A genetically engineered strain with l-tryptophan production capacity and a method for producing l-tryptophan using the genetically engineered strain

Abstract

The application discloses a genetically engineered strain with L-tryptophan production capacity and a method for producing L-tryptophan by using the genetically engineered strain, and belongs to the technical field of biotechnology. The application solves the problem that excessive fed-batch glucose is easy to cause abnormal metabolism of a production strain, and the production of acid is affected. The genetically engineered strain is obtained by knocking out a nucleotide sequence shown in SEQ ID NO:1 in an original strain. The method for producing L-tryptophan by using the genetically engineered strain comprises the following steps: preparing seed tank culture medium, fermentation culture medium and trace element mixed solution; preparing seed liquid of a ycjO gene deletion strain; sterilizing and cooling the seed tank culture medium, adjusting pH, inoculating the seed liquid of the ycjO gene deletion strain into the seed culture medium, and culturing to the middle-late logarithmic growth phase; sterilizing and cooling the fermentation tank culture medium, adjusting pH, inoculating the strain in the middle-late logarithmic growth phase into the fermentation culture medium, and fermenting and culturing to obtain L-tryptophan fermentation liquid. The genetically engineered strain is suitable for producing L-tryptophan.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a genetically engineered strain and a method for producing L-tryptophan using this genetically engineered strain. Background Technology

[0002] L-Tryptophan is one of the eight essential amino acids and has been widely used in feed, food, and pharmaceuticals due to its nutritional value. L-Tryptophan production methods include chemical synthesis, enzymatic reactions, and fermentation. However, with the gradual improvement of genetic engineering techniques, the direct fermentation method using microorganisms has rapidly developed. This method uses low-cost raw materials such as glucose as carbon sources to produce the desired product through microbial fermentation, resulting in lower production costs and a relatively simple and controllable production process.

[0003] When tryptophan-producing strains use glucose as a carbon source, there is a glucose repression effect, meaning that if the strains ingest too much glucose, they are prone to aging and death. Therefore, in production, glucose is generally supplied to the strains in a fed-batch manner to avoid the strains ingesting too much glucose and causing abnormal fermentation.

[0004] In tryptophan production, excessive glucose addition due to operational errors or other reasons can easily lead to metabolic abnormalities in the production strain, causing the fermentation broth to turn black. Testing confirmed that this blackening is due to a high accumulation of acetic acid. Generally, acetic acid levels below 5 g / L do not cause metabolic abnormalities in the cells, but levels exceeding 5 g / L greatly increase the likelihood of metabolic disturbances. Once these abnormalities occur, the biomass and acid production of the strain are severely affected, resulting in significant production losses. Therefore, obtaining production strains with high glucose tolerance is urgently needed. Summary of the Invention

[0005] This invention discloses a genetically engineered strain capable of producing L-tryptophan and a method for producing L-tryptophan using this strain. It solves the problem that excessive glucose addition can easily lead to metabolic abnormalities in the producing strain, thus affecting acid production.

[0006] The technical solution of the present invention is as follows:

[0007] A genetically engineered strain capable of producing L-tryptophan, wherein the genetically engineered strain is obtained by knocking out the nucleotide sequence shown in SEQ ID NO:1 in the original strain.

[0008] Furthermore, the original strain was Escherichia coli.

[0009] Furthermore, the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:2.

[0010] Furthermore, the method for constructing the genetically engineered strain with L-tryptophan production capacity includes the following steps:

[0011] Step 1: Based on the strain sequence information, design primers upycjO-F, as shown in SEQ ID NO.3; upycjO-R, as shown in SEQ ID NO.4; doycjO-F, as shown in SEQ ID NO.5; and doycjO-R, as shown in SEQ ID NO.6.

[0012] Step 2: Using the primers described above, amplify the gene sequences of the homologous arms flanking the ycjO gene from the strain genome to obtain two amplified fragments;

[0013] Step 3: The two amplified fragments were fused using fusion PCR technology to obtain the recombinant fragment TD1, as shown in SEQ ID NO:7;

[0014] Step 4: Based on the pTarget sequence information, design primers ycjOsgRNA-F, as shown in SEQ ID NO.8 and ycjOsgRNA-R, as shown in SEQ ID NO.9. Using primers ycjOsgRNA-F and ycjOsgRNA-R, perform PCR amplification with pTarget as a template to obtain linearized pTYC containing sgRNA.

[0015] Step 5: Ligate the linearized pTYC containing sgRNA with the recombinant fragment TD1 to construct the recombinant plasmid pTYC;

[0016] Step 6: Transform the pCas9 plasmid containing the Cas9 protein into the strain and screen out the transformed recombinant strains;

[0017] Step 7: Transform the recombinant plasmid pTYC into the recombinant strain screened in Step 6, and obtain a genetically engineered strain with L-tryptophan production capacity through screening.

[0018] The application of the genetically engineered strain with L-tryptophan production capacity in L-tryptophan production.

[0019] A method for producing L-tryptophan using a genetically engineered strain capable of producing L-tryptophan, the method comprising the following steps:

[0020] Step 1: Prepare the seed tank culture medium, fermentation culture medium, and trace element mixture;

[0021] Step 2: Prepare seed culture of strain with ycjO gene deletion;

[0022] Step 3: Sterilize and cool the seed tank culture medium, adjust the pH to neutral, inoculate the ycjO gene-deleted strain seed liquid into the seed culture medium, and culture until the mid-to-late logarithmic growth stage;

[0023] Step 4: Sterilize and cool the fermentation tank culture medium, adjust the pH to neutral, and inoculate the late logarithmic growth stage strain into the fermentation culture medium for fermentation to produce L-tryptophan fermentation broth.

[0024] Furthermore, in step 3, the seed tank culture medium is cooled to 36°C.

[0025] Furthermore, the culture conditions in step 3 are: temperature 36℃, pH neutral, dissolved oxygen 25%-30%, and tank pressure 0.02MPa.

[0026] Furthermore, in step 4, the fermentation tank culture medium is cooled to 36°C.

[0027] Furthermore, the culture conditions in step 4 are: temperature 36℃, pH neutral, dissolved oxygen 25%-30%, and tank pressure 0.02MPa.

[0028] This invention discloses a genetically engineered strain capable of producing L-tryptophan and a method for producing L-tryptophan using this strain. The genetically engineered strain improves glucose utilization efficiency due to the inactivation of the ycjO gene, thereby increasing L-tryptophan yield and improving glucose-acid conversion rate. Furthermore, the strain exhibits increased glucose tolerance, reducing the stringency of the fed-batch process. This genetically engineered strain can be applied to large-scale L-tryptophan production, exhibiting significant advantages in fermentation indicators, reducing the likelihood of metabolic abnormalities and metabolic overflow, greatly lowering production costs, and increasing production profits. Detailed Implementation

[0029] Example 1

[0030] A genetically engineered strain capable of producing L-tryptophan was constructed using homologous recombination technology. The genetically engineered strain was a strain with the ycjO gene deleted.

[0031] In this embodiment, the ycjO gene in Escherichia coli is deleted through homologous recombination.

[0032] Step 1: Design yjeO gene knockout primers using CRISPER-Cas9 technology. The primer sequences are shown in Table 1.

[0033] Table 1

[0034]

[0035] Step 2: Based on the E. coli sequence information, primers upycjO-F, upycjO-R, doycjO-F, and doycjO-R were designed. Using the above primers, the homologous arm gene sequences on both sides of the ycjO gene were amplified from the genome of E. coli CGMCC NO.11073. The two amplified fragments were fused using fusion PCR technology to obtain the recombinant fragment TD1.

[0036] Step 3: Based on the pTarget sequence information of the vector, primers ycjOsgRNA-F and ycjOsgRNA-R were designed to linearize pTYC containing sgRNA; the recombinant plasmid pTYC was constructed by ligating it with the recombinant fragment TD1, and verified by double digestion with BamHI and BsgI and sequencing to confirm that the recombinant plasmid was successfully constructed.

[0037] Step 4: Transform the pCas9 plasmid containing the Cas9 protein into Escherichia coli. The Escherichia coli has the accession number CGMCC NO.11073, is classified as Escherichia coli, was deposited on July 14, 2015, and is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Recombinant E. coli were screened and transformed using Kana resistance plates. Subsequently, the recombinant plasmid pTYC was transformed into E. coli CGMCC NO.11073-cas9. After screening to confirm successful ycjO gene knockout, the recombinant plasmid pTYC was removed. Primers were designed, and transformants were selected for colony PCR verification. After confirming the correct sequencing results, recombinant E. coli CGMCC NO.11073-ycjOT with ycjO gene deletion was obtained. After removing the plasmid pCas9, recombinant E. coli 11073△ycjO was obtained and named XYSH230901-6.

[0038] Example 2

[0039] The L-tryptophan production capacity of the ycjO gene-deleted strain was identified, and the L-tryptophan production performance of strain XYSH230901-6 was tested in shake flasks.

[0040] The seed shake flask culture medium consists of the following components: glucose 20-40 g / L, (NH4)2SO4 15 g / L, KH2PO4 1-5 g / L, MgSO4·7H2O 0.5-2 g / L, yeast extract 2-5 g / L, FeSO4·7H2O 20-40 mg / L, MnSO4·H2O 1-3 mg / L, VH 0.1-0.5 mg / L, VB1 0.5-1.0 mg / L, trace element mixture 1-3 ml / L, and the remainder is water. The pH is 7.0-7.2. The medium is sterilized in an autoclave at 115℃ for 15 min.

[0041] The fermentation shake flask culture medium consists of the following components: glucose 20-40 g / L, (NH4)2SO4 2-6 g / L, KH2PO4 1-5 g / L, MgSO4·7H2O 0.5-2 g / L, yeast extract 1-5 g / L, FeSO4·7H2O 30-60 mg / L, MnSO4·7H2O 1-5 mg / L, VH 0.1-0.5 mg / L, VB1 0.5-1.0 mg / L, trace element mixture 1-3 ml / L, phenol red 15-30 g / L, and the remainder is water. The pH is 7.0-7.2. The medium is sterilized in an autoclave at 115℃ for 15 min.

[0042] The components of the trace element mixture are: CoSO4·7H2O 0.4-0.8 g / L, ZnSO4·7H2O 6-8 g / L, CuSO4·5H2O 4-6 g / L, Al2(SO4)3·18H2O 2-4 g / L, MnSO4·H2O 4-6 g / L, Na2MoO4·2H2O 2-4 g / L, NiSO4·6H2O 2-4 g / L, and H3BO3 1-2 g / L.

[0043] After the glucose is depleted during fermentation, add 1 mL of 20 g / L glucose and 20% ammonia water to control the pH at around 6.8.

[0044] The tryptophan production of strain XYSH230901-6 was 20.1% higher than that of the control strain CGMCC NO.11073, which was a significant increase.

[0045] Table 2: Identification of L-tryptophan production capacity in strains with the ycjO gene deletion

[0046]

[0047] Example 3:

[0048] The effect of high glucose concentration on the acid production capacity of strain XYSH230901-6 was investigated by increasing the glucose concentration in the feed and identifying the tolerance of strain 11073△ycjO to high glucose.

[0049] The seed shake flask culture medium consists of the following components: glucose 20-40 g / L, (NH4)2SO4 15 g / L, KH2PO4 1-5 g / L, MgSO4·7H2O 0.5-2 g / L, yeast extract 2-5 g / L, FeSO4·7H2O 20-40 mg / L, MnSO4·H2O 1-3 mg / L, VH 0.1-0.5 mg / L, VB1 0.5-1.0 mg / L, trace element mixture 1-3 ml / L, and the remainder is water. The pH is 7.0-7.2. The medium is sterilized in an autoclave at 115℃ for 15 min.

[0050] The fermentation shake flask culture medium consists of the following components: glucose 20-40 g / L, (NH4)2SO4 2-6 g / L, KH2PO4 1-5 g / L, MgSO4·7H2O 0.5-2 g / L, yeast extract 1-5 g / L, FeSO4·7H2O 30-60 mg / L, MnSO4·7H2O 1-5 mg / L, VH 0.1-0.5 mg / L, VB1 0.5-1.0 mg / L, trace element mixture 1-3 ml / L, phenol red 15-30 g / L, and the remainder is water. The pH is 7.0-7.2. The medium is sterilized in an autoclave at 115℃ for 15 min.

[0051] The components of the trace element mixture are: CoSO4·7H2O 0.4-0.8 g / L, ZnSO4·7H2O 6-8 g / L, CuSO4·5H2O 4-6 g / L, Al2(SO4)3·18H2O 2-4 g / L, MnSO4·H2O 4-6 g / L, Na2MoO4·2H2O 2-4 g / L, NiSO4·6H2O 2-4 g / L, and H3BO3 1-2 g / L.

[0052] After glucose was depleted during fermentation, 1 mL of 20 g / L glucose was added, and 20% ammonia was added to control the pH at around 6.8. The effects of 30 g / L and 40 g / L glucose on the acid production of XYSH230901-6 shake flasks were tested.

[0053] When the glucose concentration was increased to 20%, the acid production of strain 11073 showed no significant change, while the tryptophan production of strain XYSH230901-6 gradually increased. This indicates that strain 11073△ycjO has improved tolerance to glucose.

[0054] The tryptophan production of strain XYSH230901-6 with a sugar concentration of 30 was significantly increased by 27.2% compared to the control strain CGMCC NO.11073. The tryptophan production of strain XYSH230901-6 with a sugar concentration of 30 was significantly increased by 34.4% compared to the control strain CGMCC NO.11073.

[0055] Table 3: Effects of high-concentration fed-flow sugar on acid production capacity of ycjO gene-deleted strains

[0056]

[0057] Example 4

[0058] The acid production capacity of XYSH230901-6 was tested using a 2L fermenter. The maximum sugar replenishment rate in the normal process is 10g / L. Increasing the sugar replenishment rate to 12g / L increased the L-tryptophan production, the sugar-acid conversion rate was high, and the acetic acid accumulation was low.

[0059] Seed culture preparation: Strains XYSH230901-6 and the starting strain 11073 were activated and progressively expanded from glycerol tubes to eggplant flasks. The inoculum in the eggplant flasks was then eluted with 200 mL of physiological saline to obtain the *E. coli* culture. The expansion was carried out at 36°C for 20 hours.

[0060] Seed culture medium components: glucose 40-60 g / L, yeast extract 2-6 g / L, citric acid 0.5-2 g / L, (NH4)2SO4 2-8 g / L, KH2PO4 5-7 g / L, VB1 1-2 mg / L, VH 0.3-0.5 mg / L, MgSO4·7H2O 1.5-2 g / L, FeSO4·7H2O 20-40 mg / L, and a trace element mixture solution 1-2 mL / L. Components of the trace element mixture solution: CoSO4·7H2O 0.4-0.8 g / L, ZnSO4·7H2O 6-8 g / L, CuSO4·5H2O 4-6 g / L, Al2(SO4)3· 18 H2O 2-4g / L, MnSO4·H2O 4-6g / L, Na2MoO4·2H2O 2-4g / L, NiSO4·6H2O 2-4g / L, H3BO3 1-2g / L.

[0061] Seed culture medium: Sterilize the seed culture medium, cool it to about 36℃, adjust the pH to neutral, and inoculate the *Escherichia coli* XYSH220517-04 strain into the seed culture medium for cultivation until the mid-to-late logarithmic growth stage. Culture conditions: temperature 36℃, pH neutral, dissolved oxygen 25%-30%, tank pressure 0.02 MPa.

[0062] Fermentation medium components: glucose 10-20 g / L, yeast extract 4-6 g / L, citric acid 1-4 g / L, (NH4)2SO4 2-8 g / L, KH2PO4 4-6 g / L, MgSO4·7H2O 1-2 g / L, FeSO4·7H2O 50-80 mg / L, VB1 5-7 mg / L, VH 0.2-0.6 mg / L, and a trace element mixed solution 1-2 mL / L. Components of the trace element mixed solution: CoSO4·7H2O 0.4-0.8 g / L, ZnSO4·7H2O 6-8 g / L, CuSO4·5H2O 4-6 g / L, Al2(SO4)3· 18 H2O 2-4g / L, MnSO4·H2O 4-6g / L, Na2MoO4·2H2O 2-4g / L, NiSO4·6H2O 2-4g / L, H3BO3 1-2g / L.

[0063] Fermentation tank culture: The fermentation medium was sterilized, cooled to approximately 36°C, and the pH adjusted to neutral. The inoculum cultured to the late logarithmic growth stage was inoculated into the fermentation medium for fermentation to produce L-tryptophan fermentation broth. Fermentation conditions: temperature 36°C, neutral pH, dissolved oxygen 25%-30%, tank pressure 0.02 MPa, maximum sugar addition rate controlled at 10 g / L and 14 g / L respectively during fermentation, and residual sugar content controlled at 0.04%. The culture was terminated after 35-40 hours to obtain the L-tryptophan fermentation broth.

[0064] Fermentation index determination: Tryptophan and acetic acid content were detected by liquid chromatography.

[0065] Conversion rate determination: Conversion rate calculation formula = (fermentation broth volume L × fermentation acid content g / L) / fermentation glucose dosage g × 100%.

[0066] When the maximum sugar addition rate was 10 g / L, the fermentation acid production index and conversion rate of XYSH230901-6 were slightly improved compared with the control strain. When the maximum sugar addition rate was 14 g / L, the fermentation acid production index and conversion rate of XYSH230901-6 were significantly improved compared with the control strain, with acid production increasing by 21.44% and sugar-acid conversion rate increasing by 12.94%. The acetic acid content of the original strain exceeded 5 g / L, indicating that there was an abnormality in the strain's metabolism and that metabolic overflow was affecting tryptophan production. The acetic acid content of XYSH230901-6 remained at a normal level.

[0067] Table 4: Fermentation performance of strain XYSH230901-6 tested in a 2L fermenter

[0068]

[0069]

[0070] Example 5

[0071] The acid production capacity of XYSH230901-6 was tested using a 50L fermenter, which was 25 times larger than that of Example 4. The testing process was closer to large-scale production. The maximum sugar replenishment rate of the normal process is 10g / L, and the sugar replenishment rate was increased to 14g / L. The L-tryptophan production, sugar-acid conversion rate and acetic acid accumulation were high.

[0072] Seed culture preparation: Strains XYSH230901-6 and the starting strain 11073 were activated and progressively expanded from glycerol tubes to eggplant flasks. The inoculum in the eggplant flasks was then eluted with 200 mL of physiological saline to obtain the *E. coli* culture. The expansion was carried out at 36°C for 20 hours.

[0073] Seed culture medium components: glucose 40-60 g / L, yeast extract 2-6 g / L, citric acid 0.5-2 g / L, (NH4)2SO4 2-8 g / L, KH2PO4 5-7 g / L, VB1 1-2 mg / L, VH 0.3-0.5 mg / L, MgSO4·7H2O 1.5-2 g / L, FeSO4·7H2O 20-40 mg / L, and a trace element mixture solution 1-2 mL / L. Components of the trace element mixture solution: CoSO4·7H2O 0.4-0.8 g / L, ZnSO4·7H2O 6-8 g / L, CuSO4·5H2O 4-6 g / L, Al2(SO4)3· 18 H2O 2-4 g / L, MnSO4·H2O 4-6 g / L, Na2MoO4·2H2O 2-4 g / L, NiSO4·6H2O 2-4 g / L, H3BO3 1-2 g / L. Seed tank inoculum cultivation: Sterilize the seed culture medium, cool to approximately 36℃, adjust the pH to neutral, and inoculate Escherichia coli XYSH220517-04 into the seed culture medium for cultivation until the mid-to-late logarithmic growth stage. Inoculum cultivation conditions: temperature 36℃, pH neutral, dissolved oxygen 25%-30%, tank pressure 0.02 MPa.

[0074] Fermentation medium components: glucose 10-20 g / L, yeast extract 4-6 g / L, citric acid 1-4 g / L, (NH4)2SO4 2-8 g / L, KH2PO4 4-6 g / L, MgSO4·7H2O 1-2 g / L, FeSO4·7H2O 50-80 mg / L, VB1 5-7 mg / L, VH 0.2-0.6 mg / L, and a trace element mixed solution 1-2 mL / L. Components of the trace element mixed solution: CoSO4·7H2O 0.4-0.8 g / L, ZnSO4·7H2O 6-8 g / L, CuSO4·5H2O 4-6 g / L, Al2(SO4)3· 18 H2O 2-4g / L, MnSO4·H2O 4-6g / L, Na2MoO4·2H2O 2-4g / L, NiSO4·6H2O 2-4g / L, H3BO3 1-2g / L.

[0075] Fermentation tank culture: The fermentation medium was sterilized, cooled to approximately 36°C, and the pH adjusted to neutral. The inoculum cultured to the late logarithmic growth stage was inoculated into the fermentation medium for fermentation to produce L-tryptophan fermentation broth. Fermentation conditions: temperature 36°C, neutral pH, dissolved oxygen 25%-30%, tank pressure 0.02 MPa, maximum sugar addition rate controlled at 10 g / L and 14 g / L respectively during fermentation, and residual sugar content controlled at 0.04%. The culture was terminated after 35-40 hours to obtain the L-tryptophan fermentation broth.

[0076] Fermentation index determination: Tryptophan and acetic acid content were detected by liquid chromatography.

[0077] Conversion rate determination: Conversion rate calculation formula = (fermentation broth volume L × fermentation acid content g / L) / fermentation glucose dosage g × 100%.

[0078] Three batches were tested, and the average index was used to calculate the final fermentation result.

[0079] When the maximum sugar addition rate was 10 g / L, the fermentation acid production index and conversion rate of XYSH230901-6 were slightly improved compared with the control strain. When the maximum sugar addition rate was 14 g / L, the fermentation acid production index and conversion rate of XYSH230901-6 were significantly improved compared with the control strain, with acid production increasing by 27.32% and sugar-acid conversion rate increasing by 14.54%. The acetic acid content of the original strain exceeded 5 g / L, indicating that there was an abnormality in the strain's metabolism and that metabolic overflow was affecting tryptophan production. The acetic acid content of XYSH230901-6 remained at a normal level.

[0080] Table 5: Fermentation performance of strain XYSH230901-6 tested in 50L tank

[0081]

[0082] Example 6

[0083] The acid production capacity of XYSH230901-6 was tested using a 50-ton fermenter, which is 1000 times larger than that of Example 5, and the testing process is closer to large-scale production. The maximum sugar replenishment rate of the normal process is 10 g / L, while this example increases the sugar replenishment rate to 14 g / L, resulting in increased L-tryptophan production, high sugar-acid conversion rate, and low acetic acid accumulation.

[0084] Seed culture preparation: Strains XYSH230901-6 and the starting strain 11073 were activated and progressively expanded from glycerol tubes to eggplant flasks. The inoculum in the eggplant flasks was then eluted with 200 mL of physiological saline to obtain the *E. coli* culture. The expansion was carried out at 36°C for 20 hours.

[0085] Seed culture medium components: glucose 40-60 g / L, yeast extract 2-6 g / L, citric acid 0.5-2 g / L, (NH4)2SO4 2-8 g / L, KH2PO4 5-7 g / L, VB1 1-2 mg / L, VH 0.3-0.5 mg / L, MgSO4·7H2O 1.5-2 g / L, FeSO4·7H2O 20-40 mg / L, and a trace element mixture solution 1-2 mL / L. Components of the trace element mixture solution: CoSO4·7H2O 0.4-0.8 g / L, ZnSO4·7H2O 6-8 g / L, CuSO4·5H2O 4-6 g / L, Al2(SO4)3· 18 H2O 2-4g / L, MnSO4·H2O 4-6g / L, Na2MoO4·2H2O 2-4g / L, NiSO4·6H2O 2-4g / L, H3BO3 1-2g / L.

[0086] Seed culture medium: Sterilize the seed culture medium, cool it to about 36℃, adjust the pH to neutral, and inoculate the *Escherichia coli* XYSH220517-04 strain into the seed culture medium for cultivation until the mid-to-late logarithmic growth stage. Culture conditions: temperature 36℃, pH neutral, dissolved oxygen 25%-30%, tank pressure 0.02 MPa.

[0087] Fermentation medium components: glucose 10-20 g / L, yeast extract 4-6 g / L, citric acid 1-4 g / L, (NH4)2SO4 2-8 g / L, KH2PO4 4-6 g / L, MgSO4·7H2O 1-2 g / L, FeSO4·7H2O 50-80 mg / L, VB1 5-7 mg / L, VH 0.2-0.6 mg / L, and a trace element mixed solution 1-2 mL / L. Components of the trace element mixed solution: CoSO4·7H2O 0.4-0.8 g / L, ZnSO4·7H2O 6-8 g / L, CuSO4·5H2O 4-6 g / L, Al2(SO4)3· 18 H2O 2-4g / L, MnSO4·H2O 4-6g / L, Na2MoO4·2H2O 2-4g / L, NiSO4·6H2O 2-4g / L, H3BO3 1-2g / L.

[0088] Fermentation tank culture: The fermentation medium was sterilized, cooled to approximately 36°C, and the pH adjusted to neutral. The inoculum cultured to the late logarithmic growth stage was inoculated into the fermentation medium for fermentation to produce L-tryptophan fermentation broth. Fermentation conditions: temperature 36°C, neutral pH, dissolved oxygen 25%-30%, tank pressure 0.02 MPa, maximum sugar addition rate controlled at 10 g / L and 14 g / L respectively during fermentation, and residual sugar content controlled at 0.04%. The culture was terminated after 35-40 hours to obtain the L-tryptophan fermentation broth.

[0089] Fermentation index determination: Tryptophan and acetic acid content were detected by liquid chromatography.

[0090] Conversion rate determination: Conversion rate calculation formula = (fermentation broth volume L × fermentation acid content g / L) / fermentation glucose dosage g × 100%.

[0091] Three batches were tested, and the average index was used to calculate the final fermentation result.

[0092] When the maximum sugar addition rate was 10 g / L, the fermentation acid production index and conversion rate of XYSH230901-6 were slightly improved compared with the control strain. When the maximum sugar addition rate was 14 g / L, the fermentation acid production index and conversion rate of XYSH230901-6 were significantly improved compared with the control strain, with acid production increasing by 25.35% and sugar-acid conversion rate increasing by 16.93%. The acetic acid content of the original strain exceeded 5 g / L, indicating that there was an abnormality in the strain's metabolism and that metabolic overflow was affecting tryptophan production. The acetic acid content of XYSH230901-6 remained at a normal level.

[0093] The successful completion of a 50-ton fermentation test demonstrates that strain XYSH230901-6 exhibits stable fermentation performance, significant advantages in acid production and conversion rate, and markedly enhanced tolerance to glucose, allowing for more extensive process control. Strain XYSH230901-6 significantly reduces fermentation production costs and demonstrates remarkable fermentation advantages.

[0094] Table 6: Fermentation performance of strain XYSH230901-6 tested in a 50-ton tank

[0095]

Claims

1. The application of a genetically engineered strain capable of producing L-tryptophan in L-tryptophan production, characterized in that, The genetically engineered strain was obtained by knocking out the nucleotide sequence shown in SEQ ID NO:1 in the original strain; the original strain was Escherichia coli with accession number CGMCC NO.11073.

2. A method for producing L-tryptophan using a genetically engineered strain capable of producing L-tryptophan, characterized in that, The method includes the following steps: Step 1: Prepare the seed tank culture medium and fermentation culture medium; Step 2: Prepare seed culture of the ycjO gene-deleted strain, wherein the ycjO gene-deleted strain is obtained by knocking out the nucleotide sequence shown in SEQ ID NO:1 in the original strain; the original strain is Escherichia coli with accession number CGMCC NO.11073; Step 3: Sterilize and cool the seed tank culture medium, adjust the pH to neutral, inoculate the ycjO gene-deleted strain seed liquid into the seed culture medium, and culture until the mid-to-late logarithmic growth stage; Step 4: Sterilize and cool the fermentation tank culture medium, adjust the pH to neutral, and inoculate the late logarithmic growth stage strain into the fermentation culture medium for fermentation to produce L-tryptophan fermentation broth.

3. The method according to claim 2, characterized in that, In step 3, the seed tank culture medium is cooled to 36°C.

4. The method according to claim 2, characterized in that, The culture conditions in step 3 are: temperature 36℃, pH neutral, dissolved oxygen 25%-30%, and tank pressure 0.02MPa.

5. The method according to claim 2, characterized in that, In step 4, the fermentation tank culture medium is cooled to 36°C.

6. The method according to claim 2, characterized in that, The culture conditions in step 4 are: temperature 36℃, pH neutral, dissolved oxygen 25%-30%, and tank pressure 0.02MPa.

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