Corynebacterium glutamicum for producing L-tryptophan and application of corynebacterium glutamicum
The efficient Corynebacterium glutamicum hhTRp001 obtained through natural screening solves the problems of low yield and high cost of L-tryptophan production in the prior art, and achieves efficient and green L-tryptophan fermentation production.
Patent Information
- Application Number
- CN202311744916.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-18
- Publication Date
- 2025-06-20
AI Technical Summary
The prior art has problems such as limited source of raw materials, complex process, long production cycle, high cost and large wastewater when producing L-tryptophan, resulting in low yield and low conversion rate.
A highly efficient Corynebacterium glutamicum hhTRp001 was obtained through natural screening method. This strain can stably and efficiently use glucose to directly ferment L-tryptophan, with a conversion rate of more than 24%.
It has achieved high yield of L-tryptophan, with a yield of up to 45.5g/L and a conversion rate of up to 24.3%. It also complies with the concept of green food safety production, reducing production costs.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Corynebacterium glutamicum capable of producing L-tryptophan and its application. Background Art
[0002] L-tryptophan is one of the eight essential amino acids necessary for humans and animals, also known as the second essential amino acid, which plays an important role in the growth, development and metabolism of humans and animals. At present, it is widely used in industries such as medicine, food, feed additives and agricultural environmental detection.
[0003] In the early stage of L-tryptophan production, it mainly relied on chemical synthesis method and protein hydrolysis method. Among them, in the chemical synthesis method, DL-tryptophan was mainly obtained by using phenylhydrazine or indole as raw materials, and L-tryptophan was obtained by chiral resolution of DL-tryptophan. This method requires the use of a large amount of organic solvents, and there are problems such as raw material sources and environmental pollution, so it is rarely used in production; the protein hydrolysis method uses proteins such as waste silk, hair and blood meal as raw materials, and L-tryptophan is extracted by enzymatic hydrolysis or alkaline hydrolysis of the raw materials. However, due to the disadvantages such as limited material sources, complex processes and product components, and long production cycles, the yield of L-tryptophan produced by the protein hydrolysis method is greatly limited. Based on the above technical defects, enzymatic method and fermentation method have emerged in recent years, but the enzymatic method has high cost and large amount of sewage. The fermentation method for producing L-tryptophan has the advantages of high yield, short cycle and easy control, and has become the main method for industrial production of L-tryptophan at present. In the 1930s, Japan carried out large-scale research on the fermentation method for producing L-tryptophan, mainly focusing on traditional mutagenesis breeding technology to change the characteristics of strains and improve the yield of L-tryptophan, and the highest could reach 7.2 g / L. According to the investigation, there is a big gap between the current domestic L-tryptophan fermentation level and the world advanced level. The main reasons are backward breeding technology and production process, low acid production level of strains, long fermentation cycle and low conversion rate. Preparing L-tryptophan with Corynebacterium glutamicum as the fermentation strain has the advantages of high yield, few by-products and convenient separation and extraction of products. Therefore, screening Corynebacterium glutamicum with high yield of L-tryptophan has become a research hotspot in the L-tryptophan fermentation industry. Summary of the Invention
[0004] Aiming at the deficiencies of the above-mentioned prior art, the present invention obtains a strain of Corynebacterium glutamicum hhTRp001 through natural screening. This strain can stably and efficiently utilize glucose to directly ferment and produce L-tryptophan, and the conversion rate can reach more than 24%. Moreover, the main raw material glucose used is low in price and easy to obtain, which conforms to the concept of green food safety production.
[0005] In a first aspect, the present invention provides a Corynebacterium glutamicum capable of producing L-tryptophan, which is classified and named as Corynebacterium glutamicum hhTRp001, and was deposited at the China Center for Type Culture Collection on October 30, 2023. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20232070.
[0006] In one embodiment of the present invention, the L-tryptophan fermentation yield of the Corynebacterium glutamicum can reach 45.5 g / L, and the conversion rate can reach 24.3%.
[0007] In a second aspect, the present invention provides the use of the above-mentioned Corynebacterium glutamicum hhTRp001 in the production of tryptophan.
[0008] In a third aspect, the present invention provides a method for producing L-tryptophan by fermentatively culturing the above-mentioned Corynebacterium glutamicum (Corynebacterium glutamicum hhTRp001).
[0009] According to the technical solution of the present invention, the fermentative culture includes: inoculating the above-mentioned Corynebacterium glutamicum at an inoculum size of 10-20% (v / v) into a fermentation medium and culturing with shaking and aeration.
[0010] In one embodiment of the present invention, the Corynebacterium glutamicum uses glucose as a carbon source, preferably as the sole carbon source, for fermentative production of L-tryptophan.
[0011] In one embodiment of the present invention, the fermentation medium comprises the following components: glucose 10-30 g·L -1 、yeast powder 5-15 g·L -1 、Na2HPO4 2-8 g·L -1 、KH2PO4 4-7 g·L -1 、ammonium sulfate 3-7 g.L -1 、vitamin B1 0.2-1 g.L -1 、FeSO4 0.5-1 mg.L -1 、MgSO4 2-4 g.L -1 , pH 6.0-7.5; wherein, the pH can be adjusted with ammonia water.
[0012] In a preferred embodiment of the present invention, the fermentation medium is: glucose 20 g·L -1 、yeast powder 10 g·L -1 、Na2HPO4 3 g·L -1 、KH2PO4 5 g·L-1 、4 g / L of ammonium sulfate -1 、1 g / L of vitamin B1 -1 、0.5 mg / L of FeSO4 -1 、3 g / L of MgSO4 -1 ,pH 6.9。
[0013] In one embodiment of the present invention, during the cultivation process, the dissolved oxygen is controlled to be 30 - 40%; when the glucose in the fermentation medium is exhausted, the dissolved oxygen is linked to feeding, and the feed is glucose.
[0014] In one embodiment of the present invention, when the dissolved oxygen rapidly rebounds, glucose is fed, and the concentration of the fed glucose is 50% - 80%, preferably 60%.
[0015] In one embodiment of the present invention, the oscillating aeration culture method is shake flask culture or fermenter stirring ventilation culture.
[0016] In one embodiment of the present invention, the rotational speed of the shake flask for shake flask culture is 160 - 200 rpm.
[0017] In one embodiment of the present invention, for the fermenter stirring ventilation culture, the ventilation rate is 0.5 - 2 vvm, and the stirring speed is 400 - 600 rpm.
[0018] In one embodiment of the present invention, the culture temperature is 30 - 35 °C, and the culture time is 40 - 72 h.
[0019] In a preferred embodiment of the present invention, the culture temperature is 33 °C, and the culture time is 48 h.
[0020] In one embodiment of the present invention, a method for producing L-tryptophan includes the following steps:
[0021] (1) Strain cultivation: Inoculate the well-cultured Corynebacterium glutamicum hhTRp001 on the slant into the seed medium, and cultivate it at 30 - 35 °C, ventilation rate of 0.5 - 1 vvm, rotational speed of 150 - 300 rpm for 12 - 16 h to obtain the seed liquid;
[0022] (2) Fermentation culture: Transfer the seed liquid to the fermentation medium at an inoculation amount of 10 - 20% (v / v), and perform oscillating aeration culture at 30 - 35 °C for 40 - 72 h.
[0023] In a specific embodiment of the present invention, the composition of the seed medium is: 20 g / L of glucose -1 、5 g / L of yeast powder -1 、10 g / L of peptone-1 , pH 6.5.
[0024] In one embodiment of the present invention, the method for producing L-tryptophan further includes the following steps:
[0025] (3) Separation and extraction. The separation and extraction method is not limited as long as it can separate L-tryptophan from the fermentation broth.
[0026] For example, the L-tryptophan fermentation broth can be first filtered through a membrane to remove cell proteins, the supernatant is decolorized, and the decolorized solution is successively concentrated, centrifuged, and dried to obtain the L-tryptophan product.
[0027] Advantages of the present invention:
[0028] 1. A Corynebacterium glutamicum strain hhTRp001 capable of directly fermenting and producing L-tryptophan is screened from nature. This strain is used for fermenting and producing L-tryptophan, with a yield of up to 45.5 g / L and a conversion rate of up to 24.3%.
[0029] 2. The Corynebacterium glutamicum strain hhTRp001 screened in the present invention uses glucose as the sole carbon source. Glucose is inexpensive and easily available, which conforms to the concept of green production.
[0030] 3. The Corynebacterium glutamicum strain hhTRp001 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-tryptophan. Description of the Drawings
[0031] Figure 1 It is the HPLC detection result chart of the L-tryptophan standard product in the example.
[0032] Figure 2 It is the microscopic examination cell morphology chart of the strain 13 screened in Example 1.
[0033] Figure 3 It is the tank fermentation result of the Corynebacterium glutamicum strain hhTRp001 in Example 2. Detailed Embodiments
[0034] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments 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.
[0035] The experimental methods in the following embodiments are all 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 embodiments can be obtained from commercial channels unless otherwise specified.
[0036] The Corynebacterium glutamicum with high yield of L-tryptophan of the present invention is obtained from the soil around a sugar factory through enrichment, isolation and purification, primary screening and secondary screening, and includes the following steps:
[0037] S1. A soil sample collected from a depth of 10-15 cm on the surface around a sugar factory in Funing District, Qinhuangdao City, Hebei Province is suspended with 20 mL of sterile normal saline, and the suspension is spread on the primary screening medium and cultured at 30-35 °C for 24-48 h;
[0038] Among them, the composition of the primary screening medium is: glucose 10-30 g·L -1 、yeast powder 5-15 g·L -1 、peptone 10-20 g·L -1 、sodium chloride 10-20 g·L -1 、agar 20 g.L -1 、5-methyltryptophan (5-MT) 10-20 mg / L, pH 6.0-6.2.
[0039] S2. Select strains with good growth and relatively excellent biological shapes, inoculate them into the secondary screening medium, and culture them at 30-35 °C for 24-48 h;
[0040] Among them, the secondary screening medium can be: glucose 10-30 g·L -1 、yeast powder 5-15 g·L -1 、peptone 10-20 g·L -1 、sodium chloride 10-20 g·L -1 、agar 20 g.L -1 、5-methyltryptophan (5-MT) 50-60 mg / L, pH 6.0-6.2.
[0041] S3. Pick 20 single colonies that grow well on the rescreening medium, with round, light yellow, centrally elevated, smooth and moist colony morphology, and inoculate them into the shake flask medium for fermentation culture at a fermentation temperature of 30 - 35 °C, a shake flask rotation speed of 160 - 200 rpm, and a time of 24 - 48 h; through the detection of the biomass of the fermentation broth and the yield of L-tryptophan, screen the strains with better L-tryptophan yield performance.
[0042] Among them, the composition of the shake flask medium is: glucose 10 - 30 g·L -1 , yeast powder 5 - 15 g·L -1 , Na2HPO4 2 - 8 g·L -1 , KH2PO4 4 - 7 g·L -1 , ammonium sulfate 3 - 7 g.L -1 , FeSO4 0.5 - 1 mgL -1 , biotin 0.2 - 1 g.L -1 , vitamin B1 0.2 - 1 g.L -1 , MgSO4 2 - 4 g.L -1 , pH 6.0 - 7.5.
[0043] The detection methods for L-tryptophan and glucose involved in the following examples are:
[0044] (1) Use HPLC to detect the content of L-tryptophan in the fermentation broth. The detection method is: dilute the fermentation broth 200 times with sterile water, filter it with a 0.22 μm filter membrane, and then inject it into the high-performance liquid chromatograph.
[0045] Among them, the HPLC detection conditions are:
[0046] Detection instrument: Agilent HPLC 1260-VWD; chromatographic column: Agilent TC-C18 chromatographic column, 250 * 4.6 mm, 5 μm;
[0047] Mobile phase: 10 mM KH2PO4;
[0048] Column temperature: 30 °C;
[0049] Flow rate: 1 mL / min;
[0050] UV detector, wavelength 210 nm;
[0051] Injection volume: 10 μL.
[0052] As Figure 1 shown, the retention time of the characteristic peak of L-tryptophan is about 5.3 min.
[0053] (2) Use an M-100 biosensor analyzer to detect the glucose concentration in the fermentation broth.
[0054] Example 1: Screening of Corynebacterium glutamicum hhTRp001 Producing L-Tryptophan
[0055] 1. Strain Screening
[0056] S1. Primary Screening: Soil samples were collected from a depth of 10 - 15 cm on the surface around a sugar factory in Funing District, Qinhuangdao City, Hebei Province, suspended in 20 mL of sterile normal saline, and the suspension was spread on the primary screening medium and cultured at 33 °C for 48 h.
[0057] Among them, the composition of the primary screening medium was: glucose 20 g·L -1 , yeast powder 10 g·L -1 , peptone 15 g·L -1 , sodium chloride 15 g·L -1 , agar 20 g.L -1 , 5-methyltryptophan (5-MT) 20 ppm, pH 6.0.
[0058] S2. Secondary Screening: Strains with good growth and relatively excellent biological characteristics were selected and inoculated into the secondary screening medium and cultured at 33 °C for 48 h.
[0059] Among them, the secondary screening medium was: glucose 20 g·L -1 , yeast powder 10 g·L -1 , peptone 15 g·L -1 , sodium chloride 15 g·L -1 , agar 20 g.L -1 , 5-methyltryptophan (5-MT) 50 ppm, pH 6.0.
[0060] S3. Screening of High-Yield L-Tryptophan Strains: Twenty single colonies with good growth, round colony morphology, light yellow color, central elevation, smooth and moist surface on the secondary screening medium were picked and inoculated into the shake flask medium, and cultured at a fermentation temperature of 33 °C and a shake flask rotation speed of 200 rpm for 48 h;
[0061] Among them, the composition of the shake flask medium was: glucose 30 g·L -1 , yeast powder 5 g·L -1 , Na2HPO4 5 g·L -1 , KH2PO4 4 g·L -1 , ammonium sulfate 5 g.L -1 , FeSO4 0.5 mgL -1 , biotin 0.4 g.L -1 , vitamin B1 0.6 g.L -1 , MgSO4 3 g.L -1 , pH 6.5.
[0062] Detect the OD of the fermentation broth of 20 strains of bacteria 550 value and the L-tryptophan content in the fermentation broth, screen the strains with better L-tryptophan production performance, and the results are shown in Table 1. One strain 13 with high L-tryptophan production and excellent performance was screened out.
[0063] Table 1: OD of different strains cultured for 48 h 550 value and L-tryptophan production
[0064]
[0065] 2. Strain identification
[0066] Inoculate the screened strain 13 with better production performance onto a shake flask medium and culture it at a constant temperature of 33 °C for 48 h. After Gram staining, it shows purple, the cells are short rod-shaped, slightly curved, with blunt ends, single, paired or arranged in an eight shape (see Figure 2 ), and the morphological characteristics of this strain conform to Corynebacterium glutamicum.
[0067] Perform 16S rDNA sequencing on strain 13. The sequencing results are compared in the NCBI database and found to have a similarity greater than 99% with the 16S rDNA sequence of Corynebacterium glutamicum.
[0068] Combining the above colony morphology identification and molecular identification, it is determined that strain 13 is Corynebacterium glutamicum, and it is classified and named as Corynebacterium glutamicum hhTRp001. It was deposited in the China Center for Type Culture Collection (CCTCC) on October 30, 2023, with the deposit number CCTCC NO: M 20232070. The deposit address is Wuhan University, Wuhan, China.
[0069] Example 2. Fermentation production of L-tryptophan by Corynebacterium glutamicum hhTRp001
[0070] Use Corynebacterium glutamicum hhTRp001 screened in Example 1 to ferment and produce L-tryptophan, including:
[0071] S1. Inoculate one loop of the well-cultured Corynebacterium glutamicum hhTRp001 on the slant into the seed medium and culture it at 33 °C, ventilation rate of 0.5 vvm, and stirring speed of 200 rpm for 16 h to obtain a seed solution;
[0072] Among them, the composition of the seed medium is as follows: glucose 10 g·L -1 , yeast powder 5 g·L -1 , peptone 10 g·L -1 , pH 6.5.
[0073] S2. Transfer the seed liquid to a 5-L fermenter containing 3 L of fermentation medium at a volume ratio of 10%, control the dissolved oxygen to be 40%, the fermentation temperature to be 33 °C, the ventilation rate to be 1 vvm, and the stirring speed to be 500 rpm. Ferment and culture for 48 h. After the initial sugar in the fermentation medium is exhausted, the dissolved oxygen is linked to feeding. Specifically, when the dissolved oxygen rapidly rebounds, glucose with a concentration of 60% is fed. The feeding rate is that when the dissolved oxygen is greater than 40%, 2 s of glucose feed liquid is supplemented every 20 s of the fermentation cycle.
[0074] Among them, the composition of the fermentation medium is: glucose 20 g·L -1 、yeast powder 10 g·L -1 、Na2HPO4 3 g·L -1 、KH2PO4 5 g·L -1 、ammonium sulfate 4 g.L -1 、vitamin B1 1 g.L -1 、FeSO4 0.5 mg.L -1 、MgSO4 3 g.L -1 , pH 6.9.
[0075] Detect the content of L-tryptophan (TRp), the remaining amount of glucose (i.e., the residual sugar content) and the OD 550 value in the fermentation broth, and calculate the sugar-acid conversion rate. The results are shown in Figure 3 ; It can be seen that Corynebacterium glutamicum hhTRp001 can directly ferment and produce L-tryptophan using glucose as the sole carbon source, and the yield of L-tryptophan can reach 45.5 g / L, and the conversion rate can reach 24.3%. It is suitable for industrial production and has great application value and industrial prospects.
[0076] Conversion rate = (tryptophan concentration at the end of fermentation * volume at the end of fermentation / total consumption of fermented glucose) * 100%
[0077] 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 the present invention gives specific embodiments, 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 Corynebacterium glutamicum that produces L-tryptophan, classified and named as Corynebacterium glutamicum hhTRp001, with a deposit number of CCTCC NO: M 20232070.
2. Use of the Corynebacterium glutamicum according to claim 1 in the production of tryptophan.
3. A method for producing L-tryptophan, characterized in that, Fermentatively culture the Corynebacterium glutamicum described in claim 1.
4. The method according to claim 3, characterized in that, The fermentative culture includes: inoculating the Corynebacterium glutamicum into a fermentation medium and culturing it with shaking and aeration.
5. The method according to claim 4, characterized in that, The fermentation medium comprises the following components: glucose 10 - 30 g·L -1 , yeast powder 5 - 15 g·L -1 , Na2HPO4 2 - 8 g·L -1 , KH2PO4 4 - 7 g·L -1 , ammonium sulfate 3 - 7 g.L -1 , vitamin B1 0.2 - 1 g.L -1 , FeSO4 0.5 - 1 mg.L -1 , MgSO4 2 - 4 g.L -1 , pH 6.0 - 7.
5.
6. The method according to claim 5, characterized in that, During the culture process, control the dissolved oxygen to be 30-40%; when the glucose in the fermentation medium is exhausted, the dissolved oxygen is linked to feeding, and the feed is glucose.
7. The method according to claim 6, characterized in that, When the dissolved oxygen rapidly rebounds, add glucose dropwise, and the concentration of the added glucose is 50%-80%.
8. The method according to claim 4, characterized in that, The shaking and aeration culture method is shake flask culture or fermenter stirring and ventilation culture.
9. The method according to claim 8, characterized in that, The rotation speed of the shake flask for the shake flask culture is 160-200 rpm; the ventilation rate for the fermenter stirring and ventilation culture is 0.5-2 vvm, and the stirring speed is 400-600 rpm.
10. The method according to claim 4, characterized in that, The culture temperature is 30-35 °C, and the culture time is 40-72 h.