CN115895976B - An L-tryptophan-producing Escherichia coli strain and its application in producing L-tryptophan - Google Patents

Abstract

The invention discloses an escherichia coli for producing L-tryptophan and application thereof in producing L-tryptophan, and belongs to the technical field of fermentation. The invention aims to solve the problems of large accumulation of mixed acid and high extraction difficulty in the fermentation process of the L-tryptophan strain. The invention discloses a method for high-yield L-tryptophan, which comprises the specific steps of fermenting escherichia coli XYSH220517-04 at 36 ℃ for at least 32 hours. The escherichia coli is named as XYSH220517-04 and is preserved in China general microbiological culture collection center, the preservation date is 2022, 9 and 19 days, and the strain preservation number is CGMCC NO.25753. The strain has stable fermentation performance, the content of the mixed acid in the fermentation liquor is obviously reduced, the extraction yield of the L-tryptophan is improved from 68% to 80%, and the strain has the characteristics of short period, high acid yield and high extraction yield.

Description

An L-tryptophan-producing Escherichia coli strain and its application in producing L-tryptophan

Technical field

The invention belongs to the field of fermentation technology, and specifically relates to an L-tryptophan-producing Escherichia coli strain and its application in producing L-tryptophan.

Background technique

L-tryptophan is an essential amino acid in the life activities of humans and animals. It plays an important role in the metabolism and growth and development of humans and animals. Due to its irreplaceable physiological role, it is called the second essential amino acid. At present, L-tryptophan is widely used in food, medicine, feed and other industries. With the development of technology, it plays an important role in agricultural monitoring and environmental monitoring. L-tryptophan was first produced by protein hydrolysis and chemical synthesis. However, it was gradually eliminated after the 1990s due to its shortcomings such as complex material supply, complex production processes, long cycle times, and unstable product quality. Since then, with the in-depth research on the functions of microorganisms, the production of L-tryptophan by fermentation has been gradually promoted, becoming practical and becoming mainstream.

Compared with traditional chemical synthesis and protein hydrolysis, microbial fermentation has significant advantages such as lower production cost, greener environmental protection, and easier mass production. In the process of producing L-tryptophan by microbial fermentation, the quality of the microbial strains determines the production process, production cost and even product quality in the entire fermentation production process.

At present, the problem plaguing the industry is that the synthesis pathway of L-tryptophan is complicated. During the fermentation process, a large amount of glutamic acid, organic acids and other fermentation by-products will accumulate, seriously affecting the production and conversion rate of L-tryptophan. At the same time, L - Tryptophan is easily decomposed when it coexists with miscellaneous acids, making it difficult to extract fermentation broth and resulting in low extraction yield. Therefore, breeding strains with low accumulation of glutamic acid and organic acids during metabolism is of great significance for improving L-tryptophan production, acid production efficiency, and extraction yield.

Patent CN 110643559B (Application of transport carrier gene to improve L-tryptophan production efficiency in Escherichia coli) mentions that the L-tryptophan accumulation amount of the genetically modified tryptophan strain fermented in a shake flask for 24 hours is 15.2 g/L, the shake flask adopts the method of feeding glucose and ammonia water.

Zhao Chunguang et al. reported in the 2017 article (Rational Construction of L-Tryptophan Producing Strain Deleted in the Acetate Kinase Encoding Gene) that glutamate accumulation occurs due to the imbalance of energy metabolism and reducing power metabolism during the fermentation process of the tryptophan strain. , the accumulation amount of glutamic acid is 7-9g/L, and the accumulation amount of by-product glutamic acid is relatively high, which will lead to a decrease in tryptophan production and affect the tryptophan extraction yield and purity.

Succinic acid and citric acid are organic acids in the TCA cycle. During the fermentation process of Escherichia coli, a blocked TCA cycle will lead to a large accumulation of succinic acid and citric acid. The succinic acid and citric acid in the TRTH fermentation broth of the starting strain of this patent The accumulation of citric acid is greater than 15g/L, which seriously affects the production of tryptophan and makes the extraction of fermentation broth more difficult.

The 2021 literature by Bo Xiong et al. (Flux redistribution of central carbonmetabolism for efficient production of L-tryptophan in Escherichia coli) reported that the tryptophan production of strain SX11 in a 5-liter fermenter for 40 hours was 41.7g/L, and the conversion rate was 22.7 %, the acid production rate is 1.04g/L/h.

Contents of the invention

The purpose of the present invention is to solve the problems of high accumulation of miscellaneous acids and difficulty in extraction during the fermentation process of L-tryptophan strains. The extraction yield of L-tryptophan is significantly improved and the production cost is greatly reduced.

The present invention provides Escherichia coli. The name of the Escherichia coli is XYSH220517-04, which has been deposited in the China General Microbial Culture Collection and Management Center. The deposit date is September 19, 2022. , the strain preservation number is CGMCC NO.5753.

The present invention provides a microbial preparation containing the above-mentioned Escherichia coli.

The present invention provides the use of the above-mentioned Escherichia coli or the above-mentioned microbial preparation in improving the production, conversion rate and extraction yield of L-tryptophan.

The present invention provides a method for high production of L-tryptophan. The specific steps of the method are to ferment the above-mentioned Escherichia coli at a temperature of 36°C for at least 32 hours.

To further limit, the fermentation conditions of Escherichia coli also include pH value 7.0, dissolved oxygen 25%-30%, and tank pressure 0.02MPa.

It is further limited that the culture medium used for fermentation uses glucose as the carbon source.

It is further limited that the culture medium used for fermentation uses yeast powder as the nitrogen source.

It is further limited that the fermentation medium used for fermentation contains glucose, yeast powder, citric acid, (NH4) ₂ SO ₄ , KH ₂ PO ₄ , MgSO ₄ ·7H ₂ O, FeSO ₄ ·7H ₂ O, vitamin B1, vitamin H. Trace elements.

It is further defined that the fermentation medium used for fermentation contains glucose 10-20g/L, yeast powder 4-6g/L, citric acid 1-4g/L, (NH ₄ ) ₂ SO ₄ 2-8g/L, KH ₂ PO ₄ 4-6g/L, MgSO ₄ ·7H ₂ O 1-2g/L, FeSO ₄ ·7H ₂ O 50-80mg/L, VB1 5-7mg/L, VH 0.2-0.6mg/L, trace elements mixed Solution 1-2mL/L.

The present invention provides the application of the above method in improving L-tryptophan conversion rate, extraction yield and reducing fermentation cycle.

Beneficial effect: an L-tryptophan-producing Escherichia coli strain and a method for producing L-tryptophan. The Escherichia coli is Escherichia coli XYSH220517-04. Escherichia coli XYSH220517-04 has been deposited in the China General Microbial Culture Collection Center (CGMCC). The preservation date is September 19, 2022, and the strain preservation number is CGMCC NO.25753. The present invention combines ARTP mutation breeding technology and chemical mutation screening technology of Escherichia coli TRTH to obtain the strain. Using this strain to carry out shake flask fermentation can accumulate L-tryptophan within 24 hours. Reaching 18.26g/L, which is the highest domestically reported tryptophan production in shake flasks; this strain can accumulate L-tryptophan up to 60.56g/L after fermentation in a 50L fermenter for 32 hours, which is 32.20% higher than the original strain and has short Periodic, high acid-producing characteristics. In industrial production, the fermentation performance of strain XYSH220517-04 is stable, the content of miscellaneous acids in the fermentation broth is significantly reduced, and the extraction yield of L-tryptophan is increased from 68% to 80%. The extraction yield is greatly reduced, which is a technical achievement. A breakthrough progress has been made. The strain of the present invention has the characteristics of short cycle, high acid production and high extraction yield.

[Biological Preservation]: The biological material of the present invention, (Escherichia coli) XYSH220517-04, Escherichia coli XYSH220517-04 has been deposited in the China General Microbial Culture Collection and Management Center (CGMCC), and the preservation date is 2022 On September 19, the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The strain preservation number is CGMCC NO.25753.

Description of drawings

Figure 1 is a flow chart of mutagenesis screening of Escherichia coli XYSH220517-04.

Detailed ways

The following examples are enumerated to further illustrate the present invention, but do not limit the content of the present invention.

Solid culture medium, consisting of the following components: tryptone 10g/L, beef extract 10g/L, yeast powder 5g/L, sodium chloride 5g/L, anhydrous glucose 1-4g/L, KH ₂ PO ₄ 1- 5g/L, the rest is water, pH 6.8-7.2.

Primary screening medium (deep well plate fermentation medium) consists of the following components: glucose 20-40g/L, (NH ₄ ) ₂ SO ₄ 1-5g/L, KH ₂ PO ₄ 1-5g/L, MgSO ₄ ·7H ₂ O 0.5-2g/L, FeSO ₄ ·7H ₂ O 20-40mg/L, MnSO ₄ ·H ₂ O 1-3mg/L, Na ₂ HPO ₄ 4-6g/L, yeast powder 2-4g /L, sodium citrate 2-4g/L, propanesulfonic acid 20-80g/L, the rest is water, pH 6.8-7.2. The shaker speed is 600-800rpm/min, and the culture temperature is 36-37°C.

Repeated screening medium (shake flask medium), seed shake flask medium consists of the following components: glucose 20-40g/L, (NH ₄ ) ₂ SO ₄ 1 5g/L, KH ₂ PO ₄ 1-5g/L , MgSO ₄ ·7H ₂ O 0.5-2g/L, yeast powder 2-5g/L, FeSO ₄ · _{7H 2} O20-40mg/L, MnSO ₄ ·H ₂ O 1-3mg/L, VH 0.1-0.5mg /L, VB ₁ 0.5-1.0mg/L, trace element mixture 1-3ml/L, the rest is water, pH 7.0-7.2, sterilized in a high-pressure steam cooker at 115°C for 15 minutes.

The fermentation shake flask culture medium consists of the following components: glucose 20-40g/L, (NH ₄ ) ₂ SO4 2-6g/L, KH ₂ PO ₄ 1-5g/L, MgSO ₄ · ₇ H ₂ O 0.5-2g /L, yeast powder 1-5g/L, FeSO ₄ · ₇ H ₂ O 30-60mg/L, MnSO ₄ · ₇ H ₂ O 1-5mg/L, VH0.1-0.5mg/L, VB ₁ 0.5- 1.0mg/L, trace element mixture 1-3ml/L, phenol red 15-30g/L, the rest is water, pH 7.0-7.2, sterilized in a high-pressure steam cooker at 115°C for 15 minutes.

Each component of the trace element mixture: CoSO ₄ ·7H ₂ O 0.4-0.8g/L, ZnSO ₄ ·7H ₂ O 6-8g/L, CuSO ₄ ·5H ₂ O 4-6g/L, Al ₂ (SO ₄ ) ₃ ·18H ₂ O 2-4g/L, MnSO ₄ ·H ₂ O 4-6g/L, Na ₂ MoO ₄ ·2H ₂ O 2-4g/L, NiSO ₄ ·6H ₂ O 2-4g/L, H ₃ BO ₃ 1-2g/L.

Example 1: Method for mutagenesis screening of Escherichia coli XYSH220517-04

As shown in Figure 1, the specific steps to obtain Escherichia coli XYSH220517-04 are as follows:

1. Preparation of bacterial suspension: Take fresh slants of Escherichia coli TRTH cultured at 37°C for 1 day, add 15 mL of sterile water, scrape off the strains and transfer them to a 250 mL Erlenmeyer flask with glass beads. Add 15 ml of glycerin containing 1% to prepare a bacterial suspension, and adjust the bacterial concentration to ¹⁰⁸ /mL; use an ARTP mutagenesis instrument to irradiate the bacterial suspension for 60 seconds. After the mutagenesis is completed, take the bacterial suspension, dilute it and apply Cloth solid plate culture medium, invert at 37°C for 1 day;

3. Preliminary screening: Inoculate several single colonies obtained by mutagenesis in step 1 into a 96-deep well plate containing 600 microliters of screening medium. Seal the 96-deep well plate with a sealing film, and then place it on a high-speed Cultivate on a shaking shaker with a shaking speed of 600-800rpm, a culture temperature of 36-37°C, a humidity of 80%, and culture for 24 hours. The L-tryptophan content in the fermentation broth is detected. The initial screening shows that the L-tryptophan content in the fermentation broth is greater than 2g/L single colony.

4. Re-screening: Cut out a slope for the strains preserved in the glycerol tubes initially screened, and culture them at 36°C for 20 hours. Pick a ring of bacteria from the slope into the seed shake flask, culture it at 180r/min, 36.5°C for 5 hours, and transfer it to the fermentation shake flask. Each fermentation shake flask contains 30 ml of fermentation culture medium. The shaker speed is 200 rpm. During the fermentation process, pass Add ammonia to maintain the pH at 7.0-7.2; use phenol red as an indicator. When the color of the fermentation broth no longer changes, it is considered a sugar deficiency. When there is a sugar deficiency, add 1-2mL 60% (m/v) glucose solution; fermentation cycle 20-24h, detect the L-tryptophan content in the fermentation broth, re-screen out single colonies with L-tryptophan content higher than 12g/L in the fermentation broth, and preserve them.

5. Then activate the single colony obtained by re-screening in step 4 with a slope. After culturing for 1 day, use an inoculation ring to scrape the bacteria into physiological saline to prepare a bacterial suspension. The concentration of the bacterial suspension is 10 ⁹ /L. Suspend 2 ml of bacteria. solution into a 50 ml sterile centrifuge tube, then add 4 ml of 0.5 mg/L nitrosoguanidine solution, repeat steps 3 to 4, select single colonies with acid production greater than 2.5 g/L for preservation, and re-screen to select Single colonies with acid production greater than 15g/L were preserved, including strain XYSH220517-04. The acid production of the shake flask of strain XYSH220517-04 reached 18g/L.

Use the following experiments to verify the experimental results:

Genetic Stability Test:

The high-producing tryptophan strains obtained through the above screening were isolated as single colonies and passaged continuously for 10 generations in shake flasks. Each generation of strains was first cultured in seeds, and strains with genetic stability and high acid production were selected for further research. Shake flask passaging method: transfer the tryptophan-producing strain from the slant to the shake flask, culture it to the logarithmic growth phase, and then transfer it to the next generation of shake flask.

The finally obtained tryptophan-high-producing strain XYSH220517-04 was continuously propagated ten times, and its L-tryptophan production was investigated using 500 mL shake flask fermentation culture. The result is as follows:

Table 1 Genetic stability of strain XYSH220517-04

As can be seen from Table 1, the genetic stability of mutant strain good.

Example 2. Method for producing L-tryptophan

Using Escherichia coli XYSH220517-04 and the original strain TRTH in the present invention, combined with the relevant processes in the present invention, 50L tank fermentation culture is carried out, and five batches are cultured respectively. The average calculation results of the five batches are as follows:

(1) Seed tank culture medium components: glucose 40-60g/L, yeast powder 2-6g/L, citric acid 0.5-2g/L,

(NH ₄ ) ₂ SO ₄ 2-8g/L, KH ₂ PO ₄ 5-7g/L, VB ₁ 1-2mg/L, VH 0.3-0.5mg/L, MgSO ₄ ·7H ₂ O1.5-2g/ L, FeSO ₄ · ₇ H ₂ O 20-40mg/L, trace element mixed solution 1-2mL/L.

(2) Fermentation medium components: glucose 10-20g/L, yeast powder 4-6g/L, citric acid 1-4g/L, (NH ₄ ) ₂ SO ₄ 2-8 g/L, KH ₂ PO ₄ 4-6g/L, MgSO ₄ ·7H ₂ O 1-2g/L, FeSO ₄ ·7H ₂ O 50-80mg/L, VB ₁ 5-7mg

/L, VH 0.2-0.6mg/L, trace element mixed solution 1-2mL/L.

(3) Each component of the trace element mixed solution: CoSO ₄ · ₇ H ₂ O 0.4-0.8g/L, ZnSO ₄ · ₇ H ₂ O 6-8g/L, CuSO ₄ · ₅ H ₂ O 4-6g/L , Al ₂ (SO ₄ ) ₃ · ₁₈ H ₂ O 2-4g/L, MnSO ₄ ·H ₂ O 4-6g/L, Na ₂ MoO ₄ · ₂ H ₂ O 2-4g/L, NiSO ₄ · ₆ H ₂ O 2-4g/L, H ₃ BO ₃ 1-2g/L.

(4) Cultivation of strains in seed tanks. Sterilize the seed culture medium, cool it to about 36°C, adjust the pH to about 7.0, and inoculate Escherichia coli XYSH220517-04 strain into the seed culture medium for cultivation. to the logarithmic growth phase.

Bacteria culture conditions: temperature 36°C, pH value around 7.0, dissolved oxygen 25%-30%, tank pressure 0.02MPa.

(5) Sterilize the fermentation medium, cool it to about 36°C, adjust the pH to about 7.0, insert the logarithmic growth phase bacteria obtained in step (1) into the fermentation medium for fermentation culture to prepare L Tryptophan fermentation broth. Fermentation culture conditions: temperature 36°C, pH value 7.0, dissolved oxygen 25%-30%, tank pressure 0.02MPa, and residual sugar content during fermentation controlled at 0.04%. The culture is completed at 32h-40h, and the L-tryptophan fermentation liquid is obtained.

(6) Tryptophan content, glutamic acid content, acetic acid content, citric acid content, and succinic acid content were detected using liquid chromatography, and ammonia nitrogen was detected using a Kjeldahl nitrogen analyzer.

(7) Conversion rate calculation formula = (Volume of fermentation liquid L × fermentation acid production content g/L)/fermentation glucose amount g × 100%.

Table 2 Comparison of tryptophan production performance in 50-liter fermentation tanks between mutant strains and starting strains

As can be seen from Table 2, compared with the starting strain TRTH, the strain XYSH220517-04 in the present invention has greatly improved L-tryptophan production, conversion rate and fermentation cycle, and the content of miscellaneous acids at the fermentation end point is significantly reduced. The glutamic acid content was reduced to 1.17g/L, the citric acid content was reduced to 2.12g/L, and the succinic acid content was reduced to 2.34g/L.

The extraction process of L-tryptophan is as follows: the L-tryptophan fermentation broth is filtered through membrane filtration to remove bacterial cells to obtain a filtered clear liquid. The filtered clear liquid is evaporated, concentrated and crystallized, and then cooled and cooled for crystallization. The mother liquor is further centrifuged to obtain wet crystals. , dry the wet crystals to obtain L tryptophan crystals. The membrane filtration equipment is a ceramic membrane, an organic membrane or a metal membrane, with a pore size of 8 nm, the evaporation concentration and crystallization equipment is a multi-effect evaporation crystallizer, the cooling crystallization equipment is a cold crystallizer, and the L tryptophan crystal drying equipment is a fluidized bed dryer. Machine or vacuum double cone dryer, L tryptophan granule product drying equipment is a spray granulation dryer.

In this way, a 350-ton fermentation tank is used for culture, and the fermentation is cultured for 32 hours. The tank volume is 230 tons. The output of L tryptophan is 62.56g/L, the conversion rate is 30.12%, and the acid production rate is 1.96g/L. /h. Strain XYSH220517-04 achieves the production characteristics of short cycle, high acid production and fast acid production rate. The content of miscellaneous acids in the fermentation broth was significantly reduced, with glutamic acid content reduced to 1.15g/L, citric acid content reduced to 2.18g/L, and succinic acid content reduced to 2.52g/L. The L-tryptophan fermentation broth was concentrated and then sprayed and granulated to obtain 11.51 tons of L-tryptophan granule product. The extraction yield reached 80%, and the extraction yield of the original strain TRTH was 68%. Under the premise that the extraction process remains unchanged, the L-tryptophan extraction yield of the fermentation broth of strain The national standard requirements for amino acids indicate that the production performance of the mutated strain XYSH220517-04 has made a major breakthrough and performed well in industrial production.

Table 3 Comparison of tryptophan production performance in 350-ton fermentation tanks between mutant strains and starting strains

Extraction yield formula: Extraction yield calculation formula: mass of finished tryptophan product/(final volume of fermentation broth × tryptophan concentration) × 100%.

Claims (10)

1. The Escherichia coli is characterized in that the Escherichia coli is named as XYSH220517-04 and is preserved in China general microbiological culture collection center (CGMCC) with a preservation date of 2022, 9 months and 19 days and a strain preservation number of CGMCC No.25753.

2. A microbial preparation comprising the Escherichia coli according to claim 1.

3. Use of the escherichia coli of claim 1 or the microbial preparation of claim 2 for increasing the yield, conversion rate and extraction yield of L-tryptophan.

4. A process for producing L-tryptophan at a high yield, which comprises the step of fermenting the Escherichia coli of claim 1 at 36℃for at least 32 hours.

5. The method according to claim 4, wherein the fermentation conditions of Escherichia coli further comprise pH 7.0, dissolved oxygen of 25% -30% and a tank pressure of 0.02MPa.

6. The method according to claim 4, wherein the medium for fermentation uses glucose as a carbon source.

7. The method according to claim 4, wherein the medium for fermentation uses yeast powder as nitrogen source.

8. The method according to claim 4, wherein the fermentation medium for fermentation contains glucose, yeast powder, citric acid, (NH) ₄ ) ₂ SO ₄ 、KH ₂ PO ₄ 、MgSO ₄ ·7H ₂ O、FeSO ₄ ·7H ₂ O, vitamin B1, vitamin H and trace elements.

9. The method according to claim 4, wherein the fermentation medium for fermentation contains glucose 10-20g/L, yeast powder 4-6g/L, citric acid 1-4g/L, (NH) ₄ ) ₂ SO ₄ 2-8g/L，KH ₂ PO ₄ 4-6g/L，MgSO ₄ ·7H ₂ O1-2g/L，FeSO ₄ ·7H ₂ 50-80mg/L of O, 1 5-7mg/L of VB, 0.2-0.6mg/L of VH and 1-2mL/L of trace element mixed solution.

10. Use of the process of any one of claims 4-9 to increase L-tryptophan conversion, extraction yield and decrease fermentation cycle.