Hyaluronate synthase and recombinant bacteria and application thereof

Abstract

The present application relates to a kind of hyaluronan synthase and its recombinant bacteria and application, belong to biotechnology field.The present application is by the catalytic activity region of amino acid sequence such as SEQ ID NO.1 hyaluronan synthase and the replacement modification of the type II hyaluronan synthase Has2 derived from Heterocephalus glaber, obtained the hyaluronan synthase of the amino acid sequence such as SEQ ID NO.3 shown in the present application.The hyaluronan synthase of the present application after modification is introduced into host bacteria and is carried out fermentation culture, compared with the hyaluronan synthase before unmodified, its production hyaluronic acid molecular weight is greatly improved, has very high industrial application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a hyaluronic acid synthase, its recombinant strain, and its applications. Background Technology

[0002] Hyaluronic acid is a linear, high-molecular-weight, non-sulfated glycosaminoglycan, a polymer composed of N-acetylglucosamine and D-glucuronic acid linked by alternating β-1,4- and β-1,3-glycosidic bonds. The molecular weight of natural hyaluronic acid is widely distributed, ranging from several thousand Daltons to millions or even tens of millions of Daltons, and its properties are directly related to its molecular weight. Ultra-high molecular weight hyaluronic acid (molecular weight greater than 3000 kDa) inhibits cell migration, proliferation, differentiation, and phagocytosis and can be used as a solid filler; high molecular weight hyaluronic acid (molecular weight between 1000 kDa and 3000 kDa) has good moisturizing and anti-inflammatory properties and can be used as a viscoelastic agent in ophthalmic surgery and for intra-articular injection therapy; medium molecular weight hyaluronic acid (molecular weight between 100 kDa and 1000 kDa) has good moisturizing and lubricating properties and is widely used in the cosmetics field; low molecular weight hyaluronic acid (molecular weight less than 100 kDa) and its oligosaccharides have anti-tumor, wound healing, angiogenesis, and immunomodulatory effects and have good prospects for medical applications.

[0003] Currently, the main methods for regulating the molecular weight of hyaluronic acid are physicochemical degradation and enzymatic depolymerization. Physicochemical methods primarily involve thermal degradation, ultrasound, and treatment with strong acids and alkalis to degrade hyaluronic acid. These methods result in products with uneven molecular weight distribution, low efficiency, and can damage the molecular structure of hyaluronic acid, reducing product quality. Enzymatic depolymerization of hyaluronic acid, compared to physicochemical methods, offers milder and more controllable reaction conditions and better reproducibility, but it is more expensive, and the performance of the degrading enzymes limits its further development. Direct synthesis using microorganisms is an ideal method for producing hyaluronic acid of different molecular weights. Currently, researchers are controlling the molecular weight of hyaluronic acid through enzyme modification and fermentation process optimization to achieve the synthesis of hyaluronic acid with different molecular weights. However, existing technologies for controlling the molecular weight of hyaluronic acid produced by microbial fermentation are limited to the range of 1 kDa to 3000 kDa, and cannot synthesize ultra-high molecular weight hyaluronic acid (molecular weight greater than 3000 kDa).

[0004] Early or traditional microbial fermentation relied heavily on strains such as Streptococcus vesicanthizobium. These strains are pathogenic and easily produce metabolites such as hemolysin and hyaluronidase during fermentation, which not only affect fermentation stability but also pose safety risks. Although safe strains such as Bacillus subtilis are widely used, they are prone to cell lysis during fermentation, and the released DNA can severely contaminate the hyaluronic acid product, significantly increasing the difficulty and cost of subsequent separation and purification. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a hyaluronic acid synthase, its recombinant strain, and its applications. This invention modifies the catalytic active region of the hyaluronic acid synthase, resulting in a modified hyaluronic acid synthase capable of stably synthesizing high-molecular-weight hyaluronic acid and efficiently secreting it extracellularly.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a hyaluronic acid synthase, the amino acid sequence of which is shown in SEQ ID NO.3.

[0008] The hyaluronic acid synthase described herein is a modified version of the hyaluronic acid synthase with the amino acid sequence shown in SEQ ID NO. 1.

[0009] (1) The sequence from position 54 to 320 is replaced with the sequence from position 65 to 377 of type II hyaluronic acid synthase Has2 from naked mole rat Heterocephalus glaber. Specifically, FYKPFKGRAGQYKVAAIIPSYNEDAESLLETLKSVQQQTYPLAEIYVVDDGSADETGIKRIEDYVRDTGDLSSNVIVHRSEKNQGKRHAQAWAFERSDADVFLTVDSDTYIYPDALEELLKTFNDPTVFAATGHLNVRNRQTNLLTRLTDIRYDNAFGVERAAQSVTGNILVCSGPLSVYRREVVVPNIDRYINQTFLGIPVSIGDDRCLTNYATDLGKTVYQSTAKCITDVPDKMSTYLKQQNRWNKSFFRESIISVKKIMNNPFV is replaced with LEHRKMK KSLETPIKLNKTVALCIAAYQEDPDYLRKCLQSVKRLTYPGIKVVMVIDGNSDDDLYMMDIFSEVMGRDKSATYIWKNNFHEKGPGETDESHKESSQHVTQLVLSSKSVCIMQKWGGKREVMYTAFRALGRSVDYVQVCDSDTMLDPASSVEM VKVLEEDPMVGGVGGDVQILNKYDSWISFLSSVRYWMAFNIERACQSYFGCVQCISGPLGMYRNSLLHEFVEDWYSQEFMGNQCSFGDDRHLTNRVLSLGYATKYTARSKCLTETPIEYLRWLNQQTRWSKSYFREWLYNAMWFHKHHLWMT.

[0010] (2) The sequence from position 371 to 380 is replaced with the sequence from position 423 to 429 of type II hyaluronic acid synthase Has2 from Heterocephalus glaber. Specifically, HYMLKHPLSF is replaced with FASCLRG.

[0011] (3) The sequence from position 401 to 417 is replaced with the sequence from position 451 to 475 of the type II hyaluronic acid synthase Has2 derived from Heterocephalus glaber. Specifically, SLFTIRNADWGTRKKLL is replaced with MFAATINKAGWGTSGRKTIVVNF.

[0012] A second objective of this invention is to provide a gene encoding the hyaluronic acid synthase.

[0013] A third objective of this invention is to provide a recombinant expression vector carrying the gene.

[0014] In one embodiment of the present invention, the recombinant expression vector further includes uridine diphosphate-glucose dehydrogenase; the amino acid sequence of the uridine diphosphate-glucose dehydrogenase is shown in SEQ ID NO.16.

[0015] A fourth objective of this invention is to provide a recombinant bacterium expressing the hyaluronic acid synthase.

[0016] In one embodiment of the present invention, the recombinant bacteria is a bacterium or a fungus.

[0017] In one embodiment of the present invention, the recombinant bacteria uses Corynebacterium glutamicum or Escherichia coli as the substrate bacteria.

[0018] In one embodiment of the present invention, the recombinant bacteria uses pXMJ19 as an expression vector.

[0019] The fifth objective of this invention is to provide the application of the hyaluronic acid synthase, the gene, the recombinant expression vector, or the recombinant bacteria in the synthesis of ultra-high molecular weight hyaluronic acid; wherein the ultra-high molecular weight hyaluronic acid has a molecular weight greater than 3000 kDa.

[0020] The sixth objective of this invention is to provide a method for synthesizing hyaluronic acid using the aforementioned recombinant bacteria, comprising the following steps:

[0021] The recombinant bacteria were inoculated into the fermentation medium and fermented.

[0022] In one embodiment of the present invention, the glucose feed during the fermentation process is maintained at 10 g / L-20 g / L.

[0023] In one embodiment of the present invention, the inoculation amount is 10%-20%.

[0024] In one embodiment of the present invention, the fermentation conditions are: fermentation at 20℃-60℃ for 12h-120h;

[0025] And / or, the pH of the fermentation is maintained at 7.0 to 8.0.

[0026] In one embodiment of the present invention, the fermentation culture refers to culturing in an environment containing a carbon source, a nitrogen source, inorganic salts, metal ions, and oxygen.

[0027] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0028] This invention provides a hyaluronic acid synthase, its recombinant strain, and its applications. The hyaluronic acid synthase of this invention is obtained by modifying the catalytic active region of the hyaluronic acid synthase shown in SEQ ID NO.1. When the hyaluronic acid synthase mutant of this invention is introduced into recombinant bacteria for fermentation, the molecular weight of hyaluronic acid is significantly increased compared to the unmutated hyaluronic acid synthase. Furthermore, under the premise of enhanced UDP-glucuronide dehydrogenase expression, the hyaluronic acid synthase mutant of this invention is heterologously expressed, and its yield in a 5L fermenter is approximately 6 g / L, with an average molecular weight of hyaluronic acid of 3680 kDa. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0030] Figure 1 This is a secondary structure diagram of the hyaluronic acid synthase of the present invention;

[0031] Figure 2 This is a diagram of the amino acid sequences of the two hyaluronic acid synthases of this invention;

[0032] Figure 3 This is a graph showing the hyaluronic acid production in a 250mL shake flask according to the present invention;

[0033] Figure 4 This is a diagram of the batch-feed fermentation production of ultra-high molecular weight hyaluronic acid according to the present invention. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0036] Strain: Corynebacterium glutamicum ATCC 13032.

[0037] Plasmid: pXMJ19.

[0038] LB medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0039] BHI medium: brain heart extract 37 g / L, sorbitol 91 g / L.

[0040] Fermentation medium: glucose 40 g / L, corn steep liquor powder 20 g / L, (NH4)2SO4 30 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4 25 g / L, 3-morpholine propanesulfonic acid (MOPS) 42 g / L.

[0041] Determination of hyaluronic acid production:

[0042] Relevant reagents: borax-sulfuric acid solution (weigh 4.77g sodium tetraborate and dissolve in 500mL concentrated sulfuric acid); carbazole solution (weigh 1.25g carbazole and dissolve in 500mL anhydrous ethanol); glucuronic acid solution 1g / L.

[0043] Sample purification: Take the fermentation broth and centrifuge at 12,000 rpm for 2 min. Collect the supernatant, add 4 volumes of anhydrous ethanol, place in a -20℃ freezer, and precipitate for 12 h. Centrifuge at 12,000 rpm for 5 min and discard the supernatant. After the ethanol evaporates, resuspend in water, fully dissolve, and add to the original volume. Centrifuge at 12,000 rpm for 5 min and collect the supernatant. Repeat the above operation. After the ethanol precipitation is complete, collect the supernatant. Sample purification is complete.

[0044] Borax-sulfuric acid-carbazole method: A 1 g / L glucuronic acid solution was serially diluted to 0, 10, 20, 30, 40, and 50 mg / L. 1 mL of borax-sulfuric acid solution was mixed with 200 μL of glucuronic acid solution in a glass colorimetric tube and incubated in a boiling water bath for 15 min. 50 μL of carbazole solution was added to the colorimetric tube and incubated in a boiling water bath for 10 min. The reaction system was transferred to a 96-well plate, and the absorbance of the sample at 530 nm was measured using a microplate reader. A curve was plotted with absorbance on the x-axis and glucuronic acid concentration (mg / L) on the y-axis, yielding the standard curve equation: y = 125.7x - 11.590, R0 2=0.999; Similarly, the purified sample was subjected to a borax-sulfuric acid-carbazole colorimetric reaction, and the absorbance of the sample at a wavelength of 530 nm was measured. The obtained absorbance was substituted into the above curve equation, and the hyaluronic acid content in the sample was calculated according to the following formula: Hyaluronic acid content (g / L) = (concentration measured by standard curve × dilution factor × 2.067) / 1000.

[0045] Determination of the molecular weight of hyaluronic acid:

[0046] The sample was purified by repeated alcohol precipitation according to the above purification method. An 18-angle laser light scattering gel permeation chromatography system was used. The mobile phase was ultrapure water (0.02% sodium azide), pH=6.0; the column temperature was 25℃; the flow rate was 1.0mL / min; and the chromatographic column was a Shodex OHpak SB-806HQ connected in series with an SB-804HQ.

[0047] Example 1:

[0048] Modification of the catalytic activity region of hyaluronic acid synthase: Based on the genome sequence of Streptococcus vesicae hyaluronic acid synthase (CP002904), the gene seHasA was synthesized by GENEWIZ (Suzhou), and the amino acid sequence of seHasA is shown in SEQ ID NO.1. Based on the genome sequence of naked mole rat hyaluronic acid synthase (GCA_000247695.1), the gene nmrHas2 was synthesized by GENEWIZ (Suzhou), and the amino acid sequence of nmrHas2 is shown in SEQ ID NO.2. Based on the sequence alignment of hyaluronic acid synthase (e.g.... Figure 2 As shown in Table 1), primers were designed to modify hyaluronic acid synthase. Using the synthesized seHasA and nmrHasA gene sequences as templates, primers (see Table 1 for details) seHasA-1-F / seHasA-1-R, nmrHasA-1-F / nmrHasA-1-R, seHasA-2-F / seHasA-2-R, nmrHasA-2-F / nmrHasA-2-R, seHasA-3-F / seHasA-3-R, and nmrHasA-3-F / nmrHasA-3-R were designed for PCR amplification. Then, the modified hyaluronic acid synthase gene nmr-seHasA was obtained by T5 exonuclease-dependent DNA assembly technology (TEDA) (the amino acid sequence of this gene is shown in SEQ ID NO. 3). The seHasA and nmr-seHasA gene sequences were ligated into plasmid pXMJ19 to construct recombinant expression plasmids pXMJ19-seHasA and pXMJ19-nmr-seHasA. These plasmids were then transformed into Corynebacterium glutamicum ATCC 13032 to obtain recombinant strains Corynebacterium glutamicum seHasA (as a control) and recombinant strain Corynebacterium glutamicum nmr-seHasA.

[0049] Table 1 Primers used for the modification of hyaluronic acid synthase seHasA

[0050]

[0051] Example 2: Enhancement of UDP-glucuronic acid synthesis

[0052] Primers ugd-F and ugd-R (see Table 2) were designed. Using the *Corynebacterium glutamicum* genome as a template, the UDP-glucose dehydrogenase gene ugd was amplified by PCR, and its sequence is shown in SEQ ID NO. 16. The gene ugd was ligated into plasmids pXMJ19-seHasA and pXMJ19-nmr-seHasA using the TEDA method to construct the recombinant expression plasmids pXMJ19-seHasA-ugd and pXMJ19-nmr-seHasA-ugd. The recombinant plasmids were transformed into *Corynebacterium glutamicum* by electroporation to obtain the recombinant strains *Corynebacterium glutamicum* seHasA-ugd and nmr-seHasA-ugd.

[0053] Table 2 Primers used for UDP-glucuronic acid synthesis enhancement

[0054]

[0055] Example 3: Production of ultra-high molecular weight hyaluronic acid by 250mL shake flask fermentation

[0056] The recombinant Corynebacterium glutamicum seHasA-ugd and recombinant Corynebacterium glutamicum nmr-seHasA-ugd constructed in Example 2 were inoculated into 5 mL BHI culture tubes and cultured overnight at 30°C and 220 rpm. The seed culture was set at the initial OD value. 600 An inoculum of 0.2 mg / L was transferred to a baffled Erlenmeyer flask containing 25 mL of fermentation medium. After incubation at 220 rpm and 30°C for 3.5 h, IPTG was added to a final concentration of 0.25 mM to induce gene expression. The fermentation period was 48 h. During this period, 2 M NaOH was added at 20 h and 24 h to adjust the pH of the fermentation broth to 6.5-7. The fermentation broth was collected, centrifuged at 10000 rpm for 5 min, and the supernatant was collected. After two repeated alcohol precipitations, the hyaluronic acid content in the fermentation broth was determined by the borosilicate-carbazole method and gel permeation chromatography (GPC). Figure 3 The hyaluronic acid yield of strain seHasA-ugd was 4.8 g / L, and the average molecular weight was 1880 kDa as determined by SEC; the hyaluronic acid yield of strain nmr-seHasA-ugd was 0.9 g / L, and the average molecular weight was 3800 kDa as determined by NMR.

[0057] Example 4: Feed-in fermentation of recombinant Corynebacterium glutamicum in a 5 L fermenter

[0058] The recombinant Corynebacterium glutamicum seHasA-ugd and the recombinant strain Corynebacterium glutamicum nmr-seHasA-ugd constructed in Example 2 were inoculated into 5 mL of BHI medium and cultured overnight at 30°C and 220 rpm. 600 A 0.2% inoculum was transferred to a 25 mL Erlenmeyer flask containing fermentation medium and incubated at 220 rpm and 30°C for 10 h. Then, a 10% inoculum was transferred to a 5 L fermenter. The initial temperature was set at 30°C and the fermentation speed at 3000 rpm. After 4 h of fermentation, IPTG at a final concentration of 0.25 mM was added to induce gene expression. During fermentation, the pH of the fermentation broth was controlled at approximately 7 using 14% ammonia, and glucose was added to maintain a concentration of approximately 10 g / L in the fermenter. The hyaluronic acid content in the fermentation broth was determined by the borosilicate-carbazole method and gel permeation chromatography. The final hyaluronic acid yield of the recombinant strain *Corynebacterium glutamicum* nmr-seHasA-ugd in the 5 L fermenter was 6 g / L (e.g., ...). Figure 4 As shown in the figure, the average molecular weight of hyaluronic acid is 3680 kDa. The recombinant Corynebacterium glutamicum seHasA-ugd, after fermentation in a 5L fermenter, produced 5 g / L of hyaluronic acid in the final fermentation broth, with an average molecular weight of 1800 kDa. Simultaneously, the produced hyaluronic acid accumulated intracellularly.

[0059] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hyaluronic acid synthase, characterized in that, The amino acid sequence of the hyaluronic acid synthase is shown in SEQ ID NO.

3.

2. The gene encoding the hyaluronic acid synthase of claim 1.

3. A recombinant expression vector carrying the gene of claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector also includes uridine diphosphate-glucose dehydrogenase; the amino acid sequence of the uridine diphosphate-glucose dehydrogenase is shown in SEQ ID NO.

16.

5. A recombinant bacterium expressing the hyaluronic acid synthase of claim 1.

6. The recombinant bacteria according to claim 5, characterized in that, The recombinant bacteria are bacteria or fungi.

7. The application of the hyaluronic acid synthase of claim 1, the gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant bacteria of claim 5 or 6 in the synthesis of ultra-high molecular weight hyaluronic acid; wherein the ultra-high molecular weight hyaluronic acid has a molecular weight greater than 3000 kDa.

8. A method for synthesizing hyaluronic acid using the recombinant bacteria described in claim 5 or 6, characterized in that, Includes the following steps: The recombinant bacteria were inoculated into the fermentation medium and fermented, with glucose added during the fermentation process to maintain a concentration of 10 g / L-20 g / L.

9. The method according to claim 8, characterized in that, The vaccination dose is 10%-20%.

10. The method according to claim 8, characterized in that, The fermentation conditions are: fermentation at 20℃-60℃ for 12h-120h; And / or, the pH of the fermentation is maintained at 7.0 to 8.0.

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