A fermentation method of recombinant lactococcus lactis with high yield of hyaluronic acid based on exogenous hyaluronidase regulation

Abstract

The application discloses a fermentation method for high-yield hyaluronic acid, and belongs to the technical field of microbial fermentation. szhasABC The method adopts recombinant lactococcus carrying a gene cluster from streptococcus zooepidemicus as a production strain, externally adds leech-derived hyaluronidase in a fermentation process, and dynamically controls fermentation temperature (30-37 DEG C). The strategy ingeniously couples the biosynthesis of HA with controllable enzymolysis process, effectively releases the feedback inhibition of high-molecular-weight HA capsule on cell growth, and significantly improves the yield. Meanwhile, through accurate adjustment of process parameters, precise customization of product molecular weight is realized. Experimental results show that, by using the method, the yield of hyaluronic acid can reach 4.6 g / L at most, the molecular weight can be regulated in the range of 0.4 kDa to 850 kDa as required, and the deviation rate is less than 5%. The application does not need to perform complex gene editing on the host, does not depend on the preservation of specific engineering strains, has the advantages of high safety, simple process, low cost, strong controllability and the like, and is suitable for the large-scale production demand of different specifications of hyaluronic acid in the fields of food, cosmetics and medicine.

Description

Technical Field

[0001] This invention belongs to the fields of microbial fermentation engineering, synthetic biology, and biomaterial preparation technology, specifically relating to a method utilizing recombinant lactococcus (… Lactococcus lactis This invention discloses a fermentation method for the efficient production of hyaluronic acid (HA). More specifically, it provides a synergistic optimization strategy that achieves high yield and precise molecular weight control of hyaluronic acid by exogenously adding hyaluronidase derived from leeches during fermentation, combined with dynamic control of fermentation temperature, pH, and metal ion chelating agent concentration. This method is applicable to the green, safe, and large-scale production of food-grade, cosmetic-grade, and pharmaceutical-grade hyaluronic acid. Background Technology

[0002] Hyaluronic acid (HA) is a natural linear high-molecular-weight acidic mucopolysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine linked by alternating β-1,3 and β-1,4 glycosidic bonds. Due to its excellent water-retention, lubrication, biocompatibility, and non-immunogenicity, it is widely used in ophthalmic surgery, orthopedic injections, dermal fillers, wound healing, functional foods, and high-end skincare products. HA of different molecular weights has distinctly different physiological functions: high molecular weight HA (>1000 kDa) primarily functions as a space-filler and anti-inflammatory agent; medium molecular weight HA (50–1000 kDa) promotes tissue repair; while low molecular weight HA (<50 kDa) and even oligosaccharides (<10 kDa) exhibit significant pro-angiogenic, antioxidant, and immunomodulatory activities. Therefore, developing HA production technologies that can simultaneously achieve high yields and precise molecular weight control has become a core demand in the industry.

[0003] Currently, large-scale industrial production of HA mainly relies on pathogenic streptococci (such as Streptococcus vesiculosus). Streptococcus zooepidemicus The fermentation of streptococci, despite its high yield, poses serious biosafety risks. The products may contain harmful substances such as endotoxins and hemolysins, requiring extremely complex and expensive downstream purification steps before they can be used in pharmaceuticals or high-end cosmetics. Furthermore, the high viscosity during streptococcal fermentation presents significant challenges to mass and oxygen transfer.

[0004] To address these issues, researchers are working to develop non-pathogenic alternative hosts. Among these, *Lactococcus lactis* (…) Lactococcus lactis As a food-grade microorganism recognized as "Generally Recognized As Safe" (GRAS) by both the US FDA and the European EFSA, it has outstanding advantages such as a clear genetic background, mature operating tools, simple culture medium composition, and no endotoxin production, and is regarded as a highly promising next-generation chassis cell for HA production.

[0005] However, achieving efficient HA synthesis in Lactococcus lactis faces two major bottlenecks: (1) Insufficient metabolic flux: There is a natural disconnect between the central carbon metabolism of Lactococcus lactis (mainly homolactic fermentation) and the supply pathway of the two key nucleotide precursors required for HA synthesis - UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc), resulting in limited precursor supply.

[0006] (2) Severe product feedback inhibition: HA synthase (HasA) is located on the cell membrane. The high molecular weight HA synthesized is directly secreted into the extracellular space and surrounds the cell to form a dense capsule. This capsule greatly increases the viscosity of the fermentation broth, hindering the transfer of nutrients and oxygen, severely inhibiting cell growth and metabolic activity, thus forming a strong negative feedback loop that limits the final yield. The reported HA yields based on Lactococcus lactis are generally below 1 g / L, which is difficult to meet the economic requirements for industrialization.

[0007] To address the aforementioned issues, existing technologies primarily employ two strategies: one is to enhance the synthetic pathway, for example, by co-expressing the three genes hasA, hasB, and hasC from streptococci to reconstruct a complete HA synthesis pathway; the other is to attempt co-expression of hyaluronidase within the host to reduce capsule burden by degrading some HA. However, endogenous expression of hyaluronidase has significant drawbacks: the expression level and activity of the enzyme are difficult to control precisely, easily leading to excessive degradation of HA into worthless small fragments, resulting in a decrease in total yield; simultaneously, continuous expression of exogenous enzymes may also cause unknown interference with the host cell's own metabolism, affecting genetic stability.

[0008] Furthermore, although existing patents (such as CN114107149B) have achieved ultra-high yields (>70 g / L) in non-streptococcal hosts such as Corynebacterium glutamicum through complex metabolic engineering modifications (such as knocking out competitive pathways, overexpressing precursor synthases, and introducing hemoglobin), these strategies are highly dependent on the host-specific metabolic network and powerful genetic operating system, making it difficult to directly transfer to Lactococcus lactis, a Gram-positive food-grade strain with relatively simple metabolism.

[0009] Therefore, there is an urgent need to develop a novel HA fermentation process that does not rely on complex genome editing, does not require the preservation of specific engineered strains, and can effectively eliminate product inhibition and achieve controllable molecular weight. This process should fully utilize the safety advantages of *Lactococcus lactis* while cleverly circumventing its inherent limitations as a host for HA production. Summary of the Invention

[0010] Purpose of the invention The primary objective of this invention is to provide a safe, efficient, and controllable method for producing hyaluronic acid, thereby overcoming the technical challenges of low yield and uncontrollable molecular weight in existing lactococcus fermentation systems.

[0011] Another objective of this invention is to provide a fermentation strategy that can achieve high hyaluronic acid production without complex genetic modification of the host strain (especially without integrating or expressing the hyaluronidase gene), thereby avoiding the cumbersome procedures and potential biosafety risks of strain preservation.

[0012] Another objective of this invention is to provide a process method that can precisely and flexibly control the molecular weight of the final hyaluronic acid product according to the needs of downstream applications.

[0013] Technical solution To achieve the above objectives, this invention proposes an innovative fermentation strategy that combines exogenous enzyme assistance with the coordinated regulation of process parameters. Specifically, this invention provides a fermentation method for high-yield hyaluronic acid production, comprising the following steps: (a) Provide a recombinant lactococcus engineered strain, wherein the engineered strain stably carries and expresses Streptococcus vesiculosus derived from Streptococcus pneumoniae via a plasmid vector. Streptococcus zooepidemicus The hyaluronic acid synthesis gene cluster includes the szhasA gene encoding hyaluronic acid synthase, the szhasB gene encoding UDP-glucose dehydrogenase, and the szhasC gene encoding UDP-N-acetylglucosamine pyrophosphorylase. (b) The recombinant lactococcus is inoculated into a suitable fermentation medium and fermented under anaerobic or microaerobic conditions; (c) At a specific stage of the fermentation process (preferably the late logarithmic growth stage or the early stationary stage), leeches are exogenously and quantitatively added to the fermentation system. Hirudo medicinalis Hyaluronidase from ( ) source; (d) After adding hyaluronidase, dynamically and synergistically regulate one or more of the following fermentation process parameters according to the preset target hyaluronic acid molecular weight: (i) Fermentation temperature: Gradual adjustment within the range of 30°C to 37°C; (ii) pH of fermentation broth: maintained in a slightly acidic to neutral range of 6.5 to 7.0; (iii) Metal ion chelating agents (such as EDTA): When it is necessary to inhibit enzyme activity, add a solution of EDTA with a final concentration of 0.05–0.2 mmol / L.

[0014] Through the above steps, this invention ingeniously decouples and synergizes the biosynthesis and controllable enzymatic hydrolysis of HA in both time and space. In the early stages of fermentation, the engineered bacteria synthesize high-molecular-weight HA at full capacity; in the middle and later stages, exogenously added hyaluronidase begins to function, selectively cleaving some of the long HA chains. This process not only effectively reduces the viscosity of the fermentation broth and relieves the physical inhibition of cell growth by the capsule, but also, through the feedback of degradation products (such as monosaccharides / disaccharides), may promote the regeneration of precursor substances by the flow of central carbon metabolism, thus forming a virtuous cycle of synthesis-degradation-resynthesis, ultimately achieving a significant leap in total yield. Simultaneously, by precisely controlling temperature, pH, and EDTA, the catalytic efficiency of hyaluronidase can be modulated, thereby achieving precise control over the molecular weight distribution of the final product.

[0015] Beneficial effects Compared with the prior art, the present invention has the following outstanding advantages: 1. High safety and low regulatory risk: The entire process uses GRAS-certified Lactococcus lactis as the production host, without involving any pathogenic microorganisms, resulting in high product safety and easy approval by food and pharmaceutical regulatory authorities.

[0016] 2. Significantly increased yield: By successfully eliminating product feedback inhibition through an exogenous enzyme strategy, the yield of hyaluronic acid was greatly increased from 0.894 g / L of the basic engineered bacteria to 4.6 g / L, reaching a level with industrialization potential.

[0017] 3. Precise and controllable molecular weight: Through the coordinated control of process parameters, HA products with target molecular weights can be customized on demand within a wide range from 0.4 kDa (about 1 disaccharide unit) to 850 kDa, perfectly meeting the differentiated needs of different application scenarios.

[0018] 4. Simple strain construction: The core innovation of this invention lies in the fermentation process rather than the strain itself. The engineered bacteria used are simply conventional hasABC three-gene co-expression strains, which are simple to construct and genetically stable.

[0019] 5. Strong process compatibility and easy to scale up: The M17 and other culture media used have clearly defined components and are inexpensive; the addition of exogenous enzymes and temperature / pH control are mature industrial fermentation operation units, which can be easily scaled up to production on existing fermentation equipment. Attached Figure Description

[0020] Figure 1 This is a plasmid map of the recombinant lactococcus engineered strain NFHA01 described in this invention.

[0021] Figure 2The bar chart shows the comparison of hyaluronic acid yield under different fermentation strategies. The control group was the N9000-A strain expressing only szhasA (0.438 g / L), experimental group 1 was the NFHA01 strain expressing szhasABC (0.894 g / L), and experimental group 2 was the NFHA01 + exogenous enzyme + process control strategy described in this invention (4.6 g / L).

[0022] Figure 3 The figure shows the actual molecular weight distribution of HA obtained using the method of this invention at different target molecular weights (determined by gel permeation chromatography, GPC). The figure displays the actual product spectra at four targets: 0.4 kDa, 100 kDa, 500 kDa, and 850 kDa, demonstrating the effectiveness of molecular weight regulation. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Example 1: Construction of recombinant Lactococcus lactis engineered strain

[0024] 1. Gene cloning and vector construction: using Streptococcus vesiculosus (S. veterinarian) S. zooepidemicus Using genomic DNA as a template, the szhasA, szhasB, and szhasC genes were amplified by PCR. These three genes were then assembled into a polycistronic vector in the order szhasA-szhasB-szhasC and inserted into the multiple cloning site of the *Lactococcus lactis*-*Escherichia coli* shuttle expression vector pMG36e. This vector carries the chloramphenicol resistance gene (Cm). r The recombinant plasmid was named pMG36e-szHasABC and the origin of replication of Lactococcus lactis.

[0025] 2. Obtaining the engineered strain: The recombinant plasmid pMG36e-szHasABC was introduced into Lactococcus lactis NZ9000 competent cells using electroporation. The transformed cells were plated on M17 agar plates containing 10 μg / mL chloramphenicol and incubated at 30°C for 48 hours. Single colonies were picked and verified by colony PCR and plasmid extraction to obtain a correct positive clone, named NFHA01. As a control, an engineered bacterium containing only the szhasA gene was constructed using the same method and named N9000-A. Example 2: Validation of the basic fermentation and exogenous enzyme addition strategy

[0026] 1. Seed culture: The frozen NFHA01 strain was activated and cultured in M17 liquid medium (containing 0.5% glucose and 10 μg / mL chloramphenicol) at 30°C under static conditions for 12 hours to obtain the seed culture.

[0027] 2. Shake-flask fermentation: 5 mL of seed culture was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of fresh M17 medium (containing 1% glucose) and cultured with shaking at 30℃ and 200 rpm. During fermentation, samples were taken every 4 hours, centrifuged, and the supernatant was collected. The HA concentration was determined using the phenol-sulfuric acid method.

[0028] 3. Exogenous enzyme addition experiment: When fermentation reached 24 hours (OD600 ≈ 2.0, entering the early stage of the stationary phase), a sterile filtered leech hyaluronidase solution was added to one group of fermentation broth to achieve a final activity of 5 U / mL. The other group served as a control without enzyme addition. Cultivation continued for 48 hours.

[0029] Result: As Figure 2 As shown, the yield of the control group (N9000-A) was 0.438 g / L; the yield of the NFHA01 control group expressing only szhasABC was 0.894 g / L; while the NFHA01 experimental group with added exogenous hyaluronidase showed a significant increase in HA yield to 2.8 g / L. Meanwhile, GPC analysis showed that the average molecular weight of HA in the enzyme-added group decreased from >1000 kDa to approximately 200 kDa. This preliminarily demonstrates the effectiveness of the exogenous enzyme strategy in relieving product inhibition and increasing yield. Example 3: Optimization and Validation of High-Yield and Molecular Weight Controllable Fermentation Process

[0030] Based on the results of Example 2, this example demonstrates process scale-up and optimization in a 5 L fully automated fermenter.

[0031] 1. Basic fermentation conditions: 3 L fermenter volume, M17 culture medium (containing 2% glucose), 5% (v / v) inoculum, 200 rpm stirring speed, nitrogen gas is introduced to maintain a micro-aerobic environment, initial pH 6.8 (maintained by automatic addition of 2 M NaOH or 2 M HCl), fermentation temperature 30℃.

[0032] 2. Logic of exogenous enzyme addition and parameter regulation: Enzyme addition: At 24 hours of fermentation (early stage of the stationary phase), leech hyaluronidase was added in a single flow to bring the final activity to 8 U / mL.

[0033] Temperature control: If the target is low molecular weight HA, the temperature is increased from 30℃ to 37℃ at a rate of 0.5℃ / h from the time of enzyme addition; if the target is high molecular weight HA, the temperature is kept constant at 30℃.

[0034] EDTA regulation: If online monitoring (or other rapid detection methods) finds that the molecular weight is lower than the target value, add 0.1 M EDTA solution to bring the final concentration of EDTA in the fermentation broth to 0.1 mmol / L to inhibit enzyme activity.

[0035] pH control: The pH will be strictly controlled between 6.5 and 7.0 throughout the process.

[0036] 3. Batch fermentation experiment: Following the above strategy, four batches of parallel fermentation were carried out with target molecular weights of 0.4 kDa, 100 kDa, 500 kDa and 850 kDa respectively.

[0037] 4. Results Analysis: After 48 hours of fermentation, samples were taken to determine the HA yield and molecular weight. The results are shown in Table 1.

[0038]

[0039] Conclusion: Experimental results show that the fermentation method of this invention successfully achieved precise and controllable synthesis of hyaluronic acid in the range of 401 kDa to 850 kDa, with deviation rates of less than 5% for all targets. More importantly, a maximum yield of 4.6 g / L was obtained when the target molecular weight was 100 kDa, fully verifying the excellent performance of this invention in balancing yield and quality.

Claims

1. A fermentation method for high-yield hyaluronic acid, characterized in that, Includes the following steps: (a) Provide a recombinant lactococcus strain ( Lactococcus lactis The recombinant lactococcus carries and expresses a strain derived from Streptococcus vesiculosus (S. veterinaryis). Streptococcus zooepidemicus A hyaluronic acid synthesis gene cluster, the gene cluster containing encoding hyaluronic acid synthase. szhasA Gene encoding UDP-glucose dehydrogenase szhasB Genes and encoding UDP-N-acetylglucosamine pyrophosphorylase szhasC Gene; (b) The recombinant lactococcus was inoculated into a fermentation medium and fermented at 28–32°C; (c) During the late logarithmic growth phase or early stationary phase of the fermentation process, leeches are added exogenously to the fermentation system. Hirudo medicinalis Hyaluronidase from ( ) source; (d) After step (c), the activity of the hyaluronidase is dynamically regulated according to the preset target hyaluronic acid molecular weight by at least one of the following methods: (i) The fermentation temperature was gradually increased from 30°C to 37°C at a rate of 0.3–0.8°C / h; (ii) Add the metal ion chelating agent EDTA to the fermentation system to a final concentration of 0.05–0.2 mmol / L in the fermentation broth; (iii) Control the pH of the fermentation broth within the range of 6.5–7.0; Through the aforementioned dynamic regulation, the production of hyaluronic acid and the directional control of its molecular weight are simultaneously achieved.

2. The fermentation method according to claim 1, characterized in that, The recombinant lactococcus is Lactococcus lactis NZ9000.

3. The fermentation method according to claim 1, characterized in that, The szhasA , szhasB and szhasC The genes are located on the same expression vector and are driven by the constitutive promoter P32.

4. The fermentation method according to claim 1, characterized in that, The amount of hyaluronidase derived from leeches added resulted in a final enzyme activity concentration of 3–10 U / mL in the fermentation broth.

5. The fermentation method according to claim 1, characterized in that, The fermentation medium is M17 medium, in which glucose is the carbon source and its initial concentration is 1–2% (w / v).

6. The fermentation method according to claim 1, characterized in that, The fermentation process is carried out under micro-aerobic conditions, and dissolved oxygen levels are maintained by controlling the stirring speed and / or introducing inert gas.

7. The fermentation method according to any one of claims 1 to 6, characterized in that, The final yield of hyaluronic acid is no less than 4.0 g / L.

8. The fermentation method according to any one of claims 1 to 6, characterized in that, The weight-average molecular weight of the obtained hyaluronic acid can be controlled within the range of 0.4 kDa to 850 kDa, and the deviation rate between the actual molecular weight and the target molecular weight is less than 5%.

9. The application of the fermentation method according to any one of claims 1 to 8 in the preparation of food-grade, cosmetic-grade, or pharmaceutical-grade hyaluronic acid products.

10. A hyaluronic acid product, obtained by the fermentation method according to any one of claims 1 to 8, characterized in that, The hyaluronic acid has a weight-average molecular weight between 0.4 kDa and 850 kDa, and a purity higher than 95%.

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