Hyaluronic acid synthase mutant and application thereof

Abstract

The invention provides a hyaluronic acid synthase mutant and application thereof, and relates to the technical field of biology. The hyaluronic acid synthase mutant is obtained by carrying out N-terminal and C-terminal amino acid deletion on hyaluronic acid synthase of which the amino acid sequence is as shown in SEQ ID NO.1 and carrying out point mutation and other transformation on 280 and 540 sites. The hyaluronic acid synthase mutant disclosed by the invention is introduced into host bacteria and is subjected to fermentation culture, and compared with non-modified hyaluronic acid synthase, the hyaluronic acid yield of the hyaluronic acid synthase mutant disclosed by the invention is greatly improved. In addition, on the premise of enhancing the expression of UDP-glucose dehydrogenase, the hyaluronic acid synthase and the hyaluronic acid degrading enzyme are connected through a GS linker and are subjected to heterologous expression, the yield of the hyaluronic acid synthase and the hyaluronic acid degrading enzyme in a 5L fermentation tank is about 15g / L, and the generated hyaluronic acid is hyaluronate tetrasaccharide.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a hyaluronan synthase mutant and application thereof. Background Art

[0002] Hyaluronic acid (HA) is a high molecular weight polysaccharide that is widely found in human skin, joints, eyes and connective tissues. Hyaluronic acid has excellent moisturizing properties and biocompatibility. It can form a protective film between cells, retain moisture, and promote normal cell metabolism and repair. Its main functions include moisturizing, lubricating and anti-inflammatory effects. In the fields of medicine and beauty, hyaluronic acid is widely used. In medicine, hyaluronic acid is used to treat arthritis. By injecting hyaluronic acid to replenish joint fluid, relieve 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 field of beauty, hyaluronic acid is widely used in skin care products and injectable fillers. It can deeply hydrate, improve skin elasticity and reduce wrinkles. It also shows good results in trauma repair and burn treatment. In short, 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 hyaluronic acids of different molecular weights have different functions and applications. High molecular weight hyaluronic acid has excellent lubrication, moisturizing ability and gel-forming ability, medium molecular weight hyaluronic acid has good 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, the development of low molecular weight hyaluronic acid biosynthesis technology with good uniformity is crucial to promote the widespread application of low molecular weight hyaluronic acid and its derivatives in more fields, and the prior art lacks a biosynthesis method that can produce high yields of low molecular weight hyaluronic acid. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem that the prior art lacks a high-yield biosynthetic method of low-molecular-weight hyaluronic acid.

[0005] To solve the above technical problems, the present invention provides a hyaluronan synthase mutant and its application. The hyaluronan synthase mutant of the present invention is to carry out the deletion of N-terminal and C-terminal amino acids to the hyaluronan synthase of amino acid sequence as shown in SEQ ID NO.1, and mutate the selenocysteine ​​at position 280 to serine, and mutate the cysteine ​​at position 540 to serine. The hyaluronan synthase mutant of the present invention is heterologously expressed and fermented and cultured, and its hyaluronan yield is greatly improved compared to the hyaluronan synthase of non-mutated. In addition, under the premise of strengthening the expression of UDP-glucose dehydrogenase, the hyaluronan synthase and hyaluronan degrading enzyme of the present invention are heterologously expressed, and the yield in 5L fermentation tank is about 15g / L, and the hyaluronic acid generated is hyaluronic acid tetrasaccharide, and the molecular weight is about 1100Da. The present invention provides a biosynthetic method of high-yield low-molecular-weight hyaluronic acid, which is convenient for industrial production.

[0006] The first object of the present invention is to provide a hyaluronan synthase mutant, wherein the hyaluronan synthase mutant 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) N-terminal amino acid 2-30 is missing;

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

[0009] (3) N-terminal amino acid deletion at positions 2-64;

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

[0011] (5) C-terminal amino acid deletion at position 920-972;

[0012] (6) C-terminal amino acid deletion at positions 850-972;

[0013] (7) C-terminal amino acid deletion at positions 790-972;

[0014] (8) selenocysteine ​​at position 280 was mutated to serine (U280S);

[0015] (9) Cysteine ​​at position 540 was mutated to serine (C540S).

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

[0017] MNTLSQAIKAYNSNDYQLALKLFEKSAEIYGRKIVEFQITKCKEKLSAHPSVNSAHLSVNKEEKVNVCDSPLDIATQLLLSNVKKLVLSDSEKNTLKNKWKLLTEKKSENAEVRAVALVPKDFPKDLVLAPLPDHVNDFTWYKKRKKRLGIKPEHQHVGLSIIVPTFNRPAILSITLACLVNQKTHYPFEVIVTDDGSQEDLSPIIRQYENKLDIRYVRQKDNGLQASAARNMGLRLAKYDFIGLLDCDMAPNPLWVHSYVAELLEDDLTIIGPRKYIDTQHIDPKDFLNNASLLESLPEVKTNNSVAAKGEGTVSLDWRLEQFEKTENLRLSDPFRFFAAGNVAFAKKWLNKSGFFDEEFNHWGGEDVEFGYRLFRYGSFFKTIDGIMAYHQEPPGKENETDREAGKNITLDIMREKVPYIYRKLLPIEDSHINRVPLVSIYIPAYNCANYIQRCVDSALNQTVVDLEVCICNDGSTDNTLEV INKLYGNNPRVRIMSKPNGGIASASNAAVSFAKGYYIGQLDSDDYLEPDAVELCLKEFLKDKTLACVYTTNRNVNPDGSLIANGYNWPEFSREKLTTAMIAHHFRMFTIRAVHLTDGFNEKIENAVDYDMFLKLSEVGKFKHLNKICYNRVLHGDNTSIKKLGIQKKNHFVVVNQSLNRQGITYYNYDEFDDLDESRKYIFNKTAEYQEEIDILKDIKIIQNKDAKIAVSIFYYPNTLNGLVKKLNNIIEYNKNIFVIVLHVDKNHLTPDIKKEILAFYHKHQVNILLNNDISYYTSNRLIKTEAHLSNINKLSQLNLNCEYIIFDNHDSLFVKNDSYAYMKKYDVGMNFSALTHDWIEKINAHPPFKKLIKTYFNDNDLKSNMNVKGASQGMFMTYALAHELLTIIKEVITSCQSIDSVPEYNTEDIWFQFALLILEKKTGHVFNKTSTLTYMPWERKLQWTNEQIESAKRGENIPVNKFIINSITL

[0018] The second object of the present invention is to provide a gene encoding the above hyaluronan synthase mutant.

[0019] The third object of the present invention is to provide a recombinant plasmid carrying the above gene.

[0020] The fourth object of the present invention is to provide a recombinant cell expressing the above hyaluronan synthase mutant.

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

[0022] The fifth object of the present invention is to provide a use of the above-mentioned hyaluronan synthase mutant, the above-mentioned gene, the above-mentioned recombinant plasmid or the above-mentioned recombinant cell in synthesizing hyaluronic acid.

[0023] 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.

[0024] The sixth object of the present invention is to provide a method for synthesizing hyaluronic acid, wherein the above-mentioned hyaluronic acid synthase mutant 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 fermented and cultured to synthesize hyaluronic acid.

[0025] Furthermore, the recombinant plasmid also includes gene sequences of hyaluronan degrading enzyme and UDP-glucuronic acid dehydrogenase.

[0026] Furthermore, 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 as follows:

[0027] MADRVIQRNDTAARWQSINPVLAQGELGIVSDGAKGYKIGDGVTAWNSLEYPANPASVVQELGNSETAVISQNAITNNFANTNSFLKNSIFITEIERTRLIIDGNSSNIELTSGYIKTDGNLGDIVNLTVNQSLNTRYAIIPCKEGDYFVISGNGGDLSRLFCFIDSSNRIILNAESRLVLSTYIKVPYNTSKLIVNFEQASDYIKKFNINNDSVRKYWVLNNSLNIYTNTFTPSPLYIQYDNNYIQSINPLDTTPITPSKNGFLVFNKKDSKYIFRLNSVDIDVINDIIVFVVRNNIFYNGDSLSYCLSYHVQNLVKYKTRNTYMLYSPNNDTSYTNNSVTTLGAWYLDNGTIHNHFILENGIVTPKTFVFTINSYLVYNKANYTARIVDSYKDIGDDDVIWLYYQDGKFVDGLFYSYYMASKMNSTSIESENSVETTIKKRAYQFATIPWVALKPIASTSSSTGIEIGNHVGLPYTSCMEVDKFVGYEVSLRTFMTCANNPYSLLYTEDLSRNISGYGFTYHNTGRGTIGGYMGIVCNIFGMNAISYKIPYDTGNWKFLRKKGYFKELQYQEAKYLNIGDIIVEPGHCNVITNIEKNDSGYSKIIYWGESVMDFPKINRYSEEQANNRIAERGGIIYRRDNLYKDSYYERSPFVAVEDEILDSSYVYNDDICTFAGDYAVFRENQKIVINYNLKNTNSDWNQIELYKNDELIGTYSLVDSHNYDLSSLNLKYGNYKARLKYNSTFSDYTYFQILDTEVSYTLNDSTIKIVFNSHNGEPLFIRLCKVDGGPICIVELTEDDKRKGFIEFDYNYYGNGQGYSIVNGGTYLKVYFESEYGRVTNEPILTTI

[0028] Furthermore, the amino acid sequence of the UDP-glucuronosyltransferase is shown in SEQ ID NO.3, and the sequence of SEQ ID NO.3 is as follows:

[0029] MRMTVIGTGYLGATHAACMAELSHEVLGVDVDEAKIASLKDSKVPFFEPGLPEVLERNLENGLNFTTDYAEAAAFAQVHFLGVGTPQQKGTYAADLTYVRQVVEDLVPLLEGEHIIFGKSTVPVGTAEQLQELADSLVKPGSHVEIAWNPEFLREGYAVKDTITPDRI VVGVREGATAEAIAREVYATAIAADTPFLVTDLATAELVKVSANAFLATKISFINAVAEICEQTGADVVALADAIGHDDRIGRKFLGAGLGFGGGCLPKDIRAFMARAGELGADQALTFLREVDSINMRRRDRVVQLAKEMCGGSLLGKRVTVLGAAFKPNSDDVRDSP ALSVAGSLSLQGAAVSVYDPEAMDNARRVFPTLSYASSTKEALIDAHLVVLATEWQEFRDLDPEVAGGVVEKRAIIDGRNVLDVAKWKAAGWEMEALGRNLGKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFAYGLQ CFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDVPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0030] Furthermore, the fermentation culture contains a carbon source, a nitrogen source, an inorganic salt and metal ions.

[0031] Beneficial effects of the present invention:

[0032] The hyaluronan synthase mutant of the present invention is to carry out the deletion of N-terminal and / or C-terminal amino acid to the hyaluronan synthase of amino acid sequence as shown in SEQ ID NO.1, and selenocysteine ​​at position 280 is mutated to serine, and cysteine ​​at position 540 is mutated to serine and other transformations. The hyaluronan synthase mutant of the present invention is introduced into recombinant bacteria for fermentation and culture, and its hyaluronan output is greatly improved compared to the hyaluronan synthase of unmutated. In addition, under the premise of strengthening the expression of UDP-glucose dehydrogenase, the hyaluronan synthase of the present invention and hyaluronan degrading enzyme are heterologously expressed, and the output of the hyaluronan synthase and hyaluronan degrading enzyme in 5L fermentation tank is about 15g / L, and the hyaluronic acid generated is hyaluronic acid tetrasaccharide, and the molecular weight is 1100Da. The present invention provides a convenient and feasible biosynthesis method for high-yield low molecular weight hyaluronic acid, which is convenient for industrial expansion production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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

[0034] Figure 1 yes Figure 1 This is the secondary structure diagram of hyaluronan synthase;

[0035] Figure 2 : is a graph showing the effect of truncation of the N and C termini of hyaluronan synthase on hyaluronan production; wherein, HasA represents hyaluronan synthase without truncation, HasA-N1 represents hyaluronan synthase with amino acids deleted at positions 2-30 of the N terminus, HasA-N2 represents hyaluronan synthase with amino acids deleted at positions 2-47 of the N terminus, HasA-N3 represents hyaluronan synthase with amino acids deleted at positions 2-64 of the N terminus, HasA-N4 represents hyaluronan synthase with amino acids deleted at positions 2-107 of the N terminus, HasA-C1 represents hyaluronan synthase with amino acids deleted at positions 920-972 of the C terminus, HasA-C2 represents HasA-C3 represents a hyaluronan synthase with a C-terminal amino acid deletion of 790-972, HasA-N4C1 represents a hyaluronan synthase with a N-terminal amino acid deletion of 2-107 and a C-terminal amino acid deletion of 920-972, HasA-N4C2 represents a hyaluronan synthase with a N-terminal amino acid deletion of 2-107 and a C-terminal amino acid deletion of 850-972, HasA-N4C3 represents a hyaluronan synthase with a N-terminal amino acid deletion of 2-107 and a C-terminal amino acid deletion of 790-972;

[0036] Figure 3: is a graph showing the effect of hyaluronan synthase mutations on hyaluronan production; wherein, HasA-N4C3-A represents a hyaluronan synthase with a deletion of amino acids at positions 2-107 at the N-terminus and amino acids at positions 790-972 at the C-terminus, and a mutation of U at position 280 to S; HasA-N4C3-B represents a hyaluronan synthase with a deletion of amino acids at positions 2-107 at the N-terminus and amino acids at positions 790-972 at the C-terminus, and a mutation of U at position 280 to S and a mutation of C at position 540 to S; HasA-N4C3-C represents a hyaluronan synthase with a deletion of amino acids at positions 2-107 at the N-terminus and amino acids at positions 790-972 at the C-terminus and a mutation of C at position 540 to S;

[0037] Figure 4 This is the yield graph of fed-batch fermentation in a 5L fermenter;

[0038] Figure 5 This is the mass spectrum of hyaluronic acid. DETAILED DESCRIPTION

[0039] 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.

[0040] Strain: 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, triammonium citrate: 2.0 g / ;

[0044] Preparation of Streptococcus thermophilus competent cells: Activation of strains: Take out the Streptococcus thermophilus strain stored in the -80℃ refrigerator, streak it onto MB solid medium, and culture it at 37℃ for 24-48 hours until obvious colonies appear. Seed culture: Pick a single colony from the plate and inoculate it into 5mL MB liquid medium, and culture it at 37℃, 180rpm, shaking for 8-12 hours until the OD 600Expanded culture: Take 1 mL of seed culture solution and add it to 100 mL of MB liquid culture medium. Shake and culture at 37°C and 180 rpm until OD reaches 0.4-0.6. 600 Reach 0.4-0.6. Collect bacteria by centrifugation: Transfer the culture solution to a centrifuge tube, centrifuge at 4°C, 4000rpm for 10 minutes, and discard the supernatant. Washing: Resuspend the bacteria with pre-cooled 10% glycerol solution to make the final volume about 1 / 10 of the original culture volume, mix gently, centrifuge at 4°C, 4000rpm for 10 minutes, and discard the supernatant. Resuspension and aliquoting: Resuspend the bacteria again with pre-cooled 10% glycerol solution to make the final volume about 1 / 100 of the original culture volume, mix gently, and aliquot into sterile 1.5mL centrifuge tubes, about 100μL per tube.

[0045] Purification and content detection of hyaluronic acid: To quantify hyaluronic acid, cells were removed by centrifugation at 16,000 × g for 15 minutes. The supernatants were combined and mixed with four volumes of ice-cold ethanol, and the samples were precipitated at -30°C for 8 hours. The insoluble fraction was then removed by centrifugation at 16,000 × g for 10 minutes, and the supernatant was freeze-dried. The fractions were dissolved in deionized water, and the water-insoluble fractions were removed by centrifugation. In order to remove as many impurities as possible, these steps were repeated three more times. The supernatant was purified by centrifugation with a HiPrep Q HP16 / 10 (GE Healthcare, USA) column. The crude HA samples were further purified by anion exchange chromatography using a 25 preparative chromatography system. After appropriate dilution, the NSGAGs content was determined by carbazole using glucuronic acid as an external standard.

[0046] Hyaluronan tetrasaccharide mass spectrometry: The samples were mixed with 9 volumes of methanol, and the supernatant fractions were subsequently collected by centrifugation at 16,000 × g for 20 min and filtered through a 0.22 μm membrane (ALWSCI, Shaoxing, China). The components were analyzed by LC-IT-TOF-MS (Shimadzu, Kyoto, Japan) at 40 °C using a 5 μm gasket set VP-ODS column (4.6 × 250 mm). Elution buffer A was ultrapure water, and elution buffer B was a methanol solution, both of which contained 8 mM ammonium acetate. The LC gradient elution was as follows: 0–5 min, 5% B; 5 to 15 min, 5% to 60% B; 15 to 20 min, 60% B, with a flow rate of 0.2 mL min -1 Ionization of the LC stream 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 from 100 m / z to 900 m / z.

[0047] The amino acid deletion at positions 920-972 of the C-terminus of the present invention refers to the deletion of amino acids at positions 920-972, calculated from the first amino acid at the N-terminus, and the amino acids at positions 920-972 are located at the C-terminus; the amino acid deletion at positions 850-972 of the C-terminus refers to the deletion of amino acids at positions 850-972, calculated from the first amino acid at the N-terminus, and the amino acids at positions 850-972 are located at the C-terminus; the amino acid deletion at positions 790-972 of the C-terminus refers to the deletion of amino acids at positions 790-972, calculated from the first amino acid at the N-terminus, and the amino acids at positions 790-972 are located at the C-terminus. The mutations of amino acids at positions 280 and 540 are also calculated from the N-terminus.

[0048] Example 1: Construction of hyaluronan synthase mutants

[0049] (1) Optimization of truncation of the N-terminal and C-terminal sequences of hyaluronan synthase

[0050] Based on the genome sequence of the hyaluronan synthase of Pasteurella multocida, the gene sequence of hyaluronan synthase pmHasA was synthesized by GENEWIZ (Suzhou), and the amino acid sequence of pmHasA is shown in SEQ ID NO.1. Figure 1As shown in the figure, primers were designed to truncate hyaluronan synthase, and the synthesized pmHasA gene sequence was used as a template to design primers pmHasA-N1-F and pmHasA-N1-R, pmHasA-N2-F and pmHasA-N2-R, pmHasA-N3-F and pmHasA-N3-R, pmHasA-N4-F and pmHasA-N4-R, pmHasA-C1-F and pmHasA-C1-R, pmHasA-C2-F and pmHasA-C2-R, pmHasA-C3-F and pmHasA-C3-R for PCR amplification, and then pmHasA and the truncated pmHasA gene sequence were connected to plasmid pNZ8148 by the T5 exonuclease-dependent assembly (TEDA) method to construct a recombinant expression vector. Plasmids pNZ8148-pmHasA, pNZ8148-pmHasA-N1, pNZ8148-pmHasA-N2, pNZ8148-pmHasA-N3, pNZ81 48-pmHasA-N4, pNZ8148-pmHasA-C1, pNZ8148-pmHasA-C2, pNZ8148-pmHasA-C3, pNZ8148-pm HasA-N4C1, pNZ8148-pmHasA-N4C2, pNZ8148-pmHasA-N4C3 were transferred into Streptococcus 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 shake flasks, and the hyaluronic acid production results were as follows Figure 2 shown.

[0051] Table 1 Primers used for truncation of the A and C termini of hyaluronan synthase pmHas

[0052]

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

[0054] Primers were designed to mutate hyaluronan synthase, and pNZ8148-pmHasA-N4C3 was used as a template to design primers pNZ8148-pmHasA-N4C3-280-F, pNZ8148-pmHasA-N4C3-280-R, pNZ8148-pmHasA-N4C3-540-F, and pNZ8148-pmHasA-N4C3-540-R for PCR amplification to obtain the recombinant plasmid pNZ8148-pmHasA U280S-N4C3, pNZ8148-pmHasA C540S -N4C3, pNZ8148-pmHasA U280S，C540S -N4C3, and introduced into thermophilic Streptococcus to obtain recombinant strains HasA-N4C3-A, HasA-N4C3-B, and HasA-N4C3-C. The recombinant strains were cultured in shake flasks, and the hyaluronic acid production results were as follows Figure 3 shown.

[0055] Table 2 Primers used to construct the hyaluronan synthase mutant pXMJ-HasA

[0056] Primer name Sequence (5'-3') Sequence number pNZ8148-pmHasA-N4C3-280-F TCACATTAGCCTGTTTAGTAAACC SEQ ID NO.18 pNZ8148-pmHasA-N4C3-280-R ACAGGCTAATGTAATCGATAA SEQ ID NO.19 pNZ8148-pmHasA-N4C3-540-F AGTTGAACTGTCTTTAAAAGAATT SEQ ID NO.20 pNZ8148-pmHasA-N4C3-540-R TTCTTTTAAACAGAGTTCAACTG SEQ ID NO.21

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

[0058] According to the genome sequence of Bacteroides phage 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. Using the synthesized hylase gene sequence as a template, primers pmHasA-GS-hylase-F and pmHasA-GS-hylase-R were designed for PCR amplification, and then the gene hylase was connected to the plasmid pNZ8148-pmHasA by the TEDA method. U280S,C540S -N4C3, construct the recombinant expression plasmid pNZ8148-pmHasA U280S，C540S -N4C3-GS-hylase.

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

[0060]

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

[0062] (1) Enhanced UDP-glucuronic acid synthesis

[0063] Primers ugd-F and ugd-R were designed to obtain the UDP-glucose dehydrogenase gene ugd using the genome of Corynebacterium glutamicum as a template by PCR amplification. The gene ugd was ligated to the plasmid pNZ8148-pmHasA by the TEDA method. U280S，C540S -N4C3-GS-hylase, and the recombinant expression plasmid pNZ8148-pmHasA was constructed U280S，C540S-N4C3-GS-hylase-ugd. The recombinant plasmid was transformed into Streptococcus thermophilus by electroporation to obtain the recombinant strain Streptococcus thermophilus HasA-N4C3-C-ugd. The recombinant strain was cultured in a shake flask, and the hyaluronic acid production results were as follows Figure 3 shown.

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

[0065]

[0066] (2) Fed-batch fermentation of recombinant Streptococcus thermophilus in a 5 L fermenter

[0067] 5L fermentation tank fermentation production: The recombinant strain of Streptococcus thermophilus HasA-N4C3-C-ugd constructed in Example 3 was inoculated into 5mL MB medium and cultured overnight at 220rpm and 45℃. 600 =0.1 was transferred to a baffled Erlenmeyer flask with 25mL fermentation medium, and cultured at 220rpm and 45℃ for 10h, then inoculated into a 5L fermenter with 10% inoculum. The initial temperature was set at 45℃, the rotation speed was 3000r / min, and after 4h of fermentation, IPTG was added with a final concentration of 0.2mM to induce gene expression. During the fermentation process, 14% ammonia water was used to control the pH of the fermentation liquid at around 7, and glucose was added to maintain the pH in the fermenter at around 10g / L. Figure 4 It can be seen that the final hyaluronic acid production of the recombinant strain Streptococcus thermophilus in a 5L fermenter is 15g / L. The mass spectrometry structure of the generated hyaluronic acid is identified, and the results are as follows Figure 5 As shown, the generated hyaluronic acid is hyaluronan tetrasaccharide.

[0068] 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, characterized in that: The hyaluronan synthase mutant is one or more of the following modifications to the hyaluronan synthase with the amino acid sequence shown in SEQ ID NO.1: (1) N-terminal amino acid 2-30 is missing; (2) N-terminal amino acid 2-47 deletion; (3) N-terminal amino acid deletion at positions 2-64; (4) N-terminal amino acid 2-107 deletion; (5) C-terminal amino acid deletion at position 920-972; (6) C-terminal amino acid deletion at positions 850-972; (7) C-terminal amino acid deletion at positions 790-972; (8) selenocysteine ​​at position 280 was mutated to serine; (9) Cysteine ​​at position 540 was mutated to serine.

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 recombinant cell expressing the hyaluronan synthase mutant according to claim 1.

5. The recombinant cell according to claim 4, characterized in that The recombinant 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 recombinant cell according to claim 4 or 5 in synthesizing hyaluronan.

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, characterized in that: The hyaluronan synthase mutant according to 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 fermented and cultured to synthesize hyaluronic acid.

9. The method according to claim 8, characterized in that The recombinant plasmid also includes the gene sequences of hyaluronan degrading enzyme and uridine diphosphate-glucuronic acid 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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