A transcriptional regulatory factor LysG mutant and its application

Abstract

The present invention takes a novel transcriptional regulatory factor LysG mutant and a promoter regulated by it as research objects, and particularly relates to the advantages and applications of the mutant in regulating gene expression and high-throughput screening in metabolic engineering, and belongs to the field of metabolic engineering. The present invention obtains LysG by performing point mutation on LysG. E15D , A54D , I164V The mutant, under the regulation of histidine, increased the expression intensity of downstream genes by 7.5 times compared with the wild type, that is, the concentration of histidine can be used to regulate downstream genes with higher intensity, so it can be better used for metabolic engineering regulation and high-throughput screening of histidine-producing strains.

Description

Technical field:

[0001] The present invention takes a novel transcriptional regulatory factor LysG mutant and a promoter regulated thereby as research objects, and particularly relates to the advantages and applications of the mutant in regulating gene expression and high-throughput screening in metabolic engineering, and belongs to the field of metabolic engineering. Background technology:

[0002] Transcriptional regulatory factors are widely present in organisms, one of which can be induced by specific small molecule metabolites in cells. Under different concentrations of small molecule metabolites, they can change their own structure and bind to specific DNA fragments in the promoter part of the target gene to regulate the transcription level of the target gene element. If the target gene element can also affect specific traits of the organism (such as fluorescence intensity, cell growth status, and the switch of specific metabolic pathways, etc.), then the concentration of small molecule metabolites can be used to regulate specific traits of the organism. If the target gene is a related enzyme or related gene in the metabolic pathway, the strength of the metabolic pathway can be controlled by the expression of transcriptional regulatory factors, and it can be applied to metabolic engineering transformation and other aspects; if the target gene is a fluorescent protein, resistance gene, etc., then the concentration of small molecule metabolites can be coupled with the intensity of fluorescence and the growth level of cells in the presence of antibiotics, so as to construct a biosensor that can respond to the concentration of small molecule metabolites, screen high-yield strains of small molecule metabolites, and apply it to high-throughput screening of strains and other aspects.

[0003] Studies have shown that transcriptional regulatory factors LysG and ArgP are both small molecule inducible proteins, and are homologous members of the LysR type transcriptional regulatory protein family. They are from Corynebacterium glutamicum and Escherichia coli, respectively. LysG can regulate the transcription of LysE under the induction of lysine, arginine or histidine. ArgP can only be induced by arginine to regulate the transcription of the downstream gene element ArgO. Studies have shown that after introducing LysG and its upstream regulatory sequence of LysE into Escherichia coli, the regulatory effects of ArgP and LysG do not affect each other, and the expression of LysE and ArgO cannot be mutually regulated. Therefore, after introducing LysG from Corynebacterium glutamicum and its regulated nucleic acid sequence into Escherichia coli, it is possible to construct a gene induced by lysine, arginine or histidine for metabolic regulation or high-throughput screening of related Escherichia coli.

[0004] In Corynebacterium glutamicum, a transcriptional regulator LysG was used to regulate the transcription of the target gene LysE in response to the induction of lysine, arginine and histidine. lysE, and a yellow fluorescent protein biosensor suitable for intracellular lysine detection, which uses the intensity of fluorescence to couple the concentration of lysine. Therefore, the biosensor is combined with a flow cytometer to perform high-throughput screening on a genomic mutation library, and mutants with increased yields are isolated, thereby proving the feasibility of a lysine biosensor based on the transcriptional regulator LysG in high-throughput screening (Binder et al. Genome Biology 2012, 13: R40). After lysG is regulated by lysine, arginine or histidine, it can induce P lysE Initiation of transcription, increasing P lysE The expression of downstream genes is induced by a certain concentration of lysine, arginine or histidine. lysE The higher the level of downstream gene expression, the higher the concentration of lysine, arginine or histidine in the system. Therefore, screening out lysG that can be efficiently regulated by lysine, arginine or histidine is of great significance for related metabolic engineering regulation and high-throughput screening. Summary of the invention:

[0005] In order to solve the above problems, the present invention screened out a lysG mutant with a higher degree of response to histidine by mutating the transcriptional regulatory factor lysG, so as to play a more active role in the high-throughput screening of histidine-producing strains.

[0006] One of the technical solutions provided by the present invention is a mutant of the transcriptional regulatory factor lysG, which is obtained by mutation of E15D, A54D and I164V on the basis of the wild-type lysG, and has the amino acid sequence shown in SEQ ID NO.1, and is named lysG E15D,A54D,I164V , relative to wild-type lysG, lysG E15D,A54D,I164V Histidine-induced increase in P lysE The initial transcription level is higher, and the expression of downstream genes can be more efficiently regulated by regulating the concentration of histidine.

[0007] The second technical solution provided by the present invention is lysG E15D,A54D,I164V The coding gene has the nucleotide sequence shown in SEQ ID NO.2.

[0008] The third technical solution provided by the present invention is the transcriptional regulatory factor lysG E15D,A54D,I164V The invention relates to an application of the present invention, in particular to an application in detecting histidine or screening histidine-producing strains, and more particularly to an application in constructing a biosensor for detecting histidine.

[0009] The fourth technical solution mentioned in the present invention is a biosensor, wherein the biosensor comprises lysG E15D,A54D,I164V The promoter of the gene encoding LysE lysE , and indicator genes;

[0010] Furthermore, the promoter P lysE , having the nucleotide sequence shown in SEQ ID NO.3;

[0011] Furthermore, the indicator gene includes but is not limited to a fluorescent protein encoding gene, a resistance gene, etc.;

[0012] Furthermore, the fluorescent protein includes but is not limited to yellow fluorescent protein, green fluorescent protein, red fluorescent protein, etc.;

[0013] Furthermore, the resistance gene includes but is not limited to tetracycline resistance gene, chloramphenicol resistance gene, streptomycin resistance gene, etc.;

[0014] Preferably, the biosensor comprises lysG E15D,A54D,I164V The promoter of the gene encoding LysE lysE , and tetracycline resistance gene, and expressed the above genes on plasmid pSB4K5;

[0015] Furthermore, the tetracycline resistance gene has a nucleotide sequence shown in SEQ ID NO.4.

[0016] The fifth technical solution mentioned in the present invention is the application of the biosensor described in the fourth technical solution, especially in the detection of histidine or in the screening of high-histidine producing strains.

[0017] The following definitions are used in the present invention:

[0018] 1. Nomenclature of amino acid and DNA sequences

[0019] The generally accepted IUPAC nomenclature for amino acid residues is used, either as a single letter or three letter code. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0020] 2. Identification of lysG mutants

[0021] The amino acid mutated in the lysG mutant is represented by "amino acid replaced by the original amino acid position". For example, E15D means that the amino acid at position 15 is replaced by D from E of wild-type lysG, and the position number corresponds to the amino acid sequence number of wild-type lysG in SEQ ID NO.5. The specific information is as follows:

[0022]

[0023] Beneficial effects:

[0024] The present invention obtains lysG by performing point mutation on LysG E15D,A54D,I164V The mutant, under the regulation of histidine, increased the expression intensity of downstream genes by 7.5 times compared with the wild type, that is, the concentration of histidine can be used to regulate downstream genes with higher intensity, so it can be better used for metabolic engineering regulation and high-throughput screening of histidine-producing strains. Description of the drawings:

[0025] Figure 1 Plasmid P1.

[0026] Figure 2 Plasmid P2.

[0027] Figure 3 Growth level of strain A on solid plates.

[0028] Figure 4 Growth level of strain B on solid plates. Specific implementation method:

[0029] The technical content of the present invention is further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0030] The transcriptional regulatory factor LysG can be induced by lysine, arginine or histidine. In the present application, point mutation of LysG is performed to obtain a mutant lysG E15D,A54D,I164V , regulating P lysE The initiation of transcription and the tetracycline resistance gene were coupled downstream. After the relevant genes were transformed into Escherichia coli strains, solid plates and liquid cultures were used for testing. It was found that after adding histidine, the lysG E15D,A54D,I164V The strains showed significantly enhanced resistance to tetracycline, indicating that lysG E15D,A54D,I164V The ability to respond to histidine was significantly improved. E15D,A54D,I164V A biosensor was constructed to screen mutants for their ability to produce histidine. E15D,A54D,I164V The sensor screening was more sensitive and positive for histidine, thus proving that lysG E15D,A54D,I164V Superiority in high-throughput screening of histidine-producing strains.

[0031] Mutant lysG E15D,A54D,I164V Having the amino acid sequence shown in SEQ ID NO.1:

[0032] MNPIQLDTLLSIIDDGSFEGASLALSISPSAVSQRVKALEHHVGRVLVSRTQPDK

[0033] ATEAGEVLVQAARKMVLLQAETKAQLSGRLAEIPLTIAINADSLSTWFPPVFN

[0034] EVASWGGATLTLRLEDEAHTLSLLRRGDVLGAVTREANPVAGCEVVELGTMR

[0035] HLAVATPSLRDAYMVDGKLDWAAMPVLRFGPKDVLQDRDLDGRVDGPVGRR

[0036] RVSIVPSAEGFGEAIRRGLGWGLLPETQAAPMLKAGEVILLDEIPIDTPMYWQ

[0037] RWRLESRSLARLTDAVVDAAIEGLRP*

[0038] The main culture media and antibiotics used in the embodiments of the present invention are as follows:

[0039] (1) LB liquid medium (g / L): 10% tryptone, 10% sodium chloride, 5% yeast powder, and the rest water.

[0040] (2) Fermentation medium (g / L): 40% glucose, 1% magnesium sulfate heptahydrate, 10% ammonium sulfate, 3% potassium dihydrogen phosphate, 0.01% ferrous sulfate heptahydrate, 0.01% manganese sulfate monohydrate, 10% yeast extract, 10% tryptone, and the rest water. 3 ·H 2 O adjusted pH = 7.

[0041] (3) Kanamycin was used at a concentration of 25 mg / L, and tetracycline was prepared into a 10 mg / mL stock solution and added in different amounts as needed.

[0042] The present invention will be further described below by means of specific examples.

[0043] Example 1: Construction of plasmid P1

[0044] First, plasmid pSB4K5-I52002 was used as a template (GenBank: EU496099.1) and primers carrier-F and carrier-R in Table 1 were used to PCR amplify the pSB4K5 linear vector fragment; the promoter P containing LysG and LysE was amplified using the genome of Corynebacterium glutamicum 13032 (NCBI accession number: CP025533.1) as a template and primers lysG-F and lysG-R. lysE LysG-P lysE(Also available according to LysG and P lysE The pDM1 plasmid (NCBI accession number: MN128719.1) was used as a template and the primers tet-F and tet-R were used to amplify the tetracycline resistance fragment by PCR. The PCR product was purified and recovered, and then the vector pSB4K5 linear fragment and LysG-P were cloned using seamless cloning enzyme (pEASY-Uni Seamless Cloning and Assembly Kit (Qianshijin)). lysE The tetracycline-resistant fragments were homologously recombined and transformed into E. coli DH5α competent cells, spread on LB plates containing 25 mg / L kanamycin, and cultured at 37°C, 12 h, 220 rpm. The transformants were selected for sequencing verification to obtain the recombinant plasmid pSB4K5-lysG-P containing the correct fragments. lysE -tet, referred to as plasmid P1, plasmid map as Figure 1 shown.

[0045] Table 1 Construction of plasmid pSB4K5-lysG-P lysE -tet primers used

[0046] Primer name Primer sequence 5'-3' carrier-F CGGGCCACCTCGACCTGAAACCCTTCTCACCTCGGC carrier-R TCGAGGGATTGCGGCCTTAGGCCTGGGGTGCCTAATGA lysG-F CTAAGGCCGCAATCCCTCGA lysG-R TCAGGTCGAGGTGGCCCG tet-F ATGAAATCTAACAATGCGCT tet-R TCAGGTCGAGGTGGCCCG

[0047] Example 2: Construction of plasmid P2

[0048] Design the mutation primers DTB1-F and DTB1-R for the E15D site on lysG; the mutation primers DTB2-F and DTB2-R for the A54D site; and the mutation primers DTB3-F and DTB3-R for the I164V site. Use plasmid P1 as a template for mutation to obtain a plasmid expressing lysG containing three mutation sites: E15D, A54D, and I164V.

[0049] The details are as follows:

[0050] The plasmid P1 was used as a template, and the primers DTB1-F and DTB1-R in Table 2 were used for PCR amplification and purification and recovery. The recovered product was homologously recombined by seamless cloning enzyme (pEASY-Uni Seamless Cloning and Assembly Kit (All-in-one Gold), transformed into E. coli DH5α competent cells, and spread on LB plates containing 25 mg / L kanamycin, and cultured at 37°C, 12 h, 220 rpm to obtain the plasmid pSB4K5-lysG expressing lysG containing the E15D mutation site. E15D -P lysE-tet. Using the above mutant plasmid as a template, primers DTB2-F and DTB2-R were used for amplification, fragment purification and recovery, seamless cloning enzyme connection, and the correct transformants were selected after transformation of competent cells to obtain the plasmid pSB4K5-lysG expressing lysG containing the E15D and A54D double mutation sites. E15D,A54D -P lysE -tet. The above mutant plasmid was used as a template, and primers DTB3-F and DTB3-R were used for amplification, fragment purification and recovery, seamless cloning enzyme connection, and the correct transformants were selected after transformation of competent cells to obtain the plasmid pSB4K5-lysG expressing lysG containing three mutation sites E15D, A54D and I164V. E15D,A54D,I164V -P lysE -tet, referred to as plasmid P2, plasmid map as Figure 2 shown.

[0051] Table 2 Construction of plasmid pSB4K5-lysG E15D,A54D,I164V -P lysE -tet primers used

[0052] Primer name Primer sequence 5'-3' DTB1-F GAAGCTGCCATCATCAATGAT DTB1-R ATCATTGATGATGGCAGCTTC DTB2-F GTTGCTTTGTCCGGTTGGGT DTB2-R ACCCAACCGGACAAAGCAAC DTB3-F GGGTTGCAACGGCCAAGTGG DTB3-R CCACTTGGCCGTTGCAACCC

[0053] Example 3: Testing the growth level of strain A using solid plates

[0054] The constructed plasmid P1 was introduced into the wild-type strain E. coli MG1655 to obtain strain A. Strain A was inoculated into an LB test tube containing 25 mg / L kanamycin and cultured at 37°C and 220 rpm until it grew to OD 600 Between 1.4 and 1.6, diluted 10 -5 , 100 μL was spread on a solid LB medium plate containing different concentrations of tetracycline (5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL) and 5 g / L histidine. At the same time, a plate without histidine was used as a control, and the growth status was observed after 60 hours of culture.

[0055] Growth on plates without histidine is as follows Figure 3 -A. As shown in the figure, at a tetracycline concentration of 5 μg / mL, the strain can grow significantly, and when the tetracycline concentration is higher than 10 μg / mL, the strain cannot grow.

[0056] The growth condition of the plate containing 5g / L histidine is as follows Figure 3 -B. As shown in the figure, the strain can grow significantly on the plate containing 5μg / mL or 10μg / mL tetracycline, and the strain cannot grow when the tetracycline concentration is higher than 15μg / mL.

[0057] This indicates that under the experimental test conditions, 5 g / L histidine can induce the expression of tetracycline resistance genes in strain A, increasing the strain's resistance concentration to tetracycline from 5 μg / mL to 10 μg / mL.

[0058] Example 4: Testing the growth level of strain B using solid plates

[0059] The constructed plasmid P2 was introduced into the wild-type strain MG1655 to obtain strain B. The same method as in Example 3 was used to test the change in the resistance level of strain B to tetracycline after the strain B was induced by histidine to express the tetracycline resistance gene.

[0060] After the strain B culture was diluted, it was spread on solid LB medium plates supplemented with 5μg / mL, 10μg / mL, 15μg / mL, 20μg / mL, and 25μg / mL tetracycline, and the growth was observed after 60 hours of culture. Figure 4 -A, it can be seen from the figure that at a tetracycline concentration of 5 μg / mL, strain B can grow significantly, and when the tetracycline concentration is higher than 10 μg / mL, strain B cannot grow.

[0061] After the strain B culture was diluted, it was spread on solid LB medium plates supplemented with 25μg / mL, 50μg / mL, 75μg / mL, 100μg / mL, and 125μg / mL tetracycline and 5g / L histidine, and the growth was observed after 60 hours of culture. Figure 4 -B, it can be seen from the figure that the strain can grow significantly on the plate containing 25μg / mL, 50μg / mL or 75μg / mL tetracycline, and when the tetracycline concentration is higher than 100μg / mL, the strain cannot grow.

[0062] The above results show that under the experimental test conditions, 5g / L histidine can induce the expression of the tetracycline resistance gene of strain B, increasing the strain's resistance to tetracycline from 5μg / mL to 75μg / mL. Compared with strain A, strain B's resistance to tetracycline concentration increased by 7.5 times.

[0063] The experimental results show that lysG mutates to lysG E15D,A54D,I164V Later, under the induction of histidine, the expression level of its downstream genes increased significantly. In metabolic engineering, it is more conducive to regulating the higher intensity expression of downstream genes; in high-throughput screening, through the huge differences in signals such as resistance, it is easier to distinguish strains with different production levels, thereby improving the efficiency of high-throughput screening.

[0064] Example 5: Testing the growth level of strains A and B using liquid deep well plates

[0065] In order to conduct a secondary verification of strains A and B, the liquid deep-well plate method was used to measure the OD using an enzyme-labeled analyzer. 600 The detection and analysis were conducted to test the changes in the resistance levels of strains A and B to tetracycline after they were induced by histidine to express the tetracycline resistance gene in liquid fermentation.

[0066] After culturing, strains A and B were inoculated into liquid LB medium with 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL tetracycline, and with or without 5 g / L histidine, respectively. The growth conditions after culturing for 24 hours are shown in Table 3. It can be seen that when histidine is not added, when the tetracycline concentration in the liquid LB medium is higher than 20 μg / mL, strains A and B cannot grow; when histidine is added, when the tetracycline concentration is higher than 20 μg / mL, strain A still cannot grow, but when the tetracycline concentration contains 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL, strain B can grow significantly.

[0067] The experiment confirmed that under the conditions tested, the expression of lysG E15D,A54D,I164V The strain B can express more antibiotic genes than the strain A expressing wild-type lysG, thus having more obvious growth. This will greatly facilitate the screening of histidine-producing strains.

[0068] Table 3 Growth levels of strains A and B in liquid deep-well plates

[0069]

[0070] The tetracycline resistance gene used in this application is only used to verify lysG and its mutant lysG E15D ,A54D,I164V This is an example of histidine-induced expression. Researchers with relevant knowledge background can easily know that this induction is independent of the gene being tested. Therefore, in practical applications such as metabolic engineering and high-throughput screening, the tetracycline resistance gene can be replaced with other genes according to specific needs, such as chloramphenicol resistance gene, streptomycin resistance gene, etc., which also has the same effect.

[0071] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A mutant of a transcriptional regulatory factor lysG, characterized in that: It is obtained by mutation of E15D, A54D and I164V on the wild-type lysG, has the amino acid sequence shown in SEQ ID NO.1, and is named as transcriptional regulatory factor lysG E15D,A54D,I164V .

2. The transcriptional regulatory factor lysG according to claim 1 E15D,A54D,I164V coding genes.

3. The coding gene according to claim 2, characterized in that It has the nucleotide sequence shown in SEQ ID NO.

2.

4. The transcriptional regulatory factor lysG according to claim 1 E15D,A54D,I164V Or the use of the encoding gene according to claim 2, characterized in that, The invention is used in detecting histidine or screening histidine-producing strains.

5. The use according to claim 4, characterized in that It is used in the construction of biosensors for detecting histidine.

6. A biosensor, characterized in that: The biosensor comprises the lysG according to claim 2 E15D ,A54D,I164V The promoter of the gene encoding LysE lysE , and indicator genes; The indicator gene is a fluorescent protein encoding gene or a resistance gene; The fluorescent protein is yellow fluorescent protein, green fluorescent protein, or red fluorescent protein; The resistance gene is a tetracycline resistance gene, a chloramphenicol resistance gene, or a streptomycin resistance gene.

7. A biosensor according to claim 6, characterized in that: The biosensor comprises lysG E15D,A54D,I164V The promoter of the gene encoding LysE lysE , and tetracycline resistance gene, and expressing the above genes on plasmid pSB4K5; The promoter P lysE , having the nucleotide sequence shown in SEQ ID NO.3; The tetracycline resistance gene has a nucleotide sequence shown in SEQ ID NO.

4.

8. Use of the biosensor according to any one of claims 6 to 7, characterized in that: It is used in the detection of histidine or in the screening of high-histidine-producing strains.

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