Oligopeptide permease mutant, genetically engineered bacterium and preparation method and application of oligopeptide permease mutant and genetically engineered bacterium

Abstract

The invention relates to an oligopeptide permease mutant, a genetically engineered bacterium and a preparation method and application of the oligopeptide permease mutant and the genetically engineered bacterium. The oligopeptide permease mutant comprises an OppA protein mutant, and an amino acid sequence of the OppA protein mutant comprises a sequence as shown in SEQ ID NO. 1. Regulation of an opp operon coding gene is closely related to amino acid metabolism, especially metabolic pathways of aromatic amino acids, it is proved that the opp A gene and the opp F gene in escherichia coli are closely related in function, an independent effect and a synergistic effect exist in the production process of L-tryptophan, and by improving the activity of OppA and OppF transporters, the L-tryptophan can be produced, so that the L-tryptophan production efficiency is improved, and the L-tryptophan production efficiency is improved. The cell energy state is regulated to promote the growth of thalli, so that the enzyme activity in the L-tryptophan synthesis route is indirectly influenced, and the synthesis of tryptophan is obviously promoted.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and microorganisms, and in particular to an oligopeptide permease mutant, a genetically engineered bacterium, and a preparation method and application thereof. Background Art

[0002] L-tryptophan, also known as β-indolylalanine, is one of the eight amino acids necessary for the growth of humans and animals. It plays an important role in their growth, development, and metabolism, and is widely used in medicine, feed additives, and other fields. L-tryptophan is an important nutritional enhancer that can relieve human fatigue and promote growth and development. Currently, the annual global production of L-tryptophan exceeds 50,000 tons, but this is still lower than the potential market demand. L-tryptophan is mainly produced industrially by microbial fermentation. Microorganisms are usually modified to increase L-tryptophan production. A variety of bacteria can be used in the production of L-tryptophan, such as mutant strains induced from wild-type Escherichia and Corynebacterium as production strains. With the increasing global demand for L-tryptophan, the construction and modification of high-yield L-tryptophan strains are particularly important.

[0003] L-tryptophan belongs to aromatic amino acids. In Escherichia coli, the synthesis of L-tryptophan involves the glycolytic pathway, the pentose phosphate pathway, and the shikimate pathway. However, due to the intricate biological metabolic pathways and feedback regulation mechanisms, the efficiency of L-tryptophan production by E. coli fermentation is at a low level. At present, in the past few decades, researchers have developed a wealth of metabolic engineering strategies to transform E. coli to obtain high-yield strains of L-tryptophan. For example, CN111926002A discloses the use of genetic engineering to remove the feedback inhibition of the key gene TrpE in the L-tryptophan metabolic branch pathway to obtain a genetically engineered bacterium with high L-tryptophan production.

[0004] Nevertheless, due to the long L-tryptophan metabolic pathway, the consumption of multiple ATPs during the reaction process, and the complex reaction process, multiple reaction steps are controlled by product feedback regulation, which makes it difficult to remove some feedback inhibition in the synthesis pathway. The oligopeptide permease (Opp) box is an oligopeptide transport system that belongs to the ATP-binding cassette (ABC) transporter superfamily and is widely distributed in bacteria. It is encoded by the opp operon containing oppA, oppB, oppC, oppD, and oppF. It participates in various physiological processes, including energy and nutrient uptake, signal transduction, and stress response. It is related to the uptake of extracellular proteins, amino acids, and peptides, and is responsible for transporting these substances from the extracellular to the intracellular.

[0005] In summary, how to increase the production of L-tryptophan by modifying microorganisms has become one of the urgent problems to be solved in this field. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides an oligopeptide permease mutant, a genetically engineered bacterium, and a preparation method and application thereof. The present invention transforms the microorganism by introducing a mutation site in the oppA encoding gene and combining it with a mutation site in the oppF encoding gene to explore the effects of these two mutation sites on L-tryptophan synthesis.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides the use of an oligopeptide permease mutant in the fermentative production of L-tryptophan, wherein the oligopeptide permease mutant comprises an OppA protein mutant, and the amino acid sequence of the OppA protein mutant comprises the sequence shown in SEQ ID NO.1.

[0009] Preferably, the oligopeptide permease mutant further includes an OppF protein mutant.

[0010] Preferably, the amino acid sequence of the OppF protein mutant includes the sequence shown in SEQ ID NO.2.

[0011] In the oligopeptide permease mutants provided herein, the OppA and OppF proteins are closely related in function, acting both independently and synergistically in the L-tryptophan production process. The amino acid 271 in the OppA protein's amino acid sequence is mutated from N to Y, while the amino acid 325 in the OppF protein's amino acid sequence is mutated from S to A. Simultaneous mutations in both the oppA and oppF genes can promote bacterial growth by regulating cellular energy status, significantly promoting tryptophan synthesis.

[0012] In the present invention, the amino acid at position 271 of the amino acid sequence of the OppA protein is mutated from N to Y, abbreviated as oppAN271Y, and the amino acid at position 325 of the amino acid sequence of the OppF protein is mutated from S to A, abbreviated as oppF S325A.

[0013] In a second aspect, the present invention provides a genetically engineered bacterium for producing L-tryptophan, wherein the amino acid sequence of the OppA protein in the genetically engineered bacterium includes the sequence shown in SEQ ID NO.1, and the amino acid sequence of the OppF protein in the genetically engineered bacterium includes the sequence shown in SEQ ID NO.2.

[0014] The regulation of genes encoded by the opp operon is closely related to amino acid metabolism, particularly the metabolic pathways of aromatic amino acids. By simultaneously mutating the OppA gene and the OppF protein, the present invention increases the activity of the OppA and OppF transporters, promoting bacterial growth by regulating cellular energy status and significantly boosting tryptophan synthesis.

[0015] Preferably, the starting strain of the genetically engineered bacteria includes any one of Escherichia coli, Corynebacterium glutamicum or Bacillus subtilis.

[0016] Preferably, the starting strain of the genetically engineered bacteria is Escherichia coli XJFF-151106S strain with a deposit number of CGMCC No. 11674, which was deposited in the General Microbiology Center of the China Culture Collection Administration on November 17, 2015, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0017] In a third aspect, the present invention provides a method for preparing the genetically engineered bacteria as described in the second aspect, the preparation method comprising: mutating the 271st amino acid in the amino acid sequence of the OppA protein of the starting strain from N to Y, and mutating the 325th amino acid in the amino acid sequence of the OppF protein of the starting strain from S to A.

[0018] The invention improves the activities of OppA transport protein and OppF transport protein by mutating the 271st amino acid in the amino acid sequence of the OppA protein of the starting strain from N to Y and the 325th amino acid in the amino acid sequence of the OppF protein from S to A.

[0019] Preferably, the mutation method comprises any one of mutagenesis, PCR-directed mutagenesis or homologous recombination, or a combination of at least two thereof.

[0020] Preferably, the mutation method specifically comprises: replacing the target regions of the oppA gene and the oppF gene of the starting strain by homologous recombination to obtain the genetically engineered bacteria.

[0021] Preferably, the nucleic acid sequences of the primers used in the homologous recombination include the sequences shown in SEQ ID NO. 3 to SEQ ID NO. 20.

[0022] In a fourth aspect, the present invention provides use of the genetically engineered bacteria described in the second aspect in the fermentation production of L-tryptophan.

[0023] In a fifth aspect, the present invention provides a method for producing L-tryptophan by fermentation, comprising: inoculating the genetically engineered bacteria described in the second aspect into a culture medium, and fermenting and culturing to obtain the L-tryptophan.

[0024] Preferably, the method for producing L-tryptophan by fermentation specifically comprises: inoculating the genetically engineered bacteria described in the second aspect into a seed culture medium to obtain a seed solution, inoculating the seed solution into a fermentation culture medium, and fermenting and culturing to obtain the L-tryptophan.

[0025] Preferably, the seed culture medium contains 60 g / L of glucose, 1 g / L of yeast extract, 5 g / L of KH2PO4, 2 g / L of sodium citrate, 2 g / L of MgSO4·7H2O, 5 g / L of (NH4)2SO4, 0.1 g / L of MnSO4·H2O, 0.1 g / L of FeSO4·7H2O, 0.1 g / L of ZnSO4·H2O, 0.1 g / L of CoCl2·6H2O, 0.03 g / L of CuSO4·5H2O and 20 g / L of CaCO3.

[0026] Preferably, the culture medium for the fermentation culture comprises LB medium.

[0027] Preferably, the fermentation culture conditions include: shaking culture at 35-40°C (for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, etc.) and 150-250rpm (for example, 150rpm, 170rpm, 190rpm, 200rpm, 210rpm, 230rpm or 250rpm, etc.).

[0028] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0029] The sequences of the nucleic acids and proteins involved in the present invention are as follows:

[0030] SEQ ID NO.1 (OppA mutant protein):

[0031] MTNITKRSLVAAGVLAALMAGNVALAADVPAGVTLAEKQTLVRNNGSEVQSLDPHKIEGVPESNISRDLFEGLLVSDLDGHPAPGVAESWDNKDAKVWTFHLRKDAKWSDGTPVTAQDFVYSWQRSVDPNTASPYASYLQYGHIAGIDEILEGKKPITDLGVKAIDDHTLEVTLSEPVPYFYKLLVHPSTSPVPKAAIEKFGEKWTQPGNIVTNGAYTLKDWVVNERIVLERSPTYWNNAKTVINQVTYLPIASEVTDVNRYRSGEIDMTYNSMPIELFQKLKKEIPDEVHVDPYLCTYYYEINNQKPPFNDVRVRTALKLGMDRDIIVNKVKAQGNMPAYGYTPPYTDGAKLTQPEWFGWSQEKRNEEAKKLLAEAGYTADKPLTINLLYNTSDLHKKLAIAASSLWKKNIGVNVKLVNQEWKTFLDTRHQGTFDVARAGWCADYNEPTSFLNTMLSNSSMNTAHYKSPAFDSIMAETLKVTDEAQRTALYTKAEQQLDKDSAIVPVYYYVNARLVKPWVGGYTGKDPLDNTYTRNMYIVKH。

[0032] SEQ ID NO.2 (OppF mutant protein):

[0033] MNAVTEGRKVLLEIADLKVHFEIKDGKQWFWQPPKTLKAVDGVTLRLYEGETLGVVGESGCGKSTFARAIIGLVKATDGHVAWLGKELLGMKPDEWRAVRSDIQMIFQDPLASLNPRMTIGEIIAEPLRTYHPKMSRQEVRERVKAMMLKVGLLPNLINRYPHEFSGGQCQRIGIARALILEPKLIICDEPVSALDVSIQAQVVNLLQQLQREMGLSLIFIAHDLAVVKHISDRVLVMYLGHAVELGTYDEVYHNPLHPYTRALMSAVPIPDPDLEKNKTIQLLEGELPSPINPPSGCVFRTRCPIAGPECAKTRPVLEGSFRHAVSCLKVDPL。

[0034] SEQ ID NO.3(Pkan-F):TGGTTAATACCAGTAATTAATTAGGGAGTCCAAAAAACAAGGCTGGAGCTGCTTC。

[0035] SEQ ID NO.4(Pkan-R):TCAGACACCGTGGAGCAGGACACTCCCTGCCCCACGTATTGCCATCCGGGGATCCGTCGACC。

[0036] SEQ ID NO.5(PpCP20-F):ATGTCTGAATTAGTTGTTTTCAAAGCAAATGAAC。

[0037] SEQ ID NO.6(PpCP20-R):GATCCTTCCGTATTTAGCCAGTATGTTCT。

[0038] SEQ ID NO.7(PoppA-F):ATGACCAACATCACCAAGAGAAGT。

[0039] SEQ ID NO.8(PoppA-R):TTAGTGCTTCACAATGTACATATTCCGGG。

[0040] SEQ ID NO.9(PoppA-F-1):TGGTTAATACCAGTAATTAATTAGGGAGTCCAAAAAAATGACCAACATCACCAAGAAGT。

[0041] SEQ ID NO.10(PoppA-R-1):TCAGACACCGTGGAGCAGGACTCCTGCCCCACGTATTGCCATTAGTGCTTCACAATGTACATATTCCGGG。

[0042] SEQ ID NO.11(Pkan-F-1):GCCGTCTGCGTGCTTGCTTTAAAACCGGTGGAGGAACTGTTAGGCTGGAGCTGCTTC。

[0043] SEQ ID NO.12(Pkan-R-1):AGCGTGAAAAAGGGCTGACAACTGTCAGCCCTTATTGTTTTCCGGGGATCCGTCGACC。

[0044] SEQ ID NO. 13 (PoppF-F): ATGAATGCTGTAACTGAAGGAAGAAAA.

[0045] SEQ ID NO. 14 (PoppF-R): TTAAAGCGGATCGACTTTCAGG.

[0046] SEQ ID NO. 15 (PoppF-F-1): GCCGTCTGCGTGCTTGCTTTAAACCGGTGGAGGAACTGTTATGAATGCTGTAACTGAAGGAAGAAAA.

[0047] SEQ ID NO. 16 (PoppF-R-1): AGCGTGAAAAAGGGCTGACAACTGTCAGCCCTTATTGTTTTTAAAGCGGATCGACTTTCAGG.

[0048] SEQ ID NO. 17 (PoppA-F-2): CCCAATCCGGGATTACACATGCT.

[0049] SEQ ID NO. 18 (PoppA-R-2): GCCTGTGCCTTCTGTAATGCGATAAAAATCA.

[0050] SEQ ID NO. 19 (PoppF-F-2): CCGCTGGAAGAGTTTACGCCT.

[0051] SEQ ID NO. 20 (PoppF-R-2): ATCATCTGGCGGGAATAAATAATCGCTT.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The present invention increases L-tryptophan production in strains by enhancing the activity of the OppA and OppF transport proteins. When the XJFF-151106S strain expresses the mutant OppA protein, the recombinant strain's L-tryptophan production increases by 11.1%. When the mutant OppF protein is expressed, the recombinant strain's L-tryptophan production increases by 9%. When both OppA and OppF proteins are expressed simultaneously, the recombinant strain's L-tryptophan production increases by 20.7%.

[0054] (2) The present invention demonstrates that the oppA and oppF genes in E. coli are closely related in function and have both independent and synergistic effects in the L-tryptophan production process.

[0055] (3) The present invention can increase the production of L-tryptophan by mutating either the oppA gene or the oppF gene. However, when both genes are mutated at the same time, the growth of the bacteria can be promoted by regulating the cellular energy state, indirectly affecting the enzyme activity in the L-tryptophan synthesis pathway, and significantly promoting the synthesis of tryptophan, thereby increasing the production of L-tryptophan even more. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a diagram for the validation of the oppAN271Y gene knock-in in Example 1.

[0057] Figure 2 This is a diagram for the validation of oppF S325A gene knock-in in Example 2.

[0058] Figure 3 This is a diagram for the validation of oppF S325A gene knock-in in Example 3. DETAILED DESCRIPTION

[0059] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0060] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0061] The amino acid mutations of the present invention include substitution, deletion, insertion, addition or inversion of one or more amino acid residues, including at least one of mutagenesis, PCR site-directed mutagenesis or homologous recombination, and the final modified sequence has no less than 90% homology with the amino acid sequence of the oppA gene and the oppF gene, which are all within the scope of protection of the present invention.

[0062] The XJFF-151106S strain involved in the present invention is classified as Escherichia coli, and the deposited unit is the General Microbiology Center of the China Culture Collection Administration of Microorganisms. The deposit date is November 17, 2015, the deposit number is CGMCC No. 11674, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0063] Example 1XJFF-151106S-oppA N271Y Construction of recombinant strains

[0064] In this example, the oppA gene in the XJFF-151106S strain was knocked out and knocked in using homologous recombination. The specific steps are as follows:

[0065] Competent E. coli cells were prepared and transformed with the pKD46 plasmid using arabinose stock solution into competent E. coli XJFF-151106S cells. The cells were then cultured and screened in a resistance medium containing ampicillin. Homologous arms were selected at either end of the target gene. Using plasmid pKD13 as a template, primers Pkan-F / Pkan-R were designed to amplify the kanamycin resistance gene kan. The purified PCR product was electroporated into a strain containing the pKD46 plasmid, generating the pKD46-kan strain. Homologous recombination occurred during culture at 30°C, resulting in a homologous recombination strain. The temperature-sensitive pKD46 plasmid was then removed by culture at 37°C. Subsequently, the pCP20 plasmid was introduced and verified by PCR amplification using the PpCP20-F / PpCP20-R primer pair. Expression of the flippase recombinase gene promoted homologous recombination within the FRT site, ultimately achieving knockout of the oppA gene. The strains were cultured simultaneously in LB medium and kanamycin resistance medium. Strains that grew normally in LB medium but not in resistance medium were considered to have successfully knocked out the oppA gene. Finally, the cells were cultured at 42°C to remove the pCP20 thermosensitive plasmid. PCR was performed using the primer pair PoppA-F / PoppA-R to verify successful removal of the target gene. The oppA knockout strain was obtained and named XJFF-151106S-△oppA.

[0066] Using the oppA N271Y mutant sequence as a template, homology primers PoppA-F-1 / PoppA-R-1 were designed to construct a PCR amplification product of the exogenous oppA N271Y gene. This PCR amplification product was then transformed into Escherichia coli XJFF-151106S-△oppA. The target region was replaced by homologous recombination using the same method as above, ultimately achieving knock-in of the oppA N271Y mutant gene. PCR verification of the recombinant strain was performed using the primer pair PoppA-F-2 / PoppA-R-2.

[0067] like Figure 1 As shown, M is DNA marker, E1 is the result of oppAN271Y gene knock-in verification, and E2 is the result of XJFF-151106S-△oppA strain amplification. Figure 1 It can be seen that the oppAN271Y gene has been successfully knocked in and sequenced to obtain the oppAN271Y mutant strain, named XJFF-151106S-oppA N271Y .

[0068] Example 2XJFF-151106S-oppF S325A Construction of recombinant strains

[0069] In this example, the oppF gene in E. coli XJFF-151106S was replaced using the method described in Example 1 using the primer pair Pkan-F-1 / Pkan-R-1. This knockout of the oppF gene was achieved by homologous recombination. PCR was then performed using the primer pair PoppF-F / PoppF-R to verify successful removal of the target gene, resulting in the oppF knockout strain XJFF-151106S-ΔoppF. The PCR amplification product was transformed into E. coli XJFF-151106S-ΔoppF using the primer pair PoppF-F-1 / PoppF-R-1. PCR verification of the recombinant strain was performed using the primer pair PoppF-F-2 / PoppF-R-2.

[0070] like Figure 2 As shown, M is DNA marker, E1 is the amplification result of XJFF-151106S-△oppF strain, and E2 is the verification result of oppFS325A gene knock-in. Figure 2 It can be seen that the oppF S325A mutant gene has been successfully knocked in and sequenced to obtain the oppF S325A mutant strain, named XJFF-151106S-oppF S325A .

[0071] Example 3XJFF-151106S-oppA N271Y -oppF S325A Construction of recombinant strains

[0072] This example refers to the method used in Example 1, using Pkan-F-1 / Pkan-R-1 as the primer pair, and using homologous recombination to replace Escherichia coli XJFF-151106S-oppA N271Y The oppF gene in the strain was knocked out, and PCR was performed using the primer pair PoppF-F / PoppF-R to verify successful removal of the target gene. Using the primer pair PoppF-F-1 / PoppF-R-1, the PCR amplification product was transformed into E. coli to finally achieve knock-in of the oppF S325A mutant gene. The recombinant strain was then verified by PCR using PoppF-F-2 / PoppF-R-2.

[0073] like Figure 3 As shown, M is a DNA marker, E1 is the result of oppF gene knockout verification, and E2 is the result of oppF S325A gene knockin verification. Figure 3It can be seen that the oppF S325A mutant gene has been successfully knocked in and sequenced to successfully obtain the oppFS325A mutant strain XJFF-151106S-oppA N271Y -oppF S325A .

[0074] Example 4: Fermentation production of L-tryptophan by recombinant strains

[0075] In this example, the recombinant Escherichia coli prepared in Examples 1-3 was inoculated into a seed culture medium for culture to obtain a seed solution. The seed culture medium contained the following components at the following concentrations: 60 g / L glucose, 2.5 g / L yeast extract, 20 g / L ammonium sulfate [(NH4)2SO4·7H2O], 1 g / L magnesium sulfate (MgSO4), 2 g / L potassium dihydrogen phosphate (KH2PO4), 5 g / L sodium citrate, 1 g / L sodium chloride (NaCl), 0.1 g / L L-tyrosine, 0.15 g / L L-phenylalanine, and 40 g / L calcium carbonate (CaCO3).

[0076] The successfully constructed strains were verified by shake flask fermentation, and the starting strain XJFF-151106S was used as a control, and they were cultured overnight in LB solid medium. The grown strains were inoculated into 250mL triangular baffle flasks containing 25mL fermentation medium and cultured at 37°C with shaking at 200rpm for 22 hours. After the culture was completed, the L-tryptophan content in the fermentation broth was determined by an amino acid analyzer. The final L-tryptophan production and conversion rate results are shown in Table 1 (the production and conversion rate results of each strain are the average of 3 results), and the conversion rate calculation method is: conversion rate = amino acid g / L produced / (amount of glucose input g / L-residual sugar g / L) × 100%.

[0077] Table 1

[0078]

[0079]

[0080] The results showed that after the asparagine at the 271st amino acid sequence of the oppA gene was replaced by tyrosine, the L-tryptophan production was significantly increased by 11.1% compared with the original strain; after the serine at the 325th amino acid sequence of the oppF gene was replaced by alanine, the L-tryptophan production was significantly increased by 9% compared with the original strain; and when the oppA N271Y mutant gene and the oppF S325A mutant gene were introduced into the original strain simultaneously, the L-tryptophan production increased more than the introduction of either mutation alone, by 20.7%.

[0081] This shows that in the L-tryptophan production process, the functions of OppA and OppF are both interdependent and independent. Mutating either the oppA gene or the oppF gene can increase the production of L-tryptophan. However, when both genes mutate at the same time, they can promote bacterial growth by regulating the cellular energy state, indirectly affecting the enzyme activity in the L-tryptophan synthesis pathway, and helping to increase L-tryptophan production.

[0082] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. Application of an oligopeptide permease mutant in the fermentation production of L-tryptophan, characterized in that: The oligopeptide permease mutant includes an OppA protein mutant, and the amino acid sequence of the OppA protein mutant includes the sequence shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The oligopeptide permease mutants also include OppF protein mutants.

3. The use according to claim 2, characterized in that The amino acid sequence of the OppF protein mutant includes the sequence shown in SEQ ID NO.

2.

4. A genetically engineered bacterium for producing L-tryptophan, characterized in that: The amino acid sequence of the OppA protein in the genetically engineered bacteria includes the sequence shown in SEQ ID NO.1, and the amino acid sequence of the OppF protein in the genetically engineered bacteria includes the sequence shown in SEQ ID NO.

2.

5. The genetically engineered bacterium according to claim 4, characterized in that The starting strain of the genetically engineered bacteria includes any one of Escherichia coli, Corynebacterium glutamicum or Bacillus subtilis.

6. The genetically engineered bacterium according to claim 4 or 5, characterized in that The starting strain of the genetically engineered bacteria is the Escherichia coli XJFF-151106S strain with a deposit number of CGMCC No. 11674, which was deposited at the General Microbiology Center of the China Culture Collection Administration on November 17, 2015, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

7. A method for preparing a genetically engineered bacterium according to any one of claims 4 to 6, characterized in that: The preparation method comprises: mutating the 271st amino acid in the amino acid sequence of the OppA protein of the starting strain from N to Y, and mutating the 325th amino acid in the amino acid sequence of the OppF protein of the starting strain from S to A.

8. The method for preparing the genetically engineered bacteria according to claim 7, characterized in that: The mutation method includes any one or a combination of at least two of mutagenesis, PCR-directed mutagenesis or homologous recombination; Preferably, the mutation method specifically comprises: replacing the target regions of the oppA gene and the oppF gene of the starting strain by homologous recombination to obtain the genetically engineered bacteria; Preferably, the nucleic acid sequences of the primers used in the homologous recombination include the sequences shown in SEQ ID NO. 3 to SEQ ID NO.

20.

9. Use of the genetically engineered bacterium according to any one of claims 4 to 6 in the fermentation production of L-tryptophan.

10. A method for producing L-tryptophan by fermentation, characterized in that: The method for producing L-tryptophan by fermentation comprises: inoculating the genetically engineered bacteria according to any one of claims 4 to 6 into a culture medium, and fermenting and culturing to obtain the L-tryptophan; Preferably, the culture medium for the fermentation culture comprises LB medium; Preferably, the fermentation culture conditions include: culture at 35-40°C and shaking at 150-250 rpm.

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