Fermentation process for improving yield of recombinant mussel mucin

Abstract

The invention discloses a fermentation process for improving the yield of recombinant mussel mucin. According to the fermentation process, the utilization efficiency of a nitrogen source is enhanced by optimizing microbial metabolism distribution, and the catalytic activity of a key enzyme is improved; cell damage caused by environmental stress is relieved by means of a cell membrane protection mechanism, and the apoptosis rate is reduced; finally, the stability of cells in the fermentation process is remarkably enhanced, the fermentation expression quantity of the recombinant mussel mucin is greatly improved, and the industrial production level of target protein is comprehensively improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological fermentation, and particularly relates to a fermentation process for improving the yield of recombinant mussel mucin. BACKGROUND

[0002] In the field of recombinant functional proteins, recombinant mussel mucin, as a new type of high-quality and safe protein material, has received extensive attention in recent years. Compared with naturally extracted mussel mucin, recombinant mussel mucin has significant advantages in terms of price, purity, biological activity and safety, and opens up a broader prospect for its wide application.

[0003] When expressing recombinant proteins using Pichia pastoris, the Buffered glycerol-complex medium (BMGY) and the Buffered methanol-complex medium (BMMY) are commonly used. However, these media are more suitable for small-scale recombinant protein expression culture. If industrial production is to be carried out, the use of these media has the problems of high cost and easy introduction of foreign impurities. Therefore, to solve the above problems, the Basal Salt medium (BSM) is commonly used as the fermentation medium in large-scale production. However, the inorganic salt ion concentration in the BSM medium is too high, which inhibits the expression of recombinant proteins.

[0004] To solve this problem, Chinese patent CN117535164A replaces H3PO4 and KOH in the BSM medium with NH4H2PO4 and NaH2PO4, uses the safer NaH2PO4 (sodium salt) to replace KOH (potassium salt), and reduces the amount of potassium salt, so that the protein expression amount is improved to some extent compared with the original BSM medium. However, the NH 4+ The ion content of this medium is significantly lower than that of the conventional BSM medium. However, in the culture process of recombinant proteins, insufficient nitrogen source will inhibit the growth of Pichia pastoris cells and the synthesis of recombinant proteins.

[0005] In addition, it has been found that when producing recombinant mussel mucin through genetic engineering, the phenomenon of cell growth inhibition due to protein accumulation occurs, resulting in low yield of recombinant mussel mucin.

[0006] Therefore, it is necessary to design a fermentation medium and fermentation process that can significantly improve the expression amount of recombinant mussel mucin in the Pichia pastoris system. SUMMARY

[0007] The technical problem solved by the present application is to provide a fermentation process for improving the yield of recombinant mussel mucin in view of the deficiencies of the prior art.

[0008] The technical solution adopted by the present application is: a fermentation process for improving the yield of recombinant mussel mucin, characterized in that the fermentation process comprises: inoculating Pichia fermenting seed liquid into a fermentation medium to start fermentation culture, starting to add glycerol when the dissolved oxygen rapidly rises, adding a supplementary nutrient solution at one time 2-5 hours after starting to add glycerol, stopping feeding 0.5-1 hours after adding glycerol to a bacterial wet weight of 180-220 g / L, then starting to add pure methanol for induction, simultaneously adding a growth factor solution at a variable speed during the addition of pure methanol, and adding a surfactant solution during the induction process; each liter of the fermentation medium comprises the following components: NH4H2PO4 15.1-19.1 g, K2HPO4 2.4-3.1 g, CaSO4·2H2O 0.31-0.38 g, K2SO4 4.6-7.3 g, MgSO4·7H2O 5.0-6.0 g, glycerol 30.0-40.0 g, and PTM1 solution 2.0-4.35 mL; the supplementary nutrient solution comprises glutamine, ammonium nitrate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate; and the growth factor solution comprises alanine, asparagine, aspartic acid, and serine.

[0009] The fermentation process for improving the yield of recombinant mussel mucin, characterized in that the rapid rise of dissolved oxygen refers to that the relative rise value of the dissolved oxygen concentration reaches more than 30% within 1 minute (based on the instantaneous dissolved oxygen saturation before the rapid rise of dissolved oxygen starts), wherein the value of the dissolved oxygen concentration is measured by a dissolved oxygen electrode.

[0010] The fermentation process for improving the yield of recombinant mussel mucin, characterized in that the temperature of the fermentation culture stage before the induction start to the 0-59 hours after the induction start is controlled to be 28-32 DEG C, and the temperature of the 60-64 hours after the induction start to the end of the induction stage is controlled to be 24-26 DEG C.

[0011] The fermentation process for improving the yield of recombinant mussel mucin, characterized in that the addition amount of the supplementary nutrient solution is 40-60 mL / L, the concentration of glutamine in the supplementary nutrient solution is 20-27 g / L, the concentration of ammonium nitrate is 200-250 g / L, the concentration of potassium dihydrogen phosphate is 130-150 g / L, and the concentration of dipotassium hydrogen phosphate is 20-30 g / L.

[0012] The fermentation process for improving the yield of recombinant Mytilus moolanicki mucin has the following characteristics: the concentration of alanine in the growth factor solution is 20.7-27.3 g / L, the concentration of asparagine is 27.3-39.3 g / L, the concentration of aspartic acid is 4.3-5.3 g / L, and the concentration of serine is 60-75 g / L; the feeding mode of the growth factor solution is as follows: 1-3 mL / (L·h) for 0-20 h, 3.1-4.3 mL / (L·h) for 21-40 h, 4.4-6.3 mL / (L·h) for 41-59 h, and 6.4-8.3 mL / (L·h) for 60 h to the end of fermentation.

[0013] The fermentation process for improving the yield of recombinant Mytilus moolanicki mucin has the following characteristics: the concentration of alanine in the growth factor solution is 20.7-27.3 g / L, the concentration of asparagine is 27.3-39.3 g / L, the concentration of aspartic acid is 4.3-5.3 g / L, and the concentration of serine is 60-75 g / L; the feeding mode of the growth factor solution is as follows: 1-3 mL / (L·h) for 0-20 h, 3.1-4.3 mL / (L·h) for 21-40 h, 4.4-6.3 mL / (L·h) for 41-59 h, and 6.4-8.3 mL / (L·h) for 60 h to the end of fermentation.

[0014] The fermentation process for improving the yield of recombinant Mytilus moolanicki mucin has the following characteristics: the concentration of alanine in the growth factor solution is 20.7-27.3 g / L, the concentration of asparagine is 27.3-39.3 g / L, the concentration of aspartic acid is 4.3-5.3 g / L, and the concentration of serine is 60-75 g / L; the feeding mode of the growth factor solution is as follows: 1-3 mL / (L·h) for 0-20 h, 3.1-4.3 mL / (L·h) for 21-40 h, 4.4-6.3 mL / (L·h) for 41-59 h, and 6.4-8.3 mL / (L·h) for 60 h to the end of fermentation.

[0015] The fermentation process for improving the yield of recombinant Mytilus moolanicki mucin has the following characteristics: the concentration of alanine in the growth factor solution is 20.7-27.3 g / L, the concentration of asparagine is 27.3-39.3 g / L, the concentration of aspartic acid is 4.3-5.3 g / L, and the concentration of serine is 60-75 g / L; the feeding mode of the growth factor solution is as follows: 1-3 mL / (L·h) for 0-20 h, 3.1-4.3 mL / (L·h) for 21-40 h, 4.4-6.3 mL / (L·h) for 41-59 h, and 6.4-8.3 mL / (L·h) for 60 h to the end of fermentation.

[0016] In the present application, the glycerol feeding rate controlled according to the Pichia pastoris expression manual is used to accurately regulate the carbon source supply; the pure methanol feeding rate is adjusted according to the strain state to induce the expression of target protein.

[0017] Compared with the prior art, the present application has the following advantages:

[0018] 1、The present application realizes significant cost reduction and efficiency improvement under the premise of maintaining biological activity through innovative medium formula design. Specifically, compared with traditional BSM medium, the use of CaSO4·2H2O is reduced by 70%, which not only reduces raw material cost but also significantly reduces the risk of calcium ion precipitation; the use of K2SO4 is reduced by 65%, effectively alleviating the inhibitory effect of high osmotic pressure on bacterial activity; the use of MgSO4·7H2O is reduced by 62%, achieving the best balance between meeting the needs of enzymatic reaction and cost control. After systematic optimization, the overall raw material procurement cost is reduced by 25-30% compared with traditional BSM medium; more importantly, the present process builds an endogenous nitrogen supply system through NH4H2PO4 in the fermentation medium and supplementary nutrient solution, revolutionizing the inherent mode of relying on exogenous ammonia water to supplement nitrogen in traditional processes.

[0019] 2、The medium formula and fermentation process of the present application not only greatly reduces the risk of ammonia gas escape, but also significantly improves the safety level of the production environment. Experimental data show that the compound control strategy successfully realizes cost reduction and efficiency improvement: cost dimension realizes breakthrough through the dual paths of raw material simplification and process optimization; efficiency is significantly improved in terms of cell density and target protein expression.

[0020] 3、The present application realizes key technological innovation in cell culture process, specifically in: during the glycerol feeding stage, a synchronous feeding strategy is adopted, and when the cells complete the adaptive regulation of glycerol metabolism (i.e. 2-5h after the start of glycerol feeding), a one-time addition of supplementary nutrient solution is added. This operation strategy has double advantages: (1) matching the dynamic demand of nitrogen source in the cell metabolic cycle, timely supplementing the necessary nutrient components consumed by biosynthesis; (2) by constructing a rich-nitrogen culture microenvironment, effectively activating the cell growth regulation network, promoting the growth and metabolic activity of microorganisms, so that the wet weight of the bacteria is increased to 415.5g / L (increased by 29.6%), significantly improving the protein synthesis efficiency in the subsequent induction expression stage.

[0021] 4、The present application adopts a gradient feeding strategy in the induction culture stage, dynamically adding specific growth factor compositions to regulate cell metabolism, so as to realize stable cell metabolic state, and then promote cell growth and protein expression. Among them, alanine as a key metabolic intermediate, participates in the tricarboxylic acid cycle, gluconeogenesis and other metabolic pathways, effectively maintaining the intracellular carbon-nitrogen balance; aspartic acid and aspartic acid as nitrogen carriers not only participate in purine and pyrimidine biosynthesis, but also connect the urea cycle and the tricarboxylic acid cycle through aspartic acid metabolic axis; at the same time, by adding serine, the present application can alleviate the disturbance of methionine cycle and single carbon metabolic bottleneck caused by its rapid depletion, and avoid the decrease of post-translational modification efficiency; in addition, the present application also adds hydrophilic polyoxyethylene polyoxypropylene ether surfactant in the induction culture stage, which can protect cells from mechanical stress damage caused by bubble rupture by reducing the concentration of cells in the foam layer.

[0022] 5. Because recombinant mussel adhesive protein is rich in lysine residues, it can generate a high density of positive charges under physiological pH conditions, leading to abnormal changes in cell membrane permeability. In the later stages of fermentation, cell senescence makes them highly susceptible to breakage and the release of proteases, resulting in the degradation of recombinant mussel adhesive protein by these proteases. To alleviate this problem, this invention employs a variable-temperature induction method in the later stages of induction and extends the induction period. This reduces the specific growth rate of cells, enhances their resistance to stress, and thus reduces the probability of protease degradation of recombinant mussel adhesive protein, ultimately increasing its expression level.

[0023] 6. This invention, by optimizing the amount and method of component addition in the fermentation process and combining it with innovative fermentation techniques, effectively optimizes the metabolic pathways of microorganisms, improves enzyme catalytic activity and substrate utilization, protects cells from damage and resulting cell death, and significantly enhances the stability and reliability of the fermentation process, thereby increasing the expression level of recombinant mussel adhesive protein. After optimization of the complete fermentation process according to this invention, the protein expression level increased from 1.57 g / L in the traditional method to 3.13 g / L, an increase of nearly 100%, laying a solid foundation for the industrial application of recombinant mussel adhesive protein.

[0024] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0025] Figure 1 The above are electrophoretic comparison images of the supernatant of fermentation in tanks from Example 1 of the present invention and Comparative Examples 1, 2(a), 3(a), and 4(a) (all samples were diluted 3 times). Detailed Implementation

[0026] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention are described in detail below with reference to examples. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] This invention provides a process for producing fermented recombinant mussel adhesive protein, wherein the amino acid sequence of the recombinant mussel adhesive protein is as follows (the recombinant mussel adhesive protein that can be produced using this method is not limited to mussel adhesive protein with this sequence):

[0029] GIVQSYDDYKGRGYCTNKGCRSGYNYFGNKGYCKYGEKSYTYNC NSYAGCCLPRNPYGKLKYYCTNKYGCPNDYYFYNNKGYYYYNKKDY FNCGSYNGCCLRSGY.

[0030] The PTM1 solution described in the present application refers to the operation manual of Invitrogen Company, and the specific formula is: CuSO4·5H2O 6.0 g / L; NaI 0.08 g / L; MnSO4·H2O 3.0 g / L; NaMoO4·2H2O 0.2 g / L; H3BO3 0.02 g / L; CoCl2 0.5 g / L; ZnCl2 20.0 g / L; FeSO4·7H2O 65.0 g / L; biotin 0.2 g / L; H2SO4 5.0 mL / L, filtered with a filter membrane of 0.22 μm to remove bacteria, and stored at 4°C.

[0031] The seed culture medium is an optimized YPD culture medium: yeast powder 10 g / L, peptone 20 g / L, glycerol 10 g / L, potassium dihydrogen phosphate 11.8 g / L, and dipotassium hydrogen phosphate 3.0 g / L.

[0032] Unless otherwise specified, in the present application, "mL / (L·h)", "mL / L" are based on the real-time volume of the fermentation broth in the fermenter; the concentration of each component in the "growth factor solution" is based on the volume of the growth factor solution; the "supplemental nutrient solution" is based on the volume of the supplemental nutrient solution; "wt%" refers to weight percentage; "v / v" refers to the real-time volume ratio of the surfactant solution to the fermentation broth in the fermenter. "Rapid increase in dissolved oxygen" refers to the relative increase in dissolved oxygen concentration within 1 min reaching more than 30%.

[0033] Example 1

[0034] The fermentation medium used in this example is as follows:

[0035] Each liter of fermentation medium contains: NH4H2PO4 17.3 g, K2HPO4 2.7 g, CaSO4·2H2O 0.35 g, K2SO4 6.4 g, MgSO4·7H2O 5.6 g, glycerol 35 g, and PTM1 solution 3.5 mL.

[0036] The supplementary nutrient solution used in the embodiment comprises glutamine, ammonium nitrate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and the concentrations of the components in the supplementary nutrient solution are 25 g / L of glutamine, 230 g / L of ammonium nitrate, 140 g / L of potassium dihydrogen phosphate and 25 g / L of dipotassium hydrogen phosphate, respectively. The prepared supplementary nutrient solution is sterilized at 121 ℃ and 0.1 MPa for 20 min and then used.

[0037] The growth factor solution used in the embodiment comprises a mixed solution of alanine, asparagine, aspartic acid and serine, and the concentrations of the components in the growth factor solution are 24.2 g / L of alanine, 33.5 g / L of asparagine, 4.8 g / L of aspartic acid and 67.5 g / L of serine, respectively. The prepared growth factor solution is sterilized by filtering through a sterile capsule filter with a pore size of 0.22 μm and stored for use;

[0038] The surfactant solution used in the embodiment is a 50 wt% Pluronic F-68 solution, which is sterilized at 121 ℃ and 0.1 MPa for 20 min;

[0039] The method for producing recombinant mussel mucus by using the fermentation medium of the embodiment comprises the following steps:

[0040] Step one: the Pichia pastoris preserved by glycerol is inoculated into a shake flask medium for activation; the activation culture conditions are as follows: the temperature is 29 ℃, the rotation speed of the shaker is 200 rpm, and the wet weight is 35 g / L after 48 h of culture;

[0041] Step two: the seed liquid activated in step one is inoculated into a 10 L fermenter containing 5 L of the fermentation medium at an inoculation amount of 6% (v / v) for culture, the temperature is 29 ℃, the pH is adjusted to 5.2 by ammonia water, the tank pressure is 0.05 MPa, and the dissolved oxygen concentration value is not less than 30%;

[0042] When the relative increase value of the dissolved oxygen concentration reaches more than 30% within 1 min, 50 wt% glycerol is started to be fed, and the supplementary nutrient solution is added at once 3.5 h after the start of glycerol feeding, the addition amount of the supplementary nutrient solution is 50 mL / L, and the glycerol feeding rate is 15 mL / (L·h);

[0043] When the wet weight of the bacterial cells is 200 g / L, the glycerol feeding is stopped, and 0.8 h after the feeding is stopped, pure methanol is fed to induce the bacteria. The feeding rate of the pure methanol is adjusted according to the state of the bacteria, and the feeding rate is in the range of 3.6-10.9 mL / (L·h). Meanwhile, the growth factor solution is fed. The feeding mode of the growth factor solution is as follows: 0-20 h of induction, the feeding rate is 2 mL / (L·h); 21-40 h of induction, the feeding rate is 3.8 mL / (L·h); 41-59 h of induction, the feeding rate is 5.5 mL / (L·h); 60 h of induction to the end of fermentation, the feeding rate is 7.6 mL / (L·h);

[0044] At 15 h of induction, 0.1% of the surfactant solution of the real-time volume of the fermentation broth in the fermenter is added at one time. At 35 h of induction, 0.2% of the surfactant solution of the real-time volume of the fermentation broth in the fermenter is added at one time. At 55 h of induction, 0.3% of the surfactant solution of the real-time volume of the fermentation broth in the fermenter is added at one time.

[0045] During the whole fermentation process, the pH of the fermenter is controlled to be 5.2. 62 h after the induction is started, the temperature is reduced to 25℃ for culture until the end of fermentation. It is detected that the content of the recombinant protein does not increase 76 h after the induction is started. The fermentation is stopped. The wet weight of the bacterial cells in the fermentation broth is 415.5 g / L. The content of the recombinant mussel mucin in the supernatant of the fermentation broth is 3.13 g / L.

[0046] Example 2

[0047] The fermentation medium used in this example is as follows:

[0048] Each liter of the fermentation medium contains: NH4H2PO4 15.1 g, K2HPO4 2.4 g, CaSO4·2H2O 0.31 g, K2SO4 4.6 g, MgSO4·7H2O 5.0 g, glycerol 30 g, and PTM1 solution 2 mL.

[0049] The supplementary nutrient solution used in this example includes glutamine, ammonium nitrate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate. The concentrations of the components in the supplementary nutrient solution are as follows: glutamine 20 g / L, ammonium nitrate 200 g / L, potassium dihydrogen phosphate 130 g / L, and dipotassium hydrogen phosphate 20 g / L. The prepared supplementary nutrient solution is sterilized at 121℃ and 0.1 MPa for 20 min.

[0050] The growth factor solution used in this example includes alanine, asparagine, aspartic acid and serine. The concentrations of the components in the growth factor solution are as follows: alanine 20.7 g / L, asparagine 27.3 g / L, aspartic acid 4.3 g / L and serine 60 g / L. The prepared growth factor solution is filtered through a sterile capsule filter with a pore size of 0.22 μm for sterilization and standby.

[0051] The surfactant solution used in this embodiment was a 40 wt% Pluronic F-38 solution, which was sterilized at 121°C and 0.1 MPa for 20 min.

[0052] The method for producing recombinant mussel adhesive protein using the fermentation medium of this embodiment specifically includes the following steps:

[0053] Step 1: Inoculate the Pichia pastoris preserved in glycerol into a shake flask culture medium for activation; the activation culture conditions are: temperature 29℃, shaker speed 200 rpm, and wet weight 35 g / L after 48 h of culture;

[0054] Step 2: Inoculate the activated seed culture from Step 1 into a 10L fermenter containing 5L of fermentation medium at an inoculation rate of 6% (v / v). The temperature is 28℃, the pH is adjusted to 5.0 with ammonia, the tank pressure is 0.05MPa, and the dissolved oxygen concentration is not less than 30%.

[0055] When the relative increase in dissolved oxygen reaches more than 30% within 1 minute, 50wt% glycerol is added. Two hours after the addition of glycerol begins, a supplementary nutrient solution is added all at once at a rate of 40mL / L. The glycerol flow rate is 12mL / (L·h).

[0056] When the wet weight of the cells reaches 180 g / L, stop adding glycerol and stop feeding for 0.5 h. Then start adding pure methanol for induction. Adjust the flow rate of pure methanol between 3.6 and 10.9 mL / (L·h) according to the condition of the cells. At the same time, add growth factor solution. The specific addition method of growth factor solution is as follows: add at a rate of 1 mL / (L·h) for induction 0-20 h, add at a rate of 3.1 mL / (L·h) for induction 21-40 h, add at a rate of 4.4 mL / (L·h) for induction 41-59 h, and add at a rate of 6.4 mL / (L·h) from induction 60 h to the end of fermentation.

[0057] Add 0.05% of the real-time volume of the fermentation broth in the fermenter at 10h of induction, add 0.15% of the real-time volume of the fermentation broth in the fermenter at 30h of induction, and add 0.2% of the real-time volume of the fermentation broth in the fermenter at 50h of induction.

[0058] Throughout the fermentation process, the pH inside the fermenter was controlled at 5.0. After 60 hours of induction, the temperature was lowered to 24℃ and cultured until the end of fermentation. After testing, the protein content stopped increasing 76 hours after induction, and fermentation was stopped. The wet weight of the fermentation broth was 405.25 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 3.01 g / L.

[0059] Example 3

[0060] The fermentation medium used in this embodiment is as follows:

[0061] Each liter of fermentation medium contains: 19.1 g NH4H2PO4, 3.1 g K2HPO4, 0.38 g CaSO4·2H2O, 7.3 g K2SO4, 6.0 g MgSO4·7H2O, 40 g glycerol, and 4.35 mL PTM1 solution;

[0062] The supplementary nutrient solution used in this embodiment includes glutamine, ammonium nitrate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. The concentrations of each component in the supplementary nutrient solution are glutamine 27 g / L, ammonium nitrate 250 g / L, potassium dihydrogen phosphate 150 g / L, and dipotassium hydrogen phosphate 30 g / L, respectively. The prepared supplementary nutrient solution is sterilized at 121°C and 0.1 MPa for 20 min before use.

[0063] The growth factor solution used in this embodiment includes alanine, asparagine, aspartic acid and serine. The concentrations of each component in the growth factor solution are 27.3 g / L for alanine, 39.3 g / L for asparagine, 5.3 g / L for aspartic acid and 75 g / L for serine. The prepared growth factor solution is sterilized by filtration through a 0.22 μm sterile capsule filter for later use.

[0064] The surfactant solution used in this embodiment was a 60wt% Pluronic F-127 solution, which was sterilized at 121°C and 0.1MPa for 20 min.

[0065] The method for producing recombinant mussel adhesive protein using the fermentation medium of this embodiment specifically includes the following steps:

[0066] Step 1: Inoculate the Pichia pastoris preserved in glycerol into a shake flask culture medium for activation; the activation culture conditions are: temperature 29℃, shaker speed 200 rpm, and wet weight 35 g / L after 48 h of culture;

[0067] Step 2: Inoculate the activated seed culture from Step 1 into a 10L fermenter containing 5L of fermentation medium at an inoculation rate of 6% (v / v). The temperature is 32℃, the pH is adjusted to 5.5 with ammonia, the tank pressure is 0.05MPa, and the dissolved oxygen concentration is not less than 30%.

[0068] When the dissolved oxygen concentration increases by more than 30% within 1 minute, 50 wt% glycerol is added. Five hours after the glycerol addition begins, a supplementary nutrient solution is added all at once at a rate of 60 mL / L. The glycerol flow rate is 18 mL / (L·h).

[0069] When the wet weight of the cells reached 220 g / L, the addition of glycerol was stopped. One hour after stopping feeding, pure methanol was added for induction. The flow rate of pure methanol was adjusted according to the condition of the cells, ranging from 3.6 to 10.9 mL / (L·h). At the same time, growth factor solution was added. The specific addition method of growth factor solution was as follows: 3 mL / (L·h) for induction 0-20 h, 4.3 mL / (L·h) for induction 21-40 h, 6.3 mL / (L·h) for induction 41-59 h, and 8.3 mL / (L·h) from induction 60 h to the end of fermentation.

[0070] At 20 hours of induction, 0.2% of the real-time volume of the fermentation broth in the fermenter was added at once; at 40 hours of induction, 0.3% of the real-time volume of the fermentation broth in the fermenter was added at once; and at 60 hours of induction, 0.4% of the real-time volume of the fermentation broth in the fermenter was added at once.

[0071] Throughout the fermentation process, the pH of the fermenter was controlled at 5.5. After 64 hours of induction, the temperature was lowered to 26℃ and cultured until the end of fermentation. After testing, the content of recombinant protein no longer increased after 76 hours of induction, and fermentation was stopped. The wet weight of the fermentation broth was 408 g / L, and the content of recombinant mussel avidin in the fermentation supernatant was 3.05 g / L.

[0072] Comparative Example 1

[0073] The fermentation medium used in this comparative example was BSM medium, which included the following components at the following concentrations: H3PO4 26.7 mL / L; CaSO4·2H2O 1.175 g / L; K2SO4 18.2 g / L; MgSO4·7H2O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM 14.35 mL / L.

[0074] The fermentation process specifically includes:

[0075] Step 1: Inoculate the Pichia pastoris genetically engineered strain into the modified YPD seed medium in a shake flask at an inoculation rate of 0.1% to obtain the primary seed;

[0076] Step 2: Inoculate the primary seed culture at a rate of 6% (v / v) into a 10L fermenter containing 5L of fermentation medium and culture it at a temperature of 29℃, adjust the pH to 5.2 with ammonia, maintain a tank pressure of 0.05MPa, and ensure that the dissolved oxygen concentration is not less than 30%.

[0077] When the dissolved oxygen concentration increased by more than 30% relative to the mean within 1 minute, 50 wt% glycerol was added at a flow rate of 15 mL / (L·h). When the cell wet weight reached 200 g / L, glycerol addition was stopped. After 1 hour of no further feeding, pure methanol was added to induce expression until fermentation ended. The flow rate of pure methanol was adjusted between 3.6 and 10.9 mL / (L·h) according to the cell condition. The fermentation process ended when protein expression no longer increased. After 66 hours of induction, the protein content no longer increased, the cell wet weight of the fermentation broth was 320.5 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 1.57 g / L.

[0078] Comparative Example 2

[0079] (a) Based on Example 1, no supplementary nutrient solution was added in this comparative example, and no other changes were made. After 68 hours of induction, the protein content no longer increased, the wet weight of the fermentation broth was 354.75 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.52 g / L.

[0080] (b) Based on Example 1, this comparative example only changed the timing of the addition of the supplementary nutrient solution. Specifically, when the dissolved oxygen concentration increased by more than 30% within 1 minute, 50 wt% glycerol was added, and the supplementary nutrient solution was added all at once. The amount of supplementary nutrient solution added was 50 mL / L, and the glycerol flow rate was 15 mL / (L·h). Other changes were not made. After 76 h of induction, the protein content no longer increased, the wet weight of the fermentation broth was 394.5 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.81 g / L.

[0081] (c) Based on Example 1, this comparative example only changed the timing of the addition of the supplementary nutrient solution. Specifically, the supplementary nutrient solution was added all at once 10 hours after the start of methanol induction. No other changes were made. After 76 hours of induction, the protein content no longer increased. The wet weight of the fermentation broth was 391.5 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.86 g / L.

[0082] Comparative Example 3

[0083] (a) Based on Example 1, no growth factor solution was added in this comparative example, and no other changes were made. After 76 hours of induction, the protein content no longer increased, the wet weight of the fermentation broth was 372.5 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.66 g / L.

[0084] (b) Based on Example 1, this comparative example only changed the feeding method of the growth factor solution, and did not change anything else. The feeding method of the growth factor solution was as follows: 0.5 mL / (L·h) for induction 0-20h, 1.5 mL / (L·h) for induction 21-40h, 2.0 mL / (L·h) for induction 41-59h, and 3.0 mL / (L·h) from induction 60h to the end of fermentation. After testing, the protein content no longer increased after 76h of induction, the wet weight of the fermentation broth was 403.5 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.78 g / L.

[0085] (c) Based on Example 1, this comparative example only changed the feeding method of the growth factor solution, and did not change anything else. The specific feeding method of the growth factor solution was as follows: 5.0 mL / (L·h) for induction 0-20h, 6.0 mL / (L·h) for induction 21-40h, 8.5 mL / (L·h) for induction 41-59h, and 10.5 mL / (L·h) from induction 60h to the end of fermentation. After testing, the protein content no longer increased after 76h of induction, the wet weight of the fermentation broth was 418.25 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.92 g / L.

[0086] (d) Based on Example 1, only the concentration of each component in the growth factor solution was changed in this comparative example. Specifically, alanine 10.2 g / L, asparagine 13.7 g / L, aspartic acid 2.2 g / L, serine 30 g / L, and other components remained unchanged. After 76 h of induction, the protein content no longer increased, the wet weight of the fermentation broth was 401.5 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.79 g / L.

[0087] Comparative Example 4

[0088] (a) Based on Example 1, no surfactant Pluronic F-68 was added in this comparative example, and no other changes were made. After 76 hours of induction, the protein content no longer increased, the wet weight of the fermentation broth was 386.25 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.84 g / L.

[0089] (b) Based on Example 1, the surfactant solution in this comparative example was 20 wt% Pluronic F-68 solution, and no other changes were made. After 76 h of induction, the protein content no longer increased, the wet weight of the fermentation broth was 395.5 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.91 g / L.

[0090] (c) Based on Example 1, this comparative example only changed the method of adding the surfactant Pluronic F-68. Specifically, 0.02% of the real-time volume of the fermentation broth in the fermenter was added once after 15 hours of induction; 0.075% of the real-time volume of the fermentation broth in the fermenter was added once after 35 hours of induction; and 0.1% of the real-time volume of the fermentation broth in the fermenter was added once after 55 hours of induction. After 76 hours of induction, the protein content no longer increased, the wet weight of the fermentation broth cells was 393.25 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.90 g / L.

[0091] Comparative Example 5

[0092] (a) Based on Example 1, the amount of nutrient solution added in this comparative example was 20 mL / L, and no other changes were made. After 76 h of induction, the wet weight of the fermentation broth was 395.5 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.83 g / L.

[0093] (b) Based on Example 1, the amount of nutrient solution added in this comparative example was 100 mL / L, and no other changes were made. After 76 h of induction, the protein content no longer increased, the wet weight of the fermentation broth was 413.75 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.96 g / L.

[0094] (c) Based on Example 1, the concentrations of each component in the supplementary nutrient solution of this comparative example were as follows: glutamine 10 g / L, ammonium nitrate 100 g / L, potassium dihydrogen phosphate 65 g / L, dipotassium hydrogen phosphate 10 g / L, with no other changes. After 76 h of induction, the protein content no longer increased, the wet weight of the fermentation broth was 391.75 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 2.81 g / L.

[0095] Experimental Example 1

[0096] After fermentation, solid-liquid separation was performed using a centrifuge. The protein content of the supernatant was determined by high-performance liquid chromatography (HPLC). The specific method was as follows: hydrophilic modified silica gel (TSK gelg2000SWxl, 7.8mm*300mm, 5μm or other suitable column) was used as the packing material; a mixture of 0.1mol / L phosphate and 0.1mol / L sodium chloride solution (21.8466g disodium hydrogen phosphate dodecahydrate, 6.0844g sodium dihydrogen phosphate dihydrate, and 5.844g sodium chloride were dissolved in 1L of water) and acetonitrile (95:5) was used as the mobile phase; the column temperature was 25℃, the sample pan temperature was 4℃, the flow rate was 0.7mL / min, and the detection wavelength was 280nm. The results are shown in Table 1.

[0097] Table 1. Yield of recombinant mussel adhesive protein

[0098]

[0099]

[0100] Table 1 shows that, compared with Examples 1-3, Comparative Examples 1-5 exhibited significant differences in the content of recombinant mussel adhesive protein in both the wet weight after fermentation and the fermentation supernatant. Examples 1-3 achieved a significant increase in the expression level of recombinant mussel adhesive protein through a series of process optimization strategies. Specifically, this was achieved by modifying the fermentation medium, precisely controlling the timing of nutrient solution addition during the glycerol feeding stage, adopting a dynamic feeding mode for growth factor and surfactant solutions during the methanol induction stage, and synergistically optimizing key process parameters such as reducing the temperature in the later stages of methanol induction, thus constructing a highly efficient fermentation expression system. The overall results show that the average wet weight after fermentation in Examples 1-3 was 410 g / L, and the average content of recombinant mussel adhesive protein in the fermentation supernatant increased to 3.06 g / L, verifying the effectiveness and stability of this fermentation process in increasing the yield of the target protein.

[0101] Comparative Example 1 used conventional BSM medium for culture and induction of expression. However, the wet weight of Comparative Example 1 after fermentation was only 320.5 g / L, and the content of recombinant mussel adhesive protein in the fermentation supernatant was 1.57 g / L. Compared with Example 1, the wet weight of Comparative Example 1 after fermentation decreased by 22.86%, and the fermentation expression level of recombinant mussel adhesive protein decreased by 49.84%.

[0102] Comparative Examples 2 and 5 focused on optimizing and validating key process parameters of the supplementary nutrient solution: Comparative Example 2(a) did not add any supplementary nutrient solution, while Comparative Examples 2(b) and 2(c) specifically adjusted the timing of its addition; Comparative Examples 5(a) and 5(b) changed the amount of supplementary nutrient solution added, while Comparative Example 5(c) adjusted the concentration of each component in the supplementary nutrient solution. Data in Table 1 shows that the detection of recombinant mussel adhesive protein content in the wet weight after fermentation and the fermentation supernatant of Comparative Examples 2 and 5 fully confirms the core regulatory value of the supplementary nutrient solution on the fermentation system. Specifically, the average wet weight after fermentation in Comparative Example 2 was 8.48% lower than that in Example 1, and the average content of recombinant mussel adhesive protein in the fermentation supernatant was 12.78% lower than that in Example 1; the average wet weight after fermentation in Comparative Example 5 was 3.65% lower than that in Example 1, and the average content of the target protein was 8.41% lower than that in Example 1.

[0103] This demonstrates the crucial role of nutrient solution supplementation in the entire fermentation system. This invention achieves metabolic adaptation regulation by pre-introducing appropriate amounts of nitrogen and glycerol into the fermentation medium, followed by a one-time injection of supplementary nutrient solution at key points. This allows for precise matching of the dynamic nitrogen demand during the cell metabolic cycle. On one hand, it avoids excessive nitrogen consumption during the rapid cell growth phase in the early stages of fermentation, which could lead to a decrease in the yield and purity of the target protein later on. On the other hand, precise supplementation at key points effectively alleviates the problems of hindered cell synthesis, reduced metabolic activity, and a double decrease in the yield and quality of the target protein caused by nitrogen deficiency throughout the fermentation process.

[0104] Comparative Example 3 systematically investigated the effects of growth factor solution-related process parameters on fermentation efficiency. The results showed that the presence or absence of growth factors, flow rate, and composition all significantly affected cell growth and recombinant protein expression. Specific data are as follows:

[0105] In Comparative Example 3(a), without the addition of growth factor solution, the lack of nutrients required for cell growth and expression resulted in a 10.35% decrease in wet weight after fermentation compared to Example 1, and a significant 20.45% reduction in the content of recombinant mussel adhesive protein in the fermentation supernatant. In Comparative Example 3(b), the low flow rate of the growth factor solution led to insufficient nutrient supply, resulting in a 2.89% decrease in wet weight after fermentation and an 11.18% reduction in recombinant protein expression. In Comparative Example 3(c), the high flow rate of the growth factor solution, while not significantly inhibiting cell growth (with minimal difference in wet weight), inhibited the expression of recombinant mussel adhesive protein by interfering with cellular metabolic pathways, leading to a 6.71% decrease in fermentation yield compared to Example 1. In Comparative Example 3(d), the altered composition of the growth factor solution resulted in an imbalance in the nutrient ratio, leading to insufficient nutrient supply. This resulted in a 3.37% decrease in wet weight after fermentation compared to Example 1, and a 10.86% reduction in recombinant protein content.

[0106] Therefore, the appropriate addition of growth factor solution, suitable flow rate and stable composition are key process conditions to ensure efficient cell growth and high expression of recombinant proteins.

[0107] Comparative Example 4 systematically verified the regulatory effect of Pluronic F-68 on cell growth and recombinant mussel adhesive protein expression by using Pluronic F-68 without the surfactant and by varying the amount of Pluronic F-68 added to the system. As shown in Table 1, the average wet weight of Comparative Example 4 after fermentation was 5.74% lower than that of Example 1, and the content of recombinant mussel adhesive protein in the average fermentation supernatant decreased by 7.88%. This indicates that maintaining an appropriate concentration of Pluronic F-68 during fermentation can optimize fermentation efficiency through multiple mechanisms: it can form a protective interface layer on the cell membrane surface, reduce the hydrophobic properties of the cell membrane, and reduce the damage to cells caused by environmental stresses such as shear force, thereby providing a stable microenvironment for cell growth. At the same time, this regulatory effect can further promote the synthesis and secretion of recombinant mussel adhesive protein, which is an important process parameter to ensure fermentation efficiency.

[0108] In summary, the core technological improvements of this invention are reflected in the multi-dimensional process optimization strategy to enhance efficiency: by optimizing the distribution of microbial metabolism, nitrogen source utilization efficiency is enhanced and the catalytic activity of key enzymes is improved; by using cell membrane protection mechanisms, cell damage caused by environmental stress is reduced and the apoptosis rate is decreased; ultimately, the stability of cells during the fermentation process is significantly enhanced, the fermentation expression level of recombinant mussel adhesive protein is greatly increased, and the industrial production level of the target protein is comprehensively improved.

[0109] Experiment Example 2

[0110] Five mg of each of the test samples from Example 1, Comparative Example 1, Comparative Example 2(a), Comparative Example 3(a) and Comparative Example 4(a) were taken and tested according to the standard operation of "Electrophoresis" in the General Rules of the Pharmacopoeia of the People's Republic of China 2020 Edition. The electrophoretic patterns were quantitatively analyzed using grayscale analysis technology. The results are shown in Table 2.

[0111] Quantitative data analysis of grayscale values ​​showed that the grayscale value of the target protein in Example 1 was significantly increased by 47.72% compared to Comparative Example 1, by 16.14% compared to Comparative Example 2(a), and by 10.66% and 10.85% compared to Comparative Examples 3(a) and 4(a), respectively. This result directly confirms at the protein level that the actual content of the recombinant mussel adhesive protein obtained by the optimized process of this invention is significantly higher than that of the control groups, further validating the effectiveness of the multi-dimensional process optimization strategy in improving the expression level of the target protein.

[0112] Table 2 Grayscale data of Example 1, Comparative Example 1, Comparative Example 2(a), Comparative Example 3(a) and Comparative Example 4(a)

[0113] Sample name Grey scale Example 1 32968.388 Comparative Example 1 17236.121 Comparative Example 2(a) 27648.711 Comparative Example 3(a) 29453.539 Comparative Example 4(a) 29390.125

[0114] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A fermentation process for increasing the yield of recombinant mussel adhesive protein, characterized in that, The fermentation process includes: inoculating the Pichia pastoris fermentation seed liquid into the fermentation medium and starting fermentation culture; when the dissolved oxygen rises rapidly, glycerol is fed in continuously; 2-5 hours after the start of glycerol feeding, a supplementary nutrient solution is added all at once; when the glycerol feeding reaches a cell wet weight of 180-220 g / L, feeding is stopped for 0.5-1 hour, and then pure methanol is fed in continuously for induction; simultaneously, a growth factor solution is fed in continuously at a variable rate, and a surfactant solution is added during the induction process; each liter of the fermentation medium contains the following components: NH4H2PO4 15.1-19.1 g, K2HPO4 2.4-3.1 g, CaSO4·2H2O 0.31-0.38 g, K2SO4 4.6-7.3 g, MgSO4·7H2O 5.0–6.0 g, glycerol 30.0–40.0 g, PTM1 solution 2.0–4.35 mL; the supplementary nutrient solution contains glutamine, ammonium nitrate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate; the growth factor solution contains alanine, asparagine, aspartic acid and serine.

2. The fermentation process for increasing the yield of recombinant mussel adhesive protein according to claim 1, characterized in that, The rapid increase in dissolved oxygen refers to a relative increase in dissolved oxygen concentration of more than 30% within 1 minute.

3. The fermentation process for increasing the yield of recombinant mussel adhesive protein according to claim 1, characterized in that, The temperature was controlled at 28–32℃ during the fermentation culture stage before induction and from 0–59 h after the induction stage started; and at 24–26℃ from 60–64 h after induction and from the end of the induction stage.

4. The fermentation process for increasing the yield of recombinant mussel adhesive protein according to claim 1, characterized in that, The amount of the supplementary nutrient solution added is 40-60 mL / L, the concentration of glutamine in the supplementary nutrient solution is 20-27 g / L, the concentration of ammonium nitrate is 200-250 g / L, the concentration of potassium dihydrogen phosphate is 130-150 g / L, and the concentration of dipotassium hydrogen phosphate is 20-30 g / L.

5. The fermentation process for increasing the yield of recombinant mussel adhesive protein according to claim 1, characterized in that, The growth factor solution contains alanine at a concentration of 20.7–27.3 g / L, asparagine at a concentration of 27.3–39.3 g / L, aspartic acid at a concentration of 4.3–5.3 g / L, and serine at a concentration of 60–75 g / L. The growth factor solution is added as follows: at a rate of 1–3 mL / (L·h) for induction 0–20 h, at a rate of 3.1–4.3 mL / (L·h) for induction 21–40 h, at a rate of 4.4–6.3 mL / (L·h) for induction 41–59 h, and at a rate of 6.4–8.3 mL / (L·h) from induction 60 h to the end of fermentation.

6. The fermentation process for increasing the yield of recombinant mussel adhesive protein according to claim 1, characterized in that, The surfactant solution is a 40-60 wt% hydrophilic polyoxyethylene polyoxypropylene ether surfactant solution. The surfactant solution is added as follows: 0.05-0.2% (v / v) of the real-time volume of the fermentation broth in the fermenter is added once 10-20 h after the start of induction; 0.15-0.3% (v / v) of the real-time volume of the fermentation broth in the fermenter is added once 30-40 h after the start of induction; and 0.2-0.4% (v / v) of the real-time volume of the fermentation broth in the fermenter is added once 50-60 h after the start of induction.

7. The fermentation process for increasing the yield of recombinant mussel adhesive protein according to claim 6, characterized in that, The surfactant solution is preferably Pluronic F-68.

8. The fermentation process for increasing the yield of recombinant mussel adhesive protein according to claim 1, characterized in that, Throughout the entire fermentation process, the pH inside the fermenter is maintained at 5.0–5.5.

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