deionized water and the water-insoluble fraction was removed by centrifugation. To remove as many impurities as possible, these steps were repeated three more times. The crude hyaluronic acid sample was further purified by anion exchange chromatography using an ÄKTA avant 25 preparative chromatography system equipped with a HiPrep Q HP 16 / 10 (GE Healthcare, USA) column. After appropriate dilution, the NSGAG content was determined by carbazole assay using glucuronic acid as an external standard.

[0046] Mass spectrometric determination of hyaluronic acid tetrasaccharide: The sample was mixed with 9 volumes of methanol, then the supernatant fraction was collected by centrifugation at 16000 x g for 20 minutes and filtered through a 0.22 μm membrane (ALWSCI, Shaoxing, China). The components were analyzed by LC-IT-TOF-MS (Kyoto Instruments, Japan) using a 5 μm frit set VP-ODS chromatographic column (4.6 x 250 mm) at 40°C. The elution buffer A was ultrapure water and the elution buffer B was a methanol solution, both containing 8 mM ammonium acetate. The LC gradient elution was as follows: 0-5 minutes, 5% B; 5 to 15 minutes, 5% to 60% B; 15 to 20 minutes, 60% B, at a flow rate of 0.2 mL min -1 . Ionization of the liquid chromatography flow was accomplished by an electrospray source (2 kV and 230°C), with nitrogen used as drying and nebulizing gas. Negative ion spectra were generated by scanning the range 100 m / z to 900 m / z.

[0047] The C-terminal 920-972 amino acid deletion refers to the deletion of the 920-972 amino acids counted from the first amino acid at the N-terminal, and the 920-972 amino acids are located at the C-terminal; the C-terminal 850-972 amino acid deletion refers to the deletion of the 850-972 amino acids counted from the first amino acid at the N-terminal, and the 850-972 amino acids are located at the C-terminal; the C-terminal 790-972 amino acid deletion refers to the deletion of the 790-972 amino acids counted from the first amino acid at the N-terminal, and the 790-972 amino acids are located at the C-terminal. The mutation of the 280th and 540th amino acids is counted from the N-terminal.

[0048] Example 1: Construction of hyaluronan synthase mutants

[0049] (1) Truncation optimization of N-terminal and C-terminal sequences of hyaluronan synthase

[0050] According to the genomic sequence of the hyaluronan synthase of Pasteurella multocida, the gene sequence of the hyaluronan synthase pmHasA was synthesized by GENEWIZ (Suzhou), and the amino acid of pmHasA is shown as SEQ ID NO. 1. According to the secondary structure of the hyaluronan synthase (as shown in FIG. 1), the N-terminal and C-terminal sequences of the hyaluronan synthase were truncated and optimized. Figure 1pmHasA and truncated pmHasA gene sequences were connected to plasmid pNZ8148 by T5 exonuclease-dependent assembly (TEDA) method, respectively, to construct recombinant expression plasmids pNZ8148-pmHasA, pNZ8148-pmHasA-N1, pNZ8148-pmHasA-N2, pNZ8148-pmHasA-N3, pNZ8148-pmHasA-N4, pNZ8148-pmHasA-C1, pNZ8148-pmHasA-C2, pNZ8148-pmHasA-C3, pNZ8148-pmHasA-N4C1, pNZ8148-pmHasA-N4C2, pNZ8148-pmHasA-N4C3, which were transformed into S. thermophilus to obtain recombinant strains HasA, HasA-N1, HasA-N2, HasA-N3, HasA-N4, HasA-C1, HasA-C2, HasA-C3, HasA-N4C1, HasA-N4C2, HasA-N4C3. The recombinant strains were cultured in a flask, and the results of hyaluronic acid production are shown in Table 2. Figure 2

[0051] Table 1 Primers for truncation of N-terminal and C-terminal of hyaluronan synthase pmHasA

[0052] .

[0053] (2) Construction of hyaluronan synthase mutants at positions 280 and 540

[0054] pmHasA-N4C3-280-F and pNZ8148-pmHasA-N4C3-280-R, pNZ8148-pmHasA-N4C3-540-F and pNZ8148-pmHasA-N4C3-540-R were designed for PCR amplification, and recombinant plasmid pNZ8148-pmHasA​D280S N4C3, pNZ8148-pmHasA C540S N4C3, pNZ8148-pmHasA D280S，C540S N4C3, pNZ8148-pmHasA Figure 3

[0055] Table 2 Primers used for construction of hyaluronan synthase mutant pXMJ-HasA

[0056] .

[0057] Example 2: Construction of hyaluronan synthase and hyaluronan-degrading enzyme fusion.

[0058] According to Bacteroides phage the genomic sequence of crAss001 hyaluronan-degrading enzyme, the gene hylase was synthesized by GENEWIZ (Suzhou), and the amino acid of hylase is shown in SEQ ID NO. 2. The synthesized hylase gene sequence was used as a template to design primers pmHasA-GS-hylase-F and pmHasA-GS-hylase-R for PCR amplification, and then the gene hylase was connected to the plasmid pNZ8148-pmHasA D280S, C540S N4C3, and the recombinant expression plasmid pNZ8148-pmHasA was constructed D280S，C540S N4C3-GS-hylase.

[0059] Table 3 Primers used for construction of hyaluronan synthase and hyaluronan-degrading enzyme fusion.

[0060] .

[0061] Example 3: Strengthening of hyaluronic acid synthesis pathway and fed-batch fermentation

[0062] (1) Strengthening of UDP-glucuronate synthesis

[0063] Primers ugd-F and ugd-R were designed to amplify the UDP-glucose dehydrogenase gene ugd from the Corynebacterium glutamicum genome as a template, and the gene ugd was connected to the plasmid pNZ8148-pmHasA D280S，C540S N4C3-GS-hylase, and the recombinant expression plasmid pNZ8148-pmHasA was constructed D280S，C540S ​-N4C3-GS-hylase-ugd. The recombinant plasmid was transformed into S. thermophilus by electroporation to obtain the recombinant strain S. thermophilus HasA-N4C3-C-ugd. The recombinant strain was cultured in a flask, and the results of hyaluronic acid production are shown in Table 4. Figure 3

[0064] Table 4 Primers for UDP-glucuronic acid synthesis

[0065] .

[0066] (2) Batch fermentation of recombinant S. thermophilus in a 5 L fermenter

[0067] Fermentation in a 5 L fermenter: The recombinant strain S. thermophilus HasA-N4C3-C-ugd constructed in Example 3 was inoculated into 5 mL of MB medium, and cultured overnight at 220 rpm and 45°C. The seed liquid was inoculated into a baffle triangular flask containing 25 mL of fermentation medium at an initial OD 600 =0.1, and cultured at 220 rpm and 45°C for 10 h. Then, the culture was inoculated into a 5 L fermenter at a 10% inoculation amount. The initial temperature was set to 45°C, and the rotation speed was set to 3000 r / min. After 4 h of fermentation, 0.2 mM IPTG was added to induce gene expression. The pH of the fermentation broth was controlled at about 7 by adding 14% ammonia water, and the glucose was fed to maintain a concentration of about 10 g / L in the fermenter. The fermentation was continued for 24 h. Figure 4 It can be seen that the final hyaluronic acid production of the recombinant strain S. thermophilus in the 5 L fermenter was 15 g / L. The structure of the generated hyaluronic acid was identified by mass spectrometry, and the results are shown in Table 4. Figure 5

[0068] Obviously, the above examples are merely examples for the purpose of clarity, and are not limiting of the embodiments. Other different forms of changes or variations can be made by those of ordinary skill in the art based on the above description. It is not necessary or possible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.​​

Claims

1. A hyaluronan synthase mutant, characterized in that, The hyaluronan synthase mutant is a hyaluronan synthase with the amino acid sequence shown in SEQ ID NO. 1, in which the N-terminal 2-107 amino acids are deleted, the C-terminal 790-972 amino acids are deleted, the aspartic acid at position 280 is mutated to serine, and the cysteine at position 540 is mutated to serine.

2. A gene encoding the hyaluronan synthase mutant of claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. A recombinant cell expressing the hyaluronan synthase mutant of claim 1.

5. The recombinant cell of claim 4, wherein, The recombinant cell is a bacterium or a fungus.

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

7. Use according to claim 6, characterized in that, The use is the addition of the hyaluronan synthase mutant or an expression system containing the hyaluronan synthase mutant to a reaction system to produce hyaluronic acid.

8. A method of synthesizing hyaluronic acid, characterized by, The hyaluronan synthase mutant of claim 1 is introduced into a plasmid to form a recombinant plasmid, the recombinant plasmid is introduced into a host cell to form a recombinant bacterium, and the recombinant bacterium is subjected to fermentation culture to synthesize hyaluronic acid, and the recombinant plasmid further comprises the gene sequence of a hyaluronan-degrading enzyme with the amino acid sequence shown in SEQ ID NO. 2 and a uridine diphosphate-glucose dehydrogenase with the amino acid sequence shown in SEQ ID NO.

3.

9. The method of claim 8, wherein, The fermentation culture contains a carbon source, a nitrogen source, inorganic salts, and metal ions.

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