Chondroitin synthase mutant and application thereof

Abstract

The invention provides a chondroitin synthase mutant and application thereof, and relates to the technical field of biology. The chondroitin synthase mutant is obtained by firstly deleting N-terminal and C-terminal amino acid sequences of chondroitin synthase with an amino acid sequence as shown in SEQ ID NO.1 and carrying out compound mutation on 200-site, 279-site and 346-site amino acids, and the chondroitin synthase mutant is introduced into a plasmid to form a recombinant plasmid. Recombinant plasmids are introduced into recombinant bacteria for fermentation culture, and it is found that by means of the chondroitin synthase mutant, the yield of chondroitin can be remarkably increased. In addition, according to the invention, N-acetyl glucosamine epimerase, 6-phosphoglucosamine synthetase, 6-phosphoglucosamine synthetase and uridine diphosphate-glucose dehydrogenase are also overexpressed, and the overexpressed enzymes are constructed into a protein polymer through CipA protein, so that the synthesis efficiency of chondroitin is improved; the yield of chondroitin obtained by fermentation in a 5L fermentation tank is about 10.5 g / L.

Description

Technical Field

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

[0002] Chondroitin is a linear acidic polysaccharide composed of glucuronic acid and N-acetylgalactosamine as disaccharide units. The sugar chain structure of chondroitin is highly similar to that of hyaluronic acid, and it also has the functions of water absorption, moisturizing, and lubrication. At the same time, because the sugar chain structure is consistent with chondroitin sulfate, it is also used as an enzymatic modification substrate of chondroitin sulfate.

[0003] Chondroitin sulfate is a sulfonated glycosaminoglycan with important functions such as lubrication, analgesia, and promotion of cartilage regeneration. It is widely used in the fields of medicine and health care products. With the aging of the population, diseases such as bone and joints have become an important problem that plagues the health of the elderly. As an effective drug for the treatment of osteoarthritis, the market demand for chondroitin sulfate continues to expand. However, the chondroitin sulfate on the market is mainly obtained by substrate tissue extraction, which mainly extracts chondroitin sulfate naturally present in animal tissues through chemical processes. This method has the following problems: limited raw material sources, raw materials causing instability between product batches, extraction process causing environmental pollution, and product purity is difficult to reach medical levels. In response to the above problems, people have developed an enzymatic synthesis process for chondroitin sulfate, using microorganisms to efficiently produce chondroitin, and catalyzing chondroitin sulfate under the action of sulfonated enzymes. The chondroitin sulfate obtained by this method has the characteristics of a single structure, controllable sulfonation modification, sustainable production without raw material restrictions, and high purity of the obtained chondroitin sulfate. However, an important problem that limits this method is how to achieve large-scale preparation of substrate chondroitin.

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

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a microbial fermentation method for increasing the yield of chondroitin.

[0006] To solve the above technical problems, the present invention provides a chondroitin synthase mutant and its application. The present invention first deletes the N-terminal and C-terminal amino acid sequences of the chondroitin synthase shown in SEQ ID NO.1 and performs compound mutations on the amino acids at positions 200, 279 and 346 to obtain a chondroitin synthase mutant, introduces the chondroitin synthase mutant of the present invention into a plasmid to form a recombinant plasmid, introduces the recombinant plasmid into a recombinant bacterium for fermentation and culture, and finds that the chondroitin synthase mutant of the present invention can significantly increase the yield of chondroitin. In addition, the present invention also overexpresses N-acetylglucosamine epimerase, 6-phosphoglucosamine synthetase, 6-phosphoglucosamine synthetase and udg enzyme, and constructs the overexpressed enzyme into a protein polymer through CipA protein, thereby improving the synthesis efficiency of chondroitin, and the chondroitin yield obtained by fermentation in a 5L fermenter is about 10.5g / L.

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

[0008] (a) N-terminal amino acids 2-117 are missing;

[0009] (b) C-terminal amino acids 688-965 are missing;

[0010] (c) Glutamic acid at position 200 was mutated to glutamine, leucine at position 279 was mutated to glutamate, and phenylalanine at position 346 was mutated to glutamate (E200Q / L279E / F346E).

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

[0012] MNTLSQAIKAYNSNDYELALKLFEKSAETYGRKIVEFQIIKCKEKLSTNSYVSEDKKNSVCDSSLDIATQLLLSNVKKLTLSESEKNSLKNKWKSITGKKSENAEIRKVELVPKDFPKDLVLAPLPDHVNDFTWYKNRKKSLGIKPVNKNIGLSIIIPTFNRSRILDITLACLVNQKTNYPFEVVVADDGSKENLLTIVQKYEQKLDIKYVRQKDYGYQLCAVRNLGLRTAKYDFVSILDCDMAPQQL WVHSYLTELLEDNDIVLIGPRKYVDTHNITAEQFLNDPYLIESLPETATNNNPSITSKGNISLDWRLEHFKKTDNLRLCDSPFRYFSCGNVAFSKEWLNKVGWFDEEFNHWGGEDVEFGYRLFAKGCFFRVIDGMAYHQEPPGKENETDREAGKSITLKIVKEKVPYIYRKLLPIEDSHIHRIPLVSIYIPAYNCANAYIQRCVDSALNQTVVDLEVCICNDGSTDNTLEVINKLYGNNPRVRIMSKPNGGIASNAAVSFAKGYYIGQLDSDDYLEPDAVELCLKEFLKDKTLACVYTTNRNVNPDGSLIANGYNWPEFSREKLTTAMIAHHFRMFTIRAHLTDGFNEKIENAVD YDMFLKLSEVGKFKHLNKICYNRVLHGDNTSIKNLDTQKKNHFVVVNQSLNRQRVSNYDEFDNLDESRKYIFNKTADYQEEIDILKDIKIVQRKDAKVAISIFYPNRLDGLVKKLNNIIEYNKNVLIIVLHIDKNHLTSDIKKEILEFHNKNQINILNLNDVSYYTNNRLIKTKAHL SNMNKLRQLNLNLEYIIFDNHDSLFIKNDSYNHIKKYDIGMNFSSLTNDWINKINAHSPFKNLIKKYFNDNDLKTINMKGASQGMFIKYTLAHDIATIMKEVITLCQSTDSVPEYNTEDIWFQFALLILEKKTGHVFNKTSTLTYMPWERKLQWTNEQIESAKRGENIPVNKFIINSITL

[0013] The second object of the present invention is to provide a gene encoding the above chondroitin synthase mutant.

[0014] The third object of the present invention is to provide a recombinant plasmid carrying the above gene.

[0015] The fourth object of the present invention is to provide a recombinant cell expressing the above chondroitin synthase mutant.

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

[0017] The fifth object of the present invention 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] Furthermore, the application is to add the chondroitin synthase mutant or the expression system containing the chondroitin synthase mutant into the reaction system to generate chondroitin.

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

[0020] Furthermore, the recombinant bacteria also overexpressed crystal inclusion protein A, glutamine-fructose-6-phosphate aminotransferase and uridine diphosphate-glucose dehydrogenase.

[0021] The seventh object of the present invention is to provide a method for synthesizing chondroitin, wherein the gene sequence encoding the chondroitin synthase mutant 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, and the recombinant bacterium is fermented and cultured.

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

[0023] Furthermore, 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 as follows:

[0024] MCGIVGAVAQRDVAEILINGLHRLEYRGYDSAGVAVVNDKQELQRIRCLGKVKALDDAVMENPLIGGTGIAHTRWATHGQPSEDNAHPHTSGNFAVVHNGIIENYEELRSELQAKGYVFLSQTDTEVIAHLVEWEMRTASNLLEAVQKVVKQLKGAYGMVVMDRETPQHLVAARSGSPLVIGLGIGENFLASDQLALLSVTRRFMFLEEGDIAEITRRSVDIYDMSGKKVEREVLDSQLANDSAEKGKFRHFMQKEIFEQPTALINTMEGRILHNSVVVESIGNGAKELLSQVEHIQIVACGTSYNAGMVARYWFEDLAGVSCDVEIASEFRYRKFVTRPNSLLITLSQSGETADTLAALRLAKEKGYMGAMTICNVAGSSLVRESDLAFMTRAGVEIGVASTKAFTTQLVTLLMLVTALGKLKGTISAEKESEIIKALHSLPADIEKALAFDSSIESLAEDFAEKHHALFLGRGEYYPIAMEASLKLKEISYIHAEAYAAGELKHGPLALIDADMPVIVVAPTNDLLEKVKSNIEEVRARGGQLYVFADKEAGFVESDGMKIITMPTVNPLVAPIFYTVPMQLLSYHVALIKGTDVDQPRNLAKAVTVE

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

[0026] MNILVTGGAGYIGSHTSLCLLNKGYNVVIIDNLINSSCESIRRIELIAKKKVTFYELNINNEKEVNQILKKHKFDCIMHFAGAKSVAESLIKPIFYYDNNVSGTLQLINCAIKNDVANFIFSSSATVYGESKIMPVTEDCHIGGTLNPYGTSKYISELMIRDIAKKYSD TNFLCLRYFNPTGAHESGMIGESPADIPSNLVPYILQVAMGKLEKLMVFGGDYPTKDGTGVRDYIHVMDLAEGHVAALSYLFRDNNTNYHVFNLGTGKGYSVLELVSTFEKISGVRIPYEIVSRRDGDIAESWSSPEKANKYLNWKAKRELETMLEDAWRWQMKNPNGYI

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

[0028] MKKITIAGAGYVGLSNAVLLAQHHNVILLDIDQNKVDLINNKKSPITDKEMEDFLQNKSLTMMATPDKEVALKNADFVINATPPDYNTETGYFNTSTVEAVIEQTLSINPQAAIIIKSTIPVGFTENMREKFNTPNLIFSPEFLREGKALYDNLYPSRIIVGSTSYQAKVFADMLTQCARKKDVTVLFTHNTEAE AVKLFANTYLAMRVAFFNELDTYASLHHLNTKDIINGISTDPRIGTHYNNPSFGYGGYCLPKDTKQLLANYADVPQNLIEAIVKSNETRKRFITHDVLNKKPKTVGIYRLIMKSGSDNFRASAILDIMPHLKENGVEIVIYEPTLNQQAFEDYPVINQLSEFINRSDVILANRSEPDLNQCSHKIYTRDIFGGDA

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

[0030] GVYIMINDMHPSLIKDKDIVDDVMLRSCKIIAMKVMPDKVMQVMVTVLMHDGVCEEMLLKWNLLDNRGMAIYKVLMEALCAKKDVKISTVGKVGPLGCDYINCVEISM

[0031] Furthermore, the fermentation culture contains a carbon source, a nitrogen source, biotin, an inorganic salt and metal ions.

[0032] Further, the host cell comprises Corynebacterium glutamicum.Two precursors of chondroitin are UDP-glucuronic acid and UDP-galactosamine respectively, but only UDP-glucuronic acid can be synthesized in Corynebacterium glutamicum, but UDP-galactosamine cannot be synthesized.KfoA can catalyze UDP-glucosamine to synthesize UDP-galactosamine, so it is necessary to overexpress kfoA.

[0033] Beneficial effects of the present invention:

[0034] The present invention provides a chondroitin synthase mutant and its application. The present invention first deletes the N-terminal and C-terminal amino acid sequences of the chondroitin synthase shown in SEQID NO.1 and performs compound mutations on the amino acids at positions 200, 279 and 346 to obtain a chondroitin synthase mutant, introduces the chondroitin synthase mutant of the present invention into a plasmid to form a recombinant plasmid, introduces the recombinant plasmid into a recombinant bacterium for fermentation culture, and finds that the chondroitin synthase mutant of the present invention can significantly increase the yield of chondroitin. In addition, the present invention also overexpresses N-acetylglucosamine epimerase, 6-phosphoglucosamine synthetase, 6-phosphoglucosamine synthetase and udg enzyme, and constructs the overexpressed enzyme into a protein polymer through CipA protein, thereby improving the synthesis efficiency of chondroitin, and the chondroitin yield obtained by fermentation in a 5L fermenter is about 10.5g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 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

[0036] Figure 1 It is the metabolic pathway of chondroitin;

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

[0038] Figure 3 It is the characterization of protein aggregates constructed by CipA;

[0039] Figure 4 This is a graph showing the production of chondroitin produced by recombinant Corynebacterium pekinensis in fed-batch fermentation. DETAILED DESCRIPTION

[0040] 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.

[0041] Vector: Corynebacterium pekinense

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

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

[0044] Extraction of chondroitin samples:

[0045] The fermentation liquid was centrifuged to collect the cells, the cells were reconstituted with water, high pressure homogenized and crushed, and the supernatant was collected by centrifugation. Three volumes of ethanol were added to the supernatant, and the precipitate was collected by centrifugation for 4 hours. The precipitate was reconstituted with water, the supernatant was collected by centrifugation, the supernatant was freeze-dried, the supernatant was reconstituted with water, the supernatant was collected by centrifugation, and the above experimental steps were repeated. The obtained sample was dried, reconstituted with water and diluted for testing.

[0046] Determination of chondroitin content:

[0047] Determination by carbazole sulfate method: add 2mL borax sulfate solution (9.54g borax dissolved in 1L concentrated sulfuric acid) to a glass test tube for precooling, add 400μL diluted sample, mix well and heat in boiling water for 15min, take out and cool in ice bath, add carbazole solution (1.25g dissolved in 1L ethanol) and mix well, heat in boiling water for 10min, take out and cool for testing. Configure 0, 10, 20, 30, 40 and 50mg / L glucuronic acid as a standard curve, read the absorbance at 530nm through the above treatment, and calculate the content of glucuronic acid in the sample, and calculate the chondroitin content by multiplying the glucuronic acid content by 2.067.

[0048] The C-terminal amino acid deletion at positions 688-965 of the present invention refers to the amino acids at positions 688-965 calculated from the first amino acid at the N-terminus, and the amino acids at positions 688-965 are located at the C-terminus; the mutations of the amino acids at positions 200, 279, and 346 of the present invention are also calculated from the N-terminus.

[0049] Example 1: Construction of recombinant Corynebacterium glutamicum

[0050] (1) Construction of chondroitin synthesis pathway

[0051] On the basis of the chondroitin synthase pmCS whose amino acid sequence is shown in SEQ ID NO.1, the amino acids at positions 200, 279 and 346 of the chondroitin synthase pmCS were subjected to compound mutation using E200Q-F, E200Q-R, L279E-F, L279E-R, F346E-F and F346E-R to obtain the mutant pmCS1. On the basis of the mutant pmCS1, the chondroitin synthase mutant pmCS1 was truncated using NpmCS1-F, NpmCS1-R, CpmCS1-F and CpmCS1-R as primers respectively to obtain the 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 ligated to plasmid pXMJ (the primers used for chondroitin synthase pmCS were pmCS-F and pmCS-R, and the primers used for N-acetylglucosamine epimerase kfoA were KfoA-F and KfoA-R, and the specific sequences are shown in Table 1). Chondroitin synthase and chondroitin synthase mutants were linked to N-acetylglucosamine epimerase kfoA by GS The plasmids obtained by linker connection were pXMJ-pmCS-kfoA, pXMJ-NpmCS1-kfoA, pXMJ-CpmCS1-kfoA and pXMJ-NCpmCS1-kfoA. The recombinant plasmids were transferred into Corynebacterium pekinensis to obtain recombinant strains BJ-01, BJ-02, BJ-03 and BJ-04, which were fermented and cultured. The chondroitin content results were shown in Figure 2 shown.

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

[0054]

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

[0056] Beijing bacterium was inoculated in 5 mL of BHIS medium and cultured at 30°C. The bacteria were collected and genomic DNA was extracted using a cell genome 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. The two were connected by a GS linker and connected to the plasmid pECXK99E to obtain the plasmid pECXK99E-ugd-glmS. The plasmid pECXK99E-ugd-glmS and plasmid pXMJ-NCpmCS1-kfoA were transformed into Beijing bacterium by electrotransformation. The electrotransformation conditions were a voltage of 1.5 KV and 5 ms (the width of the electric shock cup was 1 mm). The correct recombinant strain BJ-05 was obtained by resistance screening and sequencing and fermentation culture was carried out. The chondroitin yield was as follows: Figure 2 shown.

[0057] Table 2 Primers and their 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 and 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 connected to the C-terminus of the kfoA gene of the plasmid pXMJ-NCpmCS1-kfoA and the C-terminus of the glmS gene on pECXK99E-ugd-glmS, respectively. CipA, kfoA and glmS were connected by GS link to obtain the recombinant plasmids pXMJ-NCpmCS1-kfoA-CipA and pECXK99E-ugd-glmS-CipA.

[0061] Plasmids pXMJ-NCpmCS1-kfoA-CipA and pECXK99E-ugd-glmS-CipA were electroporated into Corynebacterium pekinensis using an electroporator, and the electroporation conditions were 1.5KV, 5ms (electric shock cup width was 1mm). The correct recombinant strain BJ-06 was obtained by resistance screening and sequencing.

[0062] The recombinant strain BJ-06 was selected and inoculated into BHIS liquid medium, cultured at 30°C for 12 hours, and inoculated into a 250 mL shake flask with 25 mL fermentation medium at a 1% inoculation rate. The culture was carried out at 220 rpm and 30°C for 48 hours. The bacterial cells were collected and crushed, and the chondroitin in the strain was purified and determined. The chondroitin yield was as follows: Figure 2 shown.

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

[0064]

[0065] Example 2: Fed-batch fermentation of recombinant Corynebacterium pekinensis

[0066] The recombinant Beijing Corynebacterium BJ-06 constructed in Example 1 was activated on a plate culture medium, and a single clone was selected and inoculated into a BHIS culture medium. The strain was cultured at 30°C for 10 hours, inoculated in a 250mL triangular shake flask with an inoculum size of 1%, cultured for 10 hours, and then inoculated in a 5L fermenter with an inoculum size of 20%. The fermenter was filled with 2.5L of liquid, the culture temperature was 30°C, the stirrer speed was 300rpm, and the ventilation volume was 3vvm. The pH of the fermentation process was controlled between 6.5-7, and the glucose content was controlled at 10g / L. After 3 hours of inoculation, 0.5mM IPTG and 0.2g / L biotin were added, and ammonia water was used to maintain pH stability during the fermentation process. Figure 4 As shown, the yield of chondroitin produced by the recombinant Corynebacterium Beijingensis constructed by the present invention reaches 10 g / L.

[0067] 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 chondroitin synthase mutant, characterized in that The chondroitin synthase mutant is one or more of the following modifications to the chondroitin synthase with the amino acid sequence shown in SEQ ID NO.1: (a) N-terminal amino acids 2-117 are missing; (b) C-terminal amino acids 688-965 are missing; (c) The glutamic acid at position 200 was mutated to glutamine, the leucine at position 279 was mutated to glutamic acid, and the phenylalanine at position 346 was mutated to glutamic acid.

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

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

4. A recombinant cell expressing the chondroitin 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 chondroitin 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 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 according to claim 1.

8. The recombinant bacterium 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.

9. A method for synthesizing chondroitin, characterized in that: The gene sequence encoding the chondroitin synthase mutant according to 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, and the recombinant bacterium is fermented and cultured to synthesize chondroitin.

10. The method according to claim 9, characterized in that The recombinant plasmid is introduced into a host cell to form a recombinant bacterium, and the recombinant bacterium is fermented and cultured to synthesize chondroitin; wherein the recombinant plasmid includes a first recombinant plasmid and a second recombinant plasmid, the first recombinant plasmid contains a gene sequence encoding the chondroitin synthase mutant according to claim 1, a gene sequence of N-acetylglucosamine epimerase, and a gene sequence of crystalline 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 crystalline inclusion protein A.

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