CN116120409A - IMP biosensor and construction method and application thereof - Google Patents

Abstract

The invention provides an IMP biosensor and a construction method and application thereof. The biosensor contains a GlxR-MUT1 coding gene and a control sequence Ptrc promoter thereof, and a reporter gene and a control sequence Pcg3195 promoter thereof, wherein the GlxR-MUT1 transcription regulator can specifically bind to the Pcg3195 promoter region and inhibit the expression of reporter protein. The biosensor shows excellent response relation to IMP, can be used for efficiently breeding IMP high-yield strains by combining with a high-throughput screening system and the like, and has the advantages of simplicity in operation, high detection efficiency and the like.

Description

IMP biosensor and construction method and application thereof

Technical Field

The invention relates to the field of bioengineering, in particular to an IMP biosensor and a construction method and application thereof.

Background

5' -inosinic acid (hereinafter referred to as IMP), which is a nucleic acid substance, is an intermediate of a nucleic acid metabolic pathway, and is used in many fields such as foods, medicines, various medical applications, and the like. IMP and its salt can produce delicate flavour, which is one of important flavour substances in meat, and it can also cooperate with sodium glutamate to make delicate flavour increase multiple times, so it is a common flavouring agent. In addition, inosinic acid is also an internationally recognized important index for measuring the freshness of meat.

The production method of IMP includes chemical synthesis method, RNA enzymolysis method, microbial fermentation method and biocatalysis method. The microbial fermentation method is the most commonly used method for industrial production at present, and mainly utilizes the biosynthesis way of microbial strains to produce IMP. The bacterial species used are mostly bacillus subtilis (Bacillus subtilis) and corynebacterium stagnant (Corynebacterium stationis). The former is mainly used for the production of nucleosides and purine nucleotides. The latter is the main strain for industrially synthesizing nucleotide and its higher derivative by microbe. The stagnant corynebacteria are gram-positive and non-pathogenic soil bacteria, have high GC content of DNA, have strong ATP regeneration activity and have enough supply of 5-phosphoribosyl-1-pyrophosphate (PRPP). Among them, the strain of Corynebacterium parvulum ATCC6872 is an original strain for the production of nucleotides and nucleosides, and its derivative strain is widely used for the production of industrially useful nucleotides and nucleosides.

Traditional mutagenesis is the main method for breeding IMP high-producing strains, and IMP does not confer a phenotype that is easy to detect on producer cells. Traditionally, mutagenesis generates a diverse pool of mutations, and the productivity of each genetic variant must be analyzed by time-consuming, laborious and expensive analytical methods such as chromatography and mass spectrometry, which lead to inefficiency of the screening process, severely impacting the speed of strain development.

In nature, the concentration of chemical substances can often be perceived by different molecular devices, such as allosteric enzymes, transcription factors and riboswitches. Artificial biosensors developed using such devices can react to chemical signals and convert them into readily detectable signals, such as fluorescence, which can be detected by Fluorescence Activated Cell Sorting (FACS). A high throughput screening platform based on biosensors can be used to overcome screening bottlenecks, enabling searches to be made among a large number of pathway/strain variant libraries to find suitable production strains. The construction of the high-efficiency target biosensor can provide a new method for realizing the rapid evolution of strains, the dynamic regulation of metabolic pathways and the high-throughput screening of mutants. Therefore, it is necessary to build an artificial biosensor that converts IMP into a signal that is easy to detect to improve breeding efficiency.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art, provides an IMP biosensor, and establishes an IMP high-yield strain and a high-throughput screening method thereof by using the biosensor.

The invention also aims to provide a construction method of the IMP biosensor.

It is a further object of the present invention to provide the use of an IMP biosensor as described above.

The aim of the invention is achieved by the following technical scheme:

a GlxR transcription factor mutant, capable of responding to IMP, being at least one of the following mutants:

GlxR-MUT1, the D18H/R68H/N195S/P221T/L222S mutation was present relative to wild-type GlxR;

GlxR-MUT2, the presence of the I53N/E69G/F87I/R163L/L172P/E192G/R208H mutation relative to wild-type GlxR;

GlxR-MUT3, the F87I/R163L/R208H mutation is present relative to wild-type GlxR;

GlxR-MUT4, there was an M29T/N70Y/S94S/E182E/I216F mutation relative to wild-type GlxR.

The GlxR transcription factor mutant, wherein:

the amino acid sequence of the GlxR-MUT1 is shown as SEQ ID NO. 6;

the amino acid sequence of the GlxR-MUT2 is shown as SEQ ID NO. 8;

the amino acid sequence of the GlxR-MUT3 is shown as SEQ ID NO. 10;

the amino acid sequence of GlxR-MUT4 is shown in SEQ ID NO. 12.

The coding gene of the GlxR transcription factor mutant is obtained according to a codon coding rule; preferably, the nucleotide sequence of GlxR-MUT1 is shown as SEQ ID NO.7, the nucleotide sequence of GlxR-MUT2 is shown as SEQ ID NO.9, the nucleotide sequence of GlxR-MUT3 is shown as SEQ ID NO.11, and the nucleotide sequence of GlxR-MUT4 is shown as SEQ ID NO. 13.

An IMP biosensor comprising a gene encoding at least one of the GlxR transcription factor mutants of claim 1 or 2 and a promoter controlling expression of the GlxR transcription factor mutant, and an expression vector comprising a gene encoding a reporter protein and a promoter controlling expression of the reporter protein.

The promoter for controlling the expression of the GlxR transcription factor mutant is Ptrc promoter, and the nucleotide sequence of the promoter is shown as SEQ ID No. 4.

The reporter protein is fluorescent protein; EGFP protein is preferred.

The promoter for controlling the expression of the reporter protein is a Pcg3195 promoter or a Pcg3195 promoter mutant; the nucleotide sequence of the Pcg3195 promoter is shown as SEQ ID No. 3.

The Pcg3195 promoter mutant is obtained by modifying a GlxR binding site on a nucleotide sequence of a wild type boom of the Pcg3195 promoter; preferably comprises at least one of Pom, pomm and Pmm; more preferably Pmmm.

The Pcg3195 promoter mutant, wherein:

the nucleotide sequence of Pom is shown as SEQ ID NO. 14;

the nucleotide sequence of Pomm is shown as SEQ ID NO. 15;

the nucleotide sequence of Pmm is shown as SEQ ID NO. 16.

The IMP biosensor also comprises a plasmid skeleton; the pEC-T18-mob2 plasmid backbone is preferred.

The IMP biosensor has the Ptrc promoter and Pcg3195 promoter opposite in direction.

A recombinant strain contains the above IMP biosensor.

The application of the IMP biosensor in screening IMP high-yield strains.

The application comprises the following specific steps:

mutant strains are obtained through mutagenesis, IMP biosensors are transferred into the mutant strains, fermentation culture is carried out, then flow cytometry is used for sorting, and cells with reduced reporter protein signals are selected, so that the IMP high-yield strains are obtained.

A construction method of an IMP biosensor comprises the following steps:

assembling at least one coding gene of the GlxR transcription factor mutant and a promoter for controlling the expression of the coding gene, and a coding gene of a reporter protein and a promoter for controlling the expression of the coding gene in sequence to obtain an IMP biosensor; preferably, the coding gene of the GlxR transcription factor mutant, the Ptrc promoter, the EGFP coding gene, the Pcg3195 promoter and the pEC-T18-mob2 plasmid skeleton are assembled in sequence to obtain the IMP biosensor.

The assembly is carried out by utilizing a one-step method high-efficiency seamless cloning technology.

The construction method is characterized in that the direction of a promoter for controlling the expression of the GlxR transcription factor mutant is opposite to that of a promoter for controlling the expression of the marker protein.

The carrier containing the GlxR transcription factor mutant and the engineering bacteria containing the biosensor also belong to the protection scope of the invention.

The invention provides an IMP biosensor, which comprises a coding gene of a GlxR transcription factor mutant and a Ptrc promoter of a control sequence thereof, and a coding gene of a reporter protein and a Pcg3195 promoter of a control sequence thereof, wherein the GlxR transcription regulator can specifically bind to the Pcg3195 promoter region so as to induce and activate the expression of the reporter marker protein. Wherein further, the GlxR-MUT1 has D18H, R68H, N195S, P221T and R222P mutation relative to wild type GlxR, wherein the amino acid sequence of the wild type GlxR is shown as SEQ ID No.1, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 2. Wherein, since the Ptrc promoter controls GlxR encoding gene expression, the Pcg3195 promoter controls GFP encoding gene expression, preferably the Ptrc promoter and the Pcg3195 promoter are in opposite directions.

The invention carries out molecular transformation on transcription regulatory factor GlxR by error-prone PCR and other methods so as to change response molecules of GlxR from cAMP to IMP, and finally obtains four efficient GlxR transcription factor mutants (D18H/R68H/N195S/P221T/L222S, I N/E69G/F87I/R163L/L172P/E192G/R208H, F I/R163L/R208H, M T/N70Y/S94S/E182E/I216F) through screening. The IMP biosensor constructed based on the GlxR transcription factor mutant has response molecules changed into IMP compared with wild type, and shows excellent response relationship at the concentration of 0-100 mM IMP.

In a specific embodiment of the present invention, the reporter protein carried therein is a fluorescent protein, more preferably the green fluorescent protein EGFP. In one embodiment, the plasmid backbone is preferably E.coli-C.glutamicum shuttle vector pEC-T18-mob2. The invention also provides a recombinant strain containing the biosensor.

Furthermore, the invention also provides application of the biosensor or recombinant strain containing the biosensor in screening IMP high-yield strains. In a particular embodiment of the invention, the expression of the enzyme concerned is reduced by mutating the start codon ATG of the purA or/and guaB gene to TTG (GTG), six recombinant strains of Corynebacterium stationis ATCC6872 purA being obtained by a combination of mutations ^TTG guaB ^TTG ，purA ^GTG guaB ^GTG ，purA ^TTG ，guaB ^TTG ，purA ^GTG ，guaB ^GTG . After 6 strains are transferred into an inosinic acid biosensor by electric shock and then fermented and cultured, the strains are mixed in equal proportion and are separated by a flow cytometer, and the cells with obviously reduced green fluorescence signals are selected, and 53.33 percent of cells are high-yield purA ^TTG guaB ^TTG . The practice shows that the IMP high-yield strain can be screened out efficiently, quickly and laborsaving based on the coupling of the improved biosensor and the high-throughput screening system.

Compared with the prior art, the invention has the following advantages and effects:

according to the structural characteristics of IMP and analog cAMP, the invention selects a transcription regulatory factor GlxR which can respond to cAMP and is derived from Corynebacterium (Corynebacterium), in particular from Corynebacterium parvulum (Corynebacterium stationis), changes a responsive effector molecule into IMP through mutation, constructs a high-efficiency IMP biosensor, couples the concentration of IMP with a fluorescence intensity signal, and monitors the yield of the strain IMP.

The invention has the advantages that the invention provides the IMP biosensor, and the transcription control factor GlxR and Ptrc promoter and reporter gene controlled by the transcription control factor GlxR are used as basic biological elements, so that the concentration of IMP is coupled with fluorescent signals, and the concentration of IMP becomes visual. The biosensor can be combined with a high-flux mutagenesis and screening system, is favorable for rapid evolution and efficient screening of IMP high-yield engineering strains, and provides technical support for producing IMP and derivatives through microbial fermentation. The construction of the transcription factor-based IMP whole cell biosensor can provide a new strategy for realizing high-throughput screening of high-yield IMP strains.

Drawings

FIG. 1 is a schematic diagram of the structure of an IMP biosensor.

FIG. 2 is a graph of the relative fluorescence signal response of pSen biosensor after addition of cAMP or IMP.

FIG. 3 is a graph of the relative fluorescence signal response of the IMP biosensors MUT1, MUT2, MUT3 and MUT 4.

FIG. 4 is a graph showing the response of relative fluorescence signals from IMP biosensors Poom, pomm, pom, pmm.

FIG. 5 is a graph showing the response of recombinant strains to fluorescent signals.

FIG. 6 is a graph showing the results of flow cytometer screening after isometric mixing of recombinant strains.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

Unless specific test conditions are noted in the following embodiments, conventional test conditions or test conditions recommended by the reagent company are generally followed. The materials, reagents and the like used are those obtained commercially unless otherwise specified.

The strain Corynebacterium stagnant (Corynebacterium stationis) ATCC6872 used in the examples was purchased from ATCC, and the E.coli Top10 competence was purchased from Shanghai Biotechnology Co.

Example 1: construction of biosensor plasmid

In the embodiment, the components of the biological element in the IMP biosensor are obtained through PCR amplification, and fragments are connected according to a certain sequence through a molecular assembly technology such as one-step high-efficiency seamless cloning (NEB Builder HiFi DNA Assembly Master Mix kit New England BioLabs, boston, MA) and the like, so that the construction of the plasmid of the IMP biosensor is completed.

Using the Corynebacterium stagnant (Corynebacterium stationis) ATCC6872 genome as a template, using primers F1 and R1, amplifying by a common PCR method to obtain a biosensor response module, and a GlxR coding gene (SEQ ID No. 2); the Pcg3195 promoter control sequence (SEQ ID No. 3) was obtained by PCR method using the Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 genome as template, using primers F2 and R2; the pEC-T18-mob2 plasmid backbone, ptr promoter control sequence (SEQ ID No. 4) and fluorescence-reported EGFP-encoding gene (SEQ ID No. 5) were amplified using the E.coli-Corynebacterium glutamicum shuttle vector pEC-T18-mob2-EGFP as template (pEC-T18-mob 2 purchased from Addgene, pEC-T18-mob2-EGFP obtained after addition of EGFP gene, which was disclosed in document Development of a Transcription Factor-Based Diamine Biosensor in Corynebacterium glutamicum), using primers F3 and R3, F4 and R4, and F5 and R5, using the conventional PCR method. The nucleotide fragments obtained above and the plasmid backbone are assembled in sequence (figure 1) by utilizing a one-step high-efficiency seamless cloning (NEB Builder HiFi DNA Assembly Master Mix kit New England BioLabs, boston, MA) technology, wherein the Ptrc promoter controls the expression of the GlxR coding gene, the Pcg3195 promoter controls the expression of the EGFP coding gene, and the Ptrc promoter and the Pcg3195 promoter are opposite in direction. After screening and verification, a preliminary biosensor plasmid pSen is finally obtained and is subjected to company sequencing confirmation. The primer sequences used in the plasmid construction process are as follows:

F1:5’-TCTCATCCGCCAAAACAGCCTTAACGGGCGCGCTTGGCGA-3’

R1:5’-CACACAGGAAACAGACCATGGAAGGCGTACAGGAAAT-3’

F2:5’-GGCAGTGAGCGCAACGCAATGGATCCATCCAGTTTTCC-3’

R2:5’-AGCTCCTCGCCCTTGCTCATAGCATCCTCCTTTAAAAAG-3’

F3:5’-ATTGCGTTGCGCTCACTGCCCGCT-3’

R3:5’-CCCATGCGAGAGTAGGGAACTGCC-3’

F4:5’-CATGGTCTGTTTCCTGTGTG-3’

R4:5’-ATTTTGCCAAAGGGTTCGTTGACAATTAATCATCCGGCTCG-3’

F5:5’-ATGAGCAAGGGCGAGGAGCT-3’

R5:5’-TTCCCTACTCTCGCATGGGTTACTTGTACAGCTCGTCCATGC-3’。

in order to ensure the sequence accuracy in the amplification, the PCR reaction is performed by using the high-efficiency super-fidelity DNA polymerase which is commercially available at present. The PCR 50. Mu.L amplification system was: 0.5. Mu.L KOD FX high-fidelity polymerase, 10. Mu.L dNTP, 1.5. Mu.L Primer-F (10. Mu.M), 1.5. Mu.L Primer-R (10. Mu.M), DNA template 50ng, ddH ₂ O was filled to 50. Mu.L. The PCR amplification conditions were: pre-denaturation at 94℃for 5min, denaturation at 98℃for 10s, annealing at 55℃for 30s, extension at 68℃for 1min,30 cycles; extending at 68℃for 10min. The annealing temperature is properly adjusted according to the primer; the extension time was adjusted to 1kb/min depending on the length of the amplified fragment.

Plasmid assembly was performed using a commercial recombinase-based efficient seamless cloning NEB Builder HiFi DNA Assembly Master Mix kit (New England BioLabs, boston, MA) technique, and the recombination reaction system was: 5 mu L2xNEB Builder HiFi DNA Assembly Master Mix,100pmol vector backbone, 300pmol target gene insert, make up ddH ₂ O to 10. Mu.L system. The recombination reaction conditions are as follows: preparing a recombination reaction system on ice, reacting for 20min at 37 ℃, and cooling to 4 DEG COr immediately cooled on ice. Then, the recombinant plasmid obtained above was introduced into E.coli Top10 competent cells according to a conventional E.coli heat shock transformation method (NEB kit was purchased from NEB corporation of China).

Example 2

The GlxR of CRP/FNR transcription factor family is a global regulatory transcription factor that regulates hundreds of genes and requires cAMP as an effector molecule to participate, whereas IMP is an analog of cAMP, we changed the effector mutation of GlxR to IMP by error-prone PCR. The Pcg3195 promoter was further engineered to increase response sensitivity.

Firstly, detecting response conditions of pSen to cAMP and IMP in the example 1, electrically converting pSen plasmid into corynebacterium stagnant ATCC6872, inoculating with BHISG liquid culture medium, inoculating with 25mL of FMAC basic culture medium added with cAMP or IMP, finally obtaining OD600 of 1, final concentration of cAMP of 0-10 mM, final concentration of IMP of 0-100 mM, controlling temperature of 30 ℃ and rotational speed of 250rpm, and detecting relative fluorescence value after shaking incubation for 44 hours; experimental results showed that pSen responded to cAMP only and that sensor IMP did not respond but rather promoted expression of fluorescent protein (fig. 2).

The formula of the BHISG liquid culture medium is as follows:

to 1000mL, 37g/L brain heart infusion (Becton, dickinson and Co.), 10g/L glucose, 9.1g/L L-sorbitol, the balance being water was added.

The formula of the FMAC minimal medium is:

every 1000mL, 20g/L glucose, 3g/L urea and 2g/L NH are added ₄ Cl，1g/L KH ₂ PO ₄ ，3g/L K ₂ HPO ₄ 5g/L L-asparagine, 0.04g/L L-cysteine, 0.001g/L MnSO ₄ ·4H ₂ O，0.001g/L ZnSO ₄ ·7H ₂ O，2×10 ^-4 g/L CuSO ₄ ·2H ₂ O,0.02g/L calcium pantothenate, 0.01g/L CaCl ₂ 0.3g/L magnesium sulfate, 0.01g/L FeSO ₄ ·7H ₂ O，6×10 ^-5 g/L biotin, 0.01g/L thiamine hydrochloride, and the balance of water.

EXAMPLE 3 abrupt engineering of biosensors

The GlxR coding gene mutation library is obtained by using the Corynebacterium stagnant ATCC6872 genome as a template and using the primers F6 and R6 through the error-prone PCR random mutation method. An Error-prone PCR Kit is used for testing Error-protein PCR Kit (Ybio, shanghai, china), and the fidelity in the PCR amplification process is further reduced by adjusting magnesium ions and manganese ions in a PCR reaction system, so that 3-8 point mutations are contained in the obtained coding gene. Using the biosensor plasmid pSen obtained in example 1 as a template, a PCR reaction was performed using primers F7 and R7, and a plasmid backbone was obtained using efficient super-fidelity KOD FX DNA polymerase. Plasmid assembly is carried out by utilizing a high-efficiency seamless cloning NEB Builder HiFi DNA Assembly Master Mix kit (New England BioLabs, boston, MA) technology based on recombinase, a GlxR coding gene nucleotide fragment obtained by mutation PCR and a plasmid skeleton are assembled in sequence to obtain a recombinant plasmid library containing diversified GlxR coding gene point mutations, and the obtained recombinant plasmid library is transformed into competent cells of escherichia coli Top10 by referring to the method of the example 1.

The error-prone PCR system adopted by the invention is as follows: 3. Mu.L of 10 Xerror-prone PCR Mix, 3. Mu.L of 10 Xerror-prone PCR-specific dNTPs, 3. Mu.L of error-prone PCR-specific MnCl2, 1. Mu.L of DNA template (10 ng/. Mu.L), 1. Mu.L of PCR primer (10. Mu.M each), 1. Mu.L of DNA polymerase (5U/. Mu.L) and ultra-pure water was added to 30. Mu.L. The PCR reaction procedure was: pre-denaturation at 94℃for 3min, denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 2min, 30 cycles, and extension at 72℃for 10min.

The primers used in this example were:

F6:5’-CTCATCCGCCAAAACAGCC-3’

R6:5’-ACACAGGAAACAGACCATG-3’

F7:5’-GGTCTGTTTCCTGTGTGAAA-3’

R7:5’-GGCTGTTTTGGCGGATGAGAGAAGATT-3’。

after obtaining a recombinant plasmid library on a 10mg/L tetracycline-resistant LB plate, all colonies were washed down with LB liquid medium (10 g/L yeast extract, 20g/L tryptone, 10g/L NaCl), and after incubation for 1 hour with shaking at 200rpm at 37℃were collected by centrifugation, the cells were collected by using a plasmid mini kit (Meiji Biological Co., ltd., guan)gzhou) to obtain plasmid library, and then the plasmid library is shocked to transform corynebacterium stagnant ATCC6872. After obtaining a library of recombinant plasmids of Corynebacterium stagnant ATCC6872 on a plate, all colonies were rinsed off with BHISG broth, incubated at 30℃with shaking at 250rpm for 2 hours. The first round of forward screening was performed according to the initial OD ₆₀₀ After incubation for 44 hours with shaking at 30 ℃ and 250rpm in FMAC minimal medium containing 10mM IMP =1. Using pH7.4 PBS buffer (8.005 g/L NaCl,0.2g/L KCl,1.42g/L Na) ₂ HPO ₄ ，0.2g/L KH ₂ PO ₄ ) After washing the cells, they were resuspended to od=0.1. The cell is sorted according to fluorescence intensity by using a flow cytometer, and 0.4% of cells with low fluorescence are selected. After single colony is obtained on a flat plate, the single colony is washed down by utilizing a BHISG liquid culture medium, and after incubation, negative selection is carried out by a second alternate cytometer, namely fermentation is carried out in FAMC basic culture medium added with 10mM cAMP, the bacterial cells are sorted by a top-flow cytometer according to fluorescence intensity, and 0.4% of cells with high fluorescence are selected. After single colonies are obtained on the plate, the plates are washed down by using a BHISG liquid culture medium, and after incubation, forward screening is performed by a third round of flow cytometry, namely fermentation is performed in FAMC basic culture medium added with 10mM IMP, bacterial cells are sorted by an up-flow cytometer according to fluorescence intensity, 0.4% cells with low fluorescence are selected, and finally, from 572 single colonies on the plate are detected and selected by the method of example 2, sequencing is performed on 8 single colonies with the best response to IMP, and sequencing shows that four GlxR transcription factor mutants are obtained in total.

Finally, four GlxR transcription factor mutants with good response to IMP are determined, wherein the GlxR transcription factor mutants are respectively:

GlxR-MUT1(D18H/R68H/N195S/P221T/L222S)、

GlxR-MUT2(I53N/E69G/F87I/R163L/L172P/E192G/R208H)、

GlxR-MUT3(F87I/R163L/R208H)、

GlxR-MUT4(M29T/N70Y/S94S/E182E/I216F)。

wherein, the amino acid sequence of the GlxR-MUT1 is shown as SEQ ID NO.6, the nucleotide sequence of the GlxR-MUT1 is shown as SEQ ID NO.7, the amino acid sequence of the GlxR-MUT2 is shown as SEQ ID NO.8, the nucleotide sequence of the GlxR-MUT2 is shown as SEQ ID NO.9, the amino acid sequence of the GlxR-MUT3 is shown as SEQ ID NO.10, the nucleotide sequence of the GlxR-MUT3 is shown as SEQ ID NO.11, the amino acid sequence of the GlxR-MUT4 is shown as SEQ ID NO.12, and the nucleotide sequence of the GlxR-MUT4 is shown as SEQ ID NO. 13.

Based on the above four GlxR transcription factor mutants, the modified IMP biosensors constructed by the method of example 1 were named pSenIMP-MUT1, pSenIMP-MUT2, pSenIMP-MUT3 and pSenIMP-MUT4, respectively, and were transformed into Corynebacterium stagnant ATCC6872 by electric shock to obtain Corynebacterium stagnant ATCC6872 containing the biosensors.

Referring to the experimental method for detecting IMP response in example 2, the extent of response of Corynebacterium parvum ATCC6872 containing the biosensors pSenIMP-MUT1, pSenIMP-MUT2, pSenIMP-MUT3 and pSenIMP-MUT4 obtained by the above screening was tested. According to the experimental results, the correlation between the relative fluorescence intensity and the IMP concentration of recombinant strains containing different biosensors is plotted by taking the exogenous IMP concentration as an abscissa and the relative fluorescence intensity as an ordinate. The results are shown in FIG. 3, which demonstrates that the present invention obtains IMP biosensors that respond to IMP by molecularly engineering GlxR transcription regulators, wherein pSenIMP-MUT1 is optimally effective.

Example 4 optimization of biosensors

To further increase the response dynamic range of pSenIMP-MUT1, the present invention engineered the Pcg3195 promoter by altering the number and location of GlxR binding sites on Pcg3195 and by modifying the-10 and-35 sites within the promoter to alter transcription levels. Three potential GlxR binding sites were considered, one upstream of the-35 site, one between the-35 and-10 sites, one downstream of the-10 site, denoted by "m" for presence and "o" for absence of the GlxR binding site naming system to describe the promoter class, pmom, pomm, pmmm, pcg3195 was constructed as wild-type boom, respectively. For example, pomm is a promoter with one GlxR binding site downstream of the-10 site and in between-35 and-10. Wherein the Pom nucleotide sequence is shown as SEQ ID NO.14, the Pomm nucleotide sequence is shown as SEQ ID NO.15, and the Pmm nucleotide sequence is shown as SEQ ID NO. 16.

Based on pSenIMP-MUT1 obtained in example 3, the Pcg3195 promoter on its plasmid was modified (for specific procedures, see document Design and Selection of a Synthetic Feedback Loop for Optimizing Biofuel Tolerance [ J)]ACS synth.biol.2018,7,1,16-23) to obtain 3 modified biosensors psenmp-MUT 1-Pmom, psenmp-MUT 1-Pomm, and psenmp-MUT 1-Pmmm. Inoculating four promoter type biosensors in BHISG liquid culture medium, shaking overnight, and using initial OD ₆₀₀ After the test is carried out by adding the material into 25mL FMAC basic culture medium containing 0-100 mM IMP and culturing at 30 ℃ for 44h at 250rpm, the relative fluorescence values are detected, and compared with the dynamic range of wild-type Poom, pomm and Pmm are improved, 34.30% and 63.91% are respectively improved, and experimental results prove that the regulation and control efficiency of transcription factors can be better improved by adding one more transcription factor binding site between the promoter region-10 region and the promoter region-35 region. Compared with the poor response range of the boom, the response range of the boom is improved by 11.35%, which shows that the regulation and control improvement of the gene are smaller by adding a transcription factor binding site on the left side of the-35 region, and the experimental result is shown in figure 4, wherein the maximum dynamic range of pSenIMP-MUT1-Pmm is also optimal.

Example 5: IMP high-yield strain screening based on improved biosensor

The improved biosensor can sense the change of the concentration of IMP and output the change as a fluorescent signal, and the high-throughput screening equipment such as a coupled flow cytometer (FACS) and the like can be used for rapidly sorting at the fluorescence level, so that the breeding speed of the strain is greatly increased.

To confirm the screening effect of pSenIMP-MUT1-Pmm, the activities of adenylosuccinate synthase and IMP dehydrogenase of the IMP degradation pathway in Corynebacterium parvulum ATCC6872 were attenuated. The expression of the relevant enzyme is reduced by mutating the ATG of the start codon of purA or/and guaB gene to TTG (or GTG), six recombinant strains purA of Corynebacterium stagnant ATCC6872 are obtained by mutation combination ^TTG guaB ^TTG ，purA ^GTG guaB ^GTG ，purA ^TTG ，guaB ^TTG ，purA ^GTG ，guaB ^GTG (methods for producing mutant strains see, for example, improved fermentative production of gamma-aminobutyric acid via the putrescine route: systems metabolic engineering for production from glucose, amino additives, and xylose [ J ]].Biotechnology and Bioengineering，Vol.114，No.4，April，2017)。

Evaluation of wild type ATCC6872 and purA by fermentation experiments ^TTG guaB ^TTG ，purA ^GTG guaB ^GTG ，purA ^TTG ，guaB ^TTG ，purA ^GTG ，guaB ^GTG IMP productivity of (c). After fermentation culture, the extracted supernatant was checked for IMP yield using HPLC.

The formula of the fermentation medium is as follows:

glucose 80g, NH per 1000mL ₄ Cl 10g，MgSO ₄ 3g，KH ₂ PO ₄ 5g，K ₂ HPO ₄ 5g, urea 3g, L-cysteine 0.1g, caCl ₂ .2H ₂ O 0.1g，MnSO ₄ 0.002g，FeSO ₄ .7H ₂ O0.01 g, thiamine hydrochloride 0.005g, d-biotin 30. Mu.g.

BHISG was used as seed solution, and after inoculation, the seeds were shaken overnight at 30℃and 250 rpm. The IMP yield was checked by HPLC after 50mL of fermentation medium was inoculated at 30℃and 250rpm for 72 hours with a final OD600 of 1.

TABLE 1 comparison of IMP yields for wild type and recombinant strains

Strain	IMP(g/L)
		Wild type	0
purA TTG guaB TTG	0.38
		purA GTG guaB GTG	0.09
purA TTG	0.31
		guaB TTG	0.11
purA GTG	0.05
		guaB GTG	0.04

The pSenIMP-MUT1-Pmm obtained in example 3 was transformed into the strain purA using electric shock transformation ^TTG guaB ^TTG ，purA ^GTG guaB ^GTG ，purA ^TTG ，guaB ^TTG ，purA ^GTG ，guaB ^GTG . The IMP response relative fluorescence values were measured with reference to the experimental procedure of example 2, and the experimental results are shown in FIG. 5. Experimental results prove that the higher the IMP yield is, the lower the relative fluorescence of the strain is, and the two are in a remarkable correlation.

Then wild type containing pSenIMP-MUT1-Pmm and purA ^TTG guaB ^TTG ，purA ^GTG guaB ^GTG ，purA ^TTG ，guaB ^TTG ，purA ^GTG ，guaB ^GTG Seven strains of bacteria were mixed in equal proportion and 0.1% of cells with low fluorescence were screened in a flow cytometer (fig. 6), and thirty single colonies were randomly selected for sequencing analysis. The results show that 53.33% of the colonies obtained by the screening method are purA with high IMP yield ^TTG guaB ^TTG Also 16.67% are the next highest yield purA ^TTG 30% are other strains.

The above examples show that the improved biosensor has excellent applicability in screening of high-yield IMP strains, and the transcription regulatory factor GlxR and Ptrc promoter and reporter gene controlled by the transcription regulatory factor GlxR are used as basic biological elements, so that the concentration of IMP is coupled with fluorescent signals, and becomes visible. The biosensor can be combined with a high-flux mutagenesis and screening system, is favorable for rapid evolution and efficient screening of IMP high-yield engineering strains, and provides technical support for producing IMP and derivatives through microbial fermentation. The construction of the transcription factor-based IMP whole cell biosensor can provide a new strategy for realizing high-throughput screening of high-yield IMP strains.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. A GlxR transcription factor mutant capable of responding to IMP, characterized by at least one of the following mutants:

GlxR-MUT1, the D18H/R68H/N195S/P221T/L222S mutation was present relative to wild-type GlxR;

GlxR-MUT2, the presence of the I53N/E69G/F87I/R163L/L172P/E192G/R208H mutation relative to wild-type GlxR;

GlxR-MUT3, the F87I/R163L/R208H mutation is present relative to wild-type GlxR;

GlxR-MUT4, there was an M29T/N70Y/S94S/E182E/I216F mutation relative to wild-type GlxR.

2. The GlxR transcription factor mutant of claim 1, wherein:

the coding gene nucleotide sequence of the GlxR transcription factor mutant is obtained according to a codon coding rule.

3. An IMP biosensor, characterized by:

the IMP biosensor is an expression vector comprising at least one coding gene of the GlxR transcription factor mutant as defined in claim 1 or 2 and a promoter for controlling the expression of the GlxR transcription factor mutant, and a coding gene of a reporter protein and a promoter for controlling the expression of the reporter protein.

4. An IMP biosensor according to claim 3, wherein:

the promoter for controlling the expression of the GlxR transcription factor mutant is Ptrc promoter;

the reporter protein is fluorescent protein;

the promoter for controlling the expression of the reporter protein is a Pcg3195 promoter or a Pcg3195 promoter mutant.

5. An IMP biosensor as claimed in claim 4, wherein:

the Pcg3195 promoter mutant is obtained by modifying a GlxR binding site on a nucleotide sequence of a wild type boom of the Pcg3195 promoter.

6. An IMP biosensor according to claim 3, wherein:

wherein the Ptrc promoter and Pcg3195 promoter are in opposite directions.

7. A recombinant strain, characterized in that:

an IMP biosensor according to any one of claims 3 to 6.

8. Use of an IMP biosensor according to any one of claims 3 to 6 for screening high-yielding strains of IMP.

9. The use according to claim 8, characterized by the specific steps of:

mutant strains are obtained through mutagenesis, the IMP biosensor in any one of claims 3-6 is transferred into the mutant strains, after fermentation culture, the mutant strains are separated by using a flow cytometer, and cells with reduced reporter protein signals are selected, namely the IMP high-yield strains.

10. A construction method of an IMP biosensor comprises the following steps:

assembling at least one coding gene and a promoter for controlling the expression of the GlxR transcription factor mutant according to claim 1 or 2, and a coding gene of a reporter protein and a promoter for controlling the expression of the reporter protein in sequence to obtain an IMP biosensor;

the construction method is characterized in that the direction of a promoter for controlling the expression of the GlxR transcription factor mutant is opposite to that of a promoter for controlling the expression of the marker protein.

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