Method for directly producing hyaluronic acid with specific molecular weight through mixed fermentation, regulating strain and application

Abstract

The invention belongs to the technical field of fermentation engineering, and discloses a method for directly producing hyaluronic acid with a specific molecular weight through mixed fermentation, a regulation strain and application, a way for producing hyaluronidase is constructed in streptococcus zooepidemicus ATCC 39920, the hyaluronidase is secreted, the regulation strain is used as the regulation strain in the process of producing hyaluronic acid HA through fermentation, and the application of the regulation strain in the process of producing hyaluronic acid HA through mixed fermentation is realized. Regulating strains with different proportions are added in the fermentation process, so that hyaluronic acid with different molecular weights can be fermented. In the fermentation process, regulation strains with different proportions are added for mixed fermentation, and HA fermentation products with high, medium and low molecular weights are obtained. The effects that the molecular weight of the fermented hyaluronic acid product is correspondingly reduced and the yield is improved along with the increase of the mixed bacterium proportion and the increase of the added regulation bacterium amount are achieved, the operation step of reducing the specific molecular weight after fermentation is completed is omitted, the production cost is reduced, and the production period is shortened at the same time.

Description

Technical Field

[0001] This invention belongs to the field of fermentation engineering technology, and in particular to a method, strain regulation and application of directly producing hyaluronic acid of a specific molecular weight through mixed fermentation. Background Technology

[0002] Hyaluronic acid (HA), also known as hyaluronic acid, is a high-molecular-weight polysaccharide composed of disaccharide units D-glucuronic acid and N-acetylglucosamine. It plays a vital role in various fields, including food, health products, cosmetics, and clinical medicine. Based on molecular weight, hyaluronic acid can be classified into four categories: high molecular weight hyaluronic acid, medium molecular weight hyaluronic acid (500 kDa-2000 kDa), low molecular weight hyaluronic acid (10 kDa-500 kDa), and oligomeric hyaluronic acid (less than 10 kDa). Different molecular weight hyaluronic acid has different applications. Hyaluronic acid produced by direct fermentation is usually high molecular weight hyaluronic acid. Specific molecular weight hyaluronic acid can be obtained through subsequent treatment of the high molecular weight hyaluronic acid fermentation broth. Common treatment methods include chemical methods, physical methods, and enzymatic hydrolysis. Specific molecular weight hyaluronic acid can also be obtained directly through enzymatic hydrolysis during fermentation, modification of the producing bacteria (called heterologous expression), and mixed fermentation (this invention).

[0003] Chemical methods typically involve acid-base degradation. Chinese patent publication CN104059166A describes a method for preparing oligomeric hyaluronic acid from hyaluronic acid fermentation broth. This method involves alcohol precipitation, dehydration, resolution, and filtration, followed by alkaline degradation of the filtrate to hyaluronic acid of a specific molecular weight. However, this method requires alcohol precipitation of high molecular weight hyaluronic acid, which is time-consuming and involves numerous steps.

[0004] Physical methods, such as those described in Chinese patent publication CN107200789A, utilize a homogenizer to control different pressures and cycle numbers, thereby altering the molecular weight of hyaluronic acid. This method avoids the negative effects of gradual efficiency loss, solution yellowing, and other physical degradation methods. However, homogenizers can only process hyaluronic acid substrates at concentrations of 1-2 mg / mL; excessively high or low concentrations can lead to operational inconvenience or instrument damage.

[0005] Enzymatic hydrolysis, for example, is reported in Chinese patent publication CN104178539A, which describes a method for the large-scale preparation of low-molecular-weight hyaluronic acid by using hyaluronidase to degrade high-molecular-weight hyaluronic acid. However, this method requires a reaction time of up to 12.5 hours at 45°C and 700 rpm to obtain 4000 kDa hyaluronic acid, which significantly increases the cost of equipment.

[0006] Furthermore, Chinese patent publication CN117701598A discloses a novel method for preparing hyaluronic acid of specific molecular weight using a novel hyaluronidase. This method utilizes hyaluronidase to degrade high molecular weight hyaluronic acid into fragments of specific molecular weights. By precisely controlling the type, concentration, reaction time, and temperature of the enzyme, hyaluronic acid of different molecular weights can be obtained. Although enzymatic hydrolysis is relatively environmentally friendly and efficient, biological enzymes are expensive and difficult to store for long periods. The enzymatic hydrolysis reaction conditions have strict requirements on temperature and pH, and enzyme residues may remain in the product, affecting product quality, resulting in low economic benefits for large-scale production.

[0007] Heterologous expression methods, such as those reported in Chinese Patent Publication CN105087456A, involve a method for producing recombinant Bacillus subtilis with specific molecular weight hyaluronic acid. This method integrates hyaluronic acid synthase hasA from Streptococcus vesiculosus into Bacillus subtilis, achieving hyaluronic acid production. Simultaneously, hyaluronidase from leeches is introduced and integrated into the Bacillus subtilis genome. A mutant library is constructed based on the ribosome binding site (RBS) sequence at the gene expression and translation level. High-throughput screening yields a series of mutants with different hyaluronidase expression levels. This technology can produce hyaluronic acid with molecular weights of 103 kDa-106 kDa, but further modification of the production strain is needed to obtain hyaluronic acid with other molecular weights. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method, strain regulation and application for directly producing hyaluronic acid of a specific molecular weight through mixed fermentation.

[0009] The technical solution adopted by this invention to solve its technical problem is:

[0010] A regulatory strain for hyaluronidase production was developed. The strain was constructed based on a novel hyaluronidase encoding gene, hylP, disclosed in patent CN115820694A. Codon optimization was performed on a third-party platform based on its amino acid sequence SEQ ID NO.1 to obtain the gene rHyal. This gene was inserted into the vector pDL278. The expression of the hyaluronidase encoding gene rHyal was driven by the PR31 promoter, and the secretion of mature hyaluronidase was controlled using the amyA signal peptide. The resulting plasmid, pDL278-PR31-amyA-rHyal, was electroporated into *Streptococcus zooepidemicus* ATCC 39920, causing it to secrete active hyaluronidase, thus obtaining a regulatory strain for hyaluronidase production during fermentation.

[0011] Furthermore, the nucleotide sequence of the hyaluronidase gene rHyal is SEQ ID NO.2; the nucleotide sequence of the PR31 promoter is SEQ ID NO.3; and the nucleotide sequence of the amyA signal peptide is SEQ ID NO.4.

[0012] The application of the aforementioned regulatory strains in the direct fermentation production of hyaluronic acid with a specific molecular weight.

[0013] A method for directly producing hyaluronic acid of a specific molecular weight through mixed-culture fermentation using the aforementioned regulatory strain, wherein the method involves constructing a pathway for producing hyaluronidase within Streptococcus vesicae and secreting hyaluronidase as a regulatory strain in the fermentation process for producing hyaluronic acid (HA). By adding different proportions of the regulatory strain during fermentation, hyaluronic acid of different molecular weights can be fermented.

[0014] Furthermore, the method for constructing a hyaluronidase-producing plasmid is to use the PR31 promoter to drive the expression of the hyaluronidase gene hylP, use the amyA signal peptide to control the secretion of mature hyaluronidase, and then clone it into the pDL278 vector to construct the hyaluronidase expression plasmid pDL278-PR31-amyA-rHyal.

[0015] Furthermore, it includes the following steps:

[0016] By adding a hyaluronidase-producing regulatory strain during fermentation, and mixing different proportions of the regulatory strain into the fermentation broth when the production strain reaches the exponential phase, the viscosity of the fermentation broth is reduced to varying degrees, and hyaluronic acid of a specific molecular weight is obtained directly through fermentation.

[0017] Furthermore, the specific steps are as follows:

[0018] Step 1: Use *S. zooepidemicus* ATCC 39920 as the production strain for hyaluronic acid; inoculate the production strain into seed culture medium THY, and culture at 37℃ and 200 r / min. After 24 h of primary seed culture, transfer it to secondary seed culture THY at a 2% inoculation rate and continue fermentation in fermentation medium FSB for 12 h. Then, transfer it to a 5 L fermenter containing 3 L of fermentation medium FSB at a 10% inoculation rate for fermentation.

[0019] Step 2: When the production strain has grown for 4-6 hours, add the control strain, which has been cultured at 37℃ and 200r / m for 24 hours, to a fermenter containing 3L of culture medium. Continue fermentation for another 24 hours.

[0020] Furthermore, the volume ratio of different production strains to control strains is 250:1, 2000:1, or 5000:1.

[0021] Furthermore, the specific components of the fermentation medium FSB are as follows:

[0022] FSB medium Each 1L contains water as the solvent. yeast extract 3.5g Casein peptone 10g NaCl 1.5g <![CDATA[K2HPO4]]> 2g <![CDATA[MgSO4]]> 0.4g glucose 60g

[0023] Sterilize at 121℃ for 20 min, and sterilize glucose at 115℃ for 30 min;

[0024] Alternatively, the specific components of THY culture medium are:

[0025] THY medium Each 1L contains water as the solvent. Beef Powder 10g trypsin 20g yeast extract 2g glucose 2g <![CDATA[NaHCO3]]> 2g NaCl 2g <![CDATA[Na2HPO4]]> 0.4g

[0026] Adjust the pH to 6.8; sterilize at 121℃ for 20 minutes.

[0027] The method described above is applied to the direct fermentation production of hyaluronic acid with a specific molecular weight.

[0028] The advantages and positive effects of this invention are as follows:

[0029] 1. This invention utilizes *Streptococcus zooepidemicus* ATCC 39920 in a 5L fermenter to produce hyaluronic acid (HA). During fermentation, different proportions of regulatory strains are added for mixed-culture fermentation, such as 5000:1, 2000:1, and 250:1, yielding HA fermentation products with high, medium, and low molecular weights. This achieves the effect of increasing the amount of regulatory bacteria added as the mixed-culture ratio increases, resulting in a corresponding decrease in the molecular weight of the fermented hyaluronic acid product and an increase in yield. It eliminates the need for post-fermentation degradation to specific molecular weights, reducing production costs and shortening the production cycle.

[0030] 2. This invention constructs a regulatory strain that secretes active hyaluronidase. The pDL278-PR31-amyA-rHyal plasmid was constructed and electroporated into *Streptococcus zooepidemicus* ATCC 39920, causing it to secrete active hyaluronidase. This strain serves as a regulatory strain during fermentation. By controlling the proportion of this regulatory strain added during fermentation, the purpose of producing hyaluronic acid of a specific molecular weight can be achieved. This method is simple to operate, operates under mild conditions, produces no environmental pollution, and does not damage the product structure. It enables the industrial production of hyaluronic acid of a specific molecular weight and has broad research and application prospects.

[0031] 3. This invention aims to provide a mixed-culture fermentation method, constructing a regulatory strain that participates in the fermentation production of hyaluronic acid. Mixed-culture fermentation utilizes the hyaluronidase metabolite of the regulatory strain to directly degrade high-molecular-weight HA during fermentation, resulting in more thorough mixing and improved fermentation efficiency. This achieves the goal of obtaining products with specific molecular weights in a short time. By controlling the proportion of mixed-culture fermentation, hyaluronic acid of specific molecular weights can be obtained. The operation is simple, saving production time and costs, and possessing significant application value and economic benefits. Attached Figure Description

[0032] Figure 1 This is a spectrum of plasmid pDL278 in this invention;

[0033] Figure 2 The image shows the spectrum of plasmid pDL278-PR31 constructed in this invention;

[0034] Figure 3 The image shows the spectrum of the plasmid pDL278-PR31-amyA constructed in this invention.

[0035] Figure 4 The image shows the spectrum of the plasmid pDL278-PR31-amyA-rHyal constructed in this invention;

[0036] Figure 5 The left image shows the bacterial P verification gel image (left image) and the right image shows the plasmid digestion verification gel image (right image) of the plasmid pDL278-PR31 constructed in this invention. The plasmid was verified by digestion with BpvuI and HindIII, and a band of 6565bp + 512bp was obtained. In this image, M represents a 5000bp or 10000bp marker, N represents a negative band, and 1 represents the target band for verification.

[0037] Figure 6 The left image shows the bacterial P verification gel image (left image) and the right image shows the plasmid digestion verification gel image (right image) of the plasmid pDL278-PR31-amyA constructed in this invention. The plasmid was verified by EcovI and HindIII digestion, and a 6682bp+546bp band was obtained. In this image, M represents a 5000bp or 10000bp marker, N represents a negative band, and 1 represents the target band for verification.

[0038] Figure 7 The left image shows the bacterial P verification gel image (left image) and the right image shows the plasmid digestion verification gel image (right image) of the plasmid pDL278-PR31-amyA-rHyal constructed in this invention. The plasmid was verified by NcoI and NarI digestion, and a band of 5071bp + 3060bp was obtained. In this image, M represents a 5000bp or 10000bp marker, N represents a negative band, and 1 represents the target band for verification.

[0039] Figure 8 The molecular verification diagram of the regulatory strain constructed in this invention shows that the fragment length was 1359bp when verified with the verification primers; where M represents a 5000bp marker, N represents a negative band, P represents a positive band, and 1 represents the target band for verification.

[0040] Figure 9 This is an enzyme activity diagram showing the OD value of the strain and the corresponding growth time in this invention. The enzyme activity stabilized at 15 μ / mL after 20 h of growth.

[0041] Figure 10 This is a gel graph showing the relationship between the mixed strain ratio and molecular weight after mixed fermentation with different proportions of regulatory strains in this invention. As the mixed strain ratio increases, the molecular weight of the product decreases.

[0042] Figure 11 This is an HPLC data graph showing the relationship between the mixed bacterial ratio and specific molecular weight after mixed fermentation with different proportions of regulatory strains in this invention.

[0043] Figure 12 This is a graph showing the yield data after fermentation of different mixed bacteria ratios in this invention. Detailed Implementation

[0044] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0045] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0046] A regulatory strain for hyaluronidase production was developed. The hyaluronidase gene *rHyal* was synthesized using a third-party platform and inserted into the vector pDL278. The expression of the hyaluronidase-encoding gene *hylP* was driven by the PR31 promoter, and the secretion of mature hyaluronidase was controlled using the *amyA* signal peptide. The resulting plasmid, pDL278-PR31-amyA-rHyal, was electroporated into *Streptococcus zooepidemicus* ATCC 39920, stimulating the secretion of active hyaluronidase. This strain was then used as a regulatory strain for hyaluronidase production during fermentation.

[0047] Preferably, the nucleotide sequence of the hyaluronidase gene rHyal is SEQ ID NO.2; the nucleotide sequence of the PR31 promoter is SEQ ID NO.3; and the nucleotide sequence of the amyA signal peptide is SEQ ID NO.4.

[0048] The application of the aforementioned regulatory strains in the direct fermentation production of hyaluronic acid with a specific molecular weight.

[0049] A method for directly producing hyaluronic acid of a specific molecular weight through mixed-culture fermentation using the aforementioned regulatory strain, wherein the method involves constructing a pathway for producing hyaluronidase within Streptococcus vesicae and secreting hyaluronidase as a regulatory strain in the fermentation process for producing hyaluronic acid (HA). By adding different proportions of the regulatory strain during fermentation, hyaluronic acid of different molecular weights can be fermented.

[0050] Preferredly, the method for constructing a hyaluronidase-producing plasmid is to use the PR31 promoter to drive the expression of the hyaluronidase gene rHyal, use the amyA signal peptide to control the secretion of mature hyaluronidase, and then clone the plasmid into the pDL278 vector to construct the hyaluronidase expression plasmid pDL278-PR31-amyA-rHyal.

[0051] Preferably, the steps include:

[0052] By adding a hyaluronidase-producing regulatory strain during fermentation, and mixing different proportions of the regulatory strain into the fermentation broth when the production strain reaches the exponential phase, the viscosity of the fermentation broth is reduced to varying degrees, and hyaluronic acid of a specific molecular weight is obtained directly through fermentation.

[0053] Preferably, the specific steps are as follows:

[0054] Step 1: Use *S. zooepidemicus* ATCC 39920 as the production strain for hyaluronic acid; inoculate the production strain into seed culture medium THY, and culture at 37℃ and 200 r / min. After 24 h of primary seed culture, transfer it to secondary seed culture THY at a 2% inoculation rate and continue fermentation in fermentation medium FSB for 12 h. Then, transfer it to a 5 L fermenter containing 3 L of fermentation medium FSB at a 10% inoculation rate for fermentation.

[0055] Step 2: When the production strain has grown for 4-6 hours, add the control strain, which has been cultured at 37℃ and 200r / m for 24 hours, to a fermenter containing 3L of culture medium according to the mixed strain ratio of fermentation system: control strain volume ratio, and continue fermentation for 24 hours.

[0056] Preferably, the fermentation system has a volume ratio of 250:1, 2000:1, or 5000:1 for the control strains.

[0057] Preferably, the specific components of the fermentation medium FSB are:

[0058] FSB medium Each 1L contains water as the solvent. yeast extract 3.5g Casein peptone 10g NaCl 1.5g <![CDATA[K2HPO4]]> 2g <![CDATA[MgSO4]]> 0.4g glucose 60g

[0059] Sterilize at 121℃ for 20 min, and sterilize glucose at 115℃ for 30 min;

[0060] Alternatively, the specific components of THY culture medium are:

[0061]

[0062]

[0063] Adjust the pH to 6.8; sterilize at 121℃ for 20 minutes.

[0064] The method described above is applied to the direct fermentation production of hyaluronic acid with a specific molecular weight.

[0065] This invention employs a mixed-culture fermentation strategy, adding regulatory strains during the fermentation process to degrade high molecular weight HA, and by adjusting the proportion of mixed cultures, achieving the goal of obtaining HA with a specific molecular weight.

[0066] Figure 1 This is the carrier used in this invention. Figure 2 Is Figure 1 The vector pDL278-PR31, constructed based on the vector, Figure 3 Is Figure 2 The vector pDL278-PR31-amyA, constructed based on this, Figure 4 Is Figure 3 pDL278-PR31-amyA-rHyal was constructed based on this. Figure 5-7 This is a gel image of the molecular verification constructed from the carrier. Figure 8 This is a gel image demonstrating the molecular verification of the vector pDL278-PR31-amyA-rHyal being electroporated into Streptococcus zooepidemicus ATCC 39920. Figure 9 To construct a good growth curve and graph of enzyme activity changes, the enzyme activity stabilized at 15 μ / mL after 20 hours of cell growth. Figure 10 This is a gel graph showing the relationship between the mixed bacteria ratio and the molecular weight of the product in an embodiment of the present invention. The graph shows that the molecular weight of the product decreases as the mixed bacteria ratio increases.

[0067] Specifically, the relevant preparation and testing methods are as follows:

[0068] Example 1: Obtaining the regulatory strain

[0069] 1.1 Source of the gene encoding hyaluronidase: Based on a novel hyaluronidase encoding gene hylP disclosed in patent CN115820694A, the gene rHyal was obtained by codon optimization by a third-party platform according to its amino acid sequence SEQ ID NO.1.

[0070] 1.2 Construction of recombinant plasmids: Expression element fragments were obtained by amplifying the PR31 promoter and amyA signal peptide, followed by purification and recovery. Subsequently, the vector pDL278 (e.g., Figure 1 The expression element fragments were digested with enzymes (as shown), and the corresponding fragments were recovered by gel extraction. The recovered expression element fragments were ligated with the linearized vector pDL278 using a one-step cloning kit to obtain the vector pDL278-PR31 (as shown). Figure 2 (as shown), pDL278-PR31-amyA (as shown) Figure 3 (As shown) The final plasmid obtained was pDL278-PR31-amyA-rHyal (as shown) Figure 4 (As shown), the specific steps are as follows:

[0071] Table 1

[0072] carrier Enzyme cleavage sites Insert fragment pDL278 SphI PR31 pDL278-PR31 BamHI amyA pDL278-PR31-amyA SacI, EcoRI rHyal

[0073] Table 2

[0074]

[0075] ① After the fragment amplification system is prepared, PCR, gel electrophoresis, purification and fragment recovery are performed.

[0076] ② The vector was digested with enzymes, then run on a gel, and purified to recover the vector.

[0077] ③ Homologous recombination ligation between fragments and vectors.

[0078] ④ Transform the cultured plasmid into competent E. coli cells JM109, plate it on LB solid medium containing 0.1 mg / mL of SPCC resistance, and incubate at 37°C for 12 h.

[0079] ⑤ Select single colonies from the culture medium for molecular verification. After passing the molecular verification, select single colonies on LB liquid medium containing SPC antibiotic at a final concentration of 0.1 mg / mL, incubate at 37℃ and 200 rpm for 6 hours, and then extract and preserve the plasmid using the Tiangen reagent kit.

[0080] The relevant PCR system is as follows:

[0081] Table 3

[0082] Expanded fragment system 50μL 2×PhantaMaxBuffer 25μL dNTPs (2mM) 1μL Phantaenzyme 1μL Primer-F (10μM) 2μL Primer-R (10μM) 2μL Genome 0.5μL <![CDATA[ddH2O]]> 19μL

[0083] The PCR reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s; 68℃ extension for 5 min; 35 cycles.

[0084] Table 4

[0085] Enzyme digestion vector system 50μL 10×Buffer 5μL pDL278 1μL Enzyme I 2.5μL Enzyme II 2.5μL <![CDATA[ddH2O]]> 39μL

[0086] The reaction conditions were: enzyme digestion at 37℃ for 30 minutes.

[0087] Table 5

[0088] Connection system 10μL carrier 2μL Excerpt 3μL One-step ligase 5μL

[0089] The reaction conditions were: 50℃ for 15 min.

[0090] 1.3 Construction of the regulatory strain: The constructed plasmid pDL278-PR31-amyA-rHyal was electroporated into competent cells of *S. zooepidemicus* ATCC 39920 to obtain the regulatory strain. The specific steps are as follows:

[0091] Step 1: Preparation of competent Streptococcus veterinaria cells:

[0092] ① Inoculate *S. zooepidemicus* ATCC 39920 from glycerol tubes stored at -80℃ into THY liquid medium and incubate at 37℃ and 200r / min for 18h. Then, dilute the culture in a series and plate it onto THY plates and incubate at 37℃ for 24h.

[0093] ② Pick a single colony and inoculate it into THY liquid culture medium, and incubate at 37℃ and 200r / min for 3.5h.

[0094] ③ Inoculate it into 100 mL of fresh THY liquid culture medium at a 2% inoculation rate and culture at 37℃ and 200 r / min until the OD value is about 0.5. At this time, add 250 μL of hyaluronidase with a final concentration of 0.2 g / mL and continue to culture for 30 min.

[0095] ④ Remove the Erlenmeyer flask and place it on the prepared ice. Incubate on ice for 5 minutes, then aseptically dispense the bacterial solution from the Erlenmeyer flask into 50mL dispensing tubes.

[0096] ⑤ Centrifuge at 4℃ and 8000 r / min for 10 min, discard the supernatant, and suspend the bacterial cells in 10 mL of 0.5 mol / L sucrose solution in a sterile operating table. Repeat this step twice.

[0097] ⑥ Resuspend the bacterial cells in 250 μL of 0.5 M sucrose solution containing 15% glycerol (v / v) and dispense into 1.5 mL EP tubes for storage.

[0098] Step 2: Electroporation of Streptococcus veterinaria

[0099] ① Take 2 μL of plasmid pDL278-PR31-amyA-rHyal and add it to 50 μL of Streptococcus pyogenes competent cells.

[0100] ② Transfer the well-mixed bacterial culture to a fully cooled 2mm electric transfer cup.

[0101] ③ Adjust the voltage to 250V and the resistance to 200Ω, and click for 5ms.

[0102] ④ Take 1 mL of THY liquid culture medium from an ice bath and place it in an electroporation cup.

[0103] ⑤ Transfer the thoroughly mixed bacterial solution to a sterile 1.5 mL EP tube and incubate at 37 °C and 200 r / min for 3 h.

[0104] ⑥ Spread the revived bacterial culture onto THY solid medium containing SPC resistance at a final concentration of 0.1 mg / mL, invert the plate in a 37°C constant temperature incubator, and incubate until colonies grow to obtain the regulated strain.

[0105] The specific components of the THY culture medium are shown in Table 6.

[0106] Table 6

[0107] THY medium 1L (solvent is water) Beef Powder 10g trypsin 20g yeast extract 2g glucose 2g <![CDATA[NaHCO3]]> 2g NaCl 2g <![CDATA[Na2HPO4]]> 0.4g

[0108] Adjust the pH to 6.8; sterilize at 121℃ for 20 minutes.

[0109] 1.3 Enzyme activity detection of regulatory strains:

[0110] ① Select a single colony of the control strain and incubate it in 50 mL of THY liquid medium at 37℃ and 200 r / min for 16 h.

[0111] ② Mix 20 μL of the culture medium of the control strain with 30 μL of the substrate solution and incubate at 37°C for 15 min.

[0112] Table 7

[0113] substrate solution 200mL (solvent is water) <![CDATA[Na2HPO4]]> 3.53g <![CDATA[NaH2PO4]]> 4.77g HA 0.1g

[0114] Adjust the pH to 6.8.

[0115] ③ Add 100 μL of CTAB solution containing 2% NaOH to the plate, transfer it to a 96-well plate, and detect the absorbance at 400 nm using a multi-functional microplate reader.

[0116] The relevant verification results are as follows Figures 5 to 8 As shown.

[0117] Figure 9 To construct a good growth curve and graph of enzyme activity changes, the enzyme activity stabilized at 15 μ / mL after 20 hours of cell growth. Figure 10This is a gel graph showing the relationship between the mixed bacteria ratio and the molecular weight of the product in an embodiment of the present invention. The graph shows that the molecular weight of the product decreases as the mixed bacteria ratio increases.

[0118] Example 2: Obtaining HA with a specific molecular weight through mixed-culture fermentation

[0119] This method involves adding a regulatory strain during the fermentation process of the production bacteria. The hyaluronic acid lysin secreted by this strain directly degrades high-molecular-weight HA into low-molecular-weight HA during production. By adjusting the mixed bacterial ratio, HA of a specific molecular weight can be obtained. The specific fermentation steps are as follows:

[0120] Step 1: Use Streptococcus vesicularis as the production strain for HA; inoculate the production strain into seed culture medium THY, and culture at 37℃ and 200r / min. After 24h of primary seed culture, transfer it to secondary seed culture THY at a 2% inoculation rate and continue fermentation in fermentation medium FSB for 12h. Then, transfer it to a 5L fermenter containing 3L of fermentation medium FSB at a 10% inoculation rate for fermentation.

[0121] Step 2: When the production strain has grown for 4-6 hours, add the control strain that has been cultured at 37°C and 200 rpm for 24 hours to a fermenter containing 3L of culture medium at different volume ratios, for example: fermentation system: control strain volume ratio of 250:1 (12mL control strain), 2000:1 (1.5mL control strain), 5000:1 (0.6mL control strain), and continue fermentation for 24 hours.

[0122] The specific components of the fermentation medium FSB are as follows:

[0123] Table 8

[0124] FSB medium 1L (solvent is water) yeast extract 3.5g Casein peptone 10g NaCl 1.5g <![CDATA[K2HPO4]]> 2g <![CDATA[MgSO4]]> 0.4g glucose 60g

[0125] Sterilize at 121℃ for 20 minutes, and sterilize glucose at 115℃ for 30 minutes.

[0126] Step 3: Treatment method for fermentation broth

[0127] ① Take 4 mL of fermentation broth with different mixed bacterial ratios and place them in 10 mL tubes respectively. Add an equal volume of 4 mL of aqueous solution containing 0.1% SDS, mix well and let stand at 4℃ for 10 min to separate HA from the bacterial cells. Then centrifuge at 8000 r / min for 15 min.

[0128] ② Take 2 mL of the supernatant containing HA and add it to 6 mL of anhydrous ethanol. Mix thoroughly. At this point, you can see white flocculent HA. Place at 4℃ for 6 h, centrifuge at 8000 r / min for 15 min, and discard the supernatant.

[0129] ③ After air-drying the HA precipitate, add 100 μL of deionized water to fully dissolve the precipitate, and store the fermentation sample at 4℃ for subsequent molecular weight and yield detection.

[0130] Agarose gel electrophoresis for molecular weight determination:

[0131] ① Take 0.1g of HA standards with molecular weights of 2000KDa, 1000KDa, 500KDa and 100KDa respectively, dissolve them in 10mL of deionized water, and store at 4℃ to prepare molecular weight standard solutions.

[0132] ② Weigh 0.5g of agarose into a beaker, add 90mL of distilled water, and heat to dissolve.

[0133] ③ Measure 10 mL of 10×TAE buffer and place it in a water bath at 48°C. After heating in the water bath for 15 min, pour the 10×TAE buffer into the agarose solution and shake thoroughly to mix.

[0134] ④ Pour the melted adhesive into the glue tank.

[0135] ⑤ Add an appropriate amount of fresh 1×TAE buffer to the electrophoresis tank to ensure that the electrophoresis solution completely soaks the gel surface and is more than 2 mm above it.

[0136] ⑥ Add 50 μL of the sample to be tested to each well. After the sample is spotted, allow it to settle for 5-10 minutes, then perform electrophoresis at 40V and 35mA for 1 hour; then adjust the electrophoresis apparatus to 65V and 80mA.

[0137] ⑦ Place the gel into 50 mL of prepared staining solution containing 0.005% Stains-All, gently shake in a dark environment, and stain for 12 h; then place the gel in water to decolorize for 48 h; then decolorize under natural light for about 3 h, and record the experimental results under suitable light.

[0138] HLPC Molecular Weight Determination: The molecular weight of HA was determined using high performance liquid chromatography (HPLC). The chromatographic conditions were: Ultrahv drogel linear column (10 μm, 7.8 mm × 300 mm); mobile phase: 0.1 M NaNO3; flow rate: 0.6 ml / min; column temperature and detector temperature: 35 °C; injection volume: 20 μL; the determination was performed using a differential optical analyzer, and the peak elution time was recorded. The peak elution time showed a linear relationship with the logarithm of the HA molecular weight.

[0139] HA yield detection method:

[0140] ① Preparation of standard: Weigh 5 mg of HA sample using an analytical balance, dissolve it in 10 mL of deionized water to prepare a stock solution of 5 mg / mL, and then dilute the stock solution to prepare standard solutions of 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, and 0.45 mg / mL. Store at 4℃ for later use.

[0141] ② Sample enzymatic digestion: Take 1 mL of sample and standard solution respectively, add 500 μL of hyaluronic acid lysin liquid with an enzyme activity of 2000 U, incubate at 37℃ for 30 min, and repeat three times.

[0142] ③HPLC detection conditions: Detection column: Aminex HPX-87P column, 300×7.8mm organic acid column, mobile phase: 5mM H2SO4, injection volume: 20μL, flow rate: 0.6mL / min, wavelength: 232nm, detector and column temperature: 65℃.

[0143] Figure 11-12 The results showed that adding different mixed-culture ratios of regulatory strains could yield HA fermentation products with different molecular weights. As the mixed-culture ratio increased, the molecular weight of the product decreased accordingly. The molecular weight of HA produced by pure-culture fermentation in a 5L fermenter was 2402.3 kDa, and the yield was 3.22 g / L. In this invention, a mixed-culture ratio of 5000:1 can theoretically yield HA products with a molecular weight of 1000 kDa-1500 kDa. In this experiment, HA with a molecular weight of approximately 1564.3 kDa was obtained, and the yield was 3.66 g / L. A mixed-culture ratio of 2000:1 can theoretically yield HA products with a molecular weight of 100 kDa-500 kDa. In this experiment, HA with a molecular weight of approximately 703.4 kDa was obtained, and the yield was 3.61 g / L. A mixed-culture ratio of 250:1 can theoretically yield HA products with a molecular weight of approximately 100 kDa. In this experiment, HA products with a molecular weight of approximately 218.6 kDa were obtained, and the yield was 3.38 g / L.

[0144] Experimental results have confirmed that producing hyaluronic acid (HA) of specific molecular weights using mixed-strain fermentation is entirely feasible. This invention is not limited to the three specific mixed-strain ratios mentioned in this patent, and an increase in HA yield was observed compared to single-strain fermentation. In actual industrial production, by precisely controlling the mixed-strain ratio, hyaluronic acid products of specific molecular weights can be effectively obtained. Furthermore, the experimental results reveal the significant advantages of mixed-strain fermentation in increasing hyaluronic acid yield. Compared to pure-strain fermentation, mixed-strain fermentation not only achieves controllability of product molecular weight but also further increases the HA content per unit volume of fermentation broth. This discovery is of great significance for the large-scale industrial production of hyaluronic acid, as it provides a more efficient and flexible production method. In the future, with in-depth research into the mechanism of mixed-strain fermentation, we expect to develop more types of mixed-strain combinations to meet specific requirements for the molecular weight and yield-related properties of hyaluronic acid.

[0145] The sequences used in this invention are as follows:

[0146] The amino acid sequence (302AA) of the novel hyaluronidase SEQ ID NO.1

[0147] MTSRRLFLGAFTAGAVTVAAGASEAAAAEAEGVVEGDTTFTGAVKATSFHTDSAAMS

[0148] SFAGTAATAHTHTLTVRQAGTVVDSVALNVTSTNPNDSAMWVSGKEKARGTLKVTHQGY

[0149] ADGSDYEAAAISIWLSTADGVEGTRAQGIFMRPAPGNGPTKGNLITLRNNEDKVDDFVVKA

[0150] NGRVGLGLPYGMNPRARIEIAQRPGDTMGLMLQANPESTAHLADFRNSQDVSQTRVLNDG

[0151] TLASRNVYLGGSGSPQFGGGDAVVGIRNRALKPTTNPANGGVLYAENGALVWHGSNGTVT

[0152] TIAPA*

[0153] SEQ ID NO.2: 909bp sequence of the rHyal gene encoding hyaluronidase.

[0154] ATGACTTCTAGAAGACTTTTTCTTGGAGCTTTTACTGCTGGAGCTGTTACTGTTGCTGCTGGAGCTTCTGAAGCTGCTGCTGCTGAAGCTGAAGGAGTTGTTGAAGGAGATACTACTTTTACTGGAGCTGTTAAGGCTACTTCTTTTCATACTGATTCTGCTGCTATGTCTTCTTTTGCTGGAACTGCTGCTACTGCTCATACTCATACTCTTACTGTTAGACAAGCTGGAACTGTTGTTGATTCTGTTGCTCTTAATGTTACTTCTACTAATCCAAATGATTCTGCTATGTGGGTTTCTGGAAAGGAAAAGGCTAGAGGAACTCTTAAGGTTACTCATCAAGGATATGCTGATGGATCTGATTATGAAGCTGCTGCTATTTCTATTTGGCTTTCTACTGCTGATGGAGTTGAAGGAACTAGAGCTCAAGGAATTTTTATGAGACCAGCTCCAGGAAATGGACCAACTAAGGGAAATCTTATTACTCTTAGAAATAATGAAGATAAGGTTGATGATTTTGTTGTTAAGGCTAATGGAAGAGTTGGACTTGGACTTCCATATGGAATGAATCCAAGAGCTAGAATTGAAATTGCTCAAAGACCAGGAGATACTATGGGACTTATGCTTCAAGCTAATCCAGAATCTACTGCTCATCTTGCTGATTTTAGAAATTCTCAAGATGTTTCTCAAACTAGAGTTCTTAATGATGGAACTCTTGCTTCTAGAAATGTTTATCTTGGAGGATCTGGATCTCCACAATTTGGAGGAGGAGATGCTGTTGTTGGAATTAGAAATAGAGCTCTTAAGCCAACTACTAATCCAGCTAATGGAGGAGTTCTTTATGCTGAAAATGGAGCTCTTGTTTGGCATGGATCTAATGGAACTGTTACTACTATTGCTCCAGCTTAA

[0155] SEQ ID NO.3 Nucleotide sequence of PR31 promoter (340 bp)

[0156] TTTATTCCCCTTTTGTTTTTATCTAGACATGATGTCTCCTAATTATCAACGGCAGATAATTATACCTCTATTGTAAACGTTATTGCCAATTAAATCAATATTAAAACCTGTTTTTTATTTATTTCTGTTTTTTCAATTATATGTCAACCATTACTTTAGCAAGTTACTT GTCATCGTAAGTATAGTAAGTGTTTTTGAGGAATCATCAGTATTTTATTTATTTTCTATAAATTAAAAATAAAGAAATATTAAATGCCGGTAAAAGGAACTTTTGAGATAATTTAGAGCAGCTAAAAAGCCCTGCTATAGCGCTAGAGCAGAGCTTTTCCTATTCGAT

[0157] SEQ ID NO.4 amyA signal peptide nucleotide sequence (147bp)

[0158] ATGCAGCAGGACGGCACACAGCAGGACCGGATCAAGCAGAGTCCCGCCCCTCTCAACGGAATGAGCCGACGAGGCTTCCTCGGTGGCGCCGGCACCCTCGCGCTCGCTACCGCGTCCGGGCTGCTGCTGCCCGGCACAGCCCACGCC

[0159] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A regulatory strain for producing hyaluronidase, characterized in that: The construction of the regulatory strain was based on the hyaluronidase encoding gene hylP. Codon optimization was performed using a third-party platform based on its amino acid sequence to obtain the gene rHyal. This gene was inserted into the vector pDL278. The expression of the hyaluronidase encoding gene rHyal was driven by the PR31 promoter, and the secretion of mature hyaluronidase was controlled by the amyA signal peptide. The resulting plasmid pDL278-PR31-amyA-rHyal was obtained. The plasmid was electroporated into *Streptococcus zooepidemicus* ATCC 39920 to induce the secretion of active hyaluronidase, thus obtaining a regulatory strain for hyaluronidase production during fermentation.

2. The regulatory strain according to claim 1, characterized in that: The hyaluronidase encoding gene hylP is a novel hyaluronidase encoding gene hylP disclosed in Chinese Patent Publication CN115820694A.

3. The regulatory strain according to claim 1 or 2, characterized in that: The amino acid sequence of the hyaluronidase encoding gene hylPhylP is shown in SEQ ID NO.1, the nucleotide sequence of the gene rHyal is shown in SEQ ID NO.2, the nucleotide sequence of the PR31 promoter is shown in SEQ ID NO.3, and the nucleotide sequence of the amyA signal peptide is shown in SEQ ID NO.

4.

4. A method for directly producing hyaluronic acid of a specific molecular weight through mixed fermentation using the regulatory strain as described in any one of claims 1 to 3, characterized in that: The method involves constructing a pathway for hyaluronidase production within Streptococcus zooepidemicus ATCC 39920, secreting hyaluronidase, and using it as a regulatory strain in the fermentation process for producing hyaluronic acid (HA). Adding different proportions of the regulatory strain during fermentation allows for the fermentation of hyaluronic acid with different molecular weights.

5. The method according to claim 4, characterized in that: Includes the following steps: By adding a hyaluronidase-producing regulatory strain during fermentation, and mixing different proportions of the regulatory strain into the fermentation broth when the production strain reaches the exponential phase, the viscosity of the fermentation broth is reduced to varying degrees, and hyaluronic acid of a specific molecular weight is obtained directly through fermentation.

6. The method according to claim 4, characterized in that: The method for constructing a hyaluronidase-producing plasmid is to use the PR31 promoter to drive the expression of the hyaluronidase gene rHyal, use the amyA signal peptide to control the secretion of mature hyaluronidase, and then clone it into the pDL278 vector to construct the hyaluronidase expression plasmid pDL278-PR31-amyA-rHyal.

7. The method according to any one of claims 4 to 6, characterized in that: The specific steps are as follows: Step 1: Use *S. zooepidemicus* ATCC 39920 as the production strain for hyaluronic acid; inoculate the production strain into seed culture medium THY, and culture at 37℃ and 200 r / min. After 24 h of primary seed culture, transfer it to secondary seed culture THY at a 2% inoculation rate and continue fermentation in fermentation medium FSB for 12 h. Then, transfer it to fermentation medium FSB at a 10% inoculation rate for further fermentation. Step 2: When the production strain has grown for 4-6 hours, add the control strain that has been cultured at 37℃ and 200r / m for 24 hours to the fermentation medium FSB according to different volume ratios: the mixed volume ratio of the control strain and continue fermentation for 24 hours.

8. The method according to claim 7, characterized in that: Fermentation system: The volume ratio of the mixed strains is 250:1, 2000:1 or 5000:

1.

9. The method according to claim 7, characterized in that: The specific components of the fermentation medium FSB are: Sterilize at 121℃ for 20 min, and sterilize glucose at 115℃ for 30 min; Alternatively, the regulatory strain can be cultured as follows: A single colony of the regulatory strain is picked and cultured in THY liquid medium at 37°C and 200 rpm for 24 hours; the specific THY liquid medium is as follows: Adjust the pH to 6.8; sterilize at 121℃ for 20 minutes.

10. The application of the method according to any one of claims 4 to 9 in the direct fermentation production of hyaluronic acid of a specific molecular weight.

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