Chondroitin synthase mutants and uses thereof

Abstract

The application provides a chondroitin synthase mutant and application thereof, and relates to the technical field of biology.The chondroitin synthase mutant is obtained by firstly performing deletion on N-terminal and C-terminal amino acid sequences of chondroitin synthase with an amino acid sequence as shown in SEQ ID NO.1 and performing compound mutation on amino acids at positions 200, 279 and 346, introducing the chondroitin synthase mutant into a plasmid to form a recombinant plasmid, introducing the recombinant plasmid into a recombinant bacterium for fermentation culture, and finding that the chondroitin synthase mutant can significantly improve the yield of chondroitin.In addition, the application also overexpresses N-acetylglucosamine epimerase, 6-phosphoglucosamine synthetase, 6-phosphoglucosamine synthetase and uridine diphosphate-glucose dehydrogenase, and constructs the overexpressed enzymes into a protein polymer through CipA protein, so that the synthesis efficiency of chondroitin is improved, and the yield of chondroitin obtained through fermentation in a 5L fermentation tank is about 10.5g / L.

Description

TECHNICAL FIELD

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

[0002] Chondroitin is a linear acidic polysaccharide composed of glucuronic acid and N-acetylgalactosamine as disaccharide units. Chondroitin sugar chain structure is highly similar to hyaluronic acid, also has water absorption, moisturizing, lubricating and other functions, at the same time, due to the same sugar chain structure as chondroitin sulfate, it is also used as the enzyme modification substrate of chondroitin sulfate.

[0003] Chondroitin sulfate is a sulfonated modified glycosaminoglycan, which has important functions such as lubrication, analgesia and promotion of cartilage regeneration, and has wide application in the fields of medicine and health care. With the aggravation of population aging, bone and joint diseases have become an important problem that affects the health of the elderly. Chondroitin sulfate, as an effective drug for treating osteoarthritis, has a growing market demand. However, the chondroitin sulfate on the market is mainly obtained by substrate tissue extraction method. The substrate tissue method mainly extracts the naturally existing chondroitin sulfate in animal tissues through chemical process. This method has the following problems: raw material source limitation, raw material leading to batch instability of product, extraction process leading to environmental pollution, and product purity difficult to reach medical level. In view of the above problems, people have developed an enzyme synthesis process for synthesizing chondroitin sulfate, which uses microorganisms to produce chondroitin sulfate in large quantities, and catalyzes chondroitin sulfate under the action of sulfonated enzyme. The chondroitin sulfate obtained by this method has the characteristics of single structure, controllable sulfonated modification, sustainable production without raw material restriction, and high purity of obtained chondroitin sulfate. However, an important problem limiting this method is how to realize large-scale preparation of chondroitin substrate.

[0004] In order to realize the production of chondroitin, people hope to use synthetic biology technology to construct a chondroitin synthesis pathway in some microbial strains, and prepare chondroitin in large quantities through microbial fermentation. However, the yield of chondroitin prepared by microbial fermentation in the prior art is still low, which cannot realize large-scale industrial production. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to overcome the problem that there is no microbial fermentation method for improving the yield of chondroitin in the prior art.

[0006] To solve the above technical problems, the application provides a chondroitin synthase mutant and application thereof. The application first performs deletion of N-terminal and C-terminal amino acid sequences of chondroitin synthase with an amino acid sequence as shown in SEQ ID NO. 1 and performs complex mutation of amino acids at positions 200, 279 and 346 to obtain a chondroitin synthase mutant. The chondroitin synthase mutant of the application is introduced into a plasmid to form a recombinant plasmid, and the recombinant plasmid is introduced into a recombinant bacterium for fermentation culture. It is found that the chondroitin synthase mutant of the application can significantly improve the yield of chondroitin. In addition, the application also overexpresses N-acetylglucosamine epimerase, 6-phosphoglucosamine synthetase, 6-phosphoglucosamine synthetase and udg enzyme, and constructs the overexpressed enzymes into a protein polymer through CipA protein, thereby improving the synthesis efficiency of chondroitin. The yield of chondroitin obtained by fermentation in a 5L fermenter is about 10.5 g / L.

[0007] The first object of the application is to provide a chondroitin synthase mutant, which is one or more of the following modifications to chondroitin synthase with an amino acid sequence as shown in SEQ ID NO. 1:

[0008] (a) deletion of amino acids at positions 2-117 at the N-terminal;

[0009] (b) deletion of amino acids at positions 688-965 at the C-terminal;

[0010] (c) mutation of glutamine at position 200 to glutamic acid, mutation of alanine at position 279 to glutamic acid, and mutation of leucine at position 346 to glutamic acid (Q200E / A279E / L346E).

[0011] Further, the sequence of SEQ ID NO. 1 is as shown below:

[0012] MNTLSQAIKAYNSNDYELALKLFEKSAETYGRKIVEFQIIKCKEKLSTNSYVSEDKKNSVCDSSLDIATQLLLSNVKKLTLSESEKNSLKNKWKSITGKKSENAEIRKVELVPKDFPKDLVLAPLPDHVNDFTWYKNRKKSLGIKPVNKNIGLSIIIPTFNRSRILDITLACLVNQKTNYPFEVVVADDGSKENLLTIVQKYEQKLDIKYVRQKDYGYQLCAVRNLGLRTAKYDFVSILDCDMAPQQLWVHSYLTELLEDNDIVLIGPRKYVDTHNITAEQFLNDPYLIESLPETATNNNPSITSKGNISLDWRLEHFKKTDNLRLCDSPFRYFSCGNVAFSKEWLNKVGWFDEEFNHWGGEDVEFGYRLFAKGCFFRVIDGGMAYHQEPPGKENETDREAGKSITLKIVKEKVPYIYRKLLPIEDSHIHRIPLVSIYIPAYNCANYIQRCVDSALNQTVVDLEVCICNDGSTDNTLEVINKLYGNNPRVRIMSKPNGGIASASNAAVSFAKGYYIGQLDSDDYLEPDAVELCLKEFLKDKTLACVYTTNRNVNPDGSLIANGYNWPEFSREKLTTAMIAHHFRMFTIRAWHLTDGFNEKIENAVDYDMFLKLSEVGKFKHLNKICYNRVLHGDNTSIKNLDTQKKNHFVVVNQSLNRQRVSNYNYDEFDNLDESRKYIFNKTADYQEEIDILKDIKIVQRKDAKVAISIFYPNRLDGLVKKLNNIIEYNKNVLIIVLHIDKNHLTSDIKKEILEFHNKNQINILLNNDVSYYTNNRLIKTKAHLSNMNKLRQLNLNLEYIIFDNHDSLFIKNDSYNHIKKYDIGMNFSSLTNDWINKINAHSPFKNLIKKYFNDNDLKTINMKGASQGMFIKYTLAHDIATIMKEVITLCQSTDSVPEYNTEDIWFQFALLILEKKTGHVFNKTSTLTYMPWERKLQWTNEQIESAKRGENIPVNKFIINSITL.

[0013] A second object of the present application is to provide a gene encoding the above-mentioned chondroitin synthase mutant.

[0014] A third object of the present application is to provide a recombinant plasmid carrying the above-mentioned gene.

[0015] A fourth object of the present application is to provide a recombinant cell expressing the above-mentioned chondroitin synthase mutant.

[0016] Further, the recombinant cell is a bacterium or a fungus.

[0017] A fifth object of the present application is to provide a use of the above-mentioned chondroitin synthase mutant, the above-mentioned gene, the above-mentioned recombinant plasmid or the above-mentioned recombinant cell in the preparation of chondroitin.

[0018] Further, the use is to add the chondroitin synthase mutant or an expression system containing the chondroitin synthase mutant into a reaction system to generate chondroitin.

[0019] A sixth object of the present application is to provide a recombinant bacterium for synthesizing chondroitin, which heterologously expresses the above-mentioned chondroitin synthase mutant and N-acetylglucosamine epimerase.

[0020] Further, the recombinant bacterium further overexpresses crystallization inclusion protein A, glutamine-fructose-6-phosphate aminotransferase and uridine diphosphate-glucose dehydrogenase.

[0021] A seventh object of the present application is to provide a method for synthesizing chondroitin, which introduces a gene sequence encoding the chondroitin synthase mutant and a gene sequence encoding N-acetylglucosamine epimerase into a plasmid to form a recombinant plasmid, introduces the recombinant plasmid into a host cell to form a recombinant bacterium, and ferments the recombinant bacterium.

[0022] Further, the recombinant plasmid is introduced into a host cell to form a recombinant bacterium, and the recombinant bacterium is fermented to synthesize chondroitin, wherein the recombinant plasmid comprises a first recombinant plasmid and a second recombinant plasmid, the first recombinant plasmid comprises a gene sequence of the above-mentioned chondroitin synthase mutant, a gene sequence of N-acetylglucosamine epimerase and a gene sequence of crystallization inclusion protein A; and the second recombinant plasmid comprises a gene sequence of 6-phosphoglucomutase, a gene sequence of uridine diphosphate-glucose dehydrogenase and a gene sequence of crystallization inclusion protein A.

[0023] Further, the amino acid sequence of glutamine-fructose-6-phosphate aminotransferase is shown in SEQ ID NO. 2, and the sequence of SEQ ID NO. 2 is shown as follows:

[0024] MCGIVGAVAQRDVAEILINGLHRLEYRGYDSAGVAVVNDKQELQRIRCLGKVKALDDAVMENPLIGGTGIAHTRWATHGQPSEDNAHPHTSGNFAVVHNGIIENYEELRSELQAKGYVFLSQTDTEVIAHLVEWEMRTASNLLEAVQKVVKQLKGAYGMVVMDRETPQHLVAARSGSPLVIGLGIGENFLASDQLALLSVTRRFMFLEEGDIAEITRRSVDIYDMSGKKVEREVLDSQLANDSAEKGKFRHFMQKEIFEQPTALINTMEGRILHNSVVVESIGNGAKELLSQVEHIQIVACGTSYNAGMVARYWFEDLAGVSCDVEIASEFRYRKFVTRPNSLLITLSQSGETADTLAALRLAKEKGYMGAMTICNVAGSSLVRESDLAFMTRAGVEIGVASTKAFTTQLVTLLMLVTALGKLKGTISAEKESEIIKALHSLPADIEKALAFDSSIESLAEDFAEKHHALFLGRGEYYPIAMEASLKLKEISYIHAEAYAAGELKHGPLALIDADMPVIVVAPTNDLLEKVKSNIEEVRARGGQLYVFADKEAGFVESDGMKIITMPTVNPLVAPIFYTVPMQLLSYHVALIKGTDVDQPRNLAKAVTVE.

[0025] Further, the amino acid sequence of the N-acetylglucosamine epimerase is shown as SEQ ID NO. 3, and the sequence of SEQ ID NO. 3 is shown as follows:

[0026] MNILVTGGAGYIGSHTSLCLLNKGYNVVIIDNLINSSCESIRRIELIAKKKVTFYELNINNEKEVNQILKKHKFDCIMHFAGAKSVAESLIKPIFYYDNNVSGTLQLINCAIKNDVANFIFSSSATVYGESKIMPVTEDCHIGGTLNPYGTSKYISELMIRDIAKKYSDTNFLCLRYFNPTGAHESGMIGESPADIPSNLVPYILQVAMGKLEKLMVFGGDYPTKDGTGVRDYIHVMDLAEGHVAALSYLFRDNNTNYHVFNLGTGKGYSVLELVSTFEKISGVRIPYEIVSRRDGDIAESWSSPEKANKYLNWKAKRELETMLEDAWRWQMKNPNGYI.

[0027] Further, the amino acid sequence of UDP-glucose dehydrogenase is shown as SEQ ID NO. 4, and the sequence of SEQ ID NO. 4 is shown as follows:

[0028] MKKITIAGAGYVGLSNAVLLAQHHNVILLDIDQNKVDLINNKKSPITDKEMEDFLQNKSLTMMATPDKEVALKNADFVINATPPDYNTETGYFNTSTVEAVIEQTLSINPQAAIIIKSTIPVGFTENMREKFNTPNLIFSPEFLREGKALYDNLYPSRIIVGSTSYQAKVFADMLTQCARKKDVTVLFTHNTEAEAVKLFANTYLAMRVAFFNELDTYASLHHLNTKDIINGISTDPRIGTHYNNPSFGYGGYCLPKDTKQLLANYADVPQNLIEAIVKSNETRKRFITHDVLNKKPKTVGIYRLIMKSGSDNFRASAILDIMPHLKENGVEIVIYEPTLNQQAFEDYPVINQLSEFINRSDVILANRSEPDLNQCSHKIYTRDIFGGDA.

[0029] Further, the amino acid sequence of CipA protein is shown as SEQ ID NO. 5, and the sequence of SEQ ID NO. 5 is shown as follows:

[0030] GVYIMINDMHPSLIKDKDIVDDVMLRSCKIIAMKVMPDKVMQVMVTVLMHDGVCEEMLLKWNLLDNRGMAIYKVLMEALCAKKDVKISTVGKVGPLGCDYINCVEISM.

[0031] Further, the fermentation culture contains a carbon source, a nitrogen source, biotin, inorganic salts and metal ions.

[0032] Further, the host cell comprises Corynebacterium glutamicum. Two precursors of chondroitin are UDP-glucuronic acid and UDP-galactosamine, but only UDP-glucuronic acid can be synthesized in Corynebacterium glutamicum, but UDP-galactosamine cannot be synthesized. kfoA can catalyze the synthesis of UDP-galactosamine from UDP-glucosamine, so kfoA needs to be overexpressed.

[0033] Advantages of the present application:

[0034] The present application provides a chondroitin synthase mutant and application thereof. The present application firstly performs deletion of N-terminal and C-terminal amino acid sequences of chondroitin synthase with amino acid sequence shown in SEQ ID NO. 1 and compound mutation of amino acids at positions 200, 279 and 346 to obtain a chondroitin synthase mutant. The chondroitin synthase mutant of the present application is introduced into a plasmid to form a recombinant plasmid, and the recombinant plasmid is introduced into a recombinant bacteria for fermentation culture. It is found that the chondroitin synthase mutant of the present application can significantly improve the yield of chondroitin. In addition, the present application overexpresses N-acetylglucosamine epimerase, 6-phosphoglucosamine synthetase, 6-phosphoglucosamine synthetase and udg enzyme, and constructs the overexpressed enzymes into a protein polymer through CipA protein, thereby improving the synthesis efficiency of chondroitin. The yield of chondroitin obtained by fermentation in a 5L fermenter is about 10.5g / L. BRIEF DESCRIPTION OF DRAWINGS

[0035] 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 combination with the drawings, in which

[0036] Figure 1 is a metabolic pathway of chondroitin;

[0037] Figure 2Figure 1 is the effect of different modifications on chondroitin production, wherein BJ-01 represents recombinant bacteria containing pXMJ-pmCS-kfoA, BJ-02 represents recombinant bacteria containing pXMJ-NpmCS1-kfoA, BJ-03 represents recombinant bacteria containing pXMJ-CpmCS1-kfoA, BJ-04 represents recombinant bacteria containing pXMJ-NCpmCS1-kfoA, BJ-05 represents recombinant bacteria containing plasmid pECXK99E-ugd-glmS and plasmid pXMJ-NCpmCS1-kfoA, and BJ-06 represents recombinant bacteria containing plasmid pXMJ-NCpmCS1-kfoA-CipA and plasmid pECXK99E-ugd-glmS-CipA;

[0038] Figure 3 Figure 2 is the characterization of protein aggregates of CipA construction;

[0039] Figure 4 Figure 3 is the production graph of chondroitin synthesized by batch fed-batch fermentation of recombinant C. pekinense. DETAILED DESCRIPTION

[0040] The present application is further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand the present application and implement it. The examples are not intended to limit the present application.

[0041] Vector: C. pekinense

[0042] BHIS medium: brain heart infusion broth 17.5 g / L -1 , sorbitol 91 g / L -1

[0043] Fermentation medium: glucose: 60 g / L -1 , corn syrup dry powder: 30 g / L, (NH4)2SO4: 10 g / L -1 , MgSO4: 25 g / L -1 .

[0044] Extraction of chondroitin samples:

[0045] The fermentation broth was centrifuged to collect the bacterial cells, the bacterial cells were resuspended in water, and high-pressure homogenization was performed to break the cells, and the supernatant was collected by centrifugation. The supernatant was added with 3 times the volume of ethanol, and alcohol precipitation was performed for 4 h, and the precipitate was collected by centrifugation. The precipitate was resuspended in water, and the supernatant was collected by centrifugation. The supernatant was freeze-dried, resuspended in water, and the supernatant was collected by centrifugation. The above experimental steps were repeated. The obtained sample was dried, resuspended in water, and diluted for testing.

[0046] Determination of chondroitin content:

[0047] Carbazole sulfate method: 2 mL of boric acid sulfate solution (9.54 g of boric acid is dissolved in 1 L of concentrated sulfuric acid) is added into a glass test tube, pre-cooled, and 400 μL of diluted sample is added, mixed, heated in boiling water for 15 min, removed from the ice bath, 1.25 g of carbazole solution (dissolved in 1 L of ethanol) is added, mixed, heated in boiling water for 10 min, removed and cooled for testing. Prepare 0, 10, 20, 30, 40 and 50 mg / L of glucuronic acid as a standard curve, read the absorbance value at 530 nm by the above treatment, and calculate the content of glucuronic acid in the sample. The content of chondroitin is calculated by multiplying the content of glucuronic acid by 2.067.

[0048] The C-terminal 688-965 amino acid deletion of the present application refers to the 688-965 amino acids counted from the N-terminal 1st amino acid, and the 688-965 amino acids are located at the C-terminal; the mutation of the 200th, 279th and 346th amino acids of the present application is also counted from the N-terminal.

[0049] Example 1: Construction of recombinant Corynebacterium glutamicum

[0050] (1) Construction of chondroitin synthesis pathway

[0051] On the basis of chondroitin synthase pmCS with amino acid sequence shown in SEQ ID NO. 1, the 200th, 279th and 346th amino acids of chondroitin synthase pmCS were subjected to complex mutation by Q200E-F, Q200E-R, A279E-F, A279E-R, L346E-F and L346E-R to obtain mutant pmCS1. On the basis of mutant pmCS1, chondroitin synthase mutant pmCS1 was subjected to truncation by NpmCS1-F, NpmCS1-R and CpmCS1-F and CpmCS1-R as primers to obtain chondroitin synthase mutants NpmCS1, CpmCS1 and NCpmCS1.

[0052] The gene sequences of chondroitin synthase pmCS and chondroitin synthase mutants NpmCS1, CpmCS1, NCpmCS1 and N-acetylglucosamine epimerase kfoA were respectively connected to plasmid pXMJ (wherein the primers used for chondroitin synthase pmCS are pmCS-F and pmCS-R, and the primers used for N-acetylglucosamine epimerase kfoA are KfoA-F and KfoA-R, and the specific sequences are shown in Table 1), and the chondroitin synthase and chondroitin synthase mutants were connected with N-acetylglucosamine epimerase kfoA through GS Linker, to obtain plasmids pXMJ-pmCS-kfoA, pXMJ-NpmCS1-kfoA, pXMJ-CpmCS1-kfoA and pXMJ-NCpmCS1-kfoA respectively; the recombinant plasmids were respectively transformed into C. beijingense to obtain recombinant strains BJ-01, BJ-02, BJ-03 and BJ-04 and perform fermentation culture, and the chondroitin content results are shown in Table 1. Figure 2

[0053] Table 1 Primers and sequences involved in Example 1 (1)

[0054]

[0055] (2) Expression of the synthesis pathway of precursor substances UDP-N-acetylgalactosamine and / or UDP-glucuronate

[0056] C. beijingense was inoculated in 5 mL BHIS medium and cultured at 30°C, and the bacterial cells were collected to extract genomic DNA by using a cell genomic extraction kit. Primers glmS-F, glmS-R and ugd-F, ugd-R were designed to amplify glutamine-fructose-6-phosphate aminotransferase and UDP-glucose dehydrogenase, and the two were connected through GS linker and connected to plasmid pECXK99E to obtain plasmid pECXK99E-ugd-glmS. Plasmid pECXK99E-ugd-glmS and plasmid pXMJ-NCpmCS1-kfoA were transformed into C. beijingense by electroporation, and the electroporation conditions were voltage 1.5 KV and 5 ms (the width of the shock cup was 1 mm). Through resistance screening and sequencing, the correct recombinant strain BJ-05 was obtained and fermentation culture was performed, and the chondroitin yield is shown in Table 1. Figure 2

[0057] Table 2 Primers and sequences involved in Example 1 (2)

[0058]

[0059] (3) Construction of protein aggregates using CipA

[0060] ​​According to the reported CipA sequence, the gene sequence was synthesized, which was amplified by CipA-F and CipA-R, and the plasmid was amplified by primers pXMJ-CipA-F / pXMJ-CipA-R and PEC-CipA-F / PEC-CipA-R, and then was connected to the C-terminal of the kfoA gene of the plasmid pXMJ-NCpmCS1-kfoA and the C-terminal of the glmS gene of the plasmid pECXK99E-ugd-glmS, and the CipA, kfoA and glmS were connected by GS link to obtain the recombinant plasmids pXMJ-NCpmCS1-kfoA-CipA and pECXK99E-ugd-glmS-CipA.

[0061] The plasmids pXMJ-NCpmCS1-kfoA-CipA and pECXK99E-ugd-glmS-CipA were transformed into Beijing fastidiosa by electroporation using an electroporator, and the transformation conditions were voltage 1.5 KV and 5 ms (the width of the shock cup was 1 mm). The correct recombinant strain BJ-06 was obtained by resistance screening and sequencing.

[0062] The recombinant strain BJ-06 was inoculated in a single colony in a BHIS liquid culture medium and cultured at 30°C for 12 h, and then was inoculated in a 250 mL flask with 25 mL of fermentation medium at a percentage of 1%. The flask was cultured at 220 rpm and 30°C for 48 h. The bacterial cells were collected and broken, and the chondroitin in the strain was purified and determined, and the yield of chondroitin was as shown in Table 1. Figure 2

[0063] Table 3 Primers involved in Example 1 (3) and their sequences

[0064]

[0065] Example 2: Fed-batch fermentation of recombinant Beijing fastidiosa

[0066] The recombinant Beijing fastidiosa BJ-06 constructed in Example 1 was activated on a plate medium, and a single colony was inoculated in a BHIS culture medium. The strain was cultured at 30°C for 10 h, and then was inoculated in a 250 mL flask at a percentage of 1%, and was cultured for 10 h, and then was inoculated in a 5 L fermenter at a percentage of 20%, and the liquid volume of the fermenter was 2.5 L, the culture temperature was 30°C, the stirring speed was 300 rpm, and the aeration amount was 3 vvm. The pH was controlled between 6.5 and 7, and the glucose content was controlled at 10 g / L. After 3 h of inoculation, 0.5 mM of IPTG and 0.2 g / L of biotin were added, and the pH was maintained by ammonia during the fermentation process. The yield of chondroitin was as shown in Table 2. Figure 4 ​As shown, the yield of the chondroitin produced by the recombinant B. beijingensis constructed by the application reaches 10 g / L.

[0067] Obviously, the above embodiments are only examples for clearly illustrating the present application, and are not intended to limit the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A chondroitin synthase mutant, characterized in that, The chondroitin synthase mutant is a chondroitin synthase with the amino acid sequence shown in SEQ ID NO.1 modified as follows: amino acids 2-117 at the N-terminus are deleted, amino acids 688-965 at the C-terminus are deleted, and glutamine at position 200 is mutated to glutamic acid, alanine at position 279 is mutated to glutamic acid, and leucine at position 346 is mutated to glutamic acid.

2. The gene encoding the chondroitin synthase mutant of claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. Recombinant cells expressing the chondroitin synthase mutant of claim 1.

5. The recombinant cell according to claim 4, characterized in that, The recombinant cells are bacteria or fungi.

6. The use of the chondroitin 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 preparation of chondroitin.

7. A recombinant bacterium for synthesizing chondroitin, characterized in that, The recombinant bacteria heterologously express the chondroitin synthase mutant and N-acetylglucosamine epimerase as described in claim 1, wherein the amino acid sequence of the N-acetylglucosamine epimerase is shown in SEQ ID NO.

3.

8. The recombinant bacteria according to claim 7, characterized in that, The recombinant bacteria also overexpressed crystal inclusion protein A, glutamine-fructose-6-phosphate aminotransferase, and uridine diphosphate-glucose dehydrogenase, wherein the amino acid sequence of glutamine-fructose-6-phosphate aminotransferase is shown in SEQ ID NO.2, the amino acid sequence of uridine diphosphate-glucose dehydrogenase is shown in SEQ ID NO.4, and the amino acid sequence of crystal inclusion protein A is shown in SEQ ID NO.

5.

9. A method for synthesizing chondroitin, characterized in that, The gene sequence encoding the chondroitin synthase mutant of claim 1 and the gene sequence encoding N-acetylglucosamine epimerase are introduced into a plasmid to form a recombinant plasmid. The recombinant plasmid is introduced into a host cell to form a recombinant bacterium. The recombinant bacterium is fermented and cultured to synthesize chondroitin. The amino acid sequence of the N-acetylglucosamine epimerase is shown in SEQ ID NO.

3.

10. The method according to claim 9, characterized in that, A recombinant plasmid is introduced into a host cell to form a recombinant bacterium, which is then fermented to synthesize chondroitin. The recombinant plasmid comprises a first recombinant plasmid and a second recombinant plasmid. The first recombinant plasmid contains a gene sequence encoding the chondroitin synthase mutant of claim 1, a gene sequence of N-acetylglucosamine epimerase, and a gene sequence of crystal inclusion protein A. The second recombinant plasmid contains a gene sequence of glutamine-fructose-6-phosphate aminotransferase, a gene sequence of uridine diphosphate-glucose dehydrogenase, and a gene sequence of crystal inclusion protein A. The amino acid sequence of glutamine-fructose-6-phosphate aminotransferase is shown in SEQ ID NO. 2, the amino acid sequence of uridine diphosphate-glucose dehydrogenase is shown in SEQ ID NO. 4, and the amino acid sequence of crystal inclusion protein A is shown in SEQ ID NO. 5.

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