WO2021256582A1 - Biocapteur pour la détection de tryptophane, comprenant un facteur d'activation de transcription et un commutateur toehold - Google Patents

Biocapteur pour la détection de tryptophane, comprenant un facteur d'activation de transcription et un commutateur toehold Download PDF

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WO2021256582A1
WO2021256582A1 PCT/KR2020/007828 KR2020007828W WO2021256582A1 WO 2021256582 A1 WO2021256582 A1 WO 2021256582A1 KR 2020007828 W KR2020007828 W KR 2020007828W WO 2021256582 A1 WO2021256582 A1 WO 2021256582A1
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tryptophan
biosensor
trpribo
gfp
seq
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Korean (ko)
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정규열
황윤희
석주연
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포항공과대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

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  • the present invention relates to a biosensor for detecting tryptophan, and more particularly, to a biosensor for detecting tryptophan comprising a transcriptional activator and a toehold switch, and a use thereof.
  • the previously developed assay methods include liquid/gas chromatography (LC/GC), an assay using a multi-well plate, and an assay using a biosensor.
  • LC/GC liquid/gas chromatography
  • Liquid/gas chromatography is a method of culturing an individual strain and then analyzing the concentration of metabolites in the culture medium and the strain. This method can detect most metabolites and quantitative analysis is possible if a standard calibration curve can be obtained. However, since it can measure only one mutated strain at a time, it is inefficient to analyze a library of strains larger than a certain size due to low throughput.
  • the analysis method using multiwell plates analyzes changes in the concentration of metabolites in the sample by measuring changes in color development, absorbance, or fluorescence of a small sample after putting the mutant strain into a separate well. way. Since a small amount of sample is used and a multi-plate is used, a relatively large number of mutant strains can be analyzed simultaneously. However, the processing capacity is low to analyze the large-sized strain library made by the above-described manufacturing method. In addition, the range of application is narrow because it is applicable only to metabolites that can react with metabolites as substrates or can measure changes in absorbance or fluorescence.
  • FACS fluorescence activated cell sorting
  • Screening using a selection method is a technology that makes only the strain that produces the target metabolite in a high concentration in the strain library survive and selects this strain.
  • This method has a very high throughput and can effectively select only high-producing strains from a large strain library.
  • this technique can be applied only when the concentration of the target metabolite is involved in the growth or survival of the strain.
  • the method of screening a production strain using a genetic biosensor converts the concentration of the synthesized target metabolite into a signal that can be detected immediately and detects it.
  • screening may be performed by FACS or a selection method, respectively. Therefore, it becomes possible to screen the library with high throughput. That is, if a biosensor specific to the target metabolite is developed and applied, a change in the concentration of the target metabolite that cannot be visually detected can be observed by using a suitable detector.
  • tryptophan is an aromatic amino acid widely used as a supplement for pharmaceuticals and animal feed, and can be made from microorganisms through biological processes. At this time, since the performance of the microbial strain determines the efficiency of the production process, it is essential to secure microorganisms with excellent performance in order to pursue an economical process.
  • the present inventors have completed the present invention by developing a tryptophan biosensor including a transcriptional activator and a toehold switch, and confirming that its operating range can be controlled.
  • an object of the present invention is to provide a biosensor for detecting tryptophan comprising a riboswitch and a transcriptional activator represented by the nucleotide sequence of SEQ ID NO: 1.
  • Another object of the present invention is to provide a transformant strain for detecting tryptophan into which the biosensor for detecting tryptophan is introduced.
  • Another object of the present invention is to provide a method for screening tryptophan high-producing bacteria comprising the step of culturing the transformant and candidate strains for detecting the tryptophan.
  • the present invention provides a biosensor for detecting tryptophan comprising a riboswitch and a transcriptional activator represented by the nucleotide sequence of SEQ ID NO: 1.
  • the present invention also provides a transformant strain for detecting tryptophan into which the biosensor for detecting tryptophan is introduced.
  • the present invention also provides a method for screening tryptophan high-producing bacteria, comprising the step of culturing the transformant strain and the candidate strain for detecting the tryptophan.
  • the biosensor for detecting tryptophan comprising the transcriptional activator and toehold switch of the present invention can specifically and sensitively recognize tryptophan, and can quickly and easily select strains that produce tryptophan at a high concentration.
  • the present invention confirmed that the tryptophan operating range and fold change of the biosensor for detecting tryptophan can be adjusted. Therefore, the biosensor for detecting tryptophan according to the present invention can be used in various ways in the field of industrial strain screening.
  • 1 is a diagram illustrating a dose-response curve exemplarily prepared to confirm the performance of a tryptophan biosensor.
  • FIG. 2 is a diagram illustrating a tryptophan detection mechanism of a tryptophan biosensor into which a transcriptional activator is introduced (a: in the absence of tryptophan, b: in the presence of tryptophan).
  • Trpribo-TF16-GFP Trpribo-TF16-GFP
  • Trpribo-TF32-GFP Trpribo-TF32-GFP
  • FIG. 4 is a diagram illustrating a tryptophan detection mechanism of a biosensor into which a transcriptional activator and a toehold switch are introduced (a: no tryptophan, b: tryptophan present).
  • Figure 5 is a transgenic strain Trpribo-TF-P T7 -TSN1-GFP and Trpribo-TF-P T7 -TSN3-GFP containing a biosensor introduced with a transcriptional activator and a toehold switch dose-response curves for tryptophan.
  • Trpribo-TF-P T7- TSN1-GFP Trpribo-TF-P T7- TSN3-GFP.
  • FIG. 6 is a diagram showing a dose-response curve for tryptophan of a transformed strain containing a tryptophan biosensor in which the strength of the toehold switch promoter is regulated (a: Trpribo-TF-J23100-TSN1-GFP and Trpribo-TF- J23106-TSN1-GFP, b: Trpribo-TF-J23100-TSN3-GFP and Trpribo-TF-J23106-TSN3-GFP, c: Trpribo-TF-P tac- TSN3-GFP).
  • FIG. 7 is a diagram illustrating a tryptophan detection mechanism of a tryptophan biosensor into which a transcriptional activator, a toehold switch, and a selection marker TetA are introduced (a: in the absence of tryptophan, b: in the presence of tryptophan).
  • FIG. 8 is a diagram showing the cell growth rate of a transformed strain including a tryptophan biosensor into which the selection marker TetA is introduced according to the concentration of tetracycline.
  • a biosensor for detecting tryptophan comprising a riboswitch and a transcriptional activator represented by the nucleotide sequence of SEQ ID NO: 1.
  • a biosensor refers to a sensor that measures the state and concentration of a substance, particularly an organic compound, using the function of an organism.
  • a riboswitch refers to a regulatory region to which a low molecular weight substance such as a metabolite can specifically bind. Depending on whether a specific substance is bound to the riboswitch, the degree of protein synthesis from this mRNA is regulated.
  • Regulatory RNAs that act as trans (trans) act in the form of base pairing with a complementary region or sequestering an RNA-binding protein. Riboswitches act in cis, unlike regulatory RNAs that act in trans, and exist in some form of regulatory RNA.
  • the riboswitch is preferably represented by the nucleotide sequence of SEQ ID NO: 1.
  • the gene is 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more sequence homology with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: It refers to a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by 1.
  • the % of sequence homology to a polynucleotide is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (including additions or deletions) to the optimal alignment of the two sequences. It may contain additions or deletions (ie, gaps) compared to not).
  • a transcriptional activator refers to a protein that increases the transcription of a gene or a set of genes.
  • the transcriptional activator is usually a DNA-binding protein, and binds to an enhancer or a promoter.
  • the transcriptional activator is preferably an ECF sigma factor (extra-cytoplasmic function sigma factor) or a LysR type transcriptional regulatory factor.
  • the ECF sigma factor may be an ECF16 sigma factor represented by the nucleotide sequence of SEQ ID NO: 2 or an ECF32 sigma factor represented by the nucleotide sequence of SEQ ID NO: 3.
  • the biosensor may further include a toe hold switch.
  • the toehold switch is a trigger RNA that is trN1 represented by the nucleotide sequence of SEQ ID NO: 4 or trN3 represented by the nucleotide sequence of SEQ ID NO: 6; and a switch RNA that is swN1 represented by the nucleotide sequence of SEQ ID NO: 5 or swN3 represented by the nucleotide sequence of SEQ ID NO: 7; preferably, (i) trN1 and the sequence represented by the nucleotide sequence of SEQ ID NO: 4 swN1 represented by the nucleotide sequence of number 5; or (ii) trN3 represented by the nucleotide sequence of SEQ ID NO: 6 and swN3 represented by the nucleotide sequence of SEQ ID NO: 7.
  • the biosensor may further include a promoter.
  • the promoter is promoter T7 represented by the nucleotide sequence of SEQ ID NO: 8
  • promoter BBa_J23100 represented by the nucleotide sequence of SEQ ID NO: 9
  • promoter BBa_J23106 and SEQ ID NO: 11 represented by the nucleotide sequence of SEQ ID NO: 10
  • It may be one or more selected from the group consisting of promoter Tac represented by the nucleotide sequence of
  • the biosensor may further include a selection marker gene.
  • the selection marker gene is preferably a fluorescent protein gene or an antibiotic resistance gene, but any gene for labeling may be used without limitation.
  • the fluorescent protein is a group consisting of green fluorescent protein (GFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP). It may be one or more selected from.
  • the antibiotic resistance gene is an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, a gentamicin resistance gene, and a carbenicillin resistance gene. It may be one or more selected from, but is not limited thereto.
  • a riboswitch and a transcriptional activator are operatively linked.
  • operably linked means linked in a manner that enables gene expression when an appropriate molecule binds to an expression control sequence.
  • the tryptophan sensor has an operating range of 0.001 to 1 g/L, and there is a limit in scale-up due to a low operating range.
  • the present invention has adjusted the operating range of the tryptophan biosensor.
  • the tryptophan biosensor according to the present invention can be scaled-up and used for screening of industrial strains.
  • a transformed strain for detecting tryptophan into which the biosensor for detecting tryptophan is introduced.
  • the transformed strain means a strain transformed with the recombinant vector of the present invention.
  • transformation means introducing the vector containing the promoter or target gene according to the present invention into a host cell.
  • the transformed target gene may be inserted into the chromosome of the host cell or located outside the chromosome.
  • one or a plurality of recombinant vectors may be introduced into the transformant strain for detecting tryptophan, and the one or a plurality of recombinant vectors may be introduced, respectively.
  • the recombinant vector may be introduced into the microorganism at the same time or at the same time, may be introduced sequentially, may be introduced in a mutually changed order.
  • the transformant strain for detecting tryptophan may be characterized in that it is selected from the group consisting of bacteria, yeast, and mold, and may preferably be a microorganism of the genus Escherichia, more Preferably, it may be Escherichia coli.
  • the transformant strain for detecting tryptophan may be Escherichia coli BL21 (Escherichia coli BL21) lacking a major protease encoded by the lon gene.
  • the present invention provides a method for screening tryptophan high-producing bacteria comprising the step of culturing the transformant and candidate strains for detecting tryptophan.
  • the selection marker gene is expressed when tryptophan is present. Incubated with the transformant and candidate strains for detection of tryptophan, and when the candidate strain produces tryptophan, the selection marker gene is expressed, so tryptophan high-producing bacteria can be screened by confirming the expression of the selection marker gene.
  • the method for screening tryptophan high-producing bacteria uses a transformant strain for detecting tryptophan that can be scaled-up by controlling the operating range, so it is possible to screen tryptophan high-producing bacteria at an industrial level.
  • strains and plasmids used in Examples to be described later are shown in Table 1 below, and the sequences of the primers used are shown in Table 2.
  • the primers disclosed in Table 2 were synthesized by Cosmogenetech (Seoul, Korea), and the interaction sequences between the primers are underlined.
  • a transcriptional activator was inserted into the tryptophan biosensor including the tryptophan riboswitch (SEQ ID NO: 1) and the reporter gene GFP (SEQ ID NO: 12).
  • the transcriptional activator ECF sigma factor (extra-cytoplasmic function sigma factor) recognizes a specific promoter sequence and activates gene expression.
  • two types of transcriptional activators were used using the transcriptional activator ECF16 sigma factor (SEQ ID NO: 2) derived from Caulobacter crescentus and the transcriptional activator ECF32 sigma factor (SEQ ID NO: 3) derived from plant pathogens. .
  • the performance of the constructed tryptophan biosensor was confirmed through a dose-response curve.
  • 1 shows a dose-response curve as an example, from which (i) expression level change (ie, fold change) and (ii) an operating range of the output module according to the concentration of metabolites, and (ii) the interval in which the expression level change appears. can be checked.
  • the fold activation is a value calculated as the ratio of the maximum expression level to the minimum expression level (maximum expression level/minimum expression level).
  • a plasmid pACYC-Trpribo-ECF16-GFP which is a tryptophan biosensor operating without a toehold switch, was constructed, and a transformed strain Trpribo-TF16-GFP introduced thereto was constructed.
  • the plasmid pACYC-Trpribo-ECF16-GFP was constructed by amplifying and ligating a part of the plasmid pACYC-Trpribo-ECF16-trN1-P T7-swN1-GFP containing the toehold switch and promoter.
  • a tryptophan sensor including trigger RNA trN1 (SEQ ID NO: 4), switch RNA swN1 (SEQ ID NO: 5) and promoter T7 (SEQ ID NO: 8) was constructed. Specifically, to construct the plasmid pACYC-Trpribo-ECF16-trN1-P T7- swN1-GFP, the plasmid pACYC-Trpribo-ECF16-trN1 was constructed. Specifically, three types of amplification products 1 to 3 were prepared using Q5 High-Fidelity DNA Polymerase (New England Biolabs (NEB), Ipswich, MA, USA).
  • Amplification product 1 contains a tryptophan riboswitch sequence and was obtained by amplifying plasmid pAC108_TR585 using the primer Gib-Trpribo-Homo-F/R.
  • Amplification product 2 includes a transcriptional activator sequence and a part of a promoter sequence and a trigger sequence regulated by the transcription factor.
  • the amplification product 2 is a product obtained by amplifying plasmid pVRa16-3622 using primers Gib-TF16-over-Trpribo-In-F and TF16-over-P16-1-R, which was obtained by amplifying the primer Gib-TF16-over-Trpribo.
  • -In-F and Gib-P16-over-trN1-R were amplified to obtain an amplification product, that is, amplification product 2.
  • Amplification product 3 was obtained by amplifying plasmid pACYC-B12ribo-PhlF-trN1 using primers Gib-trN1-F and Gib-over-Trpribo-R.
  • the amplification products 1 to 3 were ligated in the Gibson assembly method using NEBuilder® HiFi DNA Assembly Master Mix (NEB) to construct plasmid pACYC-Trpribo-ECF16-trN1. Also, (i) the product of amplification of the plasmid pACYC-Trpribo-ECF16-trN1 using the primers Gib-Vec-F/R; and (ii) a product of amplification of plasmid pCOLA-swN1-GFP using primers Gib-In-F/R (including a toehold switch and a fluorescent protein sequence); pACYC-Trpribo-ECF16-trN1-P T7- swN1-GFP was constructed.
  • NEB NEBuilder® HiFi DNA Assembly Master Mix
  • the 5' ends of primers Gib-del-F and Gib-del-R were phosphorylated using T4 Polynucleotide Kinase (Takara, Shiga, Japan).
  • the plasmid pACYC-Trpribo-ECF16-trN1-P T7- swN1-GFP was amplified using Gib-del-F/R, a primer with phosphorylated 5' end.
  • the amplification product was ligated by blunt-end ligation using Quick Ligase (NEB) to construct a plasmid pACYC-Trpribo-ECF16-GFP.
  • E. coli BL21 Star (DE3) was transformed with the plasmid pACYC-Trpribo-ECF16-GFP, and the transformed strain was named Trpribo-TF16-GFP.
  • a plasmid pACYC-Trpribo-ECF32-GFP a tryptophan biosensor operating without a toehold switch, was constructed, and a transformed strain Trpribo-TF32-GFP introduced thereto was constructed.
  • the plasmid pACYC-Trpribo-ECF32-GFP was constructed by amplifying and ligating a part of the plasmid pACYC-Trpribo-ECF32-trN3-P T7-swN3-GFP containing the toehold switch and promoter.
  • a tryptophan sensor including trigger RNA trN3 (SEQ ID NO: 6), switch RNA swN3 (SEQ ID NO: 7) and promoter T7 (SEQ ID NO: 8) was constructed. Specifically, in order to construct the tryptophan sensor plasmid pACYC-Trpribo-ECF32-trN3-P T7- swN3-GFP, amplification products 1 to 3 were prepared as follows.
  • Amplification product 1 includes a transcriptional activator sequence and a promoter sequence capable of reacting therewith.
  • plasmid pVRa32-1122 was amplified using primers Gib-TF32-over-Trpribo-ln-F/TF32-over-P32-1-R.
  • the amplification product was further amplified using primers Gib-TF32-over-Trpribo-In-F and Gib-P32-over-trN3-R to finally obtain amplification product 1.
  • plasmid pACYC-B12ribo-PhlF-trN3 was amplified using primers Gib-trN3-F and Gib-over-Trpribo-R to obtain amplification product 2 containing the remaining trigger sequences.
  • Amplification products 1 and 2 were ligated in the same manner as in Example 2-1 with the same Gibson assembly to construct plasmid pACYC-Trpribo-ECF32-trN3. Also, (i) the product of amplification of the plasmid pACYC-Trpribo-ECF32-trN3 using the primer Gib-Vec-F/R; and (ii) a product of amplification of plasmid pCOLA-swN3-GFP using primers Gib-In-F/R (including a threshold switch sequence and a fluorescent protein sequence); -ECF32-trN3-P T7 -swN3-GFP was constructed.
  • a part of the vector pACYC-Trpribo-ECF32-trN3-P T7- swN3-GFP was amplified and ligated in the same manner as in Example 1-1 to construct a plasmid pACYC-Trpribo-ECF32-GFP.
  • E. coli BL21 Star (DE3) was transformed with the plasmid pACYC-Trpribo-ECF32-GFP, and the transformed strain was named Trpribo-TF32-GFP.
  • the tryptophan detection mechanism of the tryptophan biosensor introduced with the transcriptional activator is shown in FIG. 2 , and the constructed plasmids pACYC-Trpribo-ECF16-GFP and pACYC-Trpribo-ECF32-GFP operate with the same mechanism as in FIG. 2 .
  • the plasmids pACYC-Trpribo-ECF16-GFP and pACYC-Trpribo-ECF32-GFP contain transcriptional activators, but do not include a toehold switch.
  • the two plasmids are not expressed in the absence of tryptophan ( FIG. 2A ), and are expressed in the presence of tryptophan to express GFP ( FIG. 2B ).
  • the culture experiment was performed in M9 medium containing glucose (4 g / L glucose, 6.78 g / L disodium phosphate (anhydrous), 3 g / L monopotassium phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 2 mM magnesium sulfate and 0.1 mM calcium chloride) were used, and chloramphenicol 34 mg/L was added as an antibiotic to maintain the plasmid.
  • the transformant strain constructed in Example 1-2 was inoculated in M9 medium and then cultured for 24 hours.
  • the cultured strain was diluted in fresh M9 medium so that the OD 600 value was 0.05, and when the OD 600 value reached 0.8, the OD 600 value was diluted to 0.05 in the new M9 medium.
  • the strain is transferred to a medium to which various concentrations of tryptophan (0, 0.01, 0.03, 0.1, 0.3, 1, 2, 4, 6 and 8 g/L) are added to the late exponential phase It was cultured until the late-exponential phase.
  • the cultured strain was washed once with PBS (phosphate-buffered saline), and then fluorescence was measured.
  • the fluorescence was measured using a 485 nm transfer filter, a 535 nm emission filter, and a VICTOR 3 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA), and the fluorescence intensity was measured for 0.1 second.
  • the value was corrected by subtracting the PBS measurement value from the measured fluorescence value, and autofluorescence of the strain was not subtracted from the measured fluorescence value.
  • VICTOR 3 1420 Multilabel Counter PerkinElmer
  • UV-1700 spectrophotometer Shiadzu, Japan
  • the tryptophan dose-response curves of the strains Trpribo-TF16-GFP and Trpribo-TF32-GFP transformed with the plasmid, respectively, are shown in FIGS. 3A and 3B , respectively.
  • the tryptophan concentration of 8 g/L is selected in consideration of the maximum solubility of tryptophan.
  • the operating ranges of the above-mentioned transforming strains Trpribo-TF16-GFP and Trpribo-TF32-GFP increased both the minimum and maximum values compared to the operating range (0.001 to 1 g/L) of the previously developed tryptophan biosensor. In particular, it was confirmed that the maximum value increased 8-fold from 1 g/L to 8 g/L.
  • the fold change of the transgenic strains Trpribo-TF16-GFP and Trpribo-TF32-GFP was increased by up to about 32% compared to the fold change (1.58 fold) of the conventionally developed tryptophan biosensor. This means that by introducing a transcriptional activator and a promoter sequence corresponding to the tryptophan biosensor, the operating range and fold change of the tryptophan biosensor can be controlled.
  • Example 1 From Example 1, it was confirmed that the introduction of the transcriptional activator can control the operating range and fold range of the tryptophan biosensor. Accordingly, a tryptophan biosensor into which a transcriptional activator and a toehold switch system were introduced was constructed, and its performance was evaluated.
  • Plasmid pACYC-Trpribo-ECF16-trN1-P T7- swN1-GFP was introduced to construct a transformant strain
  • the plasmid pACYC-Trpribo-ECF16-trN1-P T7 -swN1-GFP constructed in Example 1-1 was introduced into E. coli BL21 Star (DE3), and the transformed strain was Trpribo-TF-P T7 -TSN1 -GFP was named.
  • Plasmid pACYC-Trpribo-ECF32-trN3-P T7- swN3-GFP was introduced to construct a transformant strain
  • the plasmid pACYC-Trpribo-ECF32-trN3-P T7 -swN3-GFP constructed in Example 1-2 was introduced into E. coli BL21 Star (DE3), and the transformed strain was Trpribo-TF-P T7 -TSN3 -GFP was named.
  • the tryptophan detection mechanism of the biosensor into which the transcriptional activator and the toehold switch were introduced is shown in FIG. 4, and the constructed plasmids pACYC-Trpribo-ECF16-trN1-P T7- swN1-GFP and pACYC-Trpribo-ECF32-trN3-P T7 -swN3-GFP operates with the same mechanism as in FIG. 4 .
  • the plasmids pACYC-Trpribo-ECF16-trN1-P T7 -swN1-GFP and pACYC-Trpribo-ECF32-trN3-P T7 -swN3-GFP contain transcriptional activators, triggers and toehold switches. do.
  • GFP when tryptophan is not present, GFP is not expressed (Fig. 4a), and when tryptophan is present, transcriptional activators are expressed to express trigger RNA, and the hairpin structure of the switch RNA is released by the expressed trigger RNA.
  • GFP is expressed (Fig. 4b).
  • the maximum value increased 8 times from 1 g/L to 8 g/L.
  • the fold change of the transformant strains Trpribo-TF-P T7- TSN1-GFP and Trpribo-TF-P T7- TSN3-GFP was confirmed to be about 2.0-fold and 1.8-fold, respectively, with respect to 8 g/L tryptophan. This is an increase of 27% and 14%, respectively, compared to the fold change (1.58 times) of the conventionally developed tryptophan biosensor.
  • Example 3 Construction and performance confirmation of tryptophan biosensor in which the strength of the toehold switch promoter is regulated
  • Example 2 From Example 2, it was confirmed that the introduction of the transcriptional activator and the toehold system can control the operating range and fold change of the tryptophan biosensor. Accordingly, a tryptophan biosensor including a transcriptional activator and a toehold switch system, in which the strength of the toehold switch promoter is regulated, was constructed, and its performance was evaluated.
  • the strength of the toehold switch promoter contained in the plasmids pACYC-Trpribo-ECF16-trN1-P T7- swN1-GFP and pACYC-Trpribo-ECF32-trN3-P T7-swN3-GFP constructed in Example 2 was adjusted.
  • BBa_J23100, BBa_J23106 and Tac promoters were inserted instead of the T7 promoter, respectively.
  • the BBa_J23106 is a promoter capable of expressing a gene at 1/2 level compared to BBa_J23100.
  • plasmids pACYC-Trpribo-ECF16-trN1-P T7 -swN1-GFP and pACYC-Trpribo-ECF32-trN3-P T7- swN3-GFP was amplified.
  • the primers used to amplify the plasmid pACYC-Trpribo-ECF16-trN1-P T7- swN1-GFP were sw1-J23100-F and Change-promoter-R with phosphorylated 5' ends, and plasmid pACYC-Trpribo-ECF32-trN3-
  • the primers used for amplification of P T7- swN3-GFP were phosphorylated sw3-J23100-F and Change-promoter-R.
  • Each amplification product was blund-end ligated to construct plasmids pACYC-Trpribo-ECF16-trN1-J23100-swN1-GFP and pACYC-Trpribo-ECF32-trN3-J23100-swN3-GFP.
  • plasmids pACYC-Trpribo-ECF16-trN1-P T7 -swN1-GFP and pACYC-Trpribo-ECF32-trN3-P T7 -swN3-GFP were amplified, respectively.
  • Primers sw1-J23106-F/Change-promoter-R and sw3-J23106-F/Change-promoter-R were used for amplification of the two plasmids, respectively.
  • the amplification products were ligated, respectively, and the constructed plasmids were pACYC-Trpribo-ECF16-trN1-J23106-swN1-GFP and pACYC-Trpribo-ECF32-trN3-J23106-swN3-GFP.
  • the Tac promoter plasmids pACYC-Trpribo-ECF16-trN1-P T7 -swN1-GFP and pACYC-Trpribo-ECF32-trN3-P T7 -swN3-GFP were amplified, respectively.
  • Primers sw1-P tac -F/Change-promoter-R and sw3-P tac -F/Change-promoter-R were used for amplification of the two plasmids, respectively.
  • the amplification products were ligated, respectively, and the constructed plasmids were pACYC-Trpribo-ECF16-trN1-P tac -swN1-GFP and pACYC-Trpribo-ECF32-trN3-P tac -swN3-GFP.
  • Plasmids constructed from E. coli W3110 strain (pACYC-Trpribo-ECF16-trN1-J23100-swN1-GFP, pACYC-Trpribo-ECF32-trN3-J23100-swN3-GFP, pACYC-Trpribo-ECF16-trN1-J23106-swN1- Transformed with GFP, pACYC-Trpribo-ECF32-trN3-J23106-swN3-GFP, pACYC-Trpribo-ECF16-trN1-P tac- swN1-GFP and pACYC-Trpribo-ECF32-trN3-P tac- swN3-GFP, respectively.
  • Transformed strains are Trpribo-TF-J23100 - TSN1 - GFP, Trpribo-TF-23100-TSN3-GFP, Trpribo-TF-J23106-TSN1-GFP, Trpribo-TF-J23106-TSN3-GFP, Trpribo-TF, respectively. They were named -P tac - TSN1-GFP and Trpribo-TF-P tac - TSN3-GFP.
  • the transformed strain Trpribo-TF-J23100-TSN1-GFP, Trpribo-TF-J23100-TSN3-GFP, Trpribo-TF-J23106-TSN1-GFP of Example 3-1 Culture experiments were performed using Trpribo-TF-J23106-TSN3-GFP, Trpribo-TF-P tac - TSN1-GFP and Trpribo-TF-P tac -TSN3-GFP. The culture experiment was performed in the same manner as in Example 1-2.
  • Trpribo-TF-P tac - TSN3-GFP the tryptophan dose-response curve of Trpribo-TF-P tac - TSN3-GFP is shown in Fig. 6c, respectively.
  • Trpribo-TF-P tac- TSN1-GFP cell growth was severely inhibited, so it was not possible to proceed with the reaction experiment to tryptophan.
  • the transformant strain Trpribo-TF-P tac - TSN3-GFP containing the inducible promoter Tac promoter was treated with IPTG 0.1 ⁇ g/ml, the transformed strain Trpribo-TF-J23106-TSN3 - It was confirmed that the performance was similar to that of GFP. Since the Tac promoter can regulate the expression level by controlling the concentration of IPTG, the transformant strain Trpribo-TF-P tac - TSN3-GFP is also thought to be able to control the performance of the tryptophan biosensor by controlling the concentration of IPTG. .
  • the pACYC-Trpribo-ECF16-trN1-J23106-swN1-GFP (trN1-swN1 combination) has fold change and operating range compared to using the trN3-swN3 combination (TSN3). was confirmed similarly, but has the advantage of high reliability because the error range is narrow. Therefore, in the Examples to be described later, pACYC-Trpribo-ECF16-trN1-J23106-swN1-GFP was used.
  • the tryptophan biosensors constructed in Examples 1 to 3 have a mechanism in which the fluorescent protein GFP is expressed when tryptophan is present.
  • the fluorescent protein GFP was replaced with TetA (tetracycline/H + bidirectional transporter, encoded by the tetA gene), and BBa_J23106 and BBa_J23105 were used as promoters for TetA expression.
  • TetA tetracycline/H + bidirectional transporter, encoded by the tetA gene
  • BBa_J23106 and BBa_J23105 were used as promoters for TetA expression.
  • a tryptophan biosensor was constructed so that the growth rate of cells can be regulated according to the concentration of tryptophan.
  • the plasmid pCDF-3HPselector was amplified using primers TetA-In-F and TetA-In-R to obtain a first amplification product.
  • the plasmid pACYC-Trpribo-ECF16-trN1-J23106-swN1-GFP was amplified using primers Vec-F and Vec-R to obtain a second amplification product.
  • the obtained first and second amplification products were ligated in a Gibson assembly manner to construct a plasmid pACYC-Trpribo-ECF16-trN1-J23106-swN1-tetA.
  • the above plasmid pACYC-Trpribo-ECF16-trN1-J23106-swN1-tetA was amplified using 5' end phosphorylated primers sw1-J23105-F and Change-promoter-R.
  • the amplification product was blund-end ligated to construct a plasmid pACYC-Trpribo-ECF16-trN1-J23105-swN1-tetA.
  • the tryptophan detection mechanism of the tryptophan biosensor into which the transcriptional activator, the toehold switch and the selection marker TetA was introduced is shown in FIG. 7 , and the constructed plasmids pACYC-Trpribo-ECF16-trN1-J23106-swN1-tetA and pACYC-Trpribo-ECF16 -trN1-J23105-swN1-tetA operates with the same mechanism as in FIG. 7 .
  • the tryptophan biosensor constructed in this example does not respond when tryptophan is not present (FIG. 7a), and when tryptophan is present, TetA is expressed, and accordingly, it has resistance to the antibiotic tetracycline, thereby preventing cell growth. becomes advantageous (Fig. 7b).
  • E. coli W3110 strain was transformed with plasmids pACYC-Trpribo-ECF16-trN1-J23106-swN1-tetA and pACYC-Trpribo-ECF16-trN1-J23105-swN1-tetA, respectively, to transform strain Trpribo-TF-J23106-TSN1 -tetA and Trpribo-TF-J23105-TSN1-tetA were constructed.
  • the expression level of the promoter BBa_J23105 included in the constructed strain is 1/2 compared to that of BBa_J23106.
  • TetA in this experiment is a membrane protein inserted into the cell membrane. When overexpressed at a certain level or more, it can inhibit cell growth, so it must be expressed at an appropriate level. Accordingly, in order to maintain the expression of the membrane protein TetA lower than that of GFP, a transgenic strain Trpribo-TF-J23106-TSN1-tetA including a relatively low-strength promoter was used in the experiment described below.
  • the culture experiment for confirming the operation of the tryptophan biosensor into which TetA was introduced used the same medium as in Example 1-2.
  • the transformant strain constructed in Example 4-1 was inoculated into M9 medium and cultured for 24 hours.
  • the cultured strain was diluted in fresh M9 medium so that the OD 600 value was 0.05, and when the OD 600 value reached 0.8, it was again diluted in the new M9 medium so that the OD 600 value was 0.05.
  • the strains were transferred and cultured in a medium to which various concentrations of tryptophan (0 and 8 g/L) and tetracycline (0, 50 and 100 ⁇ g/mL) were added.
  • the OD 600 was measured a total of 5 times at 1 hour intervals to confirm the growth of the cells. The results of confirming the cell growth rate are shown in FIG. 8 .
  • the transformed strain Trpribo-TF-J23106-TSN1-tetA expresses TetA, and thus it was confirmed that it exhibits resistance to tetracycline.
  • the transformed strain Trpribo-TF-J23106-TSN1-tetA in the presence of tryptophan had a cell growth rate of about 8.7 times higher than when tryptophan was not present.
  • the above result means that the constructed tryptophan biosensor can regulate the expression of TetA by tryptophan.
  • the tryptophan-producing strain can be screened relatively simply.
  • the present inventors have developed a tryptophan biosensor including a transcriptional activator and a toehold switch, and confirmed that the operating range of the tryptophan biosensor can be controlled. This means that the tryptophan biosensor of the present invention is suitable for screening of industrial strains.

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Abstract

La présente invention concerne un biocapteur pour la détection du tryptophane et, plus particulièrement, un biocapteur pour la détection du tryptophane, comprenant un facteur d'activation de la transcription et un commutateur Toehold et son utilisation. Il a été identifié qu'un biocapteur pour détecter le tryptophane, comprenant un facteur d'activation de la transcription et un commutateur Toehold, de la présente invention, peut reconnaître spécifiquement et de manière sensible le tryptophane et peut sélectionner rapidement et facilement une souche produisant du tryptophane à une concentration élevée. De plus, il a été identifié dans la présente invention que la plage de fonctionnement et la variation du facteur de multiplication du biocapteur pour détecter le tryptophane peuvent être régulées. Par conséquent, un biocapteur pour détecter le tryptophane, selon la présente invention, peut être utilisé de diverses manières dans le domaine du criblage de souches industrielles.
PCT/KR2020/007828 2020-06-17 2020-06-17 Biocapteur pour la détection de tryptophane, comprenant un facteur d'activation de transcription et un commutateur toehold WO2021256582A1 (fr)

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CN112941088A (zh) * 2021-02-04 2021-06-11 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 与布氏杆菌毒力相关的基因及其在布氏杆菌毒力评价及制备弱毒布氏杆菌中的应用

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CN112941088B (zh) * 2021-02-04 2023-06-16 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 与布氏杆菌毒力相关的基因及其在布氏杆菌毒力评价及制备弱毒布氏杆菌中的应用

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