Method for controlling the production of chondroitin by fermentation

Abstract

The application belongs to the field of chondroitin production, and particularly relates to a chondroitin fermentation production control method, which comprises fermenting and culturing a chondroitin-producing strain, and the molar concentration ratio of pyruvic acid and acetyl coenzyme A is controlled to be (1.5-9):1 during the fermentation and culturing process. The key of the application is to produce chondroitin by fermentation, and the molar concentration ratio of pyruvic acid and acetyl coenzyme A is controlled to be in a specific range during the fermentation process, so that the substrate conversion rate and the chondroitin content in the fermentation product can be effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of chondroitin production, specifically relating to a method for controlling the fermentation production of chondroitin. Background Technology

[0002] Chondroitin is an important glycosaminoglycan (GAG) and a major component of animal connective tissue and extracellular matrix. Chondroitin possesses various biological functions, including lubricating joints, maintaining tissue elasticity, and participating in cell signaling. Currently, both domestic and international standards for the quality of chondroitin raw materials are increasingly stringent. Manufacturers must improve their production processes and enhance product quality, particularly by developing low-pollution, low-energy-consumption, high-yield, and high-quality production processes to adapt to industry development and environmental requirements, thereby achieving greater profitability.

[0003] There are various production processes for chondroitin, including animal cartilage extraction, microbial fermentation, and chemical synthesis. Currently, commercially available chondroitin is mainly extracted from bovine, porcine, and shark cartilage. This method is limited by raw materials, and subsequent purification processes are relatively complex, making it difficult to scale up. Raw material costs are high, purity is unstable, and environmental pollution is significant. Chemical synthesis requires sophisticated operations, causes severe pollution, consumes high energy, and makes it difficult to remove impurities from the finished protein. Furthermore, it is limited to laboratory scale and cannot be scaled up for mass production. Microbial fermentation for chondroitin production offers advantages such as stable product quality, low cost, less pollution, and no scalability limitations.

[0004] In recent years, literature reports have revealed that some strains can ferment and produce chondroitin analogues, leading to increasing interest in the research of microbial fermentation for the production of chondroitin or chondroitin analogues. Fermentation for chondroitin production is not limited by raw materials and offers advantages such as low fermentation costs and minimal environmental pollution, making it the most economically viable and promising method for chondroitin production. However, research on fermentation for chondroitin production is still in its early stages. Currently, conventional processes such as dissolved oxygen feedback, pH-Stat, and intermittent fed-batch methods are used to increase chondroitin yield, but these methods suffer from low sugar and glycerol conversion rates, numerous byproducts, and low chondroitin content, failing to meet industrial-scale requirements. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing fermentation methods for producing chondroitin, which result in low chondroitin content and low conversion rate, and to provide a new method for controlling chondroitin fermentation production. This method can improve chondroitin content and conversion rate.

[0006] The chondroitin fermentation production control method provided by the present invention includes fermenting and culturing a chondroitin-producing strain, wherein the molar ratio of pyruvate to acetyl-CoA is controlled at (1.5~9):1 during the fermentation and culturing process.

[0007] After in-depth and extensive research, the inventors of this invention surprisingly discovered that the molar ratio of pyruvate to acetyl-CoA during fermentation can well reflect the fermentation process of chondroitin. Controlling the molar ratio of pyruvate to acetyl-CoA at (1.5~9):1 during fermentation can fully meet the growth requirements of the cells, improve the metabolic level of the chondroitin-producing strains, and thus effectively improve the substrate conversion rate and the chondroitin content in the fermentation product.

[0008] In a preferred embodiment, the molar ratio of pyruvate to acetyl-CoA is controlled during the fermentation process as follows: during fermentation for 0-12 hours, the molar ratio of pyruvate to acetyl-CoA is controlled at (5-9):1; during fermentation for 12-36 hours, the molar ratio of pyruvate to acetyl-CoA is controlled at (1.5-6):1; and after fermentation for 36 hours, the molar ratio of pyruvate to acetyl-CoA is controlled at (3-6):1, which is more conducive to further improving substrate conversion rate and chondroitin content. Attached Figure Description

[0009] Figure 1 This is the control curve of the pyruvate and acetyl-CoA concentration ratio during the fermentation process of Example 1; Figure 2 This is the control curve of the pyruvate and acetyl-CoA concentration ratio during the fermentation process in Example 2; Figure 3 This is the control curve of the pyruvate and acetyl-CoA concentration ratio during the fermentation process in Example 3; Figure 4 This is the control curve of the pyruvate and acetyl-CoA concentration ratio during the fermentation process in Example 4; Figure 5 This is the control curve of the pyruvate and acetyl-CoA concentration ratio during the fermentation process in Example 5; Figure 6 This is the control curve for the concentration ratio of pyruvate and acetyl-CoA during the fermentation process of Comparative Example 2; Figure 7 This is the control curve for the concentration ratio of pyruvate and acetyl-CoA during the fermentation process of Comparative Example 3; Figure 8 This is a control curve showing the ratio of pyruvate to acetyl-CoA concentrations during the fermentation process of Comparative Example 4. Detailed Implementation

[0010] The chondroitin fermentation production control method provided by this invention includes fermenting and culturing a chondroitin-producing bacterial strain. During the fermentation process, the molar ratio of pyruvate to acetyl-CoA needs to be controlled at (1.5~9):1. The core of this chondroitin fermentation production control method lies in the feedback regulation of the pyruvate to acetyl-CoA concentration ratio during fermentation, thereby effectively improving substrate conversion rate and chondroitin content. When the molar ratio of pyruvate to acetyl-CoA is lower than 1.5:1, it is conducive to cell growth, but the substrate will be excessively consumed, resulting in low yield and low substrate conversion rate. When the molar ratio of pyruvate to acetyl-CoA is higher than 9:1, it is conducive to product synthesis, but it will affect cell growth, thus leading to low chondroitin yield. The molar ratio of pyruvate to acetyl-CoA during the fermentation process can be 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, etc.

[0011] In a preferred embodiment, the molar ratio of pyruvate to acetyl-CoA is controlled during the fermentation process as follows: During the first 0-12 hours of fermentation, the molar ratio is controlled at (5-9):1; during the next 12-36 hours, the ratio is controlled at (1.5-6):1; and after 36 hours, the ratio is controlled at (3-6):1. This allows for a dynamic balance between cell growth and product synthesis at different fermentation stages, which is more conducive to improving the metabolic level and substrate conversion rate of the chondroitin-producing strain. Specifically, during the first 0-12 hours of fermentation, the molar ratio of pyruvate to acetyl-CoA is preferably controlled at ratios such as 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, and 9:1. During the fermentation process from 12 to 36 hours, the molar ratio of pyruvate to acetyl-CoA is preferably controlled at ratios such as 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, and 6:1. After 36 hours of fermentation, the molar ratio of pyruvate to acetyl-CoA is preferably controlled at ratios such as 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, and 6:1. This preferred embodiment controls the molar ratio of pyruvate to acetyl-CoA in three stages (0-12 hours, 12-36 hours, and 36 hours until the end of fermentation). It should be noted that 0~12h includes the 1st, 2nd, 3rd, 4th...9th, 10th, 11th, 12th, a total of 12 hours; 12h~36h includes the 13th, 14th, 15th, 16th...33rd, 34th, 35th, 36th, a total of 24 hours; and 36h to the end of fermentation includes the 37th, 38th...end of fermentation.

[0012] In the above-mentioned chondroitin fermentation production control process, the contents of pyruvate and acetyl-CoA can be obtained in real time by online Raman spectroscopy. Specifically, online Raman spectroscopy (such as a DXR3 SmartRaman spectrometer) is used to scan 3500 cm⁻¹. -1 ~50cm -1 With a full spectral range, it can accurately capture the characteristic Raman peaks of pyruvate and acetyl-CoA (the core peak of pyruvate is 1086 cm⁻¹). -1 The auxiliary peak is at 1280 cm⁻¹. -1 The core peak of acetyl-CoA is 1650 cm⁻¹. -1 The auxiliary peak is 2930 cm⁻¹ -1The molar concentrations of pyruvate and acetyl-CoA in the test sample can be obtained by comparing the characteristic Raman peak areas of the test sample and the standard sample (of known concentration). This is a method known to those skilled in the art and will not be elaborated here. Furthermore, the online Raman spectroscopy is preferably configured with a sterile immersion fiber optic probe, which is directly connected to the fermenter to achieve real-time online spectral acquisition without sampling, avoiding sample contamination and component loss.

[0013] In the above-mentioned chondroitin fermentation production control process, the preferred fermentation conditions include a temperature of 30℃~38℃, such as 30℃, 32℃, 34℃, 36℃, 38℃, etc.; a tank pressure of 0.02MPa~0.08MPa, such as 0.02MPa, 0.03MPa, 0.04MPa, 0.05MPa, 0.06MPa, 0.07MPa, 0.08MPa, etc.; and an aeration ratio of 0.2VVM~4VVM, such as 0.2VVM, 0.5VVM, 0.8VVM, etc. Fermentation speeds include 1 VVM, 1.2 VVM, 1.4 VVM, 1.6 VVM, 1.8 VVM, 2 VVM, 2.2 VVM, 2.5 VVM, 2.8 VVM, 3 VVM, 3.2 VVM, 3.5 VVM, 3.8 VVM, and 4 VVM; rotation speeds range from 50 rpm to 500 rpm, such as 50 rpm, 100 rpm, 150 rpm, 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, and 500 rpm. Generally, fermentation is terminated when the chondroitin growth rate significantly slows or the cell staining becomes lighter. After fermentation, the chondroitin content in the fermentation broth is measured using high-performance liquid chromatography (HPLC).

[0014] In the above-mentioned chondroitin fermentation production control process, the molar ratio of pyruvate to acetyl-CoA can be controlled by adjusting at least one of the following: rotation speed, air flow rate, and tank pressure. If adjusting these three factors fails to control the molar ratio of pyruvate to acetyl-CoA within the target range, then the feed medium addition flow rate is adjusted. Specifically, when the molar ratio of pyruvate to acetyl-CoA is higher than the target range, at least one of the following factors is increased: rotation speed, air flow rate, and tank pressure. If adjusting these three factors fails to control the molar ratio of pyruvate to acetyl-CoA within the target range, then the feed medium addition flow rate is increased for further control. When the molar ratio of pyruvate to acetyl-CoA is lower than the target range, at least one of the following factors is decreased: rotation speed, air flow rate, and tank pressure. If adjusting these three factors fails to control the molar ratio of pyruvate to acetyl-CoA within the target range, then the feed medium addition flow rate is decreased for further control. It should be noted that during the adjustment of the pyruvate to acetyl-CoA molar ratio, the rotation speed, air flow rate, and tank pressure can be adjusted in any order. Preferably, the rotation speed, air flow rate, tank pressure, and feed medium addition flow rate should be adjusted sequentially. If the current parameter cannot control the pyruvate to acetyl-CoA molar ratio within the target range, then the next parameter should be adjusted. Specifically, when the pyruvate to acetyl-CoA molar ratio is higher than the target range, the rotation speed should be gradually increased. When the increased rotation speed can control the pyruvate to acetyl-CoA molar ratio within the target range, the rotation speed should be maintained for fermentation. If increasing the rotation speed to the maximum value (500 rpm) still cannot control the pyruvate to acetyl-CoA molar ratio within the target range, the rotation speed should be set to 500 rpm and the aeration ratio gradually increased. When the increased aeration ratio can control the pyruvate to acetyl-CoA molar ratio within the target range, the aeration ratio should be maintained. Fermentation is carried out by adjusting the aeration ratio. If the molar ratio of pyruvate to acetyl-CoA cannot be controlled within the target range even when the aeration ratio is increased to the maximum value (4VVM), the aeration ratio is set to 4VVM and the tank pressure is gradually increased. If the molar ratio of pyruvate to acetyl-CoA can be controlled within the target range after the tank pressure is increased, the tank pressure is kept constant for fermentation. If the molar ratio of pyruvate to acetyl-CoA cannot be controlled within the target range even when the tank pressure is increased to the maximum value (0.08MPa), the tank pressure is set to 0.08MPa and the feed medium addition flow rate is gradually increased.Conversely, if the molar ratio of pyruvate to acetyl-CoA is lower than the target range, the fermentation speed should be gradually reduced. Once the reduced speed allows the molar ratio of pyruvate to acetyl-CoA to be controlled within the target range, the fermentation speed should be maintained. If reducing the speed to the minimum value (50 rpm) still fails to control the molar ratio of pyruvate to acetyl-CoA within the target range, the fermentation speed should be set to 50 rpm, and the aeration ratio should be gradually reduced. Once the reduced aeration ratio allows the molar ratio of pyruvate to acetyl-CoA to be controlled within the target range, the aeration ratio should be maintained. Fermentation; if the aeration ratio is reduced to the minimum value (0.2 VVM) and the molar ratio of pyruvate to acetyl-CoA still cannot be controlled within the target range, then the aeration ratio is set to 0.2 VVM and the tank pressure is gradually reduced; if the molar ratio of pyruvate to acetyl-CoA can be controlled within the target range after the tank pressure is reduced, then the tank pressure is kept constant for fermentation; if the tank pressure is reduced to the minimum value (0.02 MPa) and the molar ratio of pyruvate to acetyl-CoA still cannot be controlled within the target range, then the tank pressure is set to 0.02 MPa and the feed medium addition flow rate is gradually reduced.

[0015] This invention does not particularly limit the type of chondroitin-producing strain, as long as its fermentation product is mainly chondroitin. For example, it can be a chondroitin-producing recombinant Escherichia coli strain, specifically including at least one of K4 (ATCC 23502), K5 (ATCC23506), K12 (CGMCC No.28317), DH001 (CGMCC NO.32081), C4 (as disclosed in CN119410566A), etc.

[0016] In the above-mentioned chondroitin fermentation production control process, generally, before fermentation culture, the chondroitin-producing strain is first activated, then cultured in shake flasks, and then optionally cultured with a seed culture. When the fermentation scale is small, seed culture may not be necessary, and fermentation culture can proceed directly after the shake flask culture is completed; when the fermentation scale is large, seed culture is generally required after the shake flask culture.

[0017] In the above-mentioned chondroitin fermentation production control process, the plate culture medium used for strain activation may contain 5-8 g / L sodium chloride, 1-15 g / L peptone, 1-15 g / L yeast extract, and 10-20 g / L agar powder. Before seed activation, the plate culture medium generally needs to be sterilized. The sterilization conditions typically include a temperature of 121-123℃ and a time of 20-30 min, with the pH adjusted to 6.5-7.5 before sterilization. A small amount of bacterial solution is taken from the preservation tube and streaked onto a plate (strain activation). After the strain activation is completed, mature single colonies are obtained. The preferred conditions for strain activation include a temperature of 28℃-38℃ and a time of 24-36 h. After the strain activation is completed, 1-5 single colonies are picked from the mature plate and inoculated into shake flask culture medium for shake flask culture.

[0018] In the above-mentioned chondroitin fermentation production control process, the shake flask culture medium used for the shake flask culture may contain 5-8 g / L sodium chloride, 1-15 g / L peptone, and 1-15 g / L yeast extract. Before shake flask culture, the culture medium generally needs to be sterilized. The sterilization conditions typically include a temperature of 121-123℃, a time of 20-30 min, and adjusting the pH to 6.5-7.5 before sterilization. The preferred conditions for shake flask culture include a temperature of 28℃-38℃, a rotation speed of 150-250 rpm, and a culture period of 6-18 hours. When OD... 600nm End the shake-flask culture when the inoculum reaches 1-10. After the shake-flask culture is completed, transfer the seeds to a seed tank, with the inoculum size preferably controlled at 0.1%-2%.

[0019] In the above-mentioned chondroitin fermentation production control process, depending on the fermentation scale requirements, the seed culture needs to be expanded to three stages. The seed culture formula and process for each stage can be the same or different. The seed culture medium used for the seed expansion culture preferably contains 5-15 g / L glucose and / or glycerol, 5-8 g / L sodium chloride, 1-15 g / L peptone, and 1-15 g / L yeast extract. Before seed expansion culture, the seed culture medium generally needs to be sterilized. The sterilization conditions typically include a temperature of 121-123℃, a time of 20-30 min, and adjusting the pH to 6.5-7.5 before sterilization. The preferred conditions for seed expansion culture include a temperature of 28℃-38℃, a tank pressure of 0.025 MPa-0.08 MPa, an aeration ratio of 0.2 VVM-2 VVM, a rotation speed of 100 rpm-500 rpm, and a culture cycle of 4 h-24 h. When OD... 600nm End seed culture expansion when the inoculum reaches 2-12 hours. After seed culture expansion is complete, transfer the seeds to a fermenter, with the inoculum size preferably controlled at 5%-20%.

[0020] In the above-mentioned chondroitin fermentation production control process, the fermentation medium used for fermentation culture preferably contains 10-20 g / L glucose, 5-15 g / L potassium dihydrogen phosphate, 1-10 g / L disodium hydrogen phosphate, 0.5-5 g / L manganese sulfate, 5-10 g / L sodium chloride, and 5-15 g / L peptone. Before fermentation, the fermentation medium generally needs to be sterilized. The sterilization conditions typically include a temperature of 121-123°C and a time of 20-30 minutes, with the pH adjusted to 6.5-7.5 before sterilization. The fed-batch medium used for fermentation culture preferably contains glucose and / or glycerol, that is, it may contain glucose, glycerol, or a mixture of glucose and glycerol. During the fermentation culture, the pH is preferably controlled at 6.5-8.5 by supplementing with alkali. The alkali can be at least one of ammonia, sodium hydroxide, and potassium hydroxide.

[0021] In the above-mentioned chondroitin fermentation production control process, the volume of the fermentation tank can be 2L~300m³. 3 .

[0022] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0023] In the following examples and comparative examples, the chondroitin content in the fermentation broth was detected using a liquid chromatograph equipped with a DAD detector. The liquid chromatographic column had a diameter of 4.6 mm, a height of 250 mm, and a particle size of 5 μm. The wavelength for measuring chondroitin concentration was 232 nm. Mobile phase A was ultrapure water at pH 3.5 (pH adjusted with dilute hydrochloric acid), and mobile phase B was 2M sodium chloride aqueous solution at pH 3.5 (pH adjusted with dilute hydrochloric acid). The flow rate of the mobile phase was 1 mL / min, the measurement temperature was maintained at 40 °C, and the running time was 50 min.

[0024] In the following examples and comparative examples, the molar ratio of pyruvate and acetyl-CoA during fermentation was controlled sequentially by adjusting the rotation speed, air flow rate, tank pressure, and feed medium addition rate.

[0025] In the following examples and comparative examples, recombinant strain C4 was prepared according to the method provided in CN119410566A. This recombinant strain includes the ATPS-ST-pykA-APSK / GlmS-GlmM-GlmU-Pgi-Pgm-GalU / KfoA-KfoC-KfoF gene cluster. The specific preparation process is as follows: Preparation Example 1 This preparation example illustrates the preparation of a recombinant bacterial strain containing the KfoA-KfoC-KfoF gene cluster. The specific preparation steps include: 1. Construction of recombinant plasmid I containing the KfoA-KfoC-KfoF gene cluster: (1) Bacterial genomic DNA rapid extraction kit (purchased from Sangon Biotech (Shanghai)) was used to process Escherichia coli K4 (ATCC23502) according to the instructions to extract genomic DNA.

[0026] (2) Genomic DNA was amplified by PCR using the primer system shown in Table 1. The PCR products were subjected to gel electrophoresis. The corresponding bands were recovered using the AxyPrep DNA gel recovery kit (purchased from AxyPrep) and in accordance with the instructions. The KfoA gene (SEQ ID NO:1), KfoC gene (SEQ ID NO:2) and KfoF gene (SEQ ID NO:3) were obtained.

[0027] Table 1.

[0028] The PCR amplification reaction system included: 1 μL of DNA template, 1 μL each of upstream and downstream primers, and 25 μL of 2×TransStar. ® FastPfu Fly PCR SuperMix (Beijing TransGen Biotech, catalog number AS221-02), add ddH2O to a final volume of 50 μL.

[0029] The PCR amplification reaction program included: 95℃ for 3 min, 25 cycles (95℃ for 20 s, 55℃ for 20 s, 72℃ for 2 min), and 72℃ for 10 min.

[0030] (3) The expression vector pRSFduet-1 was digested with restriction enzymes Nde I and Xho I (purchased from New England Biolabs) according to the instructions. Then, the expression vector pRSFduet-1 was ligated with the KfoA, KfoC and KfoF genes using a homologous recombination seamless cloning kit (purchased from Beijing TransGen Biotech) according to the instructions to obtain recombinant plasmid I. Following the instructions, recombinant plasmid I was transformed into Trans5α competent cells (purchased from Beijing TransGen Biotech), then seeded in LB medium and cultured overnight at 37°C with shaking at 160 rpm. A large amount of recombinant plasmid I was extracted.

[0031] 2. Construction of recombinant strain: Recombinant plasmid I was transformed into Escherichia coli BL21 Star(DE3) chemically competent cells, then inoculated into LB medium and cultured overnight at 37°C and 160 rpm with shaking to obtain the recombinant strain, named C1.

[0032] The recombinant strain C1 obtained in this preparation example can simultaneously synthesize and express UDP-glucosamine isomerase (encoded by the KfoA gene), UDP-glucose dehydrogenase (encoded by the KfoF gene), and chondroitin polymerase (encoded by the KfoC gene), with the specific amino acid sequences shown in Table 2.

[0033] Table 2.

[0034] Preparation Example 2 This preparation example illustrates the preparation of a recombinant bacterial strain containing the GlmS-GlmM-GlmU / KfoA-KfoC-KfoF gene cluster. The specific preparation steps include: 1. Construction of recombinant plasmid II containing the GlmS-GlmM-GlmU / KfoA-KfoC-KfoF gene cluster: (1) Escherichia coli K4 (ATCC23502) was treated with a bacterial genomic DNA rapid extraction kit and the instructions were followed to extract genomic DNA.

[0035] (2) Genomic DNA was amplified by PCR using the primer system shown in Table 3. The PCR products were subjected to gel electrophoresis. The corresponding bands were recovered using the AxyPrep DNA gel recovery kit and in accordance with the instructions to obtain the GlmS gene (SEQ ID NO:13), GlmM gene (SEQ ID NO:14) and GlmU gene (SEQ ID NO:15).

[0036] Table 3.

[0037] The PCR amplification reaction system included: 1 μL of DNA template, 1 μL each of upstream and downstream primers, and 25 μL of 2×TransStar. ® FastPfu Fly PCR SuperMix, add ddH2O to a final volume of 50 μL.

[0038] The PCR amplification reaction program included: 95℃ for 3 min, 25 cycles (95℃ for 20 s, 55℃ for 20 s, 72℃ for 2 min), and 72℃ for 10 min.

[0039] (3) The recombinant plasmid I obtained in Preparation Example 1 was digested with restriction enzymes Nco I and Sac I according to the instructions. Then, the recombinant plasmid I was ligated with the GlmS, GlmM and GlmU genes using the Homologous Recombination Seamless Cloning Kit according to the instructions to obtain recombinant plasmid II. Following the instructions, recombinant plasmid II was transformed into Trans5α competent cells, then seeded in LB medium and cultured overnight at 37°C with shaking at 160 rpm. A large amount of recombinant plasmid II was then extracted.

[0040] 2. Construction of recombinant strain: Recombinant plasmid II was transformed into Escherichia coli BL21 Star(DE3) chemically competent cells, then inoculated into LB medium and cultured overnight at 37°C and 160 rpm with shaking to obtain the recombinant strain, named C2.

[0041] The recombinant strain C2 obtained in this preparation example can express glucosamine isomerase, UDP-glucosamine dehydrogenase, and chondroitin polymerase, as well as glucosamine synthase (encoded by the GlmS gene), phosphoglucosamine mutase (encoded by the GlmM gene), and UDP-N-acetylglucosamine pyrophosphorylase (encoded by the GlmU gene). The specific amino acid sequences are shown in Table 4.

[0042] Table 4.

[0043] Preparation Example 3 This preparation example illustrates the preparation of a recombinant bacterial strain containing the GlmS-GlmM-GlmU-Pgi-Pgm-GalU / KfoA-KfoC-KfoF gene cluster. The specific preparation steps include: 1. Construction of recombinant plasmid III containing the GlmS-GlmM-GlmU-Pgi-Pgm-GalU / KfoA-KfoC-KfoF gene cluster: (1) Escherichia coli K4 (ATCC23502) was treated with a bacterial genomic DNA rapid extraction kit and the instructions were followed to extract genomic DNA.

[0044] (2) Genomic DNA was amplified by PCR using the primer system shown in Table 5. The PCR products were subjected to gel electrophoresis. The corresponding bands were recovered using the AxyPrep DNA gel recovery kit and in accordance with the instructions to obtain the Pgi gene (SEQ ID NO:25), Pgm gene (SEQ ID NO:26) and GalU gene (SEQ ID NO:27).

[0045] Table 5.

[0046] The PCR amplification reaction system includes: 1 μL of DNA template, 1 μL each of upstream and downstream primers, 25 μL of 2×TransStar FastPfu® Fly PCR SuperMix, and ddH2O to bring the final volume to 50 μL.

[0047] The PCR amplification reaction program included: 95℃ for 3 min, 25 cycles (95℃ for 20 s, 55℃ for 20 s, 72℃ for 2 min), and 72℃ for 10 min.

[0048] (3) The recombinant plasmid II obtained in Preparation Example 2 was digested with restriction enzyme Sac I. Then, the recombinant plasmid II was ligated with the Pgi, Pgm and GalU genes using a homologous recombination seamless cloning kit to obtain recombinant plasmid III. Following the instructions, recombinant plasmid III was transformed into Trans5α competent cells, then seeded in LB medium and cultured overnight at 37°C with shaking at 160 rpm. A large amount of recombinant plasmid III was then extracted.

[0049] 2. Construction of recombinant strain: Recombinant plasmid III was transformed into Escherichia coli BL21 Star(DE3) chemically competent cells, then inoculated into LB medium and cultured overnight at 37°C and 160 rpm with shaking to obtain the recombinant strain, named C3.

[0050] The recombinant strain C3 obtained in this preparation example can express UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthase, phosphoglucosamine mutase, and UDP-N-acetylglucosamine pyrophosphorylase. In addition, it can also express glucose-6-phosphate isomerase (encoded by the Pgi gene), glucose phosphate mutase (encoded by the Pgm gene), and glucose-1-phosphate uridine transferase (encoded by the GalU gene). The specific amino acid sequences are shown in Table 6.

[0051] Table 6.

[0052] Preparation Example 4 This preparation example illustrates the preparation of a recombinant bacterial strain containing the ATPS-ST-pykA-APSK / GlmS-GlmM-GlmU-Pgi-Pgm-GalU / KfoA-KfoC-KfoF gene cluster. The specific preparation process includes: 1. Construction of recombinant plasmid IV containing the ATPS-ST-pykA-APSK gene cluster: (1) The ATPS gene was derived from Saccharomyces cerevisiae, and the sequence obtained after codon optimization by Escherichia coli is shown in SEQ ID NO:37; the ST gene was derived from human, and the sequence obtained after codon optimization by Escherichia coli is shown in SEQ ID NO:38; the pykA gene was derived from Escherichia coli, and the sequence is shown in SEQ ID NO:39; the APSK gene was derived from Escherichia coli, and the sequence is shown in SEQ ID NO:40. All of them were synthesized by Sangon Biotech (Shanghai) and loaded into the pET24a vector.

[0053] (2) The pET24a vector was amplified by PCR using the primer system shown in Table 7. The PCR products were subjected to gel electrophoresis. The corresponding bands were recovered using the AxyPrep DNA gel recovery kit and in accordance with the instructions to obtain the ATPS, ST, pykA and APSK genes.

[0054] Table 7.

[0055] The PCR amplification reaction system includes: 1 μL of DNA template, 1 μL each of upstream and downstream primers, 25 μL of 2×TransStar FastPfu® Fly PCR SuperMix, and ddH2O to bring the final volume to 50 μL.

[0056] The PCR amplification reaction program included: 95℃ for 3 min, 25 cycles (95℃ for 20 s, 55℃ for 20 s, 72℃ for 2 min), and 72℃ for 10 min.

[0057] (3) The expression vector pCDFduet-1 was digested with restriction enzymes Nde I and Xho I according to the instructions. Then, the digested expression vector pCDFduet-1 was ligated with the ATPS, ST, pykA and APSK genes using the homologous recombination seamless cloning kit according to the instructions to obtain recombinant plasmid IV. Following the instructions, recombinant plasmid IV was transformed into Trans5α competent cells, then seeded in LB medium and cultured overnight at 37°C with shaking at 160 rpm. A large amount of recombinant plasmid IV was then extracted.

[0058] 2. Construction of recombinant strain: Recombinant plasmid IV and recombinant plasmid III provided in Preparation Example 3 were transformed into Escherichia coli BL21 Star(DE3) chemically competent cells, and then inoculated into LB medium and cultured overnight at 37°C and 160 rpm with shaking to obtain the recombinant strain, named C4.

[0059] The recombinant strain C4 obtained in this preparation example can express UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase, glucose phosphate mutase, and glucose-1-phosphate uridine transferase. In addition, it can also express ATP sulfonase (encoded by the ATPS gene), sulfonyltransferase (encoded by the ST gene), pyruvate kinase (encoded by the pykA gene), and APS kinase (encoded by the APSK gene). The specific amino acid sequences are shown in Table 8.

[0060] Table 8.

[0061] Example 1: Fermentation process in a 15L tank (1) Seed activation: A small amount of bacterial suspension (recombinant strain C4 obtained in Example 4 of CN119410566A) was taken from the preservation tube and diluted. A small amount of bacterial suspension was then transferred to a plate and cultured at 32℃ for 24 h to obtain mature single colonies. The plate culture medium consisted of 5 g / L sodium chloride, 10 g / L peptone, 10 g / L yeast extract, and 20 g / L agar powder. The pH was adjusted to 7.5 before sterilization, and the plate was sterilized at 121℃ for 25 min.

[0062] (2) Shake flask culture: Pick 3 single colonies from the cultured plate and transfer them to a 1L Erlenmeyer flask containing 100mL of culture medium. Place the flask on a shaker and culture at 32℃ and 220rpm. After 18h of culture, the OD... 600nm ≥2, then transfer to a fermenter for cultivation. The shake flask culture medium consists of 5 g / L sodium chloride, 10 g / L peptone, and 10 g / L yeast extract. The pH is adjusted to 7.5 before sterilization, and the mixture is sterilized at 121°C for 25 min.

[0063] (3) Fermentation culture: The shake flask culture medium was transferred to a 15L fermenter at a 5% inoculum rate, with a total volume of 6L. The initial culture conditions included a culture temperature of 32℃, a rotation speed of 200rpm, an aeration ratio of 0.5VVM, and a tank pressure of 0.05MPa. During fermentation, the pH was controlled at around 7.5 by adding ammonia water. During the process, a booster culture medium (50% glucose solution) was added. The fermentation medium consisted of 10g / L glucose, 10g / L potassium dihydrogen phosphate, 5g / L disodium hydrogen phosphate, 3g / L manganese sulfate, 5g / L sodium chloride, and 5g / L peptone. The pH was adjusted to 7.5 before sterilization, and the medium was sterilized at 121℃ for 25min. During fermentation, the concentration ratio of pyruvate to acetyl-CoA in the fermentation broth is monitored in real time using online Raman spectroscopy. The process is controlled in real time by increasing or decreasing at least one of the following: rotation speed, air volume, and tank pressure. If the process requirements are not met by these operations, the flow rate of the fed medium (50% glucose solution) is increased or decreased to ensure process stability. The control of the molar concentration ratio of pyruvate to acetyl-CoA during fermentation is as follows: Figure 1 As shown, specifically: from 0 to 12 h, the molar ratio of pyruvate to acetyl-CoA is (6-9):1, where it is 9:1 at 0 h, 8.4:1 at 4 h, 7.5:1 at 8 h, and 6:1 at 12 h; from 12 to 36 h, the molar ratio of pyruvate to acetyl-CoA is (1.5-6):1, where it is 2.5:1 at 16 h, 1.8:1 at 20 h, 1.5:1 at 24 h, 1.6:1 at 28 h, 1.7:1 at 32 h, and 3:1 at 36 h; after 36 h, the molar ratio of pyruvate to acetyl-CoA is (3-6):1, where it is 4:1 at 40 h, 5.5:1 at 44 h, and 6:1 at 48 h.

[0064] When fermentation reaches 50 hours, the product growth rate slows down significantly or the cell staining becomes lighter. At this point, fermentation is stopped, and the chondroitin content in the fermentation broth is determined by HPLC. The chondroitin content reaches 27 g / L at the end of fermentation, and the conversion rate reaches 57%.

[0065] Example 2: Fermentation process in a 5m³ tank (1) Seed activation: A small amount of bacterial suspension (recombinant strain C4 obtained in Example 4 of CN119410566A) was taken from the preservation tube and diluted. A small amount of bacterial suspension was then transferred to a plate and cultured at 32℃ for 24 h to obtain mature single colonies. The plate culture medium consisted of 5 g / L sodium chloride, 10 g / L peptone, 10 g / L yeast extract, and 20 g / L agar powder. The pH was adjusted to 7.5 before sterilization, and the plate was sterilized at 121℃ for 25 min.

[0066] (2) Shake flask culture: Pick 3 single colonies from the cultured plate and transfer them to a 1L Erlenmeyer flask containing 100mL of culture medium. Place the flask on a shaker and culture at 32℃ and 220rpm. After 18h of culture, the OD... 600nm ≥2, can be transferred to a seed culture tank. The shake flask culture medium consists of 5 g / L sodium chloride, 10 g / L peptone, and 10 g / L yeast extract. The pH is adjusted to 7.5 before sterilization, and the medium is sterilized at 121°C for 25 min.

[0067] (3) Seed culture expansion: The seed culture from the shake flasks was transferred to a 15L seed tank at an inoculation rate of 1%, resulting in a total volume of 10L. Seed culture conditions included a culture temperature of 32℃, a tank pressure of 0.05MPa, an aeration ratio of 1.5VVM, and a rotation speed of 500rpm. The pH was maintained at approximately 7.5 by adding ammonia. After 12 hours of culture, the OD... 600nm If the pH is ≥2.5, it can be transferred to a fermenter for cultivation. The seed culture medium consists of 10 g / L glucose, 5 g / L sodium chloride, 10 g / L peptone, and 10 g / L yeast extract. The pH is adjusted to 7.5 before sterilization, and the culture is sterilized at 121°C for 25 min.

[0068] (4) Fermentation culture: Transfer the seed culture medium to a 5m³ inoculation volume at a 15% inoculation rate. 3 Fermentation tank, with a liquid volume of 2.5m³. 3 Initial culture conditions included a culture temperature of 32℃, a rotation speed of 150 rpm, an aeration ratio of 0.5 VVM, and a tank pressure of 0.03 MPa. During fermentation, the pH was controlled at approximately 7.2 by adding ammonia. A feedstock (55% glucose solution) was added during the process. The fermentation medium consisted of 12 g / L glucose, 12 g / L potassium dihydrogen phosphate, 7 g / L disodium hydrogen phosphate, 3.5 g / L manganese sulfate, 6 g / L sodium chloride, and 6 g / L peptone. The pH was adjusted to 7.2 before sterilization, and the medium was sterilized at 121℃ for 25 min. During fermentation, the pyruvate to acetyl-CoA ratio in the fermentation broth was monitored in real-time using online Raman spectroscopy. The process was adjusted by increasing or decreasing at least one of the rotation speed, air volume, and tank pressure. If the process requirements were not met by these measures, the flow rate of the feedstock (55% glucose solution) was increased or decreased to ensure process stability. The control of the pyruvate to acetyl-CoA ratio during fermentation was as follows: Figure 2As shown, specifically: from 0 to 12 h, the molar ratio of pyruvate to acetyl-CoA is (5-7):1, where it is 7:1 at 0 h, 6.2:1 at 4 h, 6:1 at 8 h, and 5:1 at 12 h; from 12 to 36 h, the molar ratio of pyruvate to acetyl-CoA is (1.5-5):1, where it is 3:1 at 16 h, 1.6:1 at 20 h, 1.5:1 at 24 h, 1.5:1 at 28 h, 2.5:1 at 32 h, and 3.5:1 at 36 h; after 36 h, the molar ratio of pyruvate to acetyl-CoA is (3.5-6):1, where it is 4:1 at 40 h, 5:1 at 44 h, and 6:1 at 48 h.

[0069] When fermentation reaches 50 hours, the product growth rate slows down significantly or the cell staining becomes lighter. At this point, fermentation is stopped, and the chondroitin content in the fermentation broth is determined by HPLC. The chondroitin content reaches 26 g / L at the end of fermentation, and the conversion rate reaches 55%.

[0070] Example 3: Fermentation process in a 60m³ tank (1) Seed activation: Same as in Example 2.

[0071] (2) Shake flask culture: Same as in Example 2.

[0072] (3) Seed expansion culture: Same as in Example 2.

[0073] (4) Fermentation culture: Transfer the seed culture medium to a 60m³ fermentation tank at a 20% inoculum. 3 Fermentation tank with a liquid volume of 30m³ 3 Initial culture conditions included a culture temperature of 33℃, a rotation speed of 150 rpm, an aeration ratio of 0.8 VVM, and a tank pressure of 0.04 MPa. During fermentation, the pH was controlled at approximately 6.5 by adding ammonia. A 60% glucose solution was added as a feedstock. The fermentation medium consisted of 12 g / L glucose, 12 g / L potassium dihydrogen phosphate, 8 g / L disodium hydrogen phosphate, 3.5 g / L manganese sulfate, 6 g / L sodium chloride, and 6 g / L peptone. The pH was adjusted to 7.2 before sterilization, and the medium was sterilized at 121℃ for 25 min. During fermentation, the pyruvate to acetyl-CoA ratio in the fermentation broth was monitored in real-time using online Raman spectroscopy. The process was adjusted by increasing or decreasing at least one of the rotation speed, air volume, and tank pressure. If the process requirements were not met by these measures, the flow rate of the 60% glucose solution was increased or decreased to ensure process stability. The control of the pyruvate to acetyl-CoA ratio during fermentation was as follows: Figure 3As shown, specifically: from 0 to 12 h, the molar ratio of pyruvate to acetyl-CoA is (5 to 6.5):1, where it is 6.5:1 at 0 h, 6.2:1 at 4 h, 6:1 at 8 h, and 5:1 at 12 h; from 12 to 36 h, the molar ratio of pyruvate to acetyl-CoA is (2 to 5):1, where it is 3:1 at 16 h, 2.5:1 at 20 h, 2:1 at 24 h, 2.2:1 at 28 h, 2.6:1 at 32 h, and 3.5:1 at 36 h; after 36 h, the molar ratio of pyruvate to acetyl-CoA is (3.5 to 5):1, where it is 4.2:1 at 40 h, 4.9:1 at 44 h, and 5:1 at 48 h.

[0074] When fermentation reaches 50 hours, the product growth rate slows down significantly or the cell staining becomes lighter. At this point, fermentation is stopped, and the chondroitin content in the fermentation broth is determined by HPLC. The chondroitin content reaches 30 g / L at the end of fermentation, and the conversion rate reaches 60%.

[0075] Example 4: Fermentation process in a 120m³ tank (1) Seed activation: Same as in Example 2.

[0076] (2) Shake flask culture: Same as in Example 2.

[0077] (3) Seed expansion culture: Same as in Example 2.

[0078] (4) Fermentation culture: Transfer the seed culture medium to 120m at an inoculum rate of 20%. 3 Fermentation tank with a liquid volume of 60m³ 3 Initial culture conditions included a culture temperature of 30℃, a rotation speed of 60 rpm, an aeration ratio of 0.5 VVM, and a tank pressure of 0.03 MPa. During fermentation, the pH was controlled at approximately 6.0 by adding ammonia. A 60% glucose solution was added as a feedstock. The fermentation medium consisted of 15 g / L glucose, 12 g / L potassium dihydrogen phosphate, 8 g / L disodium hydrogen phosphate, 3.5 g / L manganese sulfate, 6 g / L sodium chloride, and 6 g / L peptone. The pH was adjusted to 6.0 before sterilization, and the medium was sterilized at 121℃ for 25 min. During fermentation, the pyruvate to acetyl-CoA ratio in the fermentation broth was monitored in real-time using online Raman spectroscopy. The process was adjusted by increasing or decreasing at least one of the rotation speed, air volume, and tank pressure. If the process requirements were not met by these measures, the flow rate of the 60% glucose solution was increased or decreased to ensure process stability. The control of the pyruvate to acetyl-CoA ratio during fermentation was as follows: Figure 4As shown, specifically: from 0 to 12 h, the molar ratio of pyruvate to acetyl-CoA is (6 to 7.5):1, where it is 7.5:1 at 0 h, 7.3:1 at 4 h, 7:1 at 8 h, and 6:1 at 12 h; from 12 to 36 h, the molar ratio of pyruvate to acetyl-CoA is (3 to 6):1, where it is 3.8:1 at 16 h, 3.2:1 at 20 h, 3.1:1 at 24 h, 3:1 at 28 h, 3.2:1 at 32 h, and 3.5:1 at 36 h; after 36 h, the molar ratio of pyruvate to acetyl-CoA is (3.5 to 5):1, where it is 4.5:1 at 40 h, 4.8:1 at 44 h, and 5:1 at 48 h.

[0079] When fermentation reaches 50 hours, the product growth rate slows down significantly or the cell staining becomes light. At this point, fermentation is stopped, and the chondroitin content in the fermentation broth is determined by HPLC. The chondroitin content reaches 28 g / L at the end of fermentation, and the conversion rate reaches 58%.

[0080] Example 5: Fermentation production process in a 120m³ tank (without stage control, large-scale) (1) Seed activation: Same as in Example 2.

[0081] (2) Shake flask culture: Same as in Example 2.

[0082] (3) Seed expansion culture: Same as in Example 2.

[0083] (4) Fermentation culture: Transfer the seed culture medium to 120m at an inoculum rate of 20%. 3 Fermentation tank with a liquid volume of 60m³ 3 Initial culture conditions included a culture temperature of 30℃, a rotation speed of 60 rpm, an aeration ratio of 0.5 VVM, and a tank pressure of 0.03 MPa. During fermentation, the pH was controlled at approximately 6.0 by adding ammonia. A 60% glucose solution was added as a feedstock during the process. The fermentation medium consisted of 15 g / L glucose, 12 g / L potassium dihydrogen phosphate, 8 g / L disodium hydrogen phosphate, 3.5 g / L manganese sulfate, 6 g / L sodium chloride, and 6 g / L peptone. The pH was adjusted to 6.0 before sterilization, and the medium was sterilized at 121℃ for 25 min. During fermentation, the concentration ratio of pyruvate to acetyl-CoA in the fermentation broth was monitored in real time using online Raman spectroscopy. The process was regulated in real time by increasing or decreasing at least one of the following: rotation speed, air volume, and tank pressure. Throughout the fermentation process, the concentration ratio of pyruvate to acetyl-CoA was controlled at (1.5~8):1 (without further staged control). If the process requirements were not met through the above operations, the flow rate of the replenishing medium (60% glucose solution) was increased or decreased to ensure process stability. The monitoring of the concentration ratio of pyruvate to acetyl-CoA during fermentation is as follows: Figure 5As shown, the ratios are 7.5:1 at 0h, 6.3:1 at 4h, 5.2:1 at 8h, 2.2:1 at 12h, 1.5:1 at 16h, 1.6:1 at 20h, 1.8:1 at 24h, 3.8:1 at 28h, 4.5:1 at 32h, 6.5:1 at 36h, 7.5:1 at 40h, 7.6:1 at 44h, and 8:1 at 48h.

[0084] When fermentation reaches 50 hours, the product growth rate slows down significantly or the cell staining becomes light. At this point, fermentation is stopped, and the chondroitin content in the fermentation broth is determined by HPLC. The chondroitin content reaches 20 g / L at the end of fermentation, and the conversion rate reaches 40%.

[0085] Comparative Example 1 (Original Process Test) (1) Seed activation: Same as in Example 1.

[0086] (2) Shake flask culture: Same as in Example 1.

[0087] (3) Fermentation culture: The shake flask culture was transferred to a 15L fermenter at a 5% inoculum rate, with a total volume of 6L. The initial culture conditions included a culture temperature of 37℃, a rotation speed of 200rpm, an aeration ratio of 1VVM, and a tank pressure of 0.04MPa. During the fermentation process, the pH was controlled at around 7.0 by adding ammonia. When the dissolved oxygen rebounded, the feed medium (50% glucose solution) was added continuously to maintain the dissolved oxygen in the culture medium at a constant dissolved oxygen level of 30% to keep the dissolved oxygen in the culture medium above 25%. The fermentation medium consisted of 8g / L glucose, 8g / L potassium dihydrogen phosphate, 6g / L disodium hydrogen phosphate, 3.5g / L manganese sulfate, 5g / L sodium chloride, and 6g / L peptone. The pH was adjusted to 6.5 before sterilization, and the medium was sterilized at 121℃ for 25min.

[0088] When fermentation reached 50 hours, the product growth rate slowed significantly or the cell staining became lighter. At this point, fermentation was stopped, and the chondroitin content in the fermentation broth was determined using HPLC. The chondroitin content reached 15 g / L at the end of fermentation, with a conversion rate of 36%. Compared with Example 1, the fermentation level of chondroitin in this comparative example was significantly lower.

[0089] Comparative Example 2: Fermentation production process in a 120m³ tank (in stages, some stages are not under control). (1) Seed activation: Same as in Example 2.

[0090] (2) Shake flask culture: Same as in Example 2.

[0091] (3) Seed expansion culture: Same as in Example 2.

[0092] (4) Fermentation culture: Transfer the seed culture medium to 120m at an inoculum rate of 20%. 3 Fermentation tank with a liquid volume of 60m³3 Initial culture conditions included a culture temperature of 30℃, a rotation speed of 60 rpm, an aeration ratio of 0.5 VVM, and a tank pressure of 0.03 MPa. During fermentation, the pH was controlled at approximately 6.0 by adding ammonia. A 60% glucose solution was added as a feedstock. The fermentation medium consisted of 15 g / L glucose, 12 g / L potassium dihydrogen phosphate, 8 g / L disodium hydrogen phosphate, 3.5 g / L manganese sulfate, 6 g / L sodium chloride, and 6 g / L peptone. The pH was adjusted to 6.0 before sterilization, and the medium was sterilized at 121℃ for 25 min. During fermentation, the pyruvate to acetyl-CoA ratio in the fermentation broth was monitored in real-time using online Raman spectroscopy. The process was adjusted by increasing or decreasing at least one of the rotation speed, air volume, and tank pressure. If the process requirements were not met by these measures, the flow rate of the 60% glucose solution was increased or decreased to ensure process stability. The control of the pyruvate to acetyl-CoA ratio during fermentation was as follows: Figure 6 As shown, specifically: from 0 to 12 h, the molar ratio of pyruvate to acetyl-CoA is (5 to 7.5):1, where it is 7.5:1 at 0 h, 7.2:1 at 4 h, 6.1:1 at 8 h, and 5:1 at 12 h; from 12 to 36 h, the molar ratio of pyruvate to acetyl-CoA is (0.5 to 5):1, where it is 1.2:1 at 16 h, 0.5:1 at 20 h, 0.7:1 at 24 h, 1:1 at 28 h, 1.4:1 at 32 h, and 3:1 at 36 h; after 36 h, the molar ratio of pyruvate to acetyl-CoA is (3 to 7):1, where it is 5.5:1 at 40 h, 6.5:1 at 44 h, and 7:1 at 48 h.

[0093] When fermentation reached 50 hours, the product growth rate slowed significantly or the cell staining became lighter. At this point, fermentation was stopped, and the chondroitin content in the fermentation broth was determined using HPLC. The chondroitin content reached 16 g / L at the end of fermentation, with a conversion rate of 38%. Compared to Example 4, the fermentation level was significantly reduced when the concentration ratio of pyruvate and acetyl-CoA was outside the range at certain stages.

[0094] Comparative Example 3: Fermentation production process in a 120m³ tank (in stages, some stages are not under control). (1) Seed activation: Same as in Example 2.

[0095] (2) Shake flask culture: Same as in Example 2.

[0096] (3) Seed expansion culture: Same as in Example 2.

[0097] (4) Fermentation culture: Transfer the seed culture medium to 120m at an inoculum rate of 20%. 3 Fermentation tank with a liquid volume of 60m³ 3Initial culture conditions included a culture temperature of 30℃, a rotation speed of 60 rpm, an aeration ratio of 0.5 VVM, and a tank pressure of 0.03 MPa. During fermentation, the pH was controlled at approximately 6.0 by adding ammonia. A 60% glucose solution was added as a feedstock. The fermentation medium consisted of 15 g / L glucose, 12 g / L potassium dihydrogen phosphate, 8 g / L disodium hydrogen phosphate, 3.5 g / L manganese sulfate, 6 g / L sodium chloride, and 6 g / L peptone. The pH was adjusted to 6.0 before sterilization, and the medium was sterilized at 121℃ for 25 min. During fermentation, the pyruvate to acetyl-CoA ratio in the fermentation broth was monitored in real-time using online Raman spectroscopy. The process was adjusted by increasing or decreasing at least one of the rotation speed, air volume, and tank pressure. If the process requirements were not met by these measures, the flow rate of the 60% glucose solution was increased or decreased to ensure process stability. The control of the pyruvate to acetyl-CoA ratio during fermentation was as follows: Figure 7 As shown, specifically: from 0 to 12 h, the molar ratio of pyruvate to acetyl-CoA is (6 to 7.5):1, where it is 7.5:1 at 0 h, 7.1:1 at 4 h, 6.8:1 at 8 h, and 6:1 at 12 h; from 12 to 36 h, the molar ratio of pyruvate to acetyl-CoA is (0.5 to 6):1, where it is 2.5:1 at 16 h, 1:1 at 20 h, 0.5:1 at 24 h, 0.8:1 at 28 h, 1.3:1 at 32 h, and 3.5:1 at 36 h; after 36 h, the molar ratio of pyruvate to acetyl-CoA is (3.5 to 5):1, where it is 4:1 at 40 h, 4.7:1 at 44 h, and 5:1 at 48 h.

[0098] When fermentation reaches 50 hours, the product growth rate slows down significantly or the cell staining becomes light. At this point, fermentation is stopped, and the chondroitin content in the fermentation broth is determined by HPLC. The chondroitin content reaches 14 g / L at the end of fermentation, with a conversion rate of 34%.

[0099] Comparative Example 4: Fermentation production process in a 120m³ tank (in stages, some stages are not under control). (1) Seed activation: Same as in Example 2.

[0100] (2) Shake flask culture: Same as in Example 2.

[0101] (3) Seed expansion culture: Same as in Example 2.

[0102] (4) Fermentation culture: Transfer the seed culture medium to 120m at an inoculum rate of 20%. 3 Fermentation tank with a liquid volume of 60m³ 3Initial culture conditions included a culture temperature of 30℃, a rotation speed of 60 rpm, an aeration ratio of 0.5 VVM, and a tank pressure of 0.03 MPa. During fermentation, the pH was controlled at approximately 6.0 by adding ammonia. A 60% glucose solution was added as a feedstock. The fermentation medium consisted of 15 g / L glucose, 12 g / L potassium dihydrogen phosphate, 8 g / L disodium hydrogen phosphate, 3.5 g / L manganese sulfate, 6 g / L sodium chloride, and 6 g / L peptone. The pH was adjusted to 6.0 before sterilization, and the medium was sterilized at 121℃ for 25 min. During fermentation, the pyruvate to acetyl-CoA ratio in the fermentation broth was monitored in real-time using online Raman spectroscopy. The process was adjusted by increasing or decreasing at least one of the rotation speed, air volume, and tank pressure. If the process requirements were not met by these measures, the flow rate of the 60% glucose solution was increased or decreased to ensure process stability. The control of the pyruvate to acetyl-CoA ratio during fermentation was as follows: Figure 8 As shown, specifically: from 0 to 12 h, the molar ratio of pyruvate to acetyl-CoA is (5 to 7.5):1, where it is 7.5:1 at 0 h, 7:1 at 4 h, 6.5:1 at 8 h, and 5:1 at 12 h; from 12 to 36 h, the molar ratio of pyruvate to acetyl-CoA is (1.5 to 5):1, where it is 3:1 at 16 h, 1.8:1 at 20 h, 1.5:1 at 24 h, 2:1 at 28 h, 2.5:1 at 32 h, and 3.5:1 at 36 h; after 36 h, the molar ratio of pyruvate to acetyl-CoA is (3.5 to 10):1, where it is 5.5:1 at 40 h, 8.5:1 at 44 h, and 10:1 at 48 h.

[0103] When fermentation reaches 50 hours, the product growth rate slows down significantly or the cell staining becomes lighter. At this point, fermentation is stopped, and the chondroitin content in the fermentation broth is determined by HPLC. The chondroitin content reaches 12 g / L at the end of fermentation, and the conversion rate reaches 30%.

[0104] The comparison between Example 1 and Comparative Example 1, and between Example 4 and Comparative Examples 2-4, shows that controlling the molar ratio of pyruvate to acetyl-CoA at (1.5~9):1 during fermentation can effectively improve substrate conversion rate and chondroitin content in the fermentation product. The comparison between Example 4 and Example 5 shows that controlling the molar ratio of pyruvate to acetyl-CoA during fermentation as follows: 0~12h fermentation, controlling the molar ratio of pyruvate to acetyl-CoA at (5~9):1; 12h~36h fermentation, controlling the molar ratio of pyruvate to acetyl-CoA at (1.5~6):1; and after 36h fermentation, controlling the molar ratio of pyruvate to acetyl-CoA at (3~6):1, is more conducive to further improving substrate conversion rate and chondroitin content.

[0105] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A method for controlling chondroitin fermentation production, the method comprising fermenting and culturing a chondroitin-producing bacterial strain, characterized in that, The chondroitin-producing strain is a recombinant strain C4; The molar ratio of pyruvate to acetyl-CoA is controlled during the fermentation process in the following manner: Fermentation culture for 0~12h, with the molar ratio of pyruvate to acetyl-CoA controlled at (5~9):1; Fermentation culture for 12h~36h, with the molar ratio of pyruvate to acetyl-CoA controlled at (1.5~6):1; After fermentation for 36 hours, the molar ratio of pyruvate to acetyl-CoA was controlled at (3~6):

1.

2. The chondroitin fermentation production control method according to claim 1, characterized in that, The molar ratio of pyruvate to acetyl-CoA is controlled by adjusting at least one of the rotation speed, air flow rate and tank pressure. If adjusting the above three factors cannot control the molar ratio of pyruvate to acetyl-CoA within the target range, the feed medium is adjusted to control it.

3. The chondroitin fermentation production control method according to claim 2, characterized in that, When the molar ratio of pyruvate to acetyl-CoA is higher than the target range, increase at least one of the following: rotation speed, air flow rate, and tank pressure. If adjusting these three factors cannot control the molar ratio of pyruvate to acetyl-CoA within the target range, increase the feed medium addition flow rate. When the molar ratio of pyruvate to acetyl-CoA is lower than the target range, decrease at least one of the following: rotation speed, air flow rate, and tank pressure. If adjusting these three factors cannot control the molar ratio of pyruvate to acetyl-CoA within the target range, decrease the feed medium addition flow rate.

4. The method for controlling chondroitin fermentation production according to any one of claims 1 to 3, characterized in that, The fermentation medium used in the fermentation culture contains 10-20 g / L glucose, 5-15 g / L potassium dihydrogen phosphate, 1-10 g / L disodium hydrogen phosphate, 0.5-5 g / L manganese sulfate, 5-10 g / L sodium chloride, and 5-15 g / L peptone. The fed culture medium used in the fermentation culture contains glucose and / or glycerol; During the fermentation process, the pH value is controlled at 6.5~8.5 by supplementing alkali; The fermentation conditions include a temperature of 30℃~38℃, a tank pressure of 0.02MPa~0.08MPa, an aeration ratio of 0.2VVM~4VVM, and a rotation speed of 50rpm~500rpm.

5. The method for controlling chondroitin fermentation production according to any one of claims 1 to 3, characterized in that, Fermentation was terminated when the chondroitin growth rate slowed significantly or the cell staining became light.

6. The method for controlling chondroitin fermentation production according to any one of claims 1 to 3, characterized in that, The fermentation tank used for the fermentation culture has a volume of 2L~300m³. 3 .

7. The method for controlling chondroitin fermentation production according to any one of claims 1 to 3, characterized in that, The method further includes, before fermentation culture, sequentially activating the chondroitin-producing strain and then performing shake-flask culture; or, before fermentation culture, sequentially activating the chondroitin-producing strain, performing shake-flask culture, and then performing seed culture.

8. The chondroitin fermentation production control method according to claim 7, characterized in that, The plate culture medium used for the activation of the strain contains 5-8 g / L sodium chloride, 1-15 g / L peptone, 1-15 g / L yeast extract, and 10-20 g / L agar powder; The shake flask culture medium used in the shake flask culture contains 5-8 g / L sodium chloride, 1-15 g / L peptone and 1-15 g / L yeast extract. The seed culture medium used for the seed expansion culture contains 5-15 g / L glucose and / or glycerol, 5-8 g / L sodium chloride, 1-15 g / L peptone, and 1-15 g / L yeast extract.

9. The chondroitin fermentation production control method according to claim 7, characterized in that, The activation conditions for the strain include a temperature of 28℃~38℃ and a time of 24h~36h; The conditions for the shake flask culture included a temperature of 28℃~38℃, a rotation speed of 150rpm~250rpm, and a culture period of 6h~18h. When OD... 600nm End shake-flask culture when the value reaches 1-10. The conditions for seed expansion culture included a temperature of 28℃~38℃, a tank pressure of 0.025MPa~0.08MPa, an aeration ratio of 0.2VVM~2VVM, a rotation speed of 100rpm~500rpm, and a culture period of 4h~24h. When OD... 600nm Seed propagation culture was terminated when the temperature reached 2-12.

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