Recombinant escherichia coli for producing chondroitin sulfate A and application thereof

Abstract

The invention provides recombinant escherichia coli for producing chondroitin sulfate A and application of the recombinant escherichia coli, and relates to the technical field of genetic engineering. According to the recombinant escherichia coli disclosed by the invention, chondroitin synthase, chondroitin 4-O-sulfotransferase, N-acetyl glucosamine epimerase, UDP-glucose dehydrogenase and chondroitin sulfate degrading enzyme are overexpressed; wherein the amino acid sequence of the chondroitin sulfate degrading enzyme is as shown in SEQ ID NO. 5. The recombinant escherichia coli is inoculated into a culture medium for fermentation culture, the average molecular weight of the obtained chondroitin sulfate A is 800Da, and the yield is about 12.5 g / L. Therefore, low-molecular-weight chondroitin sulfate A can be obtained and the yield of chondroitin sulfate A can be increased by fermenting the recombinant escherichia coli disclosed by the invention.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant Escherichia coli producing chondroitin sulfate A and an application thereof. Background Art

[0002] Chondroitin sulfate is an important polysaccharide compound that is widely found in the cartilage, bones and skin of animals. It has a variety of biological functions, including promoting cartilage repair, anti-inflammation, lubricating joints, anti-coagulation and lowering blood lipids. The functions and applications of chondroitin sulfate are closely related to its molecular weight. High molecular weight chondroitin sulfate has good biological stability and lubrication properties, and is suitable for the field of joint health. It can effectively promote cartilage repair and reduce joint inflammation, and improve joint function. Low molecular weight chondroitin sulfate has higher bioavailability and permeability, and is more easily absorbed by cells. Therefore, it performs better in anti-inflammation, promoting wound healing and skin moisturizing, and is often used in cosmetics and topical drugs. Chondroitin sulfates of different molecular weights play an important role in the fields of health care, cosmetics and pharmaceuticals through their unique physical and chemical properties and biological activities. Low molecular weight chondroitin sulfate has broad application prospects in the fields of health care, cosmetics and pharmaceuticals due to its high bioavailability and versatility, and the market demand continues to grow.

[0003] At present, low molecular weight chondroitin sulfate is mainly degraded into low molecular weight fragments by enzymatic, chemical and physical degradation. Chemical degradation is carried out by degradation enzymes or acid-base treatment or oxidation-reduction, and physical degradation is carried out by ultrasound and high-pressure homogenization. In the production process, ultrafiltration, dialysis and ion exchange chromatography are widely used for purification and concentration to ensure the high purity and biological activity of the product. With the development of biotechnology and the improvement of environmental awareness, the development of green and low-cost production methods has gradually become the mainstream. Therefore, the development of microbial direct fermentation synthesis of low molecular weight chondroitin sulfate is crucial to promote the widespread application of low molecular weight chondroitin sulfate and its derivative materials in more fields.

[0004] The patent with the announcement number CN 112708571 B discloses a recombinant yeast for fermenting and producing chondroitin sulfate with controllable molecular weight and its application. The invention heterologously expresses chondroitin sulfate synthesis pathway related proteins in cells: chondroitin synthase KfoC, UDP-N-acetylglucosamine C4 isomerase KfoA, chondroitin 4-O-sulfotransferase C4ST, UDP-glucose dehydrogenase TuaD and chondroitin sulfate lyase ABCI, and obtains a production strain for synthesizing chondroitin sulfate A. The molecular weight of the chondroitin sulfate A synthesized in the patent is 1.2kDa-40kDa, and methanol is required to induce the expression of chondroitin sulfate lyase ABCI to control the molecular weight of chondroitin sulfate A. The synthesis process is complicated and toxic, and the output of chondroitin sulfate A is about 2.3g / L, which is relatively low. 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 that there is a lack of a recombinant bacterium that can efficiently synthesize low molecular weight chondroitin sulfate A and has a simple and green synthesis process.

[0006] In order to solve the above technical problems, the present invention provides a recombinant Escherichia coli producing chondroitin sulfate A and its application. The recombinant Escherichia coli of the present invention overexpresses N-acetylglucosamine epimerase kfoA, UDP-glucose dehydrogenase ugd, chondroitin synthase kfoC, chondroitin 4-O-sulfotransferase C4ST and cartilage sulfate degrading enzyme CHase, and the recombinant Escherichia coli of the present invention is fermented and cultured to generate chondroitin sulfate A with an average molecular weight of 800Da. Chondroitin synthase can catalyze the connection between glucuronic acid and N-acetylgalactosamine to generate chondroitin, chondroitin 4-O-sulfotransferase is used to sulfate chondroitin to generate chondroitin sulfate A, and then chondroitin sulfate degrading enzyme is used to degrade chondroitin sulfate A into low molecular weight chondroitin sulfate, and finally chondroitin sulfate A with an average molecular weight of about 800Da is obtained and the yield is about 12.5g / L.

[0007] The first object of the present invention is to provide a recombinant Escherichia coli producing chondroitin sulfate A, wherein the recombinant Escherichia coli is transformed using Escherichia coli as a host, and the transformation includes: overexpressing chondroitin synthase, chondroitin 4-O-sulfotransferase, N-acetylglucosamine epimerase, UDP-glucose dehydrogenase and chondroitin sulfate degrading enzyme; wherein the amino acid sequence of the chondroitin sulfate degrading enzyme is shown in SEQ ID NO.5.

[0008] Further, SEQ ID NO.5 is as follows:

[0009] MKIVNKFLVMPFLLTGFLSFSQIKNNHPARESFENETSIIHFKKDNSSELSISNKHYQFGKSSLKWEWTKGGFFETSDFRILTKKESPLSYGDFFPASPTLVISLYNEKAQEDTIKISFEKAGKEEVWFPVKLDFTGWRTIRVPFFEMSGNAPKKEDEIAIYDTFRVSSSTAKSKGELFFDDIVFSQYMDDRHPYPDVMVPFIKRDRQNGEDHWMPLTRNMQRIYDLSLKSVSETKKKDLALIEKRLDDTFIKDGGKNNNLSKAKEEYARLKLVKSETVLGPPLTFVIDEVYFDKEDVNRKSYNSVSDFGKSLKQIANYYLQSTDENRIEIEDLFITSTRYFLDQGWQEGTSGGTRHHIGYALRELVDAFYMMRAPLKKAGLLNEVGNSLQWVFNLGKVLGPENEFEANIDYFNTQSFYHLKLIFMSDNAEKQAALLDGYSNYISKTLTQNNELGVFKIDGTSWHHGGHYPAYGMGAFASVPQLIFILSGTQFRIDEAGHKNFKKSLLTTRLYSQLLDFGFGNAGRHPFEDNSI KSLKKSFLLMARSGNPDGTSKIDKEVASAYLRLWGDQDKVNSKEFKSLYAVGEDILPGYHVLPYAATAIHRRNNWAAIIKGYSKYVWSSEIYVDANRYGRYPSNGSIQINNIGGDAASGFKQEGWDWNRYPGTTVIDLPFKELEPNTELIMFRSDETFAGAVTLGNNGVFGMKLNEAKGSNADGTRENIGFPGKLKANKSVFSFGDKLICIGTGISSIDTIHPVQTNLFQNFLGDTSMPVFSSENKSISTFPYQSSMEVTGNSGKWLIDAYQNGYYILSSDKIEIRRQSQKSYNNAYSVNTGKMNPKAKNDTVTKGDYASAWIEHKKAPKEASYKYVIYPGLQRDAVNSFENKVKNDKSYQVIRADNVAHIVKDNETSTTGFVIFDTAKELDAILESVSVPSLVMIKNEATTITISAVQPDLNFVEKDKKFNNYSMPVELTITLKGKWKLKQNLMVKSIWVAGNNTITMECRHGFSNQIDLMK

[0010] Furthermore, the amino acid sequence of the chondroitin synthase is shown in SEQ ID NO.1, the amino acid sequence of the chondroitin 4-O-sulfotransferase is shown in SEQ ID NO.4, the amino acid sequence of the N-acetylglucosamine isomerase is shown in SEQ ID NO.2, and the amino acid sequence of the UDP-glucose dehydrogenase is shown in SEQ ID NO.3.

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

[0012] MSILNQAINLYKNKNYRQALSLFEKVAEIYDVSWVEANIKLCQTALNLSEEVDKLNRKAVIDIDAATKIMCSNAKAISLNEVEKNEIISKYREITAKKSERAELKEVEPIPLDWPSDLTLPPLPESTNDYVWAGKRKELDDYPRKQLIID GLSIVIPTYNRAKILAITLACLCNQKTIYDYEVIVADDGSKENIEEIVREFESLLNIKYVRQKDYGYQLCAVRNLGLRAAKYNYVAILDCDMAPNPLWVQSYMELLAVDDNVALIGPRKYIDTSKHTYLDFLSQ KSLINEIPEIITNNQVAGKVEQNKSVDWRIEHFKNTDNLRLCNTPFRFFSGGNVAFAKKWLFRAGWFDEEFTHWGGEDNEFGYRLYREGCYFRSVEGAMAYHQEPPGKENETDRAAGKNITVQLLQQKVPYFYR KKEKIESATLKRVPLVSIYIPAYNCSKYIVRCVESALNQTITDLEVCICDDGSTDDTLRILQEHYANHPRVRFISQKNKGIGSASNTAVRLCRGFYIGQLDSDDFLEPDAVELCLDEFRKDLSLACVYTTNRNI DREGNLISNGYNWPIYSREKLTSAMICHHFRMFTARAWNLTEGFNESISNAVDYDMYLKLSEVGPFKHINKICYNRVLHGENTSIKKLDIQKENHFKVVNESLSRLGIKKYKYSPLTNLNECRKYTWEKIENDL

[0013] Further, SEQ ID NO.2 is as follows:

[0014] MNILVTGGAGYIGSHTSLCLLNKGYNVVIIDNLINSSCESIRRIELIAKKKVTFYELNINNEKEVNQILKKHKFDCIMHFAGAKSVAESLIKPIFYYDNNVSGTLQLINCAIKNDVANFIFSSSATVYGESKIMPVTEDCHIGGTLNPYGTSKYISELMIRDIAKKYSDTNFLCLRYFNPTGAHESGMIGESPADIPSNLVPYILQVAMGKLEKLMVFGGDYPTKDGTGVRDYIHVMDLAEGHVAALSYLFRDNNTNYHVFNLGTGKGYSVLELVSTFEKISGVRIPYEIVSRRDGDIAESWSSPEKANKYLNWKAKRELETMLEDAWRWQMKNPNGYI

[0015] Furthermore, SEQ ID NO.3 is as follows:

[0016] MRMTVIGTGYLGATHAACMAELSHEVLGVDVDEAKIASLKDSKVPFFEPGLPEVLERNLENGRLNFTTDYAEAAAFAQVHFLGVGTPQQKGTYAADLTYVRQVVEDLVPLLEGEHIIFGKSTVPVGTAEQLQELADSLVKPGSHVEIAWNPEFLREGYAVKDTITPDRIVVGVREGATAEAIAREVYATAIAADTPFLVTDLATAELVKVSANAFLATKISFINAVAEICEQTGADVVALADAIGHDDRIGRKFLGAGLGFGGGCLPKDIRAFMARAGELGADQALTFLREVDSINMRRRDRVVQLAKEMCGGSLLGKRVTVLGAAFKPNSDDVRDSPALSVA GSLSLQGAAVSVYDPEAMDNARRVFPTLSYASSTKEALIDAHLVVLATEWQEFRDLDPEVAGGVVEKRAIIDGRNVLDVAKWKAAGWEMEALGRNL

[0017] Furthermore, SEQ ID NO.4 is as follows:

[0018] MKPALLEVMRMNRICRMVLATCLGSFILVIFYFQSMLHPVIRRNPFGMDICCRKGSRSPLQELYNPIQLELSNTAILHQMRRDQVTDTCRANSAMSRKRRVLTPNDLKHLVVDEDHELIYCYVPKVACTNWKRLMMVLSGRGKYSDPMEIPANEAHVSANLKTLNQYSIPEINHRL KSYMKFLFVREPFERLVSAYRNKFTQKYNTSFHKRYGTKIIRRQRKNATQEALRKGDDVKFEEFVAYLIDPHTQREEPFNEHWQTVHSLCHPCHIHYDLVGKYETLEEDSNYVLQLAGVSGYLKFPTYAKSTRTTDEMTTEFFQNISAEHQTQLYEVYKLDFLMFNYSVPNYLKLE

[0019] Furthermore, the Escherichia coli includes Escherichia coli K12.

[0020] The second object of the present invention is to provide a method for producing chondroitin sulfate A, wherein the recombinant Escherichia coli is inoculated into a culture medium for fermentation to obtain the chondroitin sulfate A.

[0021] Furthermore, the fermentation temperature is 20-45° C. Temperature affects the growth of recombinant E. coli, which can grow at 20-45° C., and the optimal growth temperature is 37° C. Preferably, the fermentation temperature is 35-39° C.

[0022] Furthermore, the pH of the fermentation culture is 4-9.

[0023] Furthermore, the inoculation amount of the recombinant Escherichia coli is 8-12% by volume.

[0024] Furthermore, the fermentation medium includes a carbon source, a nitrogen source, an inorganic salt and metal ions.

[0025] Furthermore, the molecular weight of the chondroitin sulfate A is 800Da-1500Da.

[0026] The third object of the present invention is to provide an application of the above-mentioned recombinant Escherichia coli in the preparation of chondroitin sulfate A.

[0027] Beneficial effects of the present invention:

[0028] The recombinant Escherichia coli of the present invention overexpresses chondroitin synthase, chondroitin 4-O-sulfotransferase, N-acetylglucosamine epimerase, UDP-glucose dehydrogenase and chondroitin sulfate degrading enzyme; wherein the amino acid sequence of the chondroitin sulfate degrading enzyme is shown in SEQ ID NO.5. The recombinant Escherichia coli of the present invention is inoculated in a culture medium for fermentation and culture, and the average molecular weight of the obtained chondroitin sulfate A is 800Da, and the yield is about 12.5g / L. Therefore, fermentation with the recombinant Escherichia coli of the present invention can obtain low-molecular-weight chondroitin sulfate A and can increase the yield of chondroitin sulfate A. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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

[0030] Figure 1 is the chondroitin synthesis output;

[0031] Figure 2 is the synthesis yield of chondroitin sulfate;

[0032] Figure 3 It is the spectrum of chondroitin sulfate;

[0033] Figure 4 is the molecular weight distribution diagram of chondroitin sulfate;

[0034] Figure 5 It is the yield of low molecular weight chondroitin sulfate fermented by recombinant E. coli in a 5L fermenter;

[0035] Figure 6 It is the molecular weight distribution diagram of low molecular weight chondroitin sulfate. DETAILED DESCRIPTION

[0036] 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.

[0037] Bacterial strain: Escherichia coli K12;

[0038] Plasmids: pXMJ19, pk18mobsacb;

[0039] LB medium: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L;

[0040] BHI: brain heart infusion 37 g / L, sorbitol 91 g / L;

[0041] Fermentation medium: KH2PO4 13.5, (NH4)2HPO4 4.0, citric acid 1.7, MgSO4 1.4, thiamine 0.1, CaCl2 2.0, ZnSO4·7H2O 2.2, MnSO4·4H2O 0.5, FeSO4·7H2O 10.0, NaB4O7·10H2O 0.02, CuSO4 5H2O 1.0, (NH4)Mo7O 24 4H2O 0.1, glucose 20.0, adjust pH to 7.0.

[0042] Preparation of competent E. coli: First, pick a single colony from a fresh plate, inoculate it into 5 mL of LB medium, and culture it at 37°C overnight with shaking. The next day, take 1 mL of overnight cultured bacteria and add it to 100 mL of fresh LB medium, and culture it at 37°C with shaking until OD 600 It is about 0.4-0.6. Transfer the culture solution to a centrifuge tube, centrifuge at 4°C and 4000rpm for 10 minutes, and discard the supernatant. Resuspend the bacteria with ice-cold 0.1M CaCl2 solution to a final volume of about 1 / 10 of the original culture volume, and then place the bacterial solution on ice for 30 minutes. Centrifuge again at 4°C and 4000rpm for 10 minutes, discard the supernatant, and resuspend the bacteria with ice-cold 0.1M CaCl2 solution to a final volume of about 1 / 100 of the original culture volume. Finally, divide the resuspended bacterial solution into sterile 1.5mL centrifuge tubes, about 100μL per tube, and quickly store them in a -80°C refrigerator for subsequent transformation experiments.

[0043] Purification of chondroitin and chondroitin sulfate: collect the fermentation liquid, centrifuge at 10000rpm for 5min. Take an appropriate amount of supernatant, add 4 times the volume of anhydrous ethanol, place at 4℃, precipitate overnight, centrifuge at 10000rpm for 5min, and discard the supernatant. After the ethanol evaporates, add the original volume of water to resuspend, fully dissolve, centrifuge at 10000rpm for 10min, collect the supernatant, and repeat the above operation. After the second alcohol precipitation, collect the supernatant, which is the purified hyaluronic acid sample.

[0044] Determination of the production of chondroitin and chondroitin sulfate: Weigh 4.77 g of sodium tetraborate decahydrate and dissolve it in 500 mL of concentrated sulfuric acid to prepare a borax-sulfuric acid solution; dissolve 1.25 g of carbazole in 500 mL of anhydrous ethanol to prepare a carbazole solution; prepare a 1 g / L glucuronic acid solution.

[0045] Take the purified chondroitin or chondroitin sulfate sample, dilute it 10-100 times, draw 200 μL into a glass colorimetric tube, add 1 mL of borax sulfuric acid solution at the same time, mix well and place in a boiling water bath for 15 minutes, and cool on ice. Add 50 μL of carbazole solution, mix well and place in a boiling water bath for 10 minutes. Take 200 μL of the reaction solution to a 96-well transparent plate, and use an enzyme reader to measure the absorbance of the sample at a wavelength of 530 nm. Dilute the 1g / L glucuronic acid solution to 0, 10, 20, 30, 40, 50 mg / L in a gradient manner. After the borax sulfuric acid-carbazole color development reaction, measure the absorbance at 530 nm. With the absorbance value as the horizontal coordinate and the glucuronic acid concentration (mg / L) as the vertical coordinate, draw a relationship curve between the absorbance value and the glucuronic acid concentration to obtain the standard curve equation: y=121.7x-6.035, R 2 =0.999.

[0046] Substitute the sample absorbance value measured above into the standard equation to calculate the sample content of chondroitin or chondroitin sulfate. Chondroitin / chondroitin sulfate content calculation formula: Chondroitin / chondroitin sulfate content (g / L) = (concentration measured by the standard curve * dilution factor * 2.067) / 1000.

[0047] Example 1: Construction and strengthening of chondroitin synthesis pathway

[0048] (1) Construction of chondroitin synthesis pathway

[0049] According to the gene sequence of Escherichia coli K4 chondroitin synthase, the gene sequence of chondroitin synthase kfoC was synthesized by GENEWIZ (Suzhou), and the amino acid of kfoC is shown in SEQ ID NO.1. Primers were designed to use the synthesized kfoC gene sequence as a template for PCR amplification, and then kfoC was connected to the plasmid pET28A by the T5 exonuclease-dependent assembly (TEDA) method to construct a recombinant expression plasmid pET28A-kfoC, which was transformed into Escherichia coli to obtain the recombinant strain KfoC-1. The recombinant strain KfoC-1 was inoculated in a shake flask for culture, and the chondroitin production results were shown in Figure 1 shown.

[0050] (2) Enhancement of UDP-N-acetylgalactosamine synthesis pathway

[0051] Primers kfoA-F, kfoA-R and ugd-F, ugd-R were designed to amplify N-acetylglucosamine epimerase kfoA (shown in SEQ ID NO.2) and UDP-glucose dehydrogenase ugd (shown in SEQ ID NO.3) and ligated to plasmid pET28A-kfoC to obtain recombinant plasmid pET28A-kfoC-kfoA-ugd. The recombinant plasmid pET28A-kfoC-kfoA-ugd was transformed into Escherichia coli to obtain recombinant strain KfoC-2. The recombinant strain KfoC-2 was inoculated in a shake flask for culture, and the chondroitin production results were shown in FIG. Figure 1 shown.

[0052] Example 2: Construction of chondroitin sulfonation modification system

[0053] (1) Construction of recombinant Corynebacterium glutamicum

[0054] According to the genomic sequence of chondroitin 4-O-sulfotransferase in Peromyscus maniculatus bairdii, the gene sequence of chondroitin 4-O-sulfotransferase C4ST was synthesized by GENEWIZ (Suzhou), and the amino acid of C4ST is shown in SEQ ID NO.4. Primers C4ST-F and C4ST-R were designed to use the synthesized C4ST gene sequence as a template for PCR amplification, and then C4ST and were connected to the plasmid pET28A-kfoC-kfoA-ugd by the TEDA method to construct a recombinant plasmid pET28A-kfoC-kfoA-ugd-C4ST, which was then transformed into Escherichia coli to obtain the recombinant strain KfoC-3. The recombinant strain KfoC-3 was inoculated in a shake flask for culture, and the chondroitin sulfate production was measured as shown in the following results. Figure 2 shown.

[0055] (2) Chondroitin sulfate detection:

[0056] In order to determine whether the chondroitin sulfate produced by the fermentation culture of the recombinant strain KfoC-3 is chondroitin sulfate A, first, a certain amount of chondroitin sulfate sample in step (1) is taken, 5 μL of chondroitin sulfate degrading enzyme CHase is added, and the sample is placed in a 37°C water bath for 10 hours to fully cleave the chondroitin sulfate. Subsequently, the cleaved solution is placed at 90°C for 10 minutes to inactivate and denature the protein, and after cooling to room temperature, centrifuged (10000rpm, 10 minutes), and the supernatant is taken for use. Next, LC-MS detection is performed using a HILIC column (3 μm, 2.0×150mm, YMC, Japan), the eluent A is ultrapure water, the eluent B is acetonitrile, and the elution gradient is set as follows: 0-2 minutes 90% B, 2-8 minutes 90% B reduced to 50% B, 8-12 minutes 50% B, 12-13 minutes back to 90% B. The column temperature was maintained at 35°C, the flow rate was 0.2 mL / min, and the mass range of m / z 100-800 was scanned and monitored in negative ion mode. Figure 3 The results showed that the mass-to-charge ratio of the disaccharide molecule of chondroitin sulfate in negative ion mode was 458.2, indicating that chondroitin sulfate A was successfully synthesized.

[0057] Example 3: One-step fermentation synthesis of low molecular weight chondroitin sulfate

[0058] (1) Construction of low molecular weight chondroitin sulfate synthesis strain

[0059] According to the genomic sequence of cartilage sulfate degrading enzyme from Pseudarcicella hirudinis E92, the gene sequence of cartilage sulfate degrading enzyme CHase was synthesized by GENEWIZ (Suzhou), and the amino acid of CHase is shown in SEQ ID NO.5. Primers CHase-F and CHase-R were designed to use the synthesized CHase gene sequence as a template for PCR amplification, and then CHase and were connected to the plasmid pET28A-kfoC-kfoA-ugd-C4ST by the TEDA method to construct a recombinant expression plasmid pET28A-kfoC-kfoA-ugd-C4ST-CHase, which was then transformed into Escherichia coli to obtain the recombinant strain KfoC-4. The recombinant strain KfoC-4 was inoculated in a shake flask for culture, and the chondroitin sulfate yield and molecular weight results are shown in Figure 5. Figure 2 and Figure 4 shown.

[0060] (2) Synthesis of low molecular weight chondroitin sulfate in a 5L fermenter

[0061] The constructed recombinant Escherichia coli KfoC-4 was activated on the plate culture medium, and a single clone was selected and inoculated into LB culture medium. The strain was cultured at 37°C for 10 hours, inoculated in a 250mL triangular shake flask with an inoculation size of 1%, cultured for 10 hours, and then inoculated in a 5L fermenter with an inoculation size of 10%. The fermenter was filled with 2.5L of liquid, the culture temperature was 37°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, 0.75mM IPTG was added, and ammonia water was used to maintain pH stability during the fermentation process. The glucose consumption and OD during the fermentation process were 600 and low molecular weight chondroitin sulfate content, the results are as follows Figure 5 As shown. The molecular weight and distribution were further detected by high performance size exclusion chromatography (HPSEC) using an Agilent 1260 system, an Ultrahydrogel Linearcolumn (7.8×300mm) column, a 0.1mol / L NaNO3 solution as the mobile phase, a flow rate of 0.9mL / min, a column temperature of 40°C, and an injection volume of 30μL. Based on the elution volume of the dextran standard sample, a standard curve between molecular weight and elution volume was prepared, the elution volume of the sample was measured, and the molecular weight distribution of the sample was calculated. The results are shown in Figure 6 As shown, the average molecular weight is 800 Da.

[0062] 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 recombinant Escherichia coli producing chondroitin sulfate A, characterized in that: The recombinant Escherichia coli is transformed using Escherichia coli as a host, and the transformation includes: overexpressing chondroitin synthase, chondroitin 4-O-sulfotransferase, N-acetylglucosamine epimerase, UDP-glucose dehydrogenase and chondroitin sulfate degrading enzyme; wherein the amino acid sequence of the chondroitin sulfate degrading enzyme is shown in SEQ ID NO.

5.

2. The recombinant Escherichia coli according to claim 1, characterized in that The amino acid sequence of the chondroitin synthase is shown in SEQ ID NO.1, the amino acid sequence of the chondroitin 4-O-sulfotransferase is shown in SEQ ID NO.4, the amino acid sequence of the N-acetylglucosamine epimerase is shown in SEQ ID NO.2, and the amino acid sequence of the UDP-glucose dehydrogenase is shown in SEQ ID NO.

3.

3. The recombinant Escherichia coli according to claim 1, characterized in that The Escherichia coli includes Escherichia coli K12.

4. A method for producing chondroitin sulfate A, characterized in that: The method comprises inoculating the recombinant Escherichia coli according to any one of claims 1 to 3 into a culture medium for fermentation to obtain the chondroitin sulfate A.

5. The method according to claim 4, characterized in that The temperature of the fermentation culture is 20-45°C.

6. The method according to claim 4, characterized in that The pH of the fermentation culture is 4-9.

7. The method according to claim 4, characterized in that The inoculation amount of the recombinant Escherichia coli is 8-12% by volume.

8. The method according to claim 4, characterized in that The culture medium comprises a carbon source, a nitrogen source, inorganic salts and metal ions.

9. The method according to claim 4, characterized in that The molecular weight of the chondroitin sulfate A is 800Da-1500Da.

10. Use of the recombinant Escherichia coli according to any one of claims 1 to 3 in the preparation of chondroitin sulfate A.

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