Mutant screening of thermus butyricus transcriptional regulatory protein trpR and its application

Abstract

The application discloses a thermus butyricum transcriptional regulatory protein TrpR mutant screening and application, and belongs to the technical field of genetic engineering. Through screening of the TrpR mutant, favorable modification of the thermus butyricum transcriptional regulatory protein TrpR is realized, so that the specificity of the response of the thermus butyricum transcriptional regulatory protein TrpR to 5,6-dihydroxyindole is enhanced, a special biosensor is constructed by combining with a fluorescent reporter gene, effective detection of the 5,6-dihydroxyindole content is realized, and the efficiency and purity of extraction of a target component are ensured, thereby laying a solid foundation for subsequent application and research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to screening of a mutant of a transcriptional regulator protein TrpR of a thermophilic bacterium and application thereof. BACKGROUND

[0002] 5,6-dihydroxyindole, as a natural organic compound, shows great potential in the development of green and safe hair dyes. Traditional chemical hair dyes contain many irritating or harmful chemicals, while 5,6-dihydroxyindole, as a natural ingredient, not only can provide coloring effects comparable to traditional chemical dyes, but also can reduce irritation to the scalp and hair, and reduce the impact of chemicals on the environment. In addition, 5,6-dihydroxyindole also has good biocompatibility and safety, making it an ideal choice for developing green and safe hair dye products.

[0003] Currently, the methods for detecting the content of 5,6-dihydroxyindole in the market mostly rely on traditional chemical analysis techniques, such as high performance liquid chromatography (HPLC) and mass spectrometry. Although these methods are accurate, they usually require expensive equipment and professional operators, and the process is cumbersome, which is not suitable for rapid on-site detection.

[0004] Due to these limitations, it is particularly important to develop a new detection method that is both rapid and sensitive. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a mutant of a transcriptional regulator protein TrpR of a thermophilic bacterium and application thereof in detecting the content of 5,6-dihydroxyindole, aiming to solve the technical problem that Lactobacillus plantarum cannot be directly used to produce 5,6-dihydroxyindole, and the yield of 5,6-dihydroxyindole produced by biological conversion is low, which limits the industrial production thereof.

[0006] The first technical solution provided by the present application is a mutant of a transcriptional regulator protein, which is a mutant of the 36th tyrosine, the 37th lysine, the 38th alanine, and the 39th leucine of a parent transcriptional regulator protein TrpR, and the amino acid sequence of the parent is shown in SEQ ID NO: 1.

[0007] The parent is derived from a thermophilic bacterium, and the nucleotide sequence of the parent gene is shown in SEQ ID NO: 2.

[0008] In some embodiments, the mutant is a mutant of the 36th tyrosine Y of the parent to lysine K, the 37th lysine K to phenylalanine F, the 38th alanine A to glycine G, and the 39th leucine L to proline P, and the amino acid sequence of the mutant is shown in SEQ ID NO: 3.

[0009] In some embodiments, the nucleotide sequence encoding the mutant gene is shown as SEQ ID NO: 4.

[0010] The second technical solution provided by the present application is a gene encoding the mutant of the first technical solution.

[0011] In some embodiments, the nucleotide sequence encoding the mutant gene is shown as SEQ ID NO: 4.

[0012] The third technical solution provided by the present application is an expression vector carrying the gene of the second technical solution.

[0013] In some embodiments, the expression vector comprises a pBAD / HisA vector.

[0014] The fourth technical solution provided by the present application is a genetically engineered bacterium expressing the mutant of the first technical solution, or containing the gene of the second technical solution, or transformed with the expression vector of the third technical solution.

[0015] In some embodiments, the genetically engineered bacterium takes E. coli as a starting strain.

[0016] Preferably, the starting strain comprises E. coli MC1061.

[0017] The fifth technical solution provided by the present application is an indicator plasmid, which is an expression vector containing a gene encoding the mutant of the first technical solution and a fluorescent reporter gene, the fluorescent reporter gene being located downstream of the mutant gene, the mutant binding being released from the transcriptional inhibition of the fluorescent reporter gene by 5,6-dihydroxyindole, and the indicator plasmid being capable of regulating the transcription intensity of the fluorescent reporter gene by sensing the concentration of 5,6-dihydroxyindole.

[0018] The sixth technical solution provided by the present application is an indicator strain, which is E. coli containing the indicator plasmid of the fifth technical solution, and is capable of responding to the concentration of 5,6-dihydroxyindole in the environment by the expression intensity of fluorescence.

[0019] The seventh technical solution provided by the present application is a method for detecting 5,6-dihydroxyindole, which uses the expression intensity of fluorescence of the indicator strain of the sixth technical solution to characterize the content level of 5,6-dihydroxyindole in the sample to be detected.

[0020] The eighth technical solution provided by the application is the application of the mutant of the first technical solution, or the indicator plasmid of the fifth technical solution, or the indicator strain of the sixth technical solution, or the method of the seventh technical solution in the construction of a biosensor responding to 5,6-dihydroxyindole.

[0021] The technical effects of the application are as follows:

[0022] Through screening of the TrpR mutant, the transcriptional regulatory protein TrpR of the butyric acid thermophilic bacteria is favorably modified, so that the specificity of the response to 5,6-dihydroxyindole is enhanced, a special biosensor is constructed by combining with the fluorescent reporter gene to realize effective detection of the content of 5,6-dihydroxyindole, and the efficiency and purity of the extraction of the target component are ensured, thereby laying a solid foundation for subsequent application and research. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a pTrpR expression plasmid map.

[0024] Figure 2 It is a performance comparison of the wild-type TrpR and the mutant Trp-M.

[0025] Figure 3 It is the induction specificity of the whole-cell biosensor specifically responding to 5,6-dihydroxyindole. DETAILED DESCRIPTION

[0026] The preferred embodiments of the application are described below, and it should be understood that the embodiments are used to better explain the application and are not used to limit the application.

[0027] The raw materials used in the embodiments are as follows:

[0028] 1. The pBAD / HisA and DsRed vectors are derived from Invitrogen.

[0029] 2. The Escherichia coli MC1061 is purchased from Genview.

[0030] 3. The formula of the LB culture medium is as follows: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and pH is adjusted to 7.4.

[0031] Example 1: Obtaining of the TrpR mutant

[0032] I. Construction of the wild-type recombinant plasmid

[0033] PCR amplification was performed with pBAD / HisA vector as template, 5'-CGGTACCCGGGGATCCACTA-3' (forward primer, SEQ ID NO: 5) and 5'-CTCGAGCTCGGATCCCCATC-3' (reverse primer, SEQ ID NO: 6) as primers. The genome of Butyricimonas virescens (GenBank: CP000493.1) was extracted as template, 5'-GATGGGGATCCGAGCTCGAGATGCCCTCCGTTAACGACTG-3' (forward primer, SEQ ID NO: 7) and 5'-TAGCTGTACTCTTCGAGCTCTTACTTGAGTCCAAGCTTTT-3' (reverse primer, SEQ ID NO: 8) as primers to amplify the trpR gene (SEQ ID NO: 2), and DsRed plasmid (GenBank: MZ332518.1) as template, 5'-CGGCGCTCAGTTGGAGATCTCTACAGGAACAGGTGGTGGC-3' (forward primer, SEQ ID NO: 9) and 5'-TAGTGGATCCCCGGGTACCGATGGCCTCCTCCGAGGACGT-3' (reverse primer, SEQ ID NO: 10) as primers to amplify the rfp gene (GenBank: QWX95862.1). The PCR system was as follows: 0.2 μM of each primer, 0.2 mM of dNTPs, 10 ng of template, 5 μL of 2x Buffer, 0.5 μL of Hieff, and deionized water to make up the volume to 100 μL. The PCR reaction conditions were as follows: 94 °C for 20 s, 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min, 4 °C for 5 min, and 2-5 cycles of the above steps for 30 cycles. The obtained PCR products were connected using a Gibson Assembly kit (Anhui General Biotechnology), and the connection product was transformed to obtain a recombinant strain. The plasmid of the recombinant strain was extracted to obtain the recombinant plasmid pBAD / HisA-TrpR-RFP. The plasmid map is as follows: Figure 1 .

[0034] II. Construction of trpR error-prone mutant library

[0035] The trpR gene (SEQ ID NO: 4) was subjected to error-prone PCR using primers 5'- ATGCCCTCCGTTAACGACTG-3' (forward primer, SEQ ID NO: 11) and 5'- TTACTTGAGTCCAAGCTTTT-3' (reverse primer, SEQ ID NO: 12). The error-prone PCR system was 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 0.025 mM MnCl2, 10 x PCR Buffer 10 μL, rTaq 0.5 μL, and deionized water to a volume of 100 μL, which was then evenly divided into 10 PCR tubes. The error-prone PCR reaction conditions were 94°C for 2 min, 94°C for 30 s, 50°C for 30 s, 72°C for 1 min, 4°C for 5 min, and 30 cycles of the above procedure. The resulting PCR product was used as a template for MEGAWHOP PCR using pBAD / HisA-TtgR-RFP as a template (pTrpR expression plasmid, SEQ ID NO: 13). The reaction system was 0.2 μg of the PCR product obtained by error-prone PCR, 1 mM dNTPs, 30 ng of template pTrpR, 5 μL of 10 x Pyrobest Buffer, 0.3 μL of Pyrobest, and deionized water to a volume of 100 μL. The PCR reaction conditions were 94°C for 2 min, 94°C for 30 s, 50°C for 30 s, 72°C for 3 min, 4°C for 5 min, and 30 cycles of the above procedure. The resulting PCR product was added to DpnI (20 U) and incubated at 37°C for 2 h, and the DpnI was inactivated at 80°C for 20 min. The trpR error-prone mutant library (mutant plasmid mixture) was obtained.

[0036] III. Mutant Screening

[0037] The error-prone mutant library was screened using a flow cytometer, according to the following procedure. The plasmid of the TrpR mutant library was electroporated into MC1061 competent cells (about 10 7 After 45 min of recovery at 37°C on a shaker, the recovered liquid was added to 50 mL of LB medium containing Amp (ampicillin resistance) and 1 mM 5,6-dihydroxyindole, and incubated at 37°C for 12 h. 1 mL of the cell suspension was centrifuged at 3,000 x g for 10 min, and the supernatant was discarded and resuspended with 1 mL of PBS buffer. 200 μL of the suspension was mixed with 1.8 mL of PBS, and then added to 5 mL of a round-bottom test tube. A flow cytometer was used for sorting (excitation wavelength 561 nm, emission wavelength 610 nm), and cells with high fluorescence intensity were selected. The sorted cells were incubated in 3 mL of LB medium for 6 h, and the final OD 600= 0.2, again transferred to 3 mL LB medium containing 1 mM 5,6-dihydroxyindole for the second round of screening. After eight rounds of screening, 10,000 cells were collected in the last round of screening, and 300 μL of cell suspension was spread on Amp R plates. Single clones were picked and retested in tubes containing 0 mM, 0.01 mM, 0.1 mM, 1 mM and 10 mM 5,6-dihydroxyindole.

[0038] A mutant of the gene trpR encoding a transcriptional regulator protein specific to 5,6-dihydroxyindole, TrpR-M (SEQ ID NO: 3), was obtained, in which the 36th amino acid was mutated from tyrosine (Tyr) to lysine (Lys), the 37th amino acid was mutated from lysine (Lys) to phenylalanine (Phe), the 38th amino acid was mutated from alanine (Ala) to glycine (Gly), and the 39th amino acid was mutated from leucine (Leu) to proline (Pro), as compared with TrpR.

[0039] The wild-type TrpR and mutant Trp-M were tested with the natural inducer of TrpR protein, tryptophan, and the target inducer, 5,6-dihydroxyindole, and the performance comparison is shown as follows. Figure 2

[0040] Example 2 Construction of a biosensor responsive to rare 5,6-dihydroxyindole

[0041] The pTrpR-M plasmid containing the gene encoding TrpR-M (SEQ ID NO: 3) was transformed into the E. coli MC1061 strain to obtain a recombinant strain MC1061-TrpRM, i.e., a whole-cell biosensor capable of specifically responding to the induction of 5,6-dihydroxyindole. The induction specificity of the biosensor was detected by a multifunctional enzyme marker: single clones were selected and inoculated in 3 mL LB medium containing 100 μg / mL Amp at 37°C overnight to serve as seed liquid. The next day, the seed liquid was inoculated at a 1% inoculation amount (final OD 600 about 0.02) and 1 mM 5,6-dihydroxyindole was added at the same time. After 37°C shaking culture for 12 hours, 200 μL of cell suspension was taken and the OD 600 and RFP fluorescence intensity (excitation wavelength 556 nm, emission wavelength 586 nm) were detected by an enzyme marker. As shown in Figure 3 , the biosensor can specifically respond to the induction of 5,6-dihydroxyindole, but not to the induction of analogs such as tyrosine, dopamine, and dopamine.

[0042] ​Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is not intended to limit the present application thereto, and various modifications and alterations can be made thereto by those skilled in the art without departing from the spirit and scope of the present application, and the scope of protection of the present application should be defined by the appended claims.

Claims

1. A mutant of a transcription regulating protein, characterized in that, The mutant is a mutant of the transcriptional regulatory protein TrpR parent with the amino acid sequence shown in SEQ ID NO: 1, in which the 36th tyrosine Y is mutated to lysine K, the 37th lysine K is mutated to phenylalanine F, the 38th alanine A is mutated to glycine G, and the 39th leucine L is mutated to proline P, and the amino acid sequence of the mutant is shown in SEQ ID NO:

3.

2. A gene encoding the mutant of claim 1.

3. An expression vector carrying the gene of claim 2.

4. A genetically engineered bacterium expressing the mutant of claim 1, or containing the gene of claim 2, or transformed with the expression vector of claim 3. 5.The genetically engineered bacteria according to claim 4, characterized in that, The genetically engineered bacterium uses Escherichia coli as the starting strain.

6. A marker plasmid, characterized in that, The indicator plasmid is an expression vector containing a gene encoding the mutant of claim 1 and a fluorescent reporter gene, wherein the fluorescent reporter gene is located downstream of the mutant gene, and the mutant binds to 5,6-dihydroxyindole to relieve the transcriptional inhibition of the fluorescent reporter gene by the mutant, and the indicator plasmid can be used to sense the concentration of 5,6-dihydroxyindole to regulate the transcription intensity of the fluorescent reporter gene.

7. A bacterial strain, characterized in that, The indicator strain is Escherichia coli containing the indicator plasmid of claim 6, which can respond to the concentration of 5,6-dihydroxyindole in the environment by the intensity of fluorescent expression.

8. A method of detecting 5,6-dihydroxyindole, characterized by, The method is to use the intensity of fluorescent expression of the indicator strain of claim 7 to characterize the content level of 5,6-dihydroxyindole in the sample to be detected.

9. Use of the mutant of claim 1, or the indicator plasmid of claim 6, or the indicator strain of claim 7, or the method of claim 8 in the construction of a biosensor responsive to 5,6-dihydroxyindole.

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