Feeding method for high-density fermentation of recombinant collagen through saccharomyces cerevisiae of galactose promoter
By supplementing fructose as a carbon source during the high-density fermentation of Saccharomyces cerevisiae, the problems of cell autolysis and rapid carbon source consumption in high-density fermentation of Saccharomyces cerevisiae were solved, resulting in higher expression levels and improved economic benefits.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-07
- Publication Date
- 2026-04-07
AI Technical Summary
Saccharomyces cerevisiae is not suitable for high-density fermentation, mainly because it cannot use glycerol as a carbon source, glucose inhibits gene expression, and carbon source is consumed quickly during galactose induction, leading to cell autolysis and affecting the yield of recombinant collagen.
During galactose induction, fructose was supplemented as the main carbon source, and the fructose concentration in the fermentation broth was maintained at 2% to avoid cell autolysis and improve strain viability and protein expression.
This improved the activity of the strain and the expression yield of recombinant collagen, reduced production costs, and achieved the economic benefits of high-density fermentation.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a feeding method for high-density fermentation of recombinant collagen by Saccharomyces cerevisiae with a galactose promoter. BACKGROUND
[0002] Saccharomyces cerevisiae is the most ideal eukaryote for foreign genes, and its gene expression regulation mechanism is relatively clear, genetic operation is relatively simple, and it has a post-translational modification processing system of eukaryotic proteins which is not possessed by prokaryotic bacteria; a specific safety gene engineering receptor system; no specific virus; can secrete foreign gene expression products into the culture medium; large-scale fermentation process is simple and low in cost. At the same time, yeast is the simplest eukaryote, and the use of yeast to express animal and plant genes can clarify the basic principles of gene expression regulation of higher eukaryotes and even humans, as well as the relationship between gene coding product structure and function to a considerable extent, so the Saccharomyces cerevisiae expression system has extremely important economic significance and academic value.
[0003] Generally, the Saccharomyces cerevisiae vector is a galactose inducible type for expressing the target gene. The GAL1 promoter and the target gene are cloned to the vector, and then the expression of the target gene is induced by adding galactose to the culture medium. The glucose in the culture medium can inhibit the expression of the target gene, and raffinose can be used as a substitute carbon source which does not activate and inhibit the GAL1 promoter.
[0004] However, the Saccharomyces cerevisiae is not easy to carry out high-density fermentation, and the main reasons are as follows: the Saccharomyces cerevisiae cannot use glycerol for growth and reproduction metabolism, and can only use glycerol for energy production, so the cheap glycerol cannot be used as a carbon source. At present, most of the culture media for Saccharomyces cerevisiae still use glucose; the Saccharomyces cerevisiae itself has sulfur metabolism, and can metabolize sulfate into hydrogen sulfide gas, so inorganic salt culture medium is not suitable for high-density fermentation; when the promoter is galactose, the glucose is consumed before induction, and high-density fermentation leads to large consumption of carbon source by the Saccharomyces cerevisiae, and the utilization efficiency of single galactose is lower than that of glucose, which leads to insufficient carbon source for the Saccharomyces cerevisiae in high-density fermentation, and the cell is easy to self-fuse, thereby affecting the yield of recombinant collagen. SUMMARY
[0005] The technical problem to be solved by the present application is to provide a feeding method for high-density fermentation of recombinant collagen by Saccharomyces cerevisiae with a galactose promoter, in view of the above-mentioned deficiencies of the prior art. The method supplements fructose as the main carbon source during induction by adding galactose, which has the advantages of higher strain activity, higher expression yield and higher fermentation density of Saccharomyces cerevisiae compared with no fructose, and the price of fructose is lower than that of glucose and much lower than that of raffinose, so supplementing fructose as the main carbon source during induction has a significant economic effect.
[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a feeding method for high-density fermentation of recombinant collagen by Saccharomyces cerevisiae with galactose promoter, characterized in that: after fermentation culture for 24 hours, when the OD of the fermentation broth reaches 100, starvation culture is carried out to consume all glucose, and then an inducer is added to the fermentation tank for induction, while fructose is added at the same time as induction.
[0007] The above-mentioned feeding method for high-density fermentation of recombinant collagen using Saccharomyces cerevisiae with a galactose promoter is characterized by the addition of fructose in the following manner: continuously adding fructose to maintain the fructose concentration in the fermentation broth at 2%.
[0008] Compared with the prior art, the present invention has the following advantages:
[0009] 1. This invention supplements fructose during the galactose induction stage of high-density fermentation, making fructose the primary carbon source and avoiding the problem of cell autolysis caused by low galactose utilization efficiency, which affects recombinant protein yield. Currently, the mainstream approach is to supplement raffinose as the primary carbon source when galactose utilization efficiency is poor, but raffinose is expensive. The principle by which Saccharomyces cerevisiae utilizes raffinose is that it enzymatically hydrolyzes raffinose into fructose, one of the products, and then utilizes fructose as a carbon source. This invention directly adds inexpensive fructose, which also solves problems such as excessive cell count, rapid carbon source consumption, low utilization efficiency of galactose as a single carbon source, and cell autolysis affecting yield and subsequent purification during high-density fermentation.
[0010] 2. This invention supplements fructose as the main carbon source during galactose induction, which has advantages such as higher strain activity, higher expression yield, and higher fermentation density of Saccharomyces cerevisiae compared to not adding fructose.
[0011] 3. The presence of glucose affects galactose induction; therefore, high-density fermentation requires starvation to deplete glucose before induction. Galactose has poor utilization efficiency, and currently, raffinose is used as the carbon source for galactose induction, but raffinose is expensive. This invention uses fructose as the carbon source, which is cheaper than glucose and significantly cheaper than raffinose. Using fructose as the primary carbon source for induction has significant economic benefits.
[0012] 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
[0013] Figure 1 This is a comparison chart of OD values between the experimental group and the control group in Example 1 of the present invention.
[0014] Figure 2 This is a Western blotting verification electrophoresis diagram of protein expression in Example 1 of the present invention.
[0015] Figure 3This is a Western blotting electrophoresis diagram to verify protein expression in Example 2 of the present invention. Detailed Implementation
[0016] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified. The following embodiments use the transformation of PYES2-α signal peptide-I67 recombinant plasmid into Saccharomyces cerevisiae BY-4741 as an example for a fed-batch fermentation experiment. This strain was provided by Xi'an Giant Biogene Technology Co., Ltd.
[0017] Example 1: Shake-flask induction of expression in Saccharomyces cerevisiae
[0018] (1) Pick positive transformants from YPD plates (transformed into PYES2 vector containing galactose promoter) and inoculate them in YPD medium and culture overnight.
[0019] (2) Collect bacterial cells by centrifugation, transfer the cells to galactose liquid medium (galactose concentration of 2%), adjust the OD600 value to 1, and induce culture by shaking. 2% fructose was added to the experimental group;
[0020] (3) Take samples at certain time intervals, 1 mL, place in a 1.5 m centrifuge tube, centrifuge at 12000 rpm for 5 min, collect the supernatant, and analyze the expression level of the target protein and the optimal collection time of the bacterial culture. The sampling time is generally 24, 48, or 72 h.
[0021] (4) The OD600 of the bacterial culture at each time point was measured and growth curves were plotted. According to the sampling results, the fructose-added group (experimental group) had better growth than the control group. The yeast in the experimental group continued to grow after 48 hours, while the OD of the control group decreased after 48 hours.
[0022] (5) Perform Western blotting on the fermentation broth samples to verify protein expression electrophoresis. See [link to electrophoresis diagram]. Figure 2 Lane 1: maker; Lanes 2 and 6: fructose-added group, sampling results at 24h and 48h; Lanes 3, 4, 5, 7, 8, and 9: control group, sampling results at 24h and 48h. Electrophoresis results show that the fructose-added group (experimental group) had significantly higher protein yields at both 24h and 48h compared to the control group.
[0023] Example 2: High-density fermentation of brewing yeast in a 5L fermenter
[0024] (1) Seed culture: Select positive transformants from YPD plates (transformed into PYES2 vector containing galactose promoter) and inoculate them in YPD medium and culture overnight;
[0025] (2) Prepare several 500mL Erlenmeyer flasks, each containing 100mL of YPD medium. Inoculate the above overnight cultured bacterial solution into several Erlenmeyer flasks at an inoculation rate of 2% and culture overnight to prepare the seed culture for fermentation tank.
[0026] (3) Equipment commissioning: Commission the pH electrode, polarized dissolved oxygen electrode, heating device and cooling device, and safety valve of the fermenter;
[0027] (4) Inoculate the seed culture in YPD into the fermenter at an inoculation rate of 10% and start the culture. The dissolved oxygen level is maintained by continuously increasing the rotation speed.
[0028] (5) After 24 hours of fermentation, when the OD of the fermentation broth reaches 100, the glucose is consumed by starvation culture, and galactose is added as an inducer to start induction; fructose is added.
[0029] (6) Fructose is continuously added during induction to maintain the fructose concentration in the fermentation broth at 2%. Fructose serves as the main carbon source, while galactose serves as the main inducer, ensuring the state and activity of the strain during induction.
[0030] (7) The OD600 of the bacterial culture at each time point was measured, and growth curves were plotted. The sampling results showed that the group with added fructose (experimental group) exhibited better growth than the control group.
[0031] (8) Perform Western blotting on the fermentation broth samples to verify protein expression electrophoresis. See [image / graph]. Figure 3 Lane 1: maker; Lanes 2 & 3: shake flask fermentation group; Lanes 4, 5 & 6: shake flask fermentation group (fructose added during induction); Lane 7: high-density fermentation group (fructose added during induction). All lanes were sampled after 48 hours of induction. Electrophoresis results showed that the protein yield of the high-density fermentation group was significantly better than the control group.
[0032] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.
Claims
1. A method for fed-batch fermentation of recombinant collagen using a galactose-promoted Saccharomyces cerevisiae at high density, characterized in that, include: After 24 hours of fermentation, when the OD of the fermentation broth reaches 100, the glucose is consumed by starvation culture. Then, an inducer is added to the fermenter for induction, and fructose is added at the same time.
2. The method for feeding recombinant collagen through high-density fermentation using a galactose-promoted Saccharomyces cerevisiae according to claim 1, characterized in that, The method of adding fructose is as follows: fructose is continuously added to maintain the fructose concentration in the fermentation broth at 2%.