Escherichia coli hhThr003 and application thereof in threonine production

Abstract

The invention discloses Escherichia coli hhThr003 and application thereof in threonine production, the Escherichia coli hhThr003 is obtained through natural screening, L-threonine can be stably and efficiently produced through direct fermentation of glucose, the conversion rate can reach 65%, the used main raw material glucose is low in price and easy to obtain, and the Escherichia coli hhThr003 is more suitable for industrial production of L-threonine.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an Escherichia bacterium hhThr003 and its application in threonine production. Background Art

[0002] Threonine is the fourth amino acid among the 8 essential amino acids required by the human body, second only to methionine, lysine, and threonine. There are 4 stereoisomers, but only the L-form has biological activity. L-Threonine can be used as a chemical reagent, food fortifier / flavor enhancer, nutritional supplement, component of compound amino acid infusion, feed additive, etc., and has been widely used in the fields of food, medicine, and feed. Especially in the feed field, L-threonine is the second limiting amino acid in pig feed and the third limiting amino acid in poultry feed. The addition of L-threonine can greatly improve the feed efficiency, provide more sufficient and comprehensive nutrition for animals, and can also reduce the feed cost, with broad application prospects.

[0003] L-Threonine cannot be synthesized by itself in the animal body and must be supplied externally. The production methods of L-threonine mainly include protein hydrolysis method, chemical synthesis method, and fermentation method. Among them, the fermentation method can directly produce L-threonine using cheap glucose raw materials, and has the advantages of simple process, low production cost, almost no other impurity amino acids in the fermentation broth, good product quality after purification, easy large-scale production, and small environmental pollution. It has now become the mainstream method for industrial production of L-threonine.

[0004] Due to the advantages of clear genetics, easy cultivation and passage, fast growth rate, and simple nutritional requirements, Escherichia coli has become the main strain for fermentative production of L-threonine. Most of the existing Escherichia coli strains producing L-threonine are obtained through genetic modification, and there are disadvantages such as easy loss of plasmids and unstable fermentation production. In Escherichia coli, the level of acid production by L-threonine-producing bacteria depends mainly on whether the feedback regulation mechanism in its metabolic pathway is released or inhibited, in addition to the highly efficient expression of key enzyme genes. Therefore, in addition to genetic modification, selecting structural analog-resistant mutants that are beneficial to the accumulation of L-threonine is also an optimal method for obtaining high-yield L-threonine-producing bacteria. The high-threonine-producing strain ACThr1032 disclosed in Patent CN109266578B uses Escherichia coli E.coli THRD as the starting strain, and is obtained through protoplast ultraviolet mutagenesis, diethyl sulfate (DES) chemical mutagenesis, nitrosoguanidine (NTG) chemical mutagenesis, and then through resistance screening, isolation, and purification. The strain has been passaged continuously for ten times, and the fermentation yield of L-threonine is stably maintained at 180 g / L, and the conversion rate can reach about 62.8%. Compared with genetically modified bacteria, it has higher stability, but there is still a gap in its conversion rate. Summary of the Invention

[0005] In view of the deficiencies of the above-mentioned existing technologies, the present invention obtained a strain of Escherichia hhThr003 through natural screening, which can directly ferment and produce L-threonine using glucose as the sole carbon source, and the conversion rate can reach 65%, which is more conducive to the industrial production of L-threonine.

[0006] In a first aspect, the present invention provides an Escherichia coli Thr003, which has been deposited in the China Center for Type Culture Collection, and its taxonomic name is Escherichia coli hhThr003, with the deposit number of CCTCC NO: M20232172, the deposit date of November 09, 2023, and the deposit address of Wuhan University, Wuhan, China.

[0007] In a second aspect, the present invention provides the use of the above-mentioned Escherichia coli hhThr003 in the following A1) or A2):

[0008] A) Producing threonine;

[0009] B) Improving the fermentation yield and / or sugar-acid conversion rate of L-threonine.

[0010] According to the technical solution of the present invention, Escherichia coli hhThr003 is used to ferment and produce L-threonine.

[0011] In an embodiment of the present invention, the fermentation for producing L-threonine includes: inoculating Escherichia coli hhThr003 into a fermentation medium and culturing it with shaking and aeration.

[0012] In an embodiment of the present invention, the shake flask fermentation medium includes: 30 - 60 g / L of glucose, 2 - 5 g / L of corn steep liquor, 1 - 5 g / L of citric acid, 5 - 10 g / L of KH2PO4, 0.2 - 0.6 g / L of betaine, 10 - 30 g / L of (NH4)2SO4, 1 - 2 mg / L of MgSO4, and the pH is 6.8 - 7.0.

[0013] In an embodiment of the present invention, the tank fermentation medium includes: 30 - 60 g / L of glucose, 2 - 5 g / L of corn steep liquor, 1 - 5 g / L of citric acid, 5 - 10 g / L of KH2PO4, 0.2 - 0.6 g / L of betaine, 5 - 10 g / L of (NH4)2SO4, 2 - 4 g / L of MgSO4, 0.2 - 0.5 mg / L of FeSO4, 0.2 - 0.5 mg / L of MnSO4, and the pH is 6.8 - 7.0;

[0014] Preferably, the fermentation medium in the tank is: 30 g / L of glucose, 20 g / L of corn steep liquor, 2 g / L of citric acid, 5 g / L of KH2PO4, 0.3 g / L of betaine, 5 g / L of (NH4)2SO4, 2 g / L of MgSO4, 0.5 mg / L of FeSO4, 0.2 mg / L of MnSO4, pH 7.0.

[0015] In one embodiment of the present invention, during the cultivation process, the dissolved oxygen is controlled at 25 - 35%, preferably 30%.

[0016] In one embodiment of the present invention, when the glucose in the fermentation medium is exhausted, glucose is added in a linked manner with the dissolved oxygen;

[0017] Preferably, when the dissolved oxygen rapidly rebounds, glucose is added dropwise at a rate of 5 - 10 g / L / h, and the glucose concentration is 40% - 60%.

[0018] In one embodiment of the present invention, the initial dissolved oxygen is 100%. Before the initial sugar (glucose) in the fermentation medium is exhausted, when the dissolved oxygen first drops to the set value of the dissolved oxygen, the dissolved oxygen can be increased by increasing the stirring speed. When the dissolved oxygen drops to the set value of the dissolved oxygen again and rapidly rebounds, it indicates that the initial sugar is exhausted. Thereafter, feeding is controlled in a linked manner with the dissolved oxygen, that is, glucose is added dropwise at a rate of 5 - 10 g / L / h, and the glucose concentration is 40 - 60%, preferably 50%.

[0019] In one embodiment of the present invention, the oscillating aerated cultivation is shake flask cultivation or stirred tank ventilation cultivation;

[0020] Preferably, when shake flask cultivation is carried out, the rotation speed of the shake flask is 150 - 200 rpm;

[0021] Preferably, when stirred tank ventilation cultivation is carried out, the ventilation rate is 1 - 2 vvm, and the stirring speed is 500 - 800 rpm.

[0022] In one embodiment of the present invention, the cultivation temperature is 35 - 37 °C, and the cultivation time is 32 - 42 h.

[0023] In one embodiment of the present invention, the fermentation for producing L - threonine further includes:

[0024] Slant culture: Inoculate Escherichia coli hhThr003 into a solid slant medium and activate and culture it at 35 - 37 °C for 18 - 24 h;

[0025] Seed culture: Inoculate the well - cultured Escherichia coli hhThr003 on the slant into a seed medium and culture it at 35 - 37 °C and a rotation speed of 150 - 200 rpm for 16 - 24 h to obtain a seed solution;

[0026] The seed liquid is transferred to the fermentation medium at 10-15% (v / v);

[0027] Preferably, the solid slant medium includes: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, glucose 1 g / L, agar powder 18 g / L;

[0028] Preferably, the seed medium includes: sodium chloride 10 g / L, yeast powder 5 g / L, peptone 10 g / L, glucose 1 g / L.

[0029] Advantages of the present invention:

[0030] 1. An Escherichia bacterium hhThr003 capable of directly fermenting and producing L-threonine using glucose as the sole carbon source is screened from nature. The yield of L-threonine can reach 160 g / L, and the conversion rate can reach 65%. Moreover, glucose is inexpensive and easily available, which can reduce costs and increase efficiency.

[0031] 2. The Escherichia bacterium hhThr003 screened in the present invention can achieve high yield without genetic modification, alleviating the impact of genetically engineered strains on production instability. Moreover, the strain has strong robustness and a simple fermentation process, which is more conducive to the industrial production of L-threonine. Description of the drawings

[0032] Figure 1 It is the HPLC detection result diagram of the L-threonine standard product in the example.

[0033] Figure 2 It is the morphological identification diagram of Escherichia bacterium hhTRp003.

[0034] Figure 3 It is the tank fermentation result diagram of Escherichia bacterium hhTRp003 in Example 2. Detailed implementation manners

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

[0036] The experimental methods used in the following examples are all conventional methods unless otherwise specified; the reagents and materials used, etc., can be obtained from commercial channels unless otherwise specified.

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

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

[0039] 2. Glucose concentration: Appropriately dilute the fermentation broth and measure the glucose concentration using an SBA-40E biosensor.

[0040] 3. L-threonine content: Detect the L-threonine content in the fermentation broth by HPLC. The detection method is as follows: Dilute the fermentation broth 200-fold with sterile water, filter it through a 0.22 μm filter membrane, and then inject it into the high-performance liquid chromatograph.

[0041] Among them, the HPLC detection conditions are as follows:

[0042] Detection instrument: LC-100 liquid chromatograph; The chromatographic column is Primesep 100 4.6*250mm, 5um;

[0043] Mobile phase: Acetonitrile: Ultra-pure water: Trifluoroacetic acid (30:70:0.1, V / V / V);

[0044] Column temperature: 30 °C;

[0045] Flow rate: 0.5 mL / min;

[0046] UV detector, wavelength 210 nm;

[0047] Injection volume: 20 μL.

[0048] Inject the L-threonine standard product (purchased from Yuanye Company, product number S66781) for detection. The retention time of the characteristic peak of L-threonine is about 25.89 min (see Figure 1 ).

[0049] Example 1. Screening and identification of Escherichia coli producing L-threonine

[0050] 1. Enrichment culture of strains

[0051] Soil sample source: Soil at a depth of 5-15 cm in a forest on the slope of Laoniushan Mountain, Huludao City, Liaoning Province.

[0052] Add 10 g of soil sample to a conical flask containing 100 mL of sterile normal saline, shake well and mix. Transfer the mixed solution to the enrichment medium at 10% (v / v) and culture it at 37 °C for 24 h;

[0053] 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 7.0.

[0054] 2. Screening of strains

[0055] 2.1) Gradient dilute the bacterial liquid after enrichment culture and spread it on the primary screening plate, and place it in an incubator at 37 °C for 16 h;

[0056] Among them, the primary screening plate is: glucose 5 g / L, peptone 10 g / L, bromocresol purple 0.01 g / L, agar powder 20 g / L.

[0057] 2.2) Select the strains whose color changes from purple to yellow during the selective culture, inoculate them into the seed medium, and shake-culture at 37 °C for 16 h to obtain the seed liquid;

[0058] Among them, the seed medium is: glucose 1 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, pH 7.0;

[0059] 2.3) Transfer the seed liquid to the fermentation medium at 10% (v / v), and shake-culture at 37 °C for 24 h;

[0060] Among them, the fermentation medium is: glucose 30 g / L, corn steep liquor 20 g / L, citric acid 2 g / L, KH2PO4 5 g / L, betaine 0.3 g / L, (NH4)2SO4 5 g / L, MgSO4 2 g / L, FeSO4 0.5 mg / L, MnSO4 0.2 mg / L, pH 7.0.

[0061] 2.4) Use thin layer chromatography (TLC) to detect L-threonine, and screen out Escherichia coli strains producing L-threonine.

[0062] 3. Rescreening of the strains

[0063] 3.1) Spread the single colonies of Escherichia coli producing L-threonine obtained from the primary screening on the rescreening plate, and culture at 37 °C for 24 h;

[0064] Among them, rescreening plates containing α-amino-β-hydroxyvaleric acid (a structural analogue of threonine) at different mass concentration gradients of 2 mg / L, 4 mg / L, 6 mg / L, and 8 mg / L are prepared respectively, pH 7.0;

[0065] The rescreening plate is: glucose 30 g / L, corn steep liquor 5 g / L, MgSO4 0.6 mg / L, (NH4)2SO4 6 g / L, 4-hydroxy-3-methoxybenzoic acid 100 mg / L, α-amino-β-hydroxyvaleric acid (2 mg / L / 4 mg / L / 6 mg / L / 8 mg / L), agar powder 18 g / L, pH 7.0;

[0066] 3.2) Pick the resistant single colonies with round appearance and large colonies from the rescreening plate and transfer them into the shake-flask fermentation medium, and ferment and culture at a culture temperature of 37 °C and a shake-flask rotation speed of 160 rpm for 24 h;

[0067] Among them, the shake flask fermentation medium is: glucose 30 g / L, corn steep liquor 10 g / L, KH2PO4 5 g / L, betaine 0.2 g / L, (NH 4)2 SO4 5 g / L, MgSO4 2 g / L, FeSO4 0.5 mg / L, MnSO4 0.2 mg / L, pH 7.0.

[0068] Detect the OD 600 value of the fermentation broth and the L-threonine yield in the fermentation broth. The results are shown in Table 1. One strain 20 with high L-threonine yield and excellent performance was screened out.

[0069] Table 1: OD 600 value of the fermentation broth of different strains and the L-threonine concentration contained

[0070]

[0071] 2. Identification of the strain

[0072] a. Morphological identification

[0073] The screened high-yield L-threonine strain 20 was spread on the LB solid medium and cultured at 37 °C for 16 h. As Figure 2 shown, the colony morphology was round, white and opaque, the surface was smooth and moist, and the edge was neat; microscopic examination showed that the cells were short rod-shaped, without spores, with flagella; after Gram staining, it was pink.

[0074] b. Molecular biology identification

[0075] The screened high-yield L-threonine strain 20 was subjected to 16S rDNA sequencing. The sequencing results were compared in NCBI, and the homology was greater than 99%. It was classified and named as Escherichia coli hhThr003. This strain was deposited in the China Center for Type Culture Collection on November 09, 2023, with the deposit number CCTCC NO: M 20232172, and the deposit address is Wuhan University, Hubei, China.

[0076] Example 2. Fermentation of Escherichia coli hhTRp003 to produce L-threonine

[0077] Using Escherichia coli hhThr003 screened in Example 1 for tank fermentation to produce L-threonine, including the following steps:

[0078] S1. Slant culture: Activate and culture Escherichia coli hhThr003 in the solid slant medium at 37 °C for 24 h;

[0079] Among them, the solid slant medium is: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, glucose 1 g / L, agar powder 18 g / L, pH 7.0.

[0080] S2. Seed culture: Inoculate one loop of the activated Escherichia hhThr003 into the seed medium, and culture it with shaking at 37 °C and 160 rpm for 16 h to obtain the seed liquid.

[0081] Among them, the seed medium is: sodium chloride 10 g / L, yeast powder 5 g / L, peptone 10 g / L, glucose 1 g / L, pH 7.0.

[0082] S3. Fermentation culture: Transfer the seed liquid to a 5 L fermenter containing 2 L of fermentation medium at 10% (v / v), and ferment and culture it at 37 °C, a rotation speed of 400 rpm, and an aeration rate of 1 L / min for 36 h. During the culture process, control the dissolved oxygen to be 30%.

[0083] Specifically: The initial dissolved oxygen is 100%. As the fermentation progresses, when the dissolved oxygen first drops to 30%, increase the rotation speed to 600 rpm to supplement the dissolved oxygen. When the dissolved oxygen drops to 30% again and increases instantaneously, it indicates that the initial sugar (glucose) in the fermentation medium is exhausted. At this time, control the dissolved oxygen and feed, that is, feed glucose (concentration 50%) at a feeding rate of 8 g / L / h.

[0084] Among them, the fermentation medium is: glucose 30 g / L, corn steep liquor 20 g / L, citric acid 2 g / L, KH2PO4 5 g / L, betaine 0.3 g / L, (NH4)2SO4 5 g / L, MgSO4 2 g / L, FeSO4 0.5 mg / L, MnSO4 0.2 mg / L, pH 7.0.

[0085] The feed is: 50% glucose solution.

[0086] Detect the OD 600 value, the content of L-threonine and residual sugar (remaining amount of glucose) in the fermentation broth. The results are as Figure 3 shown. It can be seen that the yield of L-threonine can reach 160 g / L, the conversion rate can reach 65%, and Escherichia hhTRp003 is suitable for industrial production.

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

[0088] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments 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 cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art.

Claims

1. A strain of Escherichia hhThr003 has been deposited with the China Center for Type Culture Collection, classified and named as Escherichia coli hhThr003, with the deposit number CCTCC NO: M 20232172, and the deposit date is November 9, 2023.

2. Use of the Escherichia hhThr003 according to claim 1 in any of the following A1) or A2): A) Producing threonine; B) Improving the fermentation yield of L-threonine and / or the sugar-acid conversion rate.

3. The use according to claim 2, characterized in that: L-Threonine is produced by fermenting Escherichia coli hhThr003.

4. The use according to claim 3, characterized in that: The fermentation for producing L-threonine includes: inoculating Escherichia coli hhThr003 into a fermentation medium and culturing it with shaking and aeration.

5. The use according to claim 4, characterized in that: The fermentation medium includes: glucose 30 - 60 g / L, corn steep liquor 2 - 5 g / L, citric acid 1 - 5 g / L, KH2PO4 5 - 10 g / L, betaine 0.2 - 0.6 g / L, (NH4)2SO4 5 - 10 g / L, MgSO4 2 - 4 g / L, FeSO4 0.2 - 0.5 mg / L, MnSO4 0.2 - 0.5 mg / L, with a pH of 6.8 - 7.0; Preferably, the fermentation medium includes: glucose 30 g / L, corn steep liquor 2 g / L, citric acid 2 g / L, KH2PO4 5 g / L, betaine 0.3 g / L, (NH4)2SO4 5 g / L, MgSO4 2 g / L, FeSO4 0.5 mg / L, MnSO4 0.2 mg / L, with a pH of 7.

0.

6. The use according to claim 4, characterized in that: During the culturing process, the dissolved oxygen is controlled at 25 - 35%.

7. The use according to claim 6, characterized in that: When the initial sugar in the fermentation medium is exhausted, glucose is added in a linked manner with the dissolved oxygen; Preferably, when the dissolved oxygen drops to the set value and then quickly rebounds, glucose is fed at a feeding rate of 5 - 10 g / L / h; More preferably, the concentration of the fed glucose is 40% - 60%.

8. The use according to claim 6, characterized in that: The culturing with shaking and aeration is flask shaking culture or stirred ventilation culture in a fermenter; When flask shaking culture is carried out, the shaking speed of the flask is 150 - 200 rpm; When stirred ventilation culture in a fermenter is carried out, the ventilation rate is 1 - 2 vvm, and the stirring speed is 500 - 800 rpm.

9. The use according to claim 4, characterized in that: The culturing temperature is 35 - 37 °C, and the culturing time is 32 - 42 h.

10. The use according to any one of claims 3-9, characterized in that: The fermentation for producing L-threonine further includes: slant culture: inoculating Escherichia coli hhThr003 into a solid slant medium and activating and culturing it at 35 - 37 °C for 18 - 24 h; Seed culture: inoculating the well-cultured Escherichia coli hhThr003 on the slant into a seed medium and culturing it at 35 - 37 °C and a rotation speed of 150 - 200 rpm for 16 - 24 h to obtain a seed solution; The seed solution is transferred to the fermentation medium at 10 - 15% (v / v); The solid slant medium includes: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, glucose 1 g / L, agar powder 18 g / L; The seed medium includes: sodium chloride 10 g / L, yeast powder 5 g / L, peptone 10 g / L, glucose 1 g / L.

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