Genetically engineered bacterium for high-yield production of L-tryptophan and application of genetically engineered bacterium

Abstract

The invention relates to a genetically engineered bacterium for high yield of L-tryptophan and application thereof. The genetically engineered bacterium is a strain for expressing mutant DedD protein and / or mutant DicA protein. The mutation of the mutant DedD protein comprises V20G; the mutant type DicA protein mutation comprises E123K (European 123K). According to the invention, the DicA and DedD proteins are mutated and expressed in the strain, so that the L-tryptophan yield of the strain can be increased.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a genetically engineered bacterium with high yield of L-tryptophan and application thereof. BACKGROUND

[0002] L-tryptophan, also known as beta-indole alanine, is one of the eight amino acids for human and animal growth, plays an important role in growth and development and metabolism of human and animals, and is widely used in medicine, feed additives and other aspects. L-tryptophan is an important nutritional supplement, has the effect of relieving fatigue and promoting growth and development. At present, the annual output of L-tryptophan in the world is more than 50,000 tons, but it is still lower than the potential market demand. L-tryptophan is mainly produced in industry by microbial fermentation method, and the microorganism is usually modified to improve the yield of L-tryptophan. Various bacteria can be used for the production of L-tryptophan, such as wild type induced mutant strains of Escherichia, corynebacterium, etc. as production strains. With the increasing demand for L-tryptophan in the world, the construction and modification of high-yield L-tryptophan strains are particularly important. However, due to the hindrance of complex biological metabolic pathways and feedback regulation mechanism, the efficiency of bacterial fermentation for producing L-tryptophan is at a low level. Through genetic engineering modification of production strains, optimization of tryptophan synthesis pathway, relief of feedback inhibition, improvement of precursor supply and enhancement of product secretion, it is an effective method for current production of L-tryptophan.

[0003] In Escherichia coli, cell division is a highly regulated process involving the coordinated action of multiple genes, mainly including Fts protein family (Filamentous temperature-sensitive proteins), Z (Z-ring) ring formation related genes, cell wall synthesis regulation genes and cell cycle checkpoint regulation genes, among which the Fts protein family is the core component of Escherichia coli controlling cell division. DedD gene encodes a cell division protein directly involved in the formation of division septum, which is essential for the activity of FtsN in the Fts protein family for cell division and survival. Both of them have similar characteristics, with a small N-terminal cytoplasmic part and a large periplasmic part, and terminate in the SPOR domain. According to the research of Liu et al., DedD can promote cell cleavage by stimulating sPG synthesis and providing positive feedback to the sPG circuit like FtsN, which plays an important role in promoting bacterial growth.

[0004] DicA protein is a temperature-sensitive repressor protein, which can control the transcription of genes involved in the process of cell division and the activation of self-expression, and belongs to a cell cycle checkpoint and regulatory gene. The dicA gene is not only related to the transcription of genes involved in the process of cell division and the activation of self-expression, but also has a certain regulatory effect on the Fts protein family. DicA protein can inhibit the production of DicB protein by binding to the promoter of dicB gene and repressing its transcription, thereby maintaining the activity of FtsZ in the Fts protein family and normal cell division. At the same time, DicA protein can also self-inhibit, that is, DicA protein binds to its own promoter to regulate the expression level of itself, thereby maintaining stable DicA concentration.

[0005] In the current research, there is no report on improving the production of tryptophan by regulating cell division, therefore, how to regulate the production of tryptophan by regulating cell division related proteins has become a problem to be solved. SUMMARY

[0006] To solve the above technical problems, the present application provides a genetically engineered bacterium with high yield of L-tryptophan and application. The present application can improve the yield of L-tryptophan of the strain by mutating DicA and DedD proteins and expressing them in the strain.

[0007] To achieve this purpose, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a genetically engineered bacterium with high yield of L-tryptophan, wherein the genetically engineered bacterium is a strain of mutant DedD protein and / or mutant DicA protein.

[0009] The mutation of the mutant DedD protein includes V20G.

[0010] The mutation of the mutant DicA protein includes E123K.

[0011] In the present application, the mutation and inactivation of DicA protein can cause the failure of repressor effect, so that the gene transcription in the process of cell division is faster, which is more conducive to cell synthesis, helps to increase the amount of bacterial cells, makes the bacteria enter the fermentation cycle in advance, shortens the fermentation cycle, and thus improves the yield of L-tryptophan.

[0012] The present application introduces the mutation site of dicA coding gene and the mutation site of dedD coding gene to modify E. coli, which promotes the accumulation of cell bacteria and improves the yield of L-tryptophan, thereby providing a new idea for the industrial production of L-tryptophan.

[0013] In the present application, the dicA gene can indirectly affect the function (such as septum formation) of the dedD gene by regulating the dicB gene, both of which play a role in the Fts protein family division pathway and have a synergistic or complementary effect in maintaining a normal division cycle. In order to explore the role of the dicA gene and the dedD gene in maintaining a normal division cycle, mutation of either the DedD protein or the DicA protein can increase the yield of L-tryptophan, but when both proteins are mutated, the cell division can be accelerated while the repression is removed, and the synergistic effect of the two proteins can promote the accumulation of cell bacteria and increase the yield of L-tryptophan.

[0014] Preferably, the amino acid sequence of the mutant DedD protein comprises the sequence shown in SEQ ID NO. 1.

[0015] SEQ ID NO. 1:

[0016] MASKFQNRLVGTIVLVALGGIVLPGLLDGQKKHYQDEFAAIPLVPKAGDRDEPDMMPAATQALPTQPPEGAAEEVRAGDAAAPSLDPATIAANNTEFEPEPAPVAPPKPKPVEPPKPKVEAPPAPKPEPKPVVEEKAAPTGKAYVVQLGALKNADKVNEIVGKLRGAGYRVYTSPSTPVQGKITRILVGPDASKDKLKGSLGELKQLSGLSGVVMGYTPN.

[0017] In the present application, the 20th amino acid in the amino acid sequence of the DedD protein is mutated from V to G, wherein the amino acid sequence of the DedD protein is shown in SEQ ID NO: 5.

[0018] SEQ ID NO: 5:

[0019] MASKFQNRLVGTIVLVALGVIVLPGLLDGQKKHYQDEFAAIPLVPKAGDRDEPDMMPAATQALPTQPPEGAAEEVRAGDAAAPSLDPATIAANNTEFEPEPAPVAPPKPKPVEPPKPKVEAPPAPKPEPKPVVEEKAAPTGKAYVVQLGALKNADKVNEIVGKLRGAGYRVYTSPSTPVQGKITRILVGPDASKDKLKGSLGELKQLSGLSGVVMGYTPN.

[0020] Preferably, the amino acid sequence of the mutant DedD protein comprises the sequence set forth in SEQ ID NO. 3.

[0021] SEQ ID NO. 3:

[0022] METKNLTIGERIRYRRKNLKHTQRSLAKALKISHVSVSQWERGDSEPTGKNLFALSKVLQCSPTWILFGDEDKQPTPPVEKPVALSPKELELLELFNALPESEQDTQLAEMRARVKNFNKLFEELLKARQRTNKR.

[0023] In the present application, the amino acid at position 123 of the DedD protein amino acid sequence is mutated from E to K, wherein the DedD protein amino acid sequence is set forth in SEQ ID NO: 6.

[0024] SEQ ID NO: 6:

[0025] METKNLTIGERIRYRRKNLKHTQRSLAKALKISHVSVSQWERGDSEPTGKNLFALSKVLQCSPTWILFGDEDKQPTPPVEKPVALSPKELELLELFNALPESEQDTQLAEMRARVKNFNKLFEELLKARQRTNKR.

[0026] Preferably, the nucleic acid sequence encoding the mutant DedD protein comprises the sequence set forth in SEQ ID NO. 3.

[0027] SEQ ID NO. 3:

[0028] gtggcaagtaagtttcagaatcggttagtgggcacgatcgtgctggtggcgctgggggggattgtacttccagggctgctggacgggcagaaaaaacattatcaggatgagttcgcggctatcccgctggtgccgaaagcgggcgatcgtgatgagcctgatatgatgccagccgccacccaggcgttaccgacgcagccgccggaaggcgcagcggaagaggtgcgggcaggtgatgccgcagcaccgtcgctcgatccggccactattgcagccaataacaccgagtttgaaccggaacctgcaccggtcgccccaccgaagccgaaaccggtggagccgcctaaaccaaaggttgaagcaccacctgcgccgaagccagaaccgaagccggtcgtggaagaaaaagctgcaccgacgggtaaagcctatgttgtgcaactgggtgcgctgaaaaatgccgataaagtgaatgagattgtcggtaagctgcgcggtgccggttatcgggtttatacgtcgccatccacgccagtgcagggtaaaattacccgtattctggttgggccggatgcctcgaaagataagctgaaaggttcgctgggtgagttgaagcaactttctggcttaagtggcgtggtaatgggctatacgccgaattaa.

[0029] In the present application, the nucleic acid sequence encoding the mutant DedD protein is mutated from the nucleotide sequence of the dedD gene, wherein the nucleic acid sequence of the dedD gene is the sequence shown in SEQ ID NO. 7.

[0030] SEQ ID NO. 7:

[0031] gtggcaagtaagtttcagaatcggttagtgggcacgatcgtgctggtggcgctgggggtgattgtacttccagggctgctggacgggcagaaaaaacattatcaggatgagttcgcggctatcccgctggtgccgaaagcgggcgatcgtgatgagcctgatatgatgccagccgccacccaggcgttaccgacgcagccgccggaaggcgcagcggaagaggtgcgggcaggtgatgccgcagcaccgtcgctcgatccggccactattgcagccaataacaccgagtttgaaccggaacctgcaccggtcgccccaccgaagccgaaaccggtggagccgcctaaaccaaaggttgaagcaccacctgcgccgaagccagaaccgaagccggtcgtggaagaaaaagctgcaccgacgggtaaagcctatgttgtgcaactgggtgcgctgaaaaatgccgataaagtgaatgagattgtcggtaagctgcgcggtgccggttatcgggtttatacgtcgccatccacgccagtgcagggtaaaattacccgtattctggttgggccggatgcctcgaaagataagctgaaaggttcgctgggtgagttgaagcaactttctggcttaagtggcgtggtaatgggctatacgccgaattaa.

[0032] Preferably, the nucleic acid sequence encoding the mutant DicA protein comprises the sequence set forth in SEQ ID NO. 4.

[0033] SEQ ID NO. 4:

[0034] atggaaacaaaaaatttaactatcggcgaacgcatcaggtatcgtcggaaaaacctcaaacacacccaaaggtctcttgctaaagccctgaaaatctcccatgtgtctgtatcacaatgggaacggggtgatagtgaacctacagggaagaacctttttgccctcagtaaagtattgcaatgctcaccaacatggattctatttggcgatgaagacaagcaaccaacaccacctgttgagaagccagttgccttatcccccaaagaactagagctccttgagctgtttaatgcactgccagaatcagaacaggatacccagctcgccgaaatgcgagctcgagtaaaaaacttcaataaactctttaaagaattactaaaagcccgtcagcggacaaataaaagataa.

[0035] In the present application, the nucleic acid sequence encoding the mutant DicA protein is mutated from the nucleotide sequence of the dicA gene, wherein the nucleic acid sequence of the dicA gene is the sequence shown in SEQ ID NO. 8.

[0036] SEQ ID NO. 8:

[0037] atggaaacaaaaaatttaactatcggcgaacgcatcaggtatcgtcggaaaaacctcaaacacacccaaaggtctcttgctaaagccctgaaaatctcccatgtgtctgtatcacaatgggaacggggtgatagtgaacctacagggaagaacctttttgccctcagtaaagtattgcaatgctcaccaacatggattctatttggcgatgaagacaagcaaccaacaccacctgttgagaagccagttgccttatcccccaaagaactagagctccttgagctgtttaatgcactgccagaatcagaacaggatacccagctcgccgaaatgcgagctcgagtaaaaaacttcaataaactctttgaagaattactaaaagcccgtcagcggacaaataaaagataa.

[0038] Preferably, the species of the strain comprises any one or a combination of at least two of Escherichia coli, Corynebacterium glutamicum or Bacillus subtilis.

[0039] In a second aspect, the present application provides a use of the genetically engineered bacteria with high yield of L-tryptophan according to the first aspect in the preparation of L-tryptophan.

[0040] In a third aspect, the present application provides a method for producing L-tryptophan, which comprises fermenting the genetically engineered bacteria with high yield of L-tryptophan according to the first aspect.

[0041] Preferably, the method comprises inoculating the genetically engineered bacteria with high yield of L-tryptophan into a seed culture medium to obtain a seed liquid, and inoculating the seed liquid into a fermentation culture medium to obtain L-tryptophan after oscillation culture.

[0042] Preferably, the seed culture medium contains glucose 50-70 g / L, yeast extract 1-5 g / L, ammonium sulfate 15-25 g / L, magnesium sulfate 0.5-3 g / L, potassium dihydrogen phosphate 0.5-4 g / L, sodium citrate 1-10 g / L, sodium chloride 0.5-3 g / L, L-tyrosine 0.05-0.3 g / L, L-phenylalanine 0.1-0.3 g / L and calcium carbonate 30-50 g / L. The 50-70 g / L, for example, can be 50 g / L, 55 g / L, 60 g / L, 65 g / L or 70 g / L, etc. The 1-5 g / L, for example, can be 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L, etc. The 15-25 g / L, for example, can be 15 g / L, 16 g / L, 18 g / L, 20 g / L, 22 g / L, 24 g / L or 25 g / L, etc. The 0.5-3 g / L, for example, can be 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L or 3 g / L, etc. The 0.5-4 g / L, for example, can be 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L or 4 g / L, etc. The 1-10 g / L, for example, can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L, etc. The 0.05-0.3 g / L, for example, can be 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L or 0.3 g / L, etc. The 0.1-0.3 g / L, for example, can be 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L or 0.3 g / L, etc. The 30-50 g / L, for example, can be 30 g / L, 35 g / L, 40 g / L, 45 g / L or 50 g / L, etc.

[0043] Preferably, the fermentation medium contains glucose 50-70 g / L, yeast extract 0.5-3 g / L, KH2PO4 1-10 g / L, sodium citrate 0.5-4 g / L, MgSO4·7H2O 0.5-4 g / L, (NH4)2SO4 1-10 g / L, MnSO4·H2O 0.05-0.3 g / L, FeSO4·7H2O 0.05-0.3 g / L, ZnSO4·H2O 0.05-0.3 g / L, CoCl2·6H2O 0.05-0.3 g / L, CuSO4·5H2O 0.01-0.08 g / L and CaCO3 15-25 g / L. The 50-70 g / L, for example, can be 50 g / L, 55 g / L, 60 g / L, 65 g / L or 70 g / L, etc. The 0.5-3 g / L, for example, can be 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L or 3 g / L, etc. The 1-10 g / L, for example, can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L, etc. The 0.5-4 g / L, for example, can be 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L or 4 g / L, etc. The 0.05-0.3 g / L, for example, can be 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L or 0.3 g / L, etc. The 0.01-0.08 g / L, for example, can be 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L or 0.08 g / L, etc. The 15-25 g / L, for example, can be 15 g / L, 16 g / L, 18 g / L, 20 g / L, 22 g / L, 24 g / L or 25 g / L, etc.

[0044] Preferably, the temperature of the shaking culture is 35-40℃, the time is 18-26 h, and the speed is 150-300 rpm. The 35-40℃, for example, can be 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃, etc. The 18-26 h, for example, can be 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h or 26 h, etc. The 150-300 rpm, for example, can be 150 rpm, 200 rpm, 250 rpm or 300 rpm, etc.

[0045] Compared with the prior art, the present application has at least the following beneficial effects:

[0046] 1.The present application can improve the production of L-tryptophan in strains by mutating DicA and / or DedD proteins. For example, after the XJFF-151106S strain expresses the mutant DicA protein, the L-tryptophan production of the recombinant strain is increased by 9.4%; after the XJFF-151106S strain expresses the mutant DedD protein, the L-tryptophan production of the recombinant strain is increased by 8.2%; and after the XJFF-151106S strain expresses both DicA and DedD proteins, the L-tryptophan production of the recombinant strain is increased by 18.8%.

[0047] 2.In the present application, either the mutant DedD protein or the mutant DicA protein can improve the production of L-tryptophan, but when both proteins are mutated, the cell division is accelerated while the repression is removed, which together promotes the accumulation of cell mass and produces a synergistic effect, resulting in a greater increase in the production of L-tryptophan. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Verification electrophoresis map of dicAE123K mutant gene knock-in.

[0049] Figure 2 Verification electrophoresis map of dedDV20G mutant gene knock-in.

[0050] Figure 3 Verification electrophoresis map of double-gene mutant strain. DETAILED DESCRIPTION

[0051] The technical solutions of the present application will be further described below in combination with the drawings and through specific embodiments. However, the following examples are only simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0052] The sources of the materials used in the following examples are as follows:

[0053] The XJFF-151106S strain is Escherichia coli, which is preserved in the China General Microbiological Culture Collection Center on November 17, 2015, has a preservation number of CGMCC No.11674, and is preserved at the China General Microbiological Culture Collection Center located at No.1, Beichen West Road, Beijing City, Chaoyang District, China.

[0054] Example 1

[0055] This example constructs a strain containing a mutant DicA protein

[0056] The dicA gene in the XJFF-151106S strain is knocked out by homologous recombination.

[0057] (1) Knockout of the dicA gene

[0058] The E. coli cells were prepared in a competent state, and the pKD46 plasmid was induced into the competent cells of the E. coli XJFF-151106S by an arabic acid stock solution. The culture was screened in an ampicillin-resistant medium. The homologous arm primers Pkan-F-1 / Pkan-R-1 were designed for the kanamycin resistance gene kan by taking the plasmid PKD13 as a template to perform PCR amplification. The purified PCR product was transformed into the strain containing the pKD46 plasmid by electroporation to obtain the pKD46-kan strain. The homologous recombination occurred during the culture at 30°C to obtain the homologous recombinant strain. The pKD46 temperature-sensitive plasmid was removed by culturing at 37°C. Subsequently, the pCP20 plasmid was introduced, and the primers PpCP20-F / PpCP20-R were used for PCR amplification verification to express the flipase recombinase gene and promote the homologous recombination of the FRT site itself, so as to finally realize the knockout of the dicA gene. The sequences of the above primers are shown in Table 1.

[0059] The strain with the knockout dicA gene was cultured in LB medium and kanamycin-resistant medium at the same time. The strain that did not grow in the resistant medium was the strain with the successful knockout of the dicA gene. Finally, the pCP20 temperature-sensitive plasmid was removed by culturing at 42°C, and the primers PdicA-F-1 / PdicA-R-1 were used for PCR verification to determine whether the target gene was removed successfully, so as to obtain the dicA-knockout strain, which was named XJFF-151106S-△dicA. The sequences of the above primers are shown in Table 1.

[0060] Table 1

[0061]

[0062]

[0063] (2) Construction of a strain containing a mutant DicA protein

[0064] The homologous arm primers PdicA-F-2 / PdicA-R-2 were designed by taking the dicA E123K mutant sequence (SEQ ID NO. 4) as a template to construct the PCR amplification product of the exogenous dicA E123K gene. The PCR amplification product was transformed into the E. coli XJFF-151106S-△dicA, and the target region was replaced by homologous recombination. The specific implementation method is the same as above. Finally, the dicAE123K mutant gene was knocked in. The above recombinant strain was verified by PCR using the primers PdicA-F-1 / PdicA-R-1. The verification result is shown in Table 2. Figure 1 Figure 1 ​M is a DNA marker, E1 lane is the verification result of dicA E123K gene knock-in, and E2 lane is the amplification result of XJFF-151106S-△dicA strain. Sequencing analysis is performed, and the sequencing result is shown as SEQ ID NO: 4, that is, the dicA E123K mutant strain is successfully obtained and named as XJFF-151106S-dicAE123K. The sequences of the above primers are shown in Table 2.

[0065] Table 2

[0066]

[0067] Example 2

[0068] This example carries out construction of DedD protein knockout strain and DedD protein mutant strain

[0069] (1) Construction of DedD protein knockout strain

[0070] According to the method used in Example 1, the dedD gene in Escherichia coli XJFF-151106S is replaced by using homologous recombination with primer pair Pkan-F-2 / Pkan-R-2, and the dedD gene is knocked out. Primer pair PdedD-F-1 / PdedD-R-1 is used for PCR to verify whether the target gene is successfully removed, and dedD knockout strain XJFF-151106S-△dedD is obtained. The sequences of the above primers are shown in Table 3.

[0071] (2) Construction of DedD protein mutant strain

[0072] The PCR amplification product is transformed into Escherichia coli XJFF-151106S-△dedD with primer pair PdedD-F-2 / PdedD-R-2, and finally the dedD V20G mutant gene (SEQ ID NO. 3) is knocked in. Primer pair PdedD-F-1 / PdedD-R-1 is used for PCR verification, and the verification result is shown in Figure 2 M is a DNA marker, E1 lane is the amplification result of XJFF-151106S-△dedD strain, and E2 is the dedD V20G gene knock-in verification result. The sequences of the above primers are shown in Table 3.

[0073] Sequencing analysis is performed, and the result is shown as SEQ ID NO: 8, that is, the dedD V20G mutant strain XJFF-151106S-dedDV20G is successfully obtained.

[0074] Table 3

[0075]

[0076]

[0077] Example 3

[0078] This example carries out the construction of a strain containing a mutant DedD and a mutant DicA protein

[0079] According to the method used in Example 1, the dicA gene in E. coli XJFF-151106S-dedDV20G was replaced by using homologous recombination with primer pair Pkan-F-1 / Pkan-R-1, the dicA gene was knocked out, primer pair PdicA-F-1 / PdicA-R-1 was used for PCR to verify whether the target gene was removed successfully, primer pair PdicA-F-2 / PdicA-R-2 was used to transform the PCR amplification product into E. coli, and finally the dicA E123K mutant gene was knocked in, and primer pair PdicA-F-1 / PdicA-R-1 was used for PCR verification, and the verification results are shown in Figure 8, wherein lane M is a DNA marker, lane E1 is the dicA E123K gene knock-in verification result, and lane E2 is the dicA gene knockout verification result, and the sequencing result is shown as SEQ ID NO: 8, i.e. the dicA E123K mutant strain XJFF-151106S-dedDV20G-dicAE123K was successfully obtained. Figure 3

[0080] Example 4

[0081] In this example, the above-mentioned strain is used for fermentation to produce L-tryptophan

[0082] The recombinant E. coli is inoculated on a seed culture medium for cultivation to obtain a seed liquid. The seed culture medium comprises the following components at the following concentrations: glucose 60 g / L, yeast extract 2.5 g / L, ammonium sulfate [(NH4)2SO4·7H2O] 20 g / L, magnesium sulfate (MgSO4) 1 g / L, potassium dihydrogen phosphate (KH2PO4) 2 g / L, sodium citrate 5 g / L, sodium chloride (NaCl) 1 g / L, L-tyrosine 0.1 g / L, L-phenylalanine 0.15 g / L, calcium carbonate (CaCO3) 40 g / L.

[0083] ​The successfully constructed strains were verified by shake flask fermentation. The starting strain XJFF-151106S was used as a control and was cultured overnight in LB solid medium. The grown strains were inoculated into 250 mL angle baffled flasks containing 25 mL fermentation medium and were cultured at 37°C and 200 rpm for 22 hours. The fermentation medium was glucose 60 g / L, yeast extract 1 g / L, KH2PO4 5 g / L, sodium citrate 2 g / L, MgSO4·7H2O 2 g / L, (NH4)2SO4 5 g / L, MnSO4·H2O 0.1 g / L, FeSO4·7H2O 0.1 g / L, ZnSO4·H2O 0.1 g / L, CoCl2·6H2O 0.1 g / L, CuSO4·5H2O 0.03 g / L, and CaCO3 20 g / L. After the culture was completed, the L-tryptophan content in the fermentation broth was determined by an amino acid analyzer.

[0084] The final L-tryptophan yield and conversion rate results are shown in Table 4 (the yield and conversion rate results of each strain are the average of three results), and the conversion rate calculation method is: conversion rate = output amino acid g / L / (input glucose amount g / L - residual sugar g / L) x 100%.

[0085] Table 4

[0086] Strains Yield (g / L) Conversion rate (%) OD XJFF-151106S 3.41 8.81 10.49 XJFF-151106S-△dicA 3.29 8.79 10.01 XJFF-151106S-△dedD 3.33 8.83 10.05 XJFF-151106S-dicA E123K ]] 3.73 8.82 11.60 XJFF-151106S-dedD V20G ]] 3.69 8.81 11.57 XJFF-151106S-dedD V20G -dicA E123K ]]> 4.05 8.84 11.79

[0087] As shown in the results, after the glutamic acid at position 123 of the DicA protein was replaced with lysine, the L-tryptophan yield relative to the original strain was significantly improved by 9.4%, and the OD value was also significantly improved. After the valine at position 20 of the amino acid sequence of the DedD protein was replaced with glycine, the L-tryptophan yield relative to the original strain was significantly improved by 8.2%, and the OD value was also significantly improved. When the E123K mutation of the DicA protein and the V20G mutation of the DedD protein were introduced into the original strain at the same time, the L-tryptophan yield was improved by 18.8%, and the OD value was improved more. Therefore, in the production of L-tryptophan, the mutation of either the DicA protein or the DedD protein can improve the yield of L-tryptophan, but when both genes are mutated, the cell grows faster, resulting in a greater improvement in the yield of L-tryptophan.

[0088] In the present application, the mutation of the amino acid includes substitution, deletion, insertion, addition or inversion of one or several amino acid residues, and at least one of the methods such as mutagenesis, PCR site-directed mutagenesis, and / or homologous recombination, and the final modified sequence has not less than 90% homology with the amino acid sequence translated from the dicA gene and the dedD gene, which are all within the protection scope of the present application.

[0089] In conclusion, the present application can improve the L-tryptophan yield of the strain by mutating DicA and DedD proteins and expressing them in the strain.

[0090] The applicant states that the above description is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and it should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by those skilled in the art, and all of them fall within the protection scope and disclosure scope of the present application.

Claims

1. A genetically engineered bacterium for high production of L-tryptophan, characterized by, The genetically engineered bacteria are strains expressing mutant DedD protein and / or mutant DicA protein. The mutation of the mutant DedD protein includes V20G. The mutation of the mutant DicA protein includes E123K. 2.The genetically engineered L-tryptophan high-yield strain according to claim 1, characterized in that, The amino acid sequence of the mutant DedD protein includes the sequence shown in SEQ ID NO.

1. 3.The genetically engineered L-tryptophan high-yield strain according to claim 1 or 2, characterized in that, The amino acid sequence of the mutant DicA protein includes the sequence shown in SEQ ID NO.

2.

4. The genetically engineered L-tryptophan high-producing bacterium according to any one of claims 1 to 3, characterized by, The nucleic acid sequence encoding the mutant DedD protein includes the sequence shown in SEQ ID NO.

3.

5. The genetically engineered L-tryptophan high-yield bacteria according to any one of claims 1-4, characterized in that, The nucleic acid sequence encoding the mutant DicA protein includes the sequence shown in SEQ ID NO.

4.

6. The genetically engineered L-tryptophan high-yield bacteria according to any one of claims 1-5, characterized in that, The strain is any one of Escherichia coli, Corynebacterium glutamicum or Bacillus subtilis or a combination of at least two thereof.

7. Use of the genetically engineered bacteria with high yield of L-tryptophan according to any one of claims 1-6 in the preparation of L-tryptophan.

8. A method for producing L-tryptophan, characterized by, The method comprises fermenting the genetically engineered bacteria with high yield of L-tryptophan according to any one of claims 1-6 to obtain L-tryptophan.

9. The method of producing L-tryptophan according to claim 7, characterized by, The method comprises inoculating the genetically engineered bacteria with high yield of L-tryptophan into a seed culture medium to obtain seed liquid, and inoculating the seed liquid into a fermentation culture medium to obtain L-tryptophan after oscillation culture. Preferably, the seed culture medium contains glucose 50-70 g / L, yeast extract 1-5 g / L, ammonium sulfate 15-25 g / L, magnesium sulfate 0.5-3 g / L, potassium dihydrogen phosphate 0.5-4 g / L, sodium citrate 1-10 g / L, sodium chloride 0.5-3 g / L, L-tyrosine 0.05-0.3 g / L, L-phenylalanine 0.1-0.3 g / L, and calcium carbonate 30-50 g / L.

10. The method of producing L-tryptophan according to claim 9, characterized by, The fermentation culture medium contains glucose 50-70 g / L, yeast extract 0.5-3 g / L, KH2PO4 1-10 g / L, sodium citrate 0.5-4 g / L, MgSO4·7H2O 0.5-4 g / L, (NH4)2SO4 1-10 g / L, MnSO4·H2O 0.05-0.3 g / L, FeSO4·7H2O 0.05-0.3 g / L, ZnSO4·H2O 0.05-0.3 g / L, CoCl2·6H2O 0.05-0.3 g / L, CuSO4·5H2O 0.01-0.08 g / L, and CaCO3 15-25 g / L. Preferably, the oscillation culture is carried out at a temperature of 35-40℃ for 18-26 h at a speed of 150-300 rpm.

Images