Heat-resistant tryptophan producing strain and application thereof

Abstract

The heat-resistant Escherichia coli hhTRp002 for producing tryptophan is obtained by combining natural screening with high-temperature domestication, the genetic stability of the strain is high, L-tryptophan can be produced through fermentation by efficiently utilizing glucose as a unique carbon source, the conversion rate can reach 35%, the yield reaches 60 g / L, the yield is efficient and stable, and the method is suitable for industrial production. Meanwhile, the method can tolerate a relatively high-temperature environment, and the cooling cost of a fermentation system is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a heat-resistant tryptophan-producing bacterium and its application. Background Art

[0002] L-tryptophan is an essential amino acid for the human body, which participates in the synthesis of human proteins and the regulation of metabolic networks, and widely exists in nature. In the pharmaceutical field, L-tryptophan and its metabolites are used as amino acid injection solutions and compound amino acid preparations; in the food field, L-tryptophan is mainly used as a food additive, flavoring agent, and antioxidant preservative, and can also be used as a nutritional fortifier for making nutritional beverages - enzymatically hydrolyzed gelatin preparations. In the field of feed additives, tryptophan, as an essential amino acid that cannot be synthesized in animals but has multiple physiological functions, plays a role in aspects such as feed intake, production performance, stress resistance, and immunity of livestock and poultry.

[0003] The production of L-tryptophan was originally mainly dependent on protein hydrolysis methods and chemical synthesis methods. However, with the continuous in-depth research on the production of L-tryptophan by microbial methods, microbial methods have become practical and dominant. Microbial methods can be further divided into direct fermentation methods, microbial transformation methods, and enzymatic methods. Among them, the direct fermentation method uses sugars as raw materials and utilizes microorganisms to produce L-tryptophan through a series of metabolic reactions. It has the advantages of low raw material prices, simple process control, and reliable product quality, and is currently the main method for producing L-tryptophan.

[0004] As one of the most commonly used model industrial microorganisms at present, Escherichia coli has the advantages of clear genetic background, simple operation, short growth cycle, mature metabolic regulation, and strong robustness in the industrial fermentation process, and has become the main engineering bacterium for producing L-tryptophan by the direct fermentation method. However, in the process of industrial microbial fermentation for producing L-tryptophan, due to the large volume of the fermentation tank, the biological metabolic heat and mechanical agitation heat cause the fermentation system to continuously heat up. Especially in the high-temperature environment in summer, it is more difficult to control the temperature of the fermentation system, and microorganisms cannot spontaneously drop to the optimal metabolic temperature. Therefore, a large amount of cooling water is required for temperature control, resulting in an increase in energy consumption costs, accounting for about 25 - 40% of the total fermentation cost. Molecular modification and high-temperature domestication are common methods for improving the heat resistance of strains, and the genetic stability of high-temperature domesticated strains is higher. Therefore, screening heat-resistant or high-temperature-resistant Escherichia coli engineering bacteria for producing L-tryptophan through high-temperature domestication means is of great significance for the industrial production of low-cost L-tryptophan products. Summary of the Invention

[0005] The object of the present invention is to screen out a heat-resistant Escherichia coli hhTRp002 that produces tryptophan from nature. This strain can efficiently utilize glucose to ferment and produce L-tryptophan, with a conversion rate of 35% and a yield of 60 g / L. At the same time, it can tolerate a relatively high-temperature environment, reducing the cooling cost of the fermentation system.

[0006] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0007] In the first aspect, the present invention provides a heat-resistant tryptophan-producing bacterium, which is taxonomically named Escherichia coli hhTRp002. It was deposited at the China Center for Type Culture Collection on November 09, 2023, with the deposit number CCTCC NO: M 20232171, and the deposit address is Wuhan University, Wuhan, China.

[0008] According to the technical solution of the present invention, the tolerance temperature of the heat-resistant tryptophan-producing bacterium is 37 - 41 °C.

[0009] In the second aspect, the present invention provides the application of the heat-resistant tryptophan-producing bacterium in the production of tryptophan.

[0010] In the third aspect, the present invention provides a method for producing L-tryptophan by fermentatively culturing the heat-resistant tryptophan-producing bacterium.

[0011] In one embodiment of the present invention, the fermentative culture includes: inoculating the heat-resistant tryptophan-producing bacterium into a fermentation medium and culturing it with shaking and aeration.

[0012] In one embodiment of the present invention, the fermentation medium includes: 10 - 15 g / L glucose, 0.1 - 0.2 g / L indole, 0.05 - 0.2 g / L K2HPO4, 0.05 - 0.2 g / L KH2PO4, 0.1 - 0.2 g / L MgSO4·7H2O, 2 - 5 mg / L FeSO4·7H2O, 2 - 5 mg / L MnSO4, pH 6.8 - 7.2;

[0013] Preferably, the fermentation medium includes: 10 g / L glucose, 0.1 g / L indole, 0.05 g / L K2HPO4, 0.05 g / L KH2PO4, 0.1 g / L MgSO4·7H2O, 2 mg / L FeSO4·7H2O, 2 mg / L MnSO4, pH 7.2.

[0014] In one embodiment of the present invention, during the culture process, the dissolved oxygen is controlled at 30 - 50%.

[0015] In one embodiment of the present invention, when the glucose in the fermentation medium is exhausted, the dissolved oxygen is linked to feeding, and the feed liquid is glucose; the glucose concentration is 50-70%.

[0016] In one embodiment of the present invention, feeding is carried out in a fed-batch manner, and the feeding rate is 7-11 g / L / h.

[0017] In one embodiment of the present invention, during the cultivation process, the ventilation rate is 1-2 vvm, and the stirring speed is 300-600 prm.

[0018] In one embodiment of the present invention, during the cultivation process, the pH is controlled at 6.8-7.2.

[0019] In one embodiment of the present invention, the dissolved oxygen can be controlled at 30-50% in the following manner: when the dissolved oxygen is lower than 30%, the feeding flow rate is controlled at 7 g / L / h; when the dissolved oxygen is higher than 50%, the feeding flow rate is controlled at 11 g / L / h; when 11 g / L / h still cannot effectively reduce the dissolved oxygen, the rotation speed can be reduced within the range of 300-600 prm.

[0020] In one embodiment of the present invention, the cultivation temperature is 37-41 °C, and the cultivation time is 45-50 h;

[0021] Preferably, the cultivation temperature is 39 °C, and the cultivation time is 48 h.

[0022] In one embodiment of the present invention, the fermentation culture includes:

[0023] Seed culture: inoculating the heat-resistant tryptophan-producing bacterium into the seed medium, and culturing at 37-41 °C and a rotation speed of 150-200 rpm for 12-24 h to obtain a seed liquid;

[0024] Transfer the seed liquid to the fermentation medium at 5-15% (v / v).

[0025] In one embodiment of the present invention, the seed medium includes: glucose 4-7 g / L, ammonium sulfate 0.4-0.8 g / L, yeast extract 0.3-0.5 g / L, indole 0.1-0.2 g / L, K2HPO4 0.1-0.5 g / L, KH2PO4 0.1-0.5 g / L, MgSO4·7H2O 0.2-0.5 g / L, FeSO4·7H2O 5-10 mg / L, MnSO4 5-10 mg / L, pH 6.8-7.2.

[0026] Preferably, the seed culture medium comprises: 4 g / L of glucose, 0.4 g / L of ammonium sulfate, 0.3 g / L of yeast extract, 0.1 g / L of indole, 0.1 g / L of K2HPO4, 0.1 g / L of KH2PO4, 0.2 g / L of MgSO4·7H2O, 5 mg / L of FeSO4·7H2O, 5 mg / L of MnSO4, and pH 7.2.

[0027] Advantages of the present invention:

[0028] (1) In the present invention, an Escherichia coli hhTRp002 producing L-tryptophan is screened from nature. It can directly ferment L-tryptophan using glucose as the sole carbon source, and the fermentation yield of L-tryptophan can reach 60 g / L, and the conversion rate can reach 35%.

[0029] (2) The Escherichia coli hhTRp002 for L-tryptophan screened in the present invention can tolerate high temperature conditions of 37 - 41 °C, reducing the requirements for the cooling system and the consumption of cooling water. Moreover, the main raw material glucose is inexpensive and easily available, further reducing the fermentation cost, providing a new idea for low-cost industrial fermentation production of L-tryptophan.

[0030] (3) The natural screening wild-type strain provided by the present invention can achieve high yield without genetic modification, alleviating the impact of genetically engineered strains on production instability. Description of the Drawings

[0031] Figure 1 It is the standard curve for detecting tryptophan by p-dimethylaminobenzaldehyde method in Example 1;

[0032] Figure 2 It is the HPLC detection result diagram of L-tryptophan standard product;

[0033] Figure 3 It is the liquid phase standard curve of tryptophan;

[0034] Figure 4 It is the morphological identification diagram of Escherichia coli hhTRp002. Detailed Embodiments

[0035] The present invention will be further described in detail below in combination with specific embodiments. The embodiments given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following embodiments provided can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0036] In the following examples, the experimental methods are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0037] The detection methods involved in the following examples:

[0038] 1. Bacterial cell concentration: Appropriately dilute the fermentation broth, and use an ultraviolet spectrophotometer to measure the absorbance OD at a wavelength of 600 nm. 600 .

[0039] 2. Glucose concentration: Centrifuge the fermentation broth at 12000 rpm for 5 min to obtain the supernatant. Dilute the supernatant to an appropriate multiple, and use an M-100 biosensor analyzer to detect the glucose concentration in the fermentation broth.

[0040] 3. L-tryptophan content: Use high performance liquid chromatography (HPLC) to detect the L-tryptophan content in the fermentation broth. The detection method is as follows:

[0041] Prepare a 1 g / L L-tryptophan standard solution, and dilute it to 0.1, 0.2, 0.3, 0.4, and 0.5 g / L. Use a high performance liquid chromatograph for detection to obtain the peak time and the peak areas corresponding to different concentrations of L-tryptophan. Take the concentration of the L-tryptophan solution as the abscissa and the peak area as the ordinate to plot a standard curve and obtain a linear regression equation, y = 40222x, R 2 = 0.9999, see Figure 1 .

[0042] Fermentation broth pretreatment: Centrifuge the fermentation broth at 12000 rpm for 5 min to obtain the supernatant; after diluting the supernatant to an appropriate multiple, filter it through a 0.22 μm filter membrane, and collect the filtrate;

[0043] L-tryptophan content detection: Detect the above fermentation filtrate using HPLC. Substitute the obtained peak area into the linear regression equation, and multiply the obtained L-tryptophan by the dilution multiple to obtain the L-tryptophan concentration in the fermentation broth.

[0044] Among them, the HPLC detection conditions:

[0045] Detection instrument: Agilent HPLC 1260-VWD; Chromatographic column: Agilent TC-C18 chromatographic column, 250 * 4.6 mm, 5 μm;

[0046] Mobile phase: 0.03% KH2PO4: pure methanol = 9:1 (V / V);

[0047] Column temperature: 39 °C;

[0048] Flow rate: 1 mL / min;

[0049] Ultraviolet detector, wavelength 276 nm;

[0050] Sample injection volume: 20 μL.

[0051] Among them, for the L-tryptophan standard product (purchased from Aladdin, CAS number: 73-22-3), the peak emergence time of L-tryptophan is about 11.9 min, see Figure 2 。

[0052] Example 1. Screening of heat-resistant tryptophan-producing bacterium hhTRp002

[0053] 1. Strain screening

[0054] S1. Sampling: Take a water sample from the sewage discharge of the post-sterilization fermentation broth in the Qinhuangdao Huaheng factory area, Qinhuangdao City, Hebei Province.

[0055] S2. Primary screening of tryptophan-producing strains: Spread the water sample taken in step S1 on the enrichment medium and culture at 38 °C for 24 h;

[0056] Pick single colonies onto the slant of the enrichment medium and culture at 38 °C for 12 h;

[0057] Inoculate the strains enriched on the slant into the shake flask seed medium and culture at 38 °C for 12 h;

[0058] Centrifuge the seed liquid, color the supernatant with p-dimethylaminobenzaldehyde method, and visually select 10 strains showing dark blue color as tryptophan-producing strains for high-temperature acclimation;

[0059] Among them, the enrichment medium is: glucose 0.8 g / L, peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.2.

[0060] The shake flask seed medium can be: glucose 4 - 7 g / L, ammonium sulfate 0.4 - 0.8 g / L, yeast extract 0.3 - 0.5 g / L, indole 0.1 - 0.2 g / L, K2HPO4 0.1 - 0.5 g / L, KH2PO4 0.1 - 0.5 g / L, MgSO4·7H2O 0.2 - 0.5 g / L, FeSO4·7H2O 5 - 10 mg / L, MnSO4 5 - 10 mg / L, pH 7.2;

[0061] In this example, the shake flask seed medium is: glucose 4 g / L, ammonium sulfate 0.4 g / L, yeast extract 0.3 g / L, indole 0.1 g / L, K2HPO4 0.1 g / L, KH2PO4 0.1 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 5 mg / L, MnSO4 5 mg / L, pH 7.2.

[0062] S3. High-temperature domestication: The tryptophan-producing strains preliminarily screened in step S2 are respectively inoculated into shake-flask seed media and cultured at 39 °C for 36 h to obtain seed liquid A0; The seed liquid A0 is continuously transferred 6 times with an inoculation amount of 1% to obtain seed liquid A6; The seed liquid A6 is cultured at 40 °C for 36 h to obtain seed liquid B0; The seed liquid B0 is continuously transferred 6 times with an inoculation amount of 1% to obtain seed liquid B6. The seed liquid B6 is spread on the plate of the seed medium and cultured at 41 °C for 24 h. Single colonies are picked and inoculated into the shake-flask seed medium. 10 strains of bacteria with higher cell concentrations are selected for re-screening;

[0063] Among them, the shake-flask seed medium is: glucose 4 g / L, ammonium sulfate 0.4 g / L, yeast extract 0.3 g / L, indole 0.1 g / L, K2HPO4 0.1 g / L, KH2PO4 0.1 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 5 mg / L, MnSO4 5 mg / L, pH 7.2.

[0064] S4. Re-screening heat-resistant tryptophan-producing strains: The 10 strains domesticated in step S3 are inoculated into shake-flask fermentation culture and cultured at 38 °C for 12 h; The fermentation broth is centrifuged, and after the supernatant is colored by the p-dimethylaminobenzaldehyde method, the OD 550 value is measured with a spectrophotometer; It is found that the OD 600 value of strain 2 is the highest at 0.601 (the sample is diluted 10 times), and according to the tryptophan standard curve y = 0.2718x (see Figure 3 ), the content of tryptophan is calculated to be 1.63 g / L;

[0065] Among them, the fermentation medium is: glucose 10 g / L, indole 0.1 g / L, K2HPO4 0.05 g / L, KH2PO4 0.05 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 2 mg / L, MnSO4 2 mg / L, pH 7.2.

[0066] Therefore, strain 2 is determined to be a heat-resistant strain with high yield of L-tryptophan.

[0067] 2. Genetic stability detection of heat-resistant tryptophan-producing strain 2

[0068] The above-screened strain 2 is continuously passaged on the plate 10 times, and each batch of bacterial strains is inoculated into the shake-flask fermentation medium. After the supernatant is colored by the p-dimethylaminobenzaldehyde method, the OD 600 value is measured with a spectrophotometer, and then according to the tryptophan standard curve y = 0.2718x, the content of L-tryptophan is calculated. The results are shown in Table 1.

[0069] Table 1: Stability of strain 2

[0070] Passage number L-Tryptophan production (g / L) 1 1.63 2 1.72 3 1.65 4 1.59 5 1.67 6 1.57 7 1.70 8 1.69 9 1.75 10 1.59

[0071] As can be seen from Table 1, the L-tryptophan yield of the screened strain 2 remained stable after 10 consecutive passages during the fermentation culture, indicating good genetic stability.

[0072] 2. Strain identification

[0073] (1) Morphological identification

[0074] The screened L-tryptophan-producing strain 2 was spread on an LB plate and cultured at 38 °C for 16 h. As Figure 4 shown, the colony morphology was round, with a neat edge and a smooth surface ( Figure 4 a), and microscopic examination showed that the cells were short rods, without spores and with flagella ( Figure 4 b).

[0075] b. Molecular biology identification

[0076] The genomic DNA of the screened L-tryptophan-producing strain 2 was extracted for 16S rDNA sequencing. The sequencing results were analyzed by Blast alignment with the existing sequences in the Genbank database and found to have a similarity of over 99% with the 16S rDNA sequence of Escherichia coli. Based on the comprehensive morphological and molecular biology test results, the strain was identified as Escherichia coli and named Escherichia coli hhTRp002. It was deposited in the China Center for Type Culture Collection on November 9, 2023, with the deposit number CCTCC NO: M20232171 and the deposit address being Wuhan University, Wuhan, Hubei, China.

[0077] Example 2. Fermentation production of L-tryptophan by Escherichia coli hhTRp002

[0078] The Escherichia coli hhTRp002 screened in Example 1 was used for 5 L tank fermentation. The steps included:

[0079] S1. Inoculate Escherichia coli hhTRp002 into the seed medium and culture at 39 °C for 12 h to obtain a seed solution;

[0080] Among them, the seed medium was: glucose 4 g / L, ammonium sulfate 0.4 g / L, yeast extract 0.3 g / L, indole 0.1 g / L, K2HPO4 0.1 g / L, KH2PO4 0.1 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 5 mg / L, MnSO4 5 mg / L, pH 7.2.

[0081] S2. Transfer the seed liquid into a 5-L fermenter containing 2 L of fermentation medium at an inoculation amount of 5% (v / v), and ferment and culture for 48 h under the conditions of an air flow rate of 1 vvm, a culture temperature of 39 °C, and a stirring speed of 500 rpm. During the fermentation process, ammonia can be used to control the pH at 6.9, and the dissolved oxygen is controlled at 30-50%.

[0082] Among them, the fermentation medium includes: 10-15 g / L of glucose, 0.1-0.2 g / L of indole, 0.05-0.2 g / L of K2HPO4, 0.05-0.2 g / L of KH2PO4, 0.1-0.2 g / L of MgSO4·7H2O, 2-5 mg / L of FeSO4·7H2O, 2-5 mg / L of MnSO4, and the pH is 6.8-7.2;

[0083] In this example, the fermentation medium is: 10 g / L of glucose, 0.1 g / L of indole, 0.05 g / L of K2HPO4, 0.05 g / L of KH2PO4, 0.1 g / L of MgSO4·7H2O, 2 mg / L of FeSO4·7H2O, 2 mg / L of MnSO4, and the pH is 7.2;

[0084] Among them, during the fermentation process, the dissolved oxygen control method is as follows:

[0085] When the initial sugar (glucose) in the fermentation medium is exhausted, the dissolved oxygen is jointly controlled for feeding, and glucose (concentration of 60%) is added at a flow rate of 7-11 g / L / h: if the initial dissolved oxygen is 100%, when the dissolved oxygen is lower than 30%, the feeding flow rate is 7 g / L / h, when the dissolved oxygen is higher than 50%, the feeding flow rate is controlled at 11 g / L / h, and when 11 g / L / h still cannot effectively reduce the dissolved oxygen, the rotation speed can be reduced to 300 rpm.

[0086] The content of L-tryptophan in the fermentation broth is detected by HPLC to be 60 g / L, and the conversion rate is 35%.

[0087] Conversion rate = (concentration of L-tryptophan in the fermentation broth at the end of fermentation * volume of the fermentation broth at the end of fermentation / total consumption of glucose at the end of fermentation) * 100%

[0088] The above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific examples of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. A heat-resistant tryptophan-producing bacterium, classified and named as Escherichia coli hhTRp002, with a deposit number of CCTCC NO: M 20232171.

2. The heat-resistant tryptophan-producing bacterium according to claim 1, characterized in that The tryptophan-producing bacterium is tolerant to temperatures ranging from 37°C to 41°C.

3. Use of the heat-resistant tryptophan-producing bacterium according to claim 1 in the production of tryptophan.

4. A method for producing L-tryptophan, characterized in that Fermentatively culture the heat-resistant tryptophan-producing bacterium described in claim 1.

5. The method according to claim 4, characterized in that The fermentative culture includes: inoculating the heat-resistant tryptophan-producing bacterium into a fermentation medium and culturing it with shaking and aeration.

6. The method according to claim 5, characterized in that The fermentation medium includes: 10 - 15 g / L of glucose, 0.1 - 0.2 g / L of indole, 0.05 - 0.2 g / L of K2HPO4, 0.05 - 0.2 g / L of KH2PO4, 0.1 - 0.2 g / L of MgSO4·7H2O, 2 - 5 mg / L of FeSO4·7H2O, 2 - 5 mg / L of MnSO4, and the pH is 6.8 - 7.

2.

7. The method according to claim 6, characterized in that During the culturing process, control the dissolved oxygen to be 30 - 50%; preferably, the method for controlling the dissolved oxygen includes: when the glucose in the fermentation medium is exhausted, the dissolved oxygen is linked to feeding, and the feed liquid is glucose; more preferably, the feeding is carried out in a fed-batch manner, and the feeding rate is 7 - 11 g / L / h.

8. The method according to claim 5, characterized in that During the culturing process, the ventilation rate is 1 - 2 vvm, and the stirring speed is 300 - 600 prm.

9. The method according to claim 4, characterized in that The culturing temperature is 37 - 41°C, and the culturing time is 45 - 50 h.

10. The method according to any one of claims 4-9, characterized in that The fermentative culture includes: Seed culture: inoculating the heat-resistant tryptophan-producing bacterium into a seed medium and culturing it at 37 - 41°C and a rotation speed of 150 - 200 rpm for 12 - 24 h to obtain a seed liquid; Preferably, the seed liquid is transferred to the fermentation medium at 5 - 15% (v / v); Preferably, during the culturing process, the pH is controlled at 6.8 - 7.2; Preferably, the seed medium includes: 4 - 7 g / L of glucose, 0.4 - 0.8 g / L of ammonium sulfate, 0.3 - 0.5 g / L of yeast extract, 0.1 - 0.2 g / L of indole, 0.1 - 0.5 g / L of K2HPO4, 0.1 - 0.5 g / L of KH2PO4, 0.2 - 0.5 g / L of MgSO4·7H2O, 5 - 10 mg / L of FeSO4·7H2O, 5 - 10 mg / L of MnSO4, and the pH is 6.8 - 7.2.

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