Escherichia coli Nissle 1917 producing food-grade hyaluronan disaccharide and its application

Abstract

The present invention relates to an Escherichia coli Nissle 1917 producing food-grade hyaluronan disaccharide and its application, belonging to the field of genetic engineering technology. After knocking out the heparin precursor synthase gene cluster in Escherichia coli Nissle 1917, the present invention uses endogenous stealth plasmids pMUT1 and pMUT2 to heterologously express hyaluronan synthase from Pasteurella multocida and hyaluronan lyase from Proteus vulgaris, and strengthens UDP-glucuronic acid and UDP-N-acetylglucosamine synthesis pathways by overexpressing UDP-glucose dehydrogenase and phosphoglucosamine mutase from Corynebacterium glutamicum ATCC13032. The obtained recombinant Escherichia coli fermentation product is hyaluronan disaccharide with uniform molecular weight distribution, and the efficient mass production of food-grade hyaluronan disaccharide is realized, and the output of 5L fermentation tank reaches 15g / L, which solves the problems of high production cost and complicated production process of hyaluronan disaccharide in the prior art, and is conducive to the application and promotion of hyaluronan disaccharide in the field of food.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to an Escherichia coli Nissle 1917 for producing food-grade hyaluronic acid disaccharide and application thereof. Background Art

[0002] Hyaluronic acid (HA) is a high molecular weight linear mucopolysaccharide widely distributed in human skin, joints, eyes and connective tissue. Based on a variety of specific and nonspecific mechanisms, HA participates in important metabolic and physiological processes in the human body, including promoting skin hydration, regulating intestinal microbial balance, and participating in cell repair and regeneration. However, with age, the level of HA in the human body gradually decreases, and by the age of 60, its content is only 25% of the normal level. The loss of HA can cause a series of health problems such as dry skin, sagging, and body aging. In view of the excellent moisturizing ability of HA, oral supplementation of HA to improve skin hydration, increase skin elasticity and reduce wrinkles has become an effective anti-aging method. In addition, HA can also interact with intestinal microorganisms to promote the growth of probiotics in the intestine. Therefore, how to supplement HA safely and efficiently has received widespread attention.

[0003] As the basic building block of hyaluronic acid, hyaluronan disaccharide has multiple effects such as moisturizing, lubricating and promoting wound healing, and is widely used in the fields of cosmetics, medicine and food. The preparation of hyaluronan disaccharide mainly adopts the enzymatic degradation method, in which hyaluronic acid is degraded by hyaluronidase to obtain the target product. However, the existing preparation methods may introduce impurities during the enzymatic degradation process, such as enzyme proteins and nucleic acids that are not completely removed. These impurities may pose a potential threat to food safety; at the same time, the cost of enzymes is high, and the stability and activity are easily affected by environmental factors, resulting in fluctuations in degradation efficiency and product purity, which in turn affects the quality and safety of hyaluronan disaccharide. Therefore, the development of safer, more efficient and more economical preparation methods to ensure the food safety and stable quality of hyaluronan disaccharide is the focus and challenge of current research.

[0004] In synthetic biology applications, plasmids usually require the selection of antibiotics to be stably inherited in cells, but the presence of antibiotics can lead to an imbalance in the human intestinal microbial community, thus limiting the application of engineered probiotics carrying plasmid vectors in intestinal treatment and oral supplements. In response to the problem of antibiotic contamination, antibiotic-free expression systems are gradually being used in the production of food-grade functional products. Among them, stealth plasmids are used as functional gene expression vectors to maintain the genetic stability of engineered plasmids without the need for antibiotics, while using their high copy number characteristics to achieve efficient synthesis of products. Therefore, the development of efficient biosynthesis technology for food-grade hyaluronic acid combined with antibiotic-free expression systems is of great significance for expanding the application scope of hyaluronic acid and its derivatives in the food industry. It helps to promote the innovative application of hyaluronic acid in more health and beauty fields. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an Escherichia coli Nissle 1917 for producing food-grade hyaluronan disaccharide and its application. The present invention uses endogenous stealth plasmids pMUT1 and pMUT2 to heterologously express hyaluronan synthase from Pasteurella multocida and hyaluronan lyase from Proteus vulgaris in Escherichia coli Nissle 1917, and strengthens the UDP-glucuronic acid and UDP-N-acetylglucosamine synthesis pathways by overexpressing UDP-glucose dehydrogenase and phosphoglucosamine mutase from Corynebacterium glutamicum ATCC13032, and the obtained recombinant Escherichia coli fermentation product is hyaluronan disaccharide with uniform molecular weight distribution.

[0006] The first object of the present invention is to provide a recombinant Escherichia coli for producing hyaluronan disaccharide, wherein hyaluronan synthase and hyaluronan lyase are heterologously expressed in Escherichia coli Nissle 1917, and UDP-glucose dehydrogenase is overexpressed, wherein the hyaluronan synthase is derived from Pasteurella multocida, the hyaluronan lyase is derived from Proteus vulgaris, and the UDP-glucose dehydrogenase is derived from Corynebacterium glutamicum ATCC13032.

[0007] Furthermore, the nucleotide sequence of the hyaluronan synthase is shown in SEQ ID NO.1, and the nucleotide sequence of the hyaluronan lyase is shown in SEQ ID NO.2.

[0008] Furthermore, the hyaluronan synthase is expressed using the pMUT1 plasmid, and the hyaluronan lyase is expressed using the pMUT2 plasmid.

[0009] Furthermore, the UDP-glucose dehydrogenase is expressed using pMUT2 plasmid.

[0010] Furthermore, the recombinant Escherichia coli overexpresses phosphoglucosamine mutase.

[0011] Furthermore, the pMUT2 plasmid is used to express the phosphoglucosamine mutase.

[0012] UDP-glucose dehydrogenase and phosphoglucosamine mutase are the key rate-limiting enzymes in the synthesis pathways of UDP-glucuronic acid and UDP-N-acetylglucosamine, respectively. The production of hyaluronic acid was increased by overexpressing UDP-glucose dehydrogenase and phosphoglucosamine mutase.

[0013] Furthermore, the above-mentioned overexpression is achieved by increasing the gene copy number.

[0014] Furthermore, the phosphoglucomutase is derived from Corynebacterium glutamicum ATCC13032.

[0015] Furthermore, the recombinant Escherichia coli is knocked out of heparin precursor synthase.

[0016] Heparin precursor and hyaluronic acid have the same precursor substance. By knocking out the endogenous heparin precursor synthase in Escherichia coli, the synthetic heparin precursor is prevented from affecting the determination of hyaluronic acid production and molecular weight.

[0017] Furthermore, the heparin precursor synthase is encoded by a gene cluster consisting of two genes, and the nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0018] The second object of the present invention is to provide a microbial preparation comprising the above-mentioned recombinant Escherichia coli.

[0019] The third object of the present invention is to provide the use of the above-mentioned recombinant Escherichia coli or the above-mentioned microbial preparation in the preparation of hyaluronic acid disaccharide.

[0020] The fourth object of the present invention is to provide a method for preparing hyaluronic acid disaccharide, wherein the above-mentioned recombinant Escherichia coli or the above-mentioned microbial preparation is fermented and cultured.

[0021] Furthermore, IPTG was added during the fermentation process to induce expression.

[0022] Further, the concentration of IPTG was 0.2 mM.

[0023] Furthermore, the fermentation culture temperature condition is 15-45°C, and the fermentation time is 24-72 hours.

[0024] Preferably, the temperature condition of the fermentation culture is 37°C.

[0025] Furthermore, the pH condition of the fermentation culture is 6.5-7.5.

[0026] Beneficial effects of the present invention:

[0027] (1) By heterologously expressing hyaluronan synthase and hyaluronan lyase in Escherichia coli Nissle 1917, the present invention constructs a recombinant Escherichia coli capable of efficiently producing hyaluronan disaccharide, wherein hyaluronan synthase is responsible for synthesizing hyaluronan, and hyaluronan lyase is responsible for degrading it into disaccharides, thereby achieving direct production of hyaluronan disaccharide;

[0028] (2) By overexpressing UDP-glucose dehydrogenase and phosphoglucosamine mutase, the synthesis pathways of UDP-glucuronic acid and UDP-N-acetylglucosamine were strengthened, and the yield of hyaluronic acid was increased. The yield of the final product hyaluronan disaccharide reached 15 g / L in a 5L fermenter;

[0029] (3) The non-resistant stealth plasmids pMUT1 and pMUT2 were used to express foreign proteins and construct an antibiotic-free expression system. While maintaining the genetic stability of the plasmids, the chassis strain Escherichia coli Nissle 1917 was used to achieve food-grade production of hyaluronan disaccharide, which will help promote the application of hyaluronan disaccharide in food-related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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

[0031] Figure 1 is a schematic diagram of the antibiotic-free expression system involved in the present invention;

[0032] Figure 2 This is a graph showing the effect of strengthening the synthesis pathway alone on the yield of food-grade hyaluronic acid in Example 2 of the present invention;

[0033] Figure 3 This is a graph showing the effect of the combined enhancement of the synthetic pathways on the yield of food-grade hyaluronic acid in Example 2 of the present invention;

[0034] Figure 4 This is a graph of the yield of food-grade hyaluronic acid in a 5L fermenter in fed-batch fermentation in Example 2 of the present invention;

[0035] Figure 5 This is the mass spectrum of food-grade hyaluronic acid disaccharide in Example 3 of the present invention. 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 Nissle 1917;

[0038] Plasmids: pMUT1, pMUT2;

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

[0040] CaCl2 solution: PIPES 6.7g / L, CaCl2 6.65g / L, glycerol 150g / L;

[0041] Fermentation medium: citric acid 1.7 g / L, yeast extract 6 g / L, tryptone 4 g / L, thiamine 0.1 g / L, glucose 20 g / L, KH2PO4 13.5 g / L, (NH4)2HPO4 4 g / L, MgSO4·7H2O1.4 g / L, NaOH 4 g / L, FeSO4·7H2O0.1 g / L, ZnSO4·7H2O 0.022 g / L, MnSO4·4H2O 0.005 g / L, CuSO4·5H2O 0.01 g / L, CaCl2 0.02 g / L, (NH4)2MoO4·4H2O: 0.001 g / L, Na2B4O7·10H2O: 0.0002 g / L, HCl: 0.05 mol / L;

[0042] Preparation of competent E. coli: Activation of strains: Take out the E. coli strain stored in -80℃ refrigerator, streak it onto LB solid medium, and culture it at 37℃ for 24-48h until obvious colonies appear; Seed culture: Pick a single colony from the plate and inoculate it into 5mL LB liquid medium, shake and culture it at 37℃ and 220rpm for 8-12h, and measure the OD 600 =0.2 Inoculate the seed culture solution into 50mL LB liquid medium, shake and culture at 37℃ and 220rpm for 1.5-2h; Collect the bacteria: transfer the culture solution to a centrifuge tube, centrifuge at 4℃ and 4000rpm for 10min, and discard the supernatant. Wash: resuspend the bacteria with 20mL pre-cooled CaCl2 solution or 10% glycerol solution, centrifuge at 4℃ and 4000rpm for 10min, and discard the supernatant. Add 2mL pre-cooled CaCl2 solution or 10% glycerol solution to resuspend the bacteria again to obtain E. coli competent state.

[0043] Purification and content determination of hyaluronic acid oligosaccharide: collect the fermentation broth, centrifuge at 8,000rpm for 2min, and discard the supernatant. Add 15mL P1 buffer (25mM Tris-HCl, 10mM EDTA, 10mg / L lysozyme, pH=8.0) to resuspend the bacteria, and incubate at 220rpm and 37℃ for 2h. Add 15mL P2 buffer (0.2M NaOH, 10g / L sodium dodecyl sulfate), mix gently, and let stand for 10min. Add 21mL P3 buffer (3M potassium acetate, pH=6.0). Centrifuge at 12,000rpm for 10min and take the supernatant. Add 3 times the volume of anhydrous ethanol, mix well and let stand at 4℃ overnight. Centrifuge at 12,000rpm for 10min, take the supernatant, wait for the ethanol to evaporate, and add the original volume of deionized water to dissolve the precipitate. Centrifuge at 12,000rpm for 10min, and the supernatant is the hyaluronic acid oligosaccharide sample. Take the purified hyaluronic acid sample and dilute it 10-50 times appropriately. Take 200 μL of diluent to a colorimetric tube, add 1 mL of borax sulfate solution, mix, boil in a boiling water bath for 15 min, and cool on ice. Add 50 μL of carbazole solution and boil in a boiling water bath for 10 min. Take the reaction solution to an ELISA plate and use an ELISA reader to measure the absorbance of the reaction solution at a wavelength of 530 nm. Take 0, 10, 20, 30, 40, 50 mg / L of glucuronic acid solution respectively, measure the absorbance according to the above operation, and draw a standard curve with the absorbance as the horizontal coordinate and the glucuronic acid concentration (mg / L) as the vertical coordinate. Substitute the above-mentioned sample absorbance values ​​measured into the standard curve equation to calculate the hyaluronic acid content.

[0044] Hyaluronic acid oligosaccharide mass spectrometry: The sample was mixed with 9 volumes of methanol, and then the supernatant fraction was collected by centrifugation at 12,000 rpm for 20 min and filtered through a 0.22 μm membrane. The components were analyzed by LC-IT-TOF-MS (Shimadzu, Kyoto, Japan) using a 5 μm gasket set VP-ODS column (4.6×250 mm) at 40°C. Elution buffer A was ultrapure water, and elution buffer B was a methanol solution, both eluents containing 8 mM ammonium acetate. The LC gradient elution was as follows: 0-5 min, 5% B; 5-15 min, 5%-60% B; 15-20 min, 60% B, with a flow rate of 0.2 mL / min. The ionization of the liquid chromatography flow was completed by an electrospray source (2 kV and 230°C), and nitrogen was used as a drying and nebulizing gas. Negative ion spectra were generated by scanning the range of 100-900 m / z.

[0045] The primer sequences involved in the embodiments are shown in the following table:

[0046] Table 1 Primer sequences

[0047]

[0048]

[0049]

[0050]

[0051] Example 1: Knockout of the endogenous heparin precursor synthase gene cluster in Escherichia coli

[0052] Genes were amplified using primers Del-kfiABC-up-F and Del-kfiABC-up-R, Del-kfiABC-down-F and Del-kfiABC-down-R, and the kanamycin resistance gene Kan was amplified from plasmid pKD13 using primers Del-kfiABC-kan-F and Del-kfiABC-kan-R, including a short flippase recognition target (FRT) site.

[0053] Transform plasmid pKD46 into E. coli, spread on LB plates containing ampicillin, and culture at 30°C for 12 hours to prepare competent cells. Mix the homologous fragment of the gene to be knocked out with the Kan resistance gene fragment with FRT site, and electroporate into the above competent cells. Pick a single clone for colony PCR, amplify the DNA fragment using the genomic DNA of the positive transformant as a template, and sequence to verify whether the knockout is successful.

[0054] The knockout strain was inoculated into a non-resistant LB medium, cultured at 37°C for 12 hours, and then transferred to an LB medium containing ampicillin and continued to be cultured. If the strain cannot grow, it means that the plasmid pKD46 has been successfully eliminated. The plasmid pCP20 was transformed into a recombinant strain that eliminated the plasmid pKD46, and a single clone was picked to a non-resistant LB medium, cultured at 30°C for 12 hours, and 250 μL of seed liquid was transferred to 25 mL of non-resistant LB medium, cultured at 30°C for 2 hours, and then transferred to 42°C for 12 hours. Streak on a non-resistant plate, pick a single clone and spot it on a non-resistant, ampicillin-containing, and kanamycin-containing plate. If it only grows on a non-resistant plate, it means that the Kan resistance gene and plasmid pCP20 have been successfully eliminated, and the recombinant strain is named ΔKfiABC.

[0055] Example 2: Construction and strengthening of food grade hyaluronic acid synthesis pathway

[0056] (1) Expression of hyaluronan synthase gene

[0057] The genomic DNA of Pasteurella multocida was extracted using a cell genome extraction kit. The genomic DNA of Pasteurella multocida was used as a template, and samn-F and samn-R were used as primers for PCR amplification to obtain a 1000bp fragment samn (nucleotide sequence as shown in SEQ ID NO.1). The recombinant plasmid pMUT1-samn was constructed using the TDEA assembly method. The strain was transformed into Escherichia coli Top 10 by heat shock method, and the transformants were picked for plasmid extraction and sequencing to verify the successful construction of the pMUT1-samn plasmid. Using an electroporator, the electroporator parameters were set as follows: 2.5kV, 25μF, 200Ω, and the recombinant plasmid pMUT1-samn was transformed into ΔKfiABC using a 2mm electroporation cup, and cultured at 37°C for 12h. The recombinant strain was named ΔKfiABC-samn.

[0058] (2) Enhanced synthesis of UDP-glucuronic acid and UDP-N-acetylglucosamine

[0059] The genomic DNA of Escherichia coli Nissle 1917 (ecn), Escherichia coli O10:K5:H4 (eco), Escherichia coli K12 MG1655 (ec), Bacillus subtilis 168 (bs), Corynebacterium glutamicum ATCC 13032 (cg), Pseudomonas putida KT 2440 (pt), Streptococcus zooepidemicus WSH.24 (se) was extracted using a cell genome extraction kit. Primers were designed to amplify the genes galU, ugdA, glmS, glmM, and glmU from the corresponding strains, where cgugdA1 and cgugdA2, bsglmS1 and bsglmS2 represent ugdA and glmS isozymes, respectively. The recombinant plasmid pMUT2-X was constructed using the TDEA assembly method. The recombinant plasmid was transformed into Escherichia coli Top 10 by heat shock method, and the transformants were picked for plasmid extraction and sequencing, and the pMUT2-X plasmid was successfully constructed.

[0060] Table 2 Synthesis pathway related enzymes and corresponding gene sequences

[0061]

[0062]

[0063] The above recombinant plasmids (concentration greater than 100 ng / μL) were transformed into ΔKfiABC-samn by electroporation to construct the recombinant strain ΔKfiABC-samn-X (where X corresponds to the above different genes). The recombinant strains ΔKfiABC-samn-X and ΔKfiABC-samn were inoculated into 5 mL LB medium and cultured at 220 rpm and 37°C overnight. The seed solution was conditioned at an initial OD 600=0.1 was transferred to 25 mL of fermentation medium and cultured at 220 rpm and 37°C for 24 h. During the fermentation period, 50 μL IPTG (100 mM) was added at 2 h. When the fermentation reached 4-8 h, 2 M NaOH was used to adjust the pH of the fermentation liquid to neutral. Figure 2 The hyaluronic acid production of the recombinant strains ΔKfiABC-samn and ΔKfiABC-samn-X in 250 mL shake flasks showed that the key rate-limiting enzymes in the synthesis pathways of UDP-glucuronic acid and UDP-N-acetylglucosamine were UDP-glucose dehydrogenase and phosphoglucosamine mutase, respectively.

[0064] Primers were designed to express the key rate-limiting enzymes in the synthesis pathway of UDP-glucuronic acid and UDP-N-acetylglucosamine. The recombinant plasmid pMUT2-cgugdA2-X was constructed using the TDEA assembly method. X corresponded to the segalU, bgglmS1, cgglmM and bsglmU screened above with high yields. The above plasmids were transformed into ΔKfiABC-samn by electroporation to construct the recombinant strain ΔKfiABC-samn-cgugdA2-X. After 250 mL shake flask fermentation of hyaluronic acid, Figure 3 As shown, the recombinant strain ΔKfiABC-samn-cgugdA2-cgglmM had the highest final hyaluronic acid production, reaching 2 g / L.

[0065] (3) Expression of hyaluronan lyase gene

[0066] The genomic DNA of Proteus vulgaris was extracted using a cell genome extraction kit. Using the genomic DNA of Proteus vulgaris as a template, primers chaseABC-F and chaseABC-R were designed to amplify the hyaluronate lyase gene (nucleotide sequence as shown in SEQ ID NO.2), and the recombinant plasmid pMUT2-chaseABC was constructed using the TDEA assembly method. The plasmid pMUT2-chaseABC was transformed into ΔKfiABC-samn-cgugdA2-cgglmM by electroporation. The recombinant strain was named ΔKfiABC-samn-ugdA-glmM-chaseABC.

[0067] Example 3: Synthesis of hyaluronic acid by fed-batch fermentation of recombinant Escherichia coli in a 5 L fermenter

[0068] The recombinant strain ΔKfiABC-samn-ugdA-glmM-chaseABC constructed in Example 2 was inoculated into 5 mL LB medium and cultured overnight at 220 rpm and 37°C. 600=0.1 was transferred to 25 mL fermentation culture, and cultured at 220 rpm and 37 °C for 10 h, and then inoculated into a 5 L fermentation tank with 10% inoculum. The initial temperature was set to 37 °C and the speed was 300 rpm / min. When the fermentation liquid OD 600 =18, IPTG with a final concentration of 0.2mM was added to induce gene expression, and the pH of the fermentation liquid was controlled at about 7 by 29% ammonia water during the fermentation process, and glucose was added to maintain the concentration in the fermentation liquid at about 20g / L. Figure 4 As shown in FIG. 1 , the final hyaluronic acid yield of the recombinant strain ΔKfiABC-samn-ugdA-glmM-chaseABC in a 5L fermenter was 15 g / L. The mass spectrometry structure of hyaluronic acid was identified, and the results were as follows: Figure 5 As shown, the products are all hyaluronic acid disaccharide, and the present invention successfully realizes the mass production of food-grade hyaluronic acid disaccharide.

[0069] 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 strain producing hyaluronan disaccharide, characterized in that: Hyaluronan synthase and hyaluronan lyase were heterologously expressed in Escherichia coli Nissle 1917, and UDP-glucose dehydrogenase was overexpressed. The hyaluronan synthase was derived from Pasteurella multocida, the hyaluronan lyase was derived from Proteus vulgaris, and the UDP-glucose dehydrogenase was derived from Corynebacterium glutamicum ATCC13032.

2. The recombinant Escherichia coli according to claim 1, characterized in that: The nucleotide sequence of the hyaluronan synthase is shown in SEQ ID NO.1, and the nucleotide sequence of the hyaluronan lyase is shown in SEQ ID NO.

2.

3. The recombinant Escherichia coli according to claim 1, characterized in that: The pMUT1 plasmid was used to express the hyaluronan synthase, and the pMUT2 plasmid was used to express the hyaluronan lyase.

4. The recombinant Escherichia coli according to claim 1, characterized in that: The recombinant Escherichia coli overexpresses phosphoglucomutase.

5. The recombinant Escherichia coli according to claim 4, characterized in that: The phosphoglucomutase is derived from Corynebacterium glutamicum ATCC13032.

6. The recombinant Escherichia coli according to claim 1, characterized in that: The recombinant Escherichia coli is knocked out of heparin precursor synthase.

7. A microbial preparation comprising the recombinant Escherichia coli according to any one of claims 1 to 6.

8. Use of the recombinant Escherichia coli according to any one of claims 1 to 6 or the microbial preparation according to claim 7 in the preparation of hyaluronic acid disaccharide.

9. A method for preparing hyaluronic acid disaccharide, characterized in that: The recombinant Escherichia coli according to any one of claims 1 to 6 or the microbial preparation according to claim 7 is fermented and cultured.

10. The method according to claim 9, characterized in that: The temperature condition of the fermentation culture is 15-45° C., and the fermentation time is 24-72 hours.

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