Mutant AcrR protein and application thereof

Abstract

The invention relates to a mutant AcrR protein and application thereof, the mutant AcrR protein is obtained by mutation of AcrR protein, and the mutation comprises D139N. According to the invention, the AcrR protein is subjected to single-point mutation, so that the activity of an AcrAB-TolC system is enhanced, the robustness of the strain in the acid production process is further improved, and finally, the accumulation of L-tryptophan is improved.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and in particular to a mutant AcrR protein and applications thereof. Background Art

[0002] L-tryptophan, also known as β-indolylalanine, is an important aromatic amino acid that not only has multiple physiological functions in organisms but also has widespread applications in food, medicine, and feed. With the development of the food, medicine, and feed industries, the market demand for tryptophan continues to grow. L-tryptophan is an essential amino acid that cannot be synthesized by humans and animals and must be consumed through diet or feed. Traditional methods for producing tryptophan rely primarily on chemical synthesis and microbial fermentation, with microbial fermentation attracting significant attention due to its environmental friendliness and cost-effectiveness. A variety of bacteria can be used for L-tryptophan production, such as mutant strains derived from wild-type Escherichia and Corynebacterium. With the increasing global demand for L-tryptophan, the development and engineering of high-yielding strains is crucial. However, the efficiency of bacterial fermentation for L-tryptophan production remains low due to the complex metabolic pathways and feedback regulation mechanisms. Genetically engineering production strains to optimize the tryptophan biosynthesis pathway, relieve feedback inhibition, increase precursor supply, and enhance product secretion is currently an effective method for producing L-tryptophan.

[0003] The AcrAB-TolC efflux pump system is one of the most important multidrug-resistant efflux pumps in Escherichia coli and other Gram-negative bacteria. It is powered by protons, capturing and transporting substrates through AcrB. Assisted by the membrane fusion protein AcrA, substrate-induced conformational changes open the AcrB and TolC channels, allowing substrates to be pumped out of the cell. This system can expel a variety of substrates (including antibiotics, toxins, dyes, and metabolites) from the cell, thereby helping bacteria resist external stress and maintain a stable intracellular environment, playing a crucial role in bacterial adaptation. The acrR gene encodes the AcrR protein (DNA-binding transcriptional repressor), a transcriptional repressor that binds to the promoter site between acrR and acrA, repressing its own expression and that of acrAB, thereby inhibiting the expression of the AcrAB-TolC system. Numerous reports have been published on the function of AcrR, including studies of clinically isolated mutants and laboratory-induced mutations. For example, Cristian Ruiz et al. found that inactivating AcrR could trigger the upregulation of acrAB expression, restoring homeostasis in the strain. Grkovic et al. summarized the then-known regulatory mechanisms for drug export gene expression, finding that acrAB expression in Escherichia coli is regulated by the repressor protein AcrR. These studies have found that overexpression of AcrR can reduce acrAB expression, that its mutational inactivation can lead to increased acrAB transcription, and that AcrR can also repress its own promoter to regulate its own expression.

[0004] However, there has been no in-depth research on modifying the acrR gene for industrial production of L-tryptophan. How to regulate L-tryptophan synthesis by modifying the acrR gene has become an urgent problem to be solved. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a mutant AcrR protein and its application, wherein the mutant AcrR protein can significantly increase the production of L-tryptophan.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a mutant AcrR protein, wherein the mutant AcrR protein is an AcrR protein mutated, and the mutation includes D139N.

[0008] In the present invention, a single-point mutation is performed on the AcrR protein to further improve the robustness of the strain during acid production, thereby ultimately increasing the accumulation of L-tryptophan.

[0009] Preferably, the amino acid sequence of the mutant AcrR protein includes the sequence shown in SEQ ID NO.1.

[0010] SEQ ID NO.1:

[0011] MARKTKQEAQETRQHILDVALRLFSQQGVSSTSLGEIAKAAGVTRGAIYWHFKDKSDLFSEIWELSESNIGELELEYQAKFPGDPLSVLREILIHVLESTVTEERRRLLMEIIFHKCEFVGEMAVVQQAQRNLCLESYNRIEQTLKHCIEAKMLPA DLMTRRAAIIMRGYISGLMENWLFAPQSFDLKKEARDYVAILLEMYLLCPTLRNPATNE.

[0012] The present invention is based on the AcrR protein and performs single-point mutation to obtain a mutant AcrR protein, wherein the nucleic acid sequence of the AcrR protein is shown in SEQ ID NO.3.

[0013] SEQ ID NO.3:

[0014] MARKTKQEAQETRQHILDVALRLFSQQGVSSTSLGEIAKAAGVTRGAIYWHFKDKSDLFSEIWELSESNIGELELEYQAKFPGDPLSVLREILIHVLESTVTEERRRL LMEIIFHKCEFVGEMAVVQQAQRNLCLESYDRIEQTLKHCIEAKMLPADLMTRAAIIMRGYISGLMENWLFAPQSFDLKKEARDYVAILLEMYLLCPTLRNPATNE.

[0015] In a second aspect, the present invention provides a nucleic acid molecule comprising the coding sequence of the mutant AcrR protein described in the first aspect.

[0016] Preferably, the nucleic acid sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO.2.

[0017] SEQ ID NO.2:

[0018] ATGGCACGAAAAACCAAACAAGAAGCGCAAGAAACGCGCCAACACATCCTCGATGTGGCTCTACGTCTTTCTCACAGCAGGGGGTATCATCCACCTCGCTGGGCGAGATTGCAAAAGCAGCTGGCGTTACGCGGTGCAATCTACTGGCATTTTAAAGACAAGTCGGATTTGTTCAGTGAGATCTGGGAACTGTCAGAATCCAATATTGGTGAACTAGAGCTTGA GTATCAGGCAAAATTCCCTGGCGATCCACTCTCAGTATTAAGAGAGATATTAATTCATGTTCTTGAATCCACGGTGACAGAAGAACGGCGTCGATTATTGAATGGAGATTATATTCCACAAATGCGAATTTGTCGGAGAAATGGCTGTTGTGCAACAGGCACAACGTAATCTCTGTCTGGAAAGTTATAACCGTATAGAACAAACGTTAAAACATTGTATTGAAGCGAA AATGTTGCCTGCGGATTTAATGACGCGTCGCGCAGCAATTATTATGCGCGGCTATATTTCCGGCCTGATGGAAAACTGGCTCTTTGCCCCGCAATCTTTTGATCTTAAAAAAGAAGCCCGCGATTACGTTGCCATCTTACTGGAGATGTATCTCCTGTGCCCCACGCTTCGTAATCCTGCCACTAACGAATAA.

[0019] In the present invention, a single-point mutation is performed on the acrR gene, and G at position 415 is replaced by A to obtain an acrR mutant gene. The nucleic acid sequence of the acrR gene is shown in SEQ ID NO.4.

[0020] SEQ ID NO.4:

[0021] .

[0022] In a third aspect, the present invention provides an expression vector, which contains the nucleic acid molecule described in the second aspect; and after transfection, transduction or transformation of a host cell, the expression vector causes the host cell to express the mutant AcrR protein described in the first aspect.

[0023] In a fourth aspect, the present invention provides a recombinant cell, which contains the nucleic acid molecule described in the second aspect and / or the recombinant vector described in the third aspect.

[0024] In a fifth aspect, the present invention provides a use of the mutant AcrR protein according to the first aspect, the nucleic acid molecule according to the second aspect, the recombinant vector according to the third aspect, or the recombinant cell according to the fourth aspect in increasing L-tryptophan production.

[0025] In a sixth aspect, the present invention provides a method for increasing L-tryptophan production, wherein the mutant AcrR protein described in the first aspect replaces the AcrR protein in the host cell, and the recombinant cells are cultured to obtain L-tryptophan.

[0026] Preferably, the replacement method comprises any one of suicide plasmid homologous recombination method or λ-Red homologous recombination method and CRISPR-Cas9 gene editing technology.

[0027] Preferably, the host cell comprises any one or a combination of at least two of Escherichia coli, Corynebacterium glutamicum or Bacillus subtilis.

[0028] Preferably, the culture medium comprises 55-65 g / L glucose, 2-3 g / L yeast extract, 15-25 g / L ammonium sulfate, 0.5-2 g / L magnesium sulfate, 1-3 g / L potassium dihydrogen phosphate, 4-6 g / L sodium citrate, 0.5-2 g / L sodium chloride, 0.05-0.2 g / L L-tyrosine, 0.1-0.2 g / L L-phenylalanine, and 38-42 g / L calcium carbonate. The 55-65 g / L may be, for example, 55 g / L, 56 g / L, 58 g / L, 60 g / L, 62 g / L, 64 g / L, or 65 g / L. The 2-3 g / L may be, for example, 2 g / L, 2.2 g / L, 2.4 g / L, 2.6 g / L, or 3 g / L. The 15-25 g / L may be, for example, 15 g / L, 16 g / L, 18 g / L, 20 g / L, 22 g / L, 24 g / L, or 25 g / L. The 0.5-2 g / L may be, for example, 0.5 g / L, 1 g / L, 1.5 g / L, or 2 g / L. The 1-3 g / L may be, for example, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, or 3 g / L. The 4-6 g / L may be, for example, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, or 6 g / L. The 0.05-0.2 g / L may be, for example, 0.05 g / L, 0.1 g / L, 0.15 g / L, or 0.2 g / L. The 0.1-0.2 g / L may be, for example, 0.1 g / L, 0.12 g / L, 0.14 g / L, 0.16 g / L, 0.18 g / L or 0.2 g / L, etc. The 38-42 g / L may be, for example, 38 g / L, 39 g / L, 40 g / L, 41 g / L or 42 g / L, etc.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects:

[0030] The present invention introduces a mutation from D to N at position 139 of the AcrR protein amino acid sequence, labeled acrRD139N, into the L-tryptophan-producing strain XJFF-151106S. The resulting strain produces a moderate L-tryptophan yield. The recombinant strain produces 3.78 g / L of tryptophan, a 10.2% increase over the wild-type strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Construct a schematic for recombinant cells.

[0032] Figure 2 This is the verification result of acrR gene knockout.

[0033] Figure 3 This is the verification result of the recombinant strain. DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0035] Various instruments, consumables, and reagents not specifically described in the examples of the present invention are conventional commercial products in the art and can be obtained through commercial channels. The specific experimental materials and their sources listed in the following examples are merely illustrative and are not intended to limit the present invention. Materials of the same or similar type, model, quality, properties, or functions as the following reagents and instruments can be used to practice the present invention.

[0036] The Escherichia coli XJFF-151106S in the following examples was deposited in the China Center for Type Culture Collection on November 17, 2015, with the deposit number CGMCC No. 11674. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0037] Example 1

[0038] This example is used to prepare recombinant cells

[0039] In the Escherichia coli multidrug efflux pump AcrAB-TolC system, the genes for AcrA and AcrB proteins are located on the same operon, such as Figure 1As shown, acrR, a regulatory gene, exists upstream of acrA. The acrR gene encodes the AcrR protein, a transcriptional repressor located 141 bp upstream of the acrAB operon, in the opposite direction of acrAB transcription. It binds to the promoter site between acrR and acrA, repressing both its own expression and that of acrAB, thereby inhibiting the expression of the AcrAB-TolC system. Mutations in the acrR gene increase acrAB expression, enhancing the activity of the AcrAB-TolC system and further improving the robustness of the strain during acid production.

[0040] By mutating the acrR gene, the present invention further promotes the system's excretion of various substrates (including antibiotics, toxins, dyes and metabolites) in Escherichia coli, thereby helping the bacteria resist external pressure and maintain the stability of the intracellular environment, improving the robustness of the strain in the acid production process, thereby promoting the accumulation of L-tryptophan and increasing production.

[0041] (1) Preparation of acrR knockout strain

[0042] Competent E. coli cells were prepared, and the pKD46 plasmid was transformed into competent E. coli XJFF-151106S cells using arabinose stock solution. The cells were cultured and screened in a resistance medium containing ampicillin. Homologous arms were selected at both ends of the target gene, and the homologous arm primers Pkan-F / Pkan-R of the kanamycin resistance gene kan were designed using plasmid PKD13 as a template for PCR amplification. The purified PCR product was transformed into a strain containing the pKD46 plasmid by electroporation to obtain the pKD46-kan strain. Homologous recombination occurred during cultivation at 30°C to obtain a homologous recombinant strain. The pKD46 temperature-sensitive plasmid was removed by cultivation at 37°C, and then the pCP20 plasmid was introduced and transformed. PCR amplification was verified using the PpCP20-F / PpCP20-R primer pair to express the flippase recombinase gene, promote homologous recombination at the FRT site itself, and ultimately achieve acrR gene knockout. The strain was cultured simultaneously in LB medium and kanamycin resistance medium. The strain that grew normally in LB medium but did not grow in resistance medium was the strain with successful acrR gene knockout. Finally, the pCP20 temperature-sensitive plasmid was removed by cultivation at 42°C, and PCR was performed using the primer pair PacrR-F / PacrR-R to verify whether the target gene was successfully removed. The primers PacrR-F-2 / PacrR-R-2 were used for PCR verification. Figure 2 It can be seen that the acrR gene has been successfully knocked out, and an acrR knockout strain was obtained, named XJFF-151106S-△acrR. Figure 2Lane M is a DNA marker, lane E1 is the acrR gene amplification result, and lane E2 is the acrR gene knockout verification result.

[0043] (2) Preparation of acrR D139N mutant strain

[0044] Using the mutant sequence of acrR D139N SEQ ID NO.2 as a template, homology arm primers PacrR-F-1 / PacrR-R-1 were designed to construct a PCR amplification product of the exogenous acrR D139N gene. The PCR amplification product was transformed into Escherichia coli XJFF-151106S-△acrR, and the target region was replaced by homologous recombination. The specific implementation method was the same as above, and the knock-in of the acrR D139N mutant gene was finally achieved. The recombinant strain was verified by PCR using the primer pair PacrR-F-2 / PacrR-R-2. Figure 3 It can be seen that the acrR D139N gene has been successfully knocked in and sequenced. The final sequencing result is shown in SEQ ID NO: 4, that is, the acrR D139N mutant strain was successfully obtained and named XJFF-151106S-acrRD139N. The nucleic acid sequences of the primers are shown in Table 1. Figure 3 In the figure, lane M is a DNA marker, lane E1 is the amplification result of the XJFF-151106S-△acrR strain, and lane E2 is the acrR D139N gene knock-in verification result.

[0045] Table 1

[0046]

[0047]

[0048] Example 2

[0049] This example tests the production of L-tryptophan in the recombinant strain.

[0050] The strains XJFF-151106S-△acrR (gene knockout group), XJFF-151106S-acrRD139N (gene mutation group) and XJFF-151106S (blank control) were respectively inoculated into seed culture medium for culture to obtain seed solution, wherein the seed culture medium contained 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).

[0051] The above strains were inoculated into LB solid medium and cultured overnight. Each strain was then inoculated into a 250 mL baffled flask containing 25 mL of fermentation medium and cultured at 37°C with shaking at 200 rpm for 22 hours. The fermentation medium consisted of 60 g / L glucose, 1 g / L yeast extract, 5 g / L KH2PO4, 2 g / L sodium citrate, 2 g / L MgSO4·7H2O, 5 g / L (NH4)2SO4, 0.1 g / L MnSO4·H2O, 0.1 g / L FeSO4·7H2O, 0.1 g / L ZnSO4·H2O, 0.1 g / L CoCl2·6H2O, 0.03 g / L CuSO4·5H2O, and 20 g / L CaCO3.

[0052] After the culture was completed, the L-tryptophan content in the fermentation broth was measured using an amino acid analyzer. The specific test results are shown in Table 2. The yield and conversion rate for each strain are the average of three results. The conversion rate was calculated as follows: Conversion rate = amino acid produced (g / L) / (glucose input (g / L) - residual sugar (g / L)) × 100%.

[0053] Table 2

[0054]

[0055]

[0056] The results showed that after the 139th amino acid aspartic acid in the amino acid sequence of the acrR gene was replaced by aspartyl, the L-tryptophan production of the strain was significantly improved, by 10.2% compared with the original strain. This indicates that during the L-tryptophan production process, the acrR gene mutation increases the expression level of acrAB, enhances the activity of the AcrAB-TolC system, further improves the robustness of the strain during acid production, and thus increases the L-tryptophan production.

[0057] In summary, the present invention significantly increases L-tryptophan production by mutating the 139th amino acid in the amino acid sequence of the AcrR protein from D to N, labeled as acrR D139N, and introducing the mutant gene into the L-tryptophan-producing strain XJFF-151106S.

[0058] 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. A mutant AcrR protein, characterized in that: The mutant AcrR protein is an AcrR protein with a mutation, and the mutation includes D139N.

2. The mutant AcrR protein according to claim 1, characterized in that The amino acid sequence of the mutant AcrR protein includes the sequence shown in SEQ ID NO.

1.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule contains the coding sequence of the mutant AcrR protein according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleic acid sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO.

2.

5. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 3 or 4; and after transfection, transduction or transformation of a host cell, the expression vector enables the host cell to express the mutant AcrR protein according to claim 1 or 2.

6. A recombinant cell, characterized in that The recombinant cell contains the nucleic acid molecule according to claim 3 or 4 and / or the recombinant vector according to claim 5.

7. Use of the mutant AcrR protein according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the recombinant vector according to claim 5, or the recombinant cell according to claim 6 for increasing L-tryptophan production.

8. A method for increasing L-tryptophan production, characterized in that: The mutant AcrR protein according to claim 1 or 2 replaces the AcrR protein in the host cell, and the recombinant cell is cultured to obtain L-tryptophan.

9. The method for increasing L-tryptophan production according to claim 8, wherein The replacement method includes any one of suicide plasmid homologous recombination method, λ-Red homologous recombination method or CRISPR-Cas9 gene editing technology. Preferably, the host cell comprises any one or a combination of at least two of Escherichia coli, Corynebacterium glutamicum or Bacillus subtilis.

10. The method for increasing L-tryptophan production according to claim 8 or 9, wherein: The culture medium includes 55-65 g / L of glucose, 2-3 g / L of yeast extract, 15-25 g / L of ammonium sulfate, 0.5-2 g / L of magnesium sulfate, 1-3 g / L of potassium dihydrogen phosphate, 4-6 g / L of sodium citrate, 0.5-2 g / L of sodium chloride, 0.05-0.2 g / L of L-tyrosine, 0.1-0.2 g / L of L-phenylalanine and 38-42 g / L of calcium carbonate.

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