Fermentation method for increasing yield of 5-hydroxytryptophan

Abstract

The invention discloses a fermentation method for increasing the yield of 5-hydroxytryptophan, and relates to the technical field of 5-hydroxytryptophan ferment.The fermentation method comprises the following steps of flat plate pouring, flat plate coating, secondary scribing, liquid strain preparation, fermentation tank fermentation, induction, yeast powder adding and fermentation. According to the method disclosed by the invention, through an innovative fermentation control means, a'feedback inhibition 'phenomenon that a product 5-HTP is continuously generated and the synthesis capability of microorganisms is reversely inhibited is successfully broken, so that the microorganisms can continuously and efficiently synthesize the 5-HTP, and finally, the stable rate that 0.5 g of 5-HTP can be produced per hour by per liter of fermentation liquor is realized; the method has the advantages that fermentation equipment in unit time and unit volume can produce more target products, the high-efficiency production capacity can quickly respond to the requirements of the market on 5-HTP, and the method has the effects of high conversion rate and stable production performance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of 5-hydroxytryptophan fermentation, and more particularly to a fermentation method for improving the yield of 5-hydroxytryptophan. BACKGROUND

[0002] 5-hydroxytryptophan (5-HTP) is an important neurotransmitter in the human body, serotonin (5-hydroxytryptamine, 5-HT), a direct precursor substance, an amino acid derivative, which cannot be synthesized in the human body itself and needs to be obtained by food intake of precursors (such as L-tryptophan) or direct supplements, and plays a key role in regulating the function of the nervous system, mood and physiological rhythm.

[0003] There are many conditions that affect the yield of 5-HTP production, such as the continuous generation of product 5-HTP, which in turn inhibits the synthesis ability of microorganisms (this phenomenon is called "feedback inhibition"), resulting in stagnation of production efficiency; tetrahydrobiopterin (BH4) is easily oxidized and consumed during fermentation, resulting in insufficient reducing power in the synthesis process; excessive accumulation of tryptophan can affect the synthesis of 5-HTP; product accumulation has toxicity to the growth of the bacteria, inhibiting the fermentation process.

[0004] Currently, 5-HTP is produced by genetic engineering to improve the yield of 5-HTP, such as the Chinese patent application for invention with publication number CN113373103A, which uses glucose as a carbon source and ferments for 65h, with a 5-HTP yield of 20.9g / L. However, this yield is still relatively low, and there is still a long way to go for industrial production.

[0005] In order to overcome the shortcomings of the above method, the present application provides a method for efficiently producing 5-HTP using a flow-adding tryptophan solution strategy with inexpensive carbon sources such as glucose as a substrate, which has the effects of high conversion rate and stable production performance, with a content of 30.5g / L after 60 hours and a specific production rate of 0.5g / (L·h). SUMMARY

[0006] The purpose of the present application is to solve the problems raised in the background art, and the present application provides a fermentation method for improving the yield of 5-hydroxytryptophan.

[0007] In order to achieve the above purpose, the present application specifically adopts the following technical solution: A fermentation method for improving the yield of 5-hydroxytryptophan, comprising the following steps: S1. Pouring a flat plate: preparing LB solid medium and LB liquid medium for standby, adding chloramphenicol and ampicillin resistance to the LB solid medium for activation, and obtaining a double-antibiotic plate for standby; S2. Plating: Take the 5-hydroxy tryptophan strain, dilute and plate on double-antibiotic plates for culture to obtain single bacteria; S3. Secondary streaking: pick the single bacteria obtained by culture and streak on double-antibiotic plates for culture to obtain bacterial cells; S4. Liquid strain preparation: take 1 single colony of the obtained bacterial cells and inoculate in 50 ml of LB liquid medium containing chloramphenicol and ampicillin, and culture at 250 rpm and 37°C, and obtain liquid strain when OD 600 = 2-4. S5. Fermentation in a fermenter: inoculate the liquid strain into a fermenter at a volume percentage of 10%-20% of the fermentation medium, control the pH at 6.5-7.2 during the fermentation process, maintain the temperature at 30°C-37°C, and maintain the dissolved oxygen at 15-30%; S6. Induction: after the DO rises and the glucose in the fermentation medium is consumed, add 60% glucose solution and maintain the glucose concentration in the fermentation medium at <0.1 g / L, OD 600 ≥ 25, cool to 28°C-32°C, add 0.1 mM-0.5 mM / L IPTG inducer, maintain the induction temperature at 28°C-32°C, and add metal ions once before induction to maintain the concentration at 0.35 g / L; S7. Add yeast powder: after adding the inducer, start adding yeast powder to maintain the yeast powder concentration in the fermentation system at 5 g-15 g / L; S8. Fermentation: 1-3 hours after the addition of the inducer is completed, add tryptophan solution with a mass percentage of 20% at a flow rate of 20 ml-40 ml / H according to the volume of the fermentation system, and adjust the amount of tryptophan solution added according to the detection of the content of 5-hydroxy tryptophan in the fermentation system to control the content of tryptophan within 100 mg-500 mg / L, and the fermentation period is 55-60 hours.

[0008] Preferably, in step S1, the formula of the LB solid culture medium is: yeast extract powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, and agar strip 15 g / L. The formula of the LB liquid culture medium is: yeast extract powder 5 g / L, peptone 10 g / L, and sodium chloride 10 g / L.

[0009] Preferably, in step S1, the concentrations of chloramphenicol and ampicillin resistance activated in the LB solid culture medium are 0.05% and 0.01%, respectively.

[0010] Preferably, in step S1, the chloramphenicol and ampicillin antibiotics are sterilized by filtering through a 0.2 μm filter membrane.

[0011] Preferably, the culture conditions in steps 2 and 3 are both 37°C for 12 hours.

[0012] Preferably, the final concentration of the step 4 chloramphenicol and ampicillin antibiotics in the LB liquid culture medium is 100 mg / L.

[0013] Preferably, in the step 5, the components of the fermentation medium are: glucose 10-20 g / L, yeast powder 5-15 g / L, KH2PO4 3-8 g / L, citric acid 0.5-3 g / L, ammonium sulfate 2-5 g / L, metal ion solution 2-10 mL, MgSO4·7H2O 0.5-5 g / L, antifoam agent 0.01%, chloramphenicol 0.1 g / L, ampicillin 0.1 g / L, and the metal ion solution comprises: FeSO4·7H2O 30 g / L, ZnCl2 2 g / L, CuSO4 1 g / L, VH 0.1 g / L, MnCl2 2 g / L, Na2B4O7 0.5 g / L, and CoCl2 1 g / L.

[0014] Preferably, in the step 5, the addition amount of the liquid strain is 15% of the volume percentage of the fermentation medium.

[0015] Preferably, in the step 6, the induction temperature is 30°C, and the added metal ion component is FeSO4·7H2O 30 g / L, ZnCl2 2 g / L, CuSO4 1 g / L, VH 0.1 g / L, MnCl2 2 g / L, Na2B4O7 0.5 g / L, and CoCl2 1 g / L.

[0016] Preferably, in the step 8, the addition time of the tryptophan solution is 1 hour after the addition of the inducer is completed.

[0017] Compared with the prior art, the present application has the beneficial effects that: 1. The present application successfully breaks the "feedback inhibition" phenomenon that the continuously generated product 5-HTP inhibits the synthesis ability of microorganisms, so that microorganisms can continuously and efficiently synthesize 5-HTP, and ultimately achieve a stable rate of "0.5 grams of 5-HTP per liter of fermentation liquid per hour". This means that the fermentation equipment can produce more target products per unit time and per unit volume. This high production efficiency can quickly respond to market demand for 5-HTP, and has the effects of high conversion rate and stable production performance.

[0018] 2. The method of this invention achieves a tryptophan substrate conversion efficiency of 99.5% within a fermentation cycle of 55-60 hours, significantly reducing raw material costs. Simultaneously, due to the high substrate conversion efficiency and low byproduct production, subsequent separation and purification processes do not require complex impurity removal steps (such as removing large amounts of unconverted tryptophan or byproducts), simplifying the purification process (e.g., reducing the number of chromatography column uses and shortening crystallization time), lowering reagent consumption and equipment wear costs, and reducing waste emissions during production. Detailed Implementation

[0019] The technical solutions 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.

[0020] The materials and instruments used in the following examples are all commercially available.

[0021] Example 1 S1. Plate preparation: Prepare LB solid medium and LB liquid medium, sterilize at 121℃ for 20 min for later use. Add 50 μL of chloramphenicol and 50 μL of ampicillin antibiotics to 50 ml of LB solid medium and store at 4℃ for later use. S2. Plate plating: Take the 5-hydroxytryptophan strain stored in a glycerol tube at -80℃, dilute it, and plate it on a double-antibiotic plate for incubation. After incubation at 37℃ for 12 hours, single bacteria are obtained. S3. Second streak: Pick about 30 single bacteria from the double-antibiotic plate and streak them onto a new double-antibiotic plate. Incubate at 37°C for 12 hours to obtain bacterial cells. S4. Preparation of liquid culture: Slice the obtained bacterial cells, take one single colony, and inoculate it into 50 ml of LB liquid medium. Add 50 mg / L chloramphenicol and 100 mg / L ampicillin. Incubate at 37°C and shake at 250 rpm for 11 h to obtain the liquid culture. After incubation, measure the OD value. 600 Approximately 4; S5. Fermentation in a fermenter: Inoculate the liquid inoculum into a 20L fermenter at an inoculation rate of 15% of the fermentation medium volume. During fermentation, control the pH at 7.2 (adjust the pH with ammonia), maintain the temperature at 37℃, and keep the dissolved oxygen at 30%. S6. Induction: After D0 recovers and the glucose in the fermentation medium is depleted, add a 60% glucose solution and maintain the glucose concentration in the fermentation medium <0.1 g / L. 600≥25℃, cool down to 30℃, add 0.5mM / L IPTG inducer, maintain the induction temperature at 30℃, add metal ions once before induction, maintain the concentration at 0.35g / L; S7. Add yeast powder: After the inducer is added, start adding yeast powder to make the yeast powder concentration in the fermentation system 15g / L; S8. Fermentation: 2 hours after the inducing agent is added, add a 20% tryptophan solution based on the volume of the fermentation system at a flow rate of 40 ml / H. Adjust the amount of tryptophan solution added based on the detected 5-hydroxytryptophan content in the fermentation system, and control the tryptophan content within 100 mg-500 mg / L. The fermentation cycle is 60 hours.

[0022] HPLC detection of tryptophan and 5-HTPH The chromatography column was Hypersil C18 (4.6×250mm, 5μm, Thermo-Fisher, MA, USA), and the detection conditions were 30℃ with a Shimadzu SPD-20A detector. The mobile phase was 0.1mol / L dipotassium phosphate and methanol in a volume ratio of 85:15; the flow rate was 1.0mL / min; the detection wavelength was 280nm; and the column oven temperature was 30℃.

[0023] Configuration of each component In step S1, the LB solid culture medium formula is: yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L, and agar strips 15g / L. In step S1, the LB liquid culture medium formula is: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride.

[0024] In step S1, the concentrations of chloramphenicol and ampicillin resistance activation in LB solid medium are 0.05% and 0.01%, respectively.

[0025] In step S1, the chloramphenicol and ampicillin antibiotics are sterilized by filtration through a 0.2 μm filter membrane.

[0026] The cultivation conditions for both steps 2 and 3 are 37℃ for 12 hours.

[0027] In step 4, the final concentration of chloramphenicol and ampicillin antibiotics in LB liquid medium is 100 mg / L.

[0028] In step 5, the fermentation medium consists of: 10-20 g / L glucose, 5-15 g / L yeast powder, 3-8 g / L KH2PO4, 0.5-3 g / L citric acid, 2-5 g / L ammonium sulfate, 2-10 mL metal ion solution, 0.5-5 g / L MgSO4·7H2O, 0.01% defoamer, 0.1 g / L chloramphenicol, and 0.1 g / L ampicillin.

[0029] The composition of the metal ion solution is as follows: FeSO4·7H2O 30 g / L, ZnCl2 2 g / L, CuSO4 1 g / L, VH 0.1 g / L, MnCl2 2 g / L, Na2B4O7 0.5 g / L, and CoCl2 1 g / L. It is dissolved using concentrated hydrochloric acid solution at a ratio of 10 mL / L between the metal ions and the concentrated hydrochloric acid.

[0030] Feeding medium: 30% yeast extract. The yeast extract is added by volume from a 30% yeast extract feeding solution.

[0031] In step 5, the amount of liquid inoculum added is 15% of the volume percentage of the fermentation medium.

[0032] In step 6, the induction temperature is 30℃, and the added metal ions are: FeSO4·7H2O 30g / L, ZnCl2 2g / L, CuSO4 1g / L, VH 0.1g / L, MnCl2 2g / L, Na2B4O7 0.5g / L, and CoCl2 1g / L.

[0033] In step 8, the tryptophan solution is added 1 hour after the inducing agent is added. The 20% tryptophan solution is prepared by dissolving 200g of tryptophan in 1000mL of 5% H2SO4 and sterilizing it in an autoclave at 121℃ for 20min.

[0034] Chloramphenicol: 50 g / L stock solution; ampicillin: 100 g / L stock solution; 0.2 μm filter membrane, sterilize by filtration, store at -20℃, working concentration 1.0 per thousand.

[0035] Large-scale preparation of IPTG inducer: 0.5M stock solution, 0.2μm filter membrane, sterilize by filtration, store at -20℃, working concentration: 0.1mM-0.5mM.

[0036] Preparation of 60% glucose solution: Add 660g of glucose monohydrate to water to a final volume of 1 liter, and sterilize in an autoclave at 115℃ for 20 minutes.

[0037] Product Characterization 4 The purified sample of the fermentation product from Example 1 was first analyzed using HPLC-UV (C18 column, 0.1 mol / L KH2PO4 / methanol 85:15, 1.0 mL / min, 280 nm). The retention time was approximately 10.1 ± 0.2 min, consistent with the standard; purity ≥ 98.2%, main peak 5HTP; the main impurity was tryptophan (≤ 1.8%), with no other significant impurity peaks. ESIMS analysis yielded [M+H]⁺ m / z = 221.1, consistent with the theoretical molecular weight; ^1H NMR (600 MHz D2O) showed clear signals at δ 7.13, 6.73, 3.46 / 3.09 ppm, highly consistent with literature data, confirming the correct structure.

[0038] Tryptophan Residual Analysis Tryptophan concentration was sampled and analyzed at key fermentation time points (0h, 24h, 36h, 48h, and 60h). Results showed that the initial concentration was approximately 10 g / L, which decreased to ≤1.4 g / L (tryptophan to 5HTP mass ratio ≤4.8%) after staged feeding and metabolic transformation, by the end of fermentation. The experimental results indicate that tryptophan accumulation was effectively avoided during high-risk stages through a pre-feeding + delayed feeding strategy and appropriate fermentation environment control.

[0039] Monitoring of L-tryptophan concentration during fermentation Using the same HPLC method, the fermentation broth was sampled at 0h, 24h, 36h, 48h and 60h after completion, and the tryptophan concentration (unit: g / L) was determined.

[0040] Example 2, Seed liquid inoculation amount Table 1 Experimental groups were set up according to the experimental conditions shown in Table 1. Except for the experimental conditions shown in Table 1, all other conditions were the same as in Example 1. The control group had a 5% inoculum and a fermenter with a fixed volume of 20L. The inoculum was gradually increased, and the time to reach OD=25 was gradually reduced. When the inoculum was increased to 18% and 20%, the cell growth rate could not be further increased. Considering that an excessively large inoculum would place a heavy demand on the fermentation equipment and that excessive nutrient consumption would be detrimental to the later stages of fermentation, an inoculum of 15% was selected.

[0041] Example 3, Induction Temperature Table 2 Experimental groups were set up according to the experimental conditions shown in Table 2. Except for the experimental conditions shown in Table 2, all other conditions were consistent with those in Example 1. The control group was induced at 37℃, and the final yield was 9.6 g / L. It showed rapid cell growth, high sugar and oxygen consumption, and a maximum OD value of 128. SDS-PAGE electrophoresis analysis showed that the intracellular protein was insoluble. Comparative verification was performed at induction temperatures of 35℃, 30℃, 28℃, and 25℃. The highest yield was 28.7 g / L at 30℃. Further decreasing the temperature significantly reduced the NR yield, decreased sugar and oxygen consumption, and inhibited cell growth.

[0042] Example 4: Time at which 20% tryptophan solution is first added Table 3 Experimental groups were set up according to the experimental conditions shown in Table 3. Except for the experimental conditions shown in Table 3, all other conditions were the same as in Example 1. Directly adding 20% ​​tryptophan solution after induction inhibited bacterial growth. In contrast, in the control group, adding 20% ​​tryptophan solution 1 hour after the inducing agent addition resulted in a final yield of 27.3 g / L. Adding tryptophan solution 1.5-2 hours after the inducing agent addition also yielded higher yields. Adding tryptophan solution at other times inhibited bacterial growth due to factors such as incomplete protein expression in the bacteria after induction, resulting in a different yield compared to adding it 1.5-2 hours after the inducing agent addition.

[0043] Example 5: Feeding after induction Table 4 Except for the experimental conditions shown in Table 4, all other experimental conditions were the same. After the inducer was added, yeast powder 35 g / L was added once according to the fermentation broth volume to bring the final concentration to 15 g / L. During fermentation for 33-45 hours, the accumulation of TRP slowed down, the conversion of 5-HTP was improved, the cell activity was enhanced in the later stage, and the final yield was 30.5 g / L.

[0044] Based on the aforementioned optimization conditions, the substrate conversion rate and production rate under optimal conditions were tested. The results showed that after 60 hours of fermentation, the 5-HTP content was 30.5 g / L, and the trp substrate conversion efficiency reached 99.5%, reducing the burden of separation and purification. The maximum 5-HTP production rate per unit time was 0.55 g / (L·H).

[0045] As can be seen from the above parameters, the method of this application can increase the 5-HTP content to 30.5 g / L after 60 hours of fermentation, effectively increasing the yield of 5-HTP. At the same time, the tryptophan substrate conversion efficiency reaches 99.5%, effectively reducing production costs, improving material utilization, and reducing the complexity of subsequent impurity removal steps.

Claims

1. A fermentation method for increasing 5-hydroxytryptophan yield, characterized in that, Includes the following steps: S1. Plate preparation: Prepare LB solid medium and LB liquid medium for later use. Add chloramphenicol and ampicillin to the LB solid medium to activate resistance and obtain double-antibiotic plates for later use. S2. Plate plating: Take the 5-hydroxytryptophan strain, dilute it, and plate it on a double-antibiotic plate for incubation to obtain single bacteria; S3. Secondary streak: Select single bacteria obtained from the culture and streak them onto a double-antibiotic plate for further culture to obtain bacterial cells; S4. Preparation of liquid culture: Slice the obtained bacterial cells, take one single colony and inoculate it into 50 ml of LB liquid medium. Add chloramphenicol and ampicillin, and incubate on a shaker at 250 rpm and 37°C. OD 600 =2-4, to obtain liquid bacterial culture; S5. Fermentation in a fermenter: Inoculate the liquid inoculum into the fermenter at an inoculation rate of 10%-20% of the fermentation medium volume. During fermentation, control the pH at 6.5-7.2, maintain the temperature at 30℃-37℃, and keep the dissolved oxygen at 15-30%. S6. Induction: After D0 recovers and the glucose in the fermentation medium is depleted, add a 60% glucose solution and maintain the glucose concentration in the fermentation medium <0.1 g / L. 600 ≥25, cool to 28℃-32℃, add 0.1mM-0.5mM / L IPTG inducer, maintain the induction temperature at 28℃-32℃, add metal ions once before induction, maintain the concentration at 0.35g / L; S7. Add yeast powder: After the inducer is added, start adding yeast powder to make the yeast powder concentration in the fermentation system 5g-15g / L; S8. Fermentation: 1-3 hours after the inducing agent is added, add a 20% tryptophan solution according to the volume of the fermentation system at a flow rate of 20-40 ml / H. Adjust the amount of tryptophan solution added based on the detected 5-hydroxytryptophan content in the fermentation system, and control the tryptophan content within 100-500 mg / L. The fermentation cycle is 55-60 hours.

2. The fermentation method for increasing 5-hydroxytryptophan yield according to claim 1, characterized in that, In step S1, the LB solid culture medium formula is: yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L, and agar strips 15g / L. The LB liquid medium formula is: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride.

3. The fermentation method for increasing 5-hydroxytryptophan yield according to claim 1, characterized in that, In step S1, the concentrations of chloramphenicol and ampicillin resistance activation in LB solid medium were 0.05% and 0.01%, respectively.

4. The fermentation method for increasing 5-hydroxytryptophan yield according to claim 1, characterized in that, In step S1, the chloramphenicol and ampicillin antibiotics are sterilized by filtration through a 0.2 μm filter membrane.

5. The fermentation method for increasing 5-hydroxytryptophan yield according to claim 1, characterized in that, The cultivation conditions for both steps 2 and 3 are 37℃ for 12 hours.

6. The fermentation method for increasing 5-hydroxytryptophan yield according to claim 1, characterized in that, In step 4, the final concentration of chloramphenicol and ampicillin antibiotics in LB liquid medium is 100 mg / L.

7. The fermentation method for increasing 5-hydroxytryptophan yield according to claim 1, characterized in that, In step 5, the fermentation medium consists of: glucose 10g-20g / L, yeast powder 5g-15g / L, KH2PO4 3g-8g / L, citric acid 0.5g-3g / L, ammonium sulfate 2g-5g / L, metal ion solution 2mL-10mL, MgSO4·7H2O 0.5g-5g / L, defoamer 0.01%, chloramphenicol 0.1g / L, and ampicillin 0.1g / L. The metal ion solution consists of: FeSO4·7H2O 30g / L, ZnCl2 2g / L, CuSO4 1g / L, VH 0.1g / L, MnCl2 2g / L, Na2B4O7 0.5g / L, and CoCl2 1g / L.

8. The fermentation method for increasing 5-hydroxytryptophan yield according to claim 1, characterized in that, In step 5, the amount of liquid inoculum added is 15% of the volume percentage of the fermentation medium.

9. The fermentation method for increasing 5-hydroxytryptophan yield according to claim 1, characterized in that, In step 6, the induction temperature is 30℃, and the added metal ions are: FeSO4·7H2O 30g / L, ZnCl2 2g / L, CuSO4 1g / L, VH 0.1g / L, MnCl2 2g / L, Na2B4O7 0.5g / L, and CoCl2 1g / L.

10. The fermentation method for increasing 5-hydroxytryptophan yield according to claim 1, characterized in that, In step 8, the tryptophan solution is added 1 hour after the inducing agent is added.

Images