WO2013191473A1 - Method for screening for high l-tryptophan producing microorganisms using riboswitch - Google Patents

Description

L-Tryptophan High Production Bacteria Screening Using Ribo Switch

The present invention relates to a method for screening L-tryptophan high-producing bacteria using riboswitch, and more specifically, for L-tryptophan-producing bacteria screening, which comprises tryptophan aptamer, a DNA sequence consisting of 1 to 20 bases, and a selection marker gene. It relates to a switch and a screening method using the same.

In order to increase the price competitiveness of the method of producing metabolites using microorganisms, strains have been continuously developed. A traditional and effective, combinatorial approach to strain development consists of creating a strain library based on production strains and then screening for improved strains in the library. An example is the development of a monosodium glutamate (MSG) producing strain whose production method has been converted from fermentation using microorganisms in the 1960s.

In order to generate a strain library, UV irradiation, chemical mutations such as adding a chemical mutagen such as NTG (nitrosoguanidine), etc. have been used. In modern times, where molecular biological tools and biochemical knowledge are accumulated, genome shuffling and transposons can be caused by mutations in genes based on polymerase chain reaction (PCR) or through protoplast fusion. A variety of strain library generation methods have been proposed, including random insertion. The larger the size of the strain library is, the higher the likelihood that the target metabolite high-producing bacteria is contained therein, so that the strain library can be generated by using the above-described library preparation method in multiple.

Various assays are used to determine the target metabolite productivity of the strain. Liquid / gas chromatography (LC / GC) is a method of culturing individual strains and then analyzing the metabolite concentrations in the culture and strain. This method is capable of quantitative analysis if most metabolites can be detected and a standard calibration curve can be obtained. However, since only one variation of strain can be measured at a time, the throughput is low, making it inefficient for analyzing a library of strains of a certain size or more.

In the metabolite analysis method using multi plates, the variation of metabolite concentration in the sample is analyzed by measuring the change in color development, absorbance or fluorescence of a small amount of sample after putting the mutant strain in a separate well. That's how. By using a small amount of sample and using a multiplate, a relatively large number of variant strains can be analyzed simultaneously. However, the processing capacity is low to analyze a large strain library produced by the above-described preparation method. In addition, the scope of application is narrow because it is applicable only to metabolites capable of color reaction using metabolites as substrates or to measure changes in absorbance or fluorescence.

A method for screening production strains using genetic biosensors is detected by converting the concentration of the synthesized desired metabolite into a signal that can be detected immediately. If a biosensor specific to the target metabolite is developed and applied, the concentration of the target metabolite that cannot be detected visually can be observed by using a suitable detector.

The fluorescence activated cell sorting (FACS) technique detects the fluorescence emitted from individual strains by flowing the mutant strains to the detector. Through the flow of large numbers of cells simultaneously and fluorescence can be detected very quickly, the throughput is more than 10 ⁹ . When the target metabolite fluoresces, large libraries can be analyzed relatively quickly and easily, effectively screening high producing bacteria. However, there is a limitation that it can be applied only to the metabolite which shows fluorescence.

Finally, a selection method can be used. This is a technique that allows only strains that produce high concentrations of the desired metabolite in the strain library to survive. This method is so high throughput that only high production strains can be effectively selected from a large strain library. However, this technique can only be applied if the concentration of the desired metabolite is involved in the growth or survival of the strain.

On the other hand, riboswitch (riboswitch) is a biosensor that detects the concentration of a specific metabolite in the cell to regulate the expression of the gene located downstream, substrate specificity and substrate affinity is very high. In addition, techniques for generating aptamers that bind to specific metabolites using the Systematic Evolution of Ligand by Exponential Enrichment (SELEX) technology and making riboswitches based thereon have been developed. Therefore, we can develop riboswitches that regulate the expression level of downstream genes by recognizing only and sensitively the metabolites we want to screen.

By inserting the selection marker gene downstream of the riboswitch developed in this way, it is possible to obtain an RNA device that regulates the expression of the selection marker gene according to the concentration of the target metabolite. When RNA devices are introduced into strain libraries generated by various methods, the expression level of the selection marker gene is changed according to the concentration of the target metabolite in each strain. At this time, when the strain selection group transformed by the artificial selection circuit to the appropriate selection pressure corresponding to the selection marker gene of the RNA device will only survive the strain producing a high concentration of the target metabolite.

The present invention can be applied to a variety of target metabolites in order to solve the problems of the prior art as described above, and to develop a screening technique having a high throughput.

To this end, the present invention is to provide a ribo switch for screening L-tryptophan high production bacteria.

Another object of the present invention is to provide a method for effectively screening L-tryptophan high producing bacteria using the ribo switch.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides riboswitches for screening L-tryptophan high-producing bacteria comprising tryptophan aptamers, DNA sequences consisting of 1 to 20 bases, and selection marker genes.

In the present invention, the tryptophan aptamer is preferably described by SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 15.

In the present invention, the DNA sequence consisting of 1 to 20 bases is preferably a DNA sequence consisting of 10 bases, and may include those described in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, but are not limited thereto. Do not.

In the present invention, the selection marker gene may include, but is not limited to, the tetA gene.

The present invention also provides a method for screening L-tryptophan high-producing bacteria using a ribo switch comprising a tryptophan aptamer, a DNA sequence consisting of 1 to 20 bases, and a selection marker gene.

In the present invention, the tryptophan aptamer is preferably described by SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 15.

In the present invention, the DNA sequence consisting of 1 to 20 bases is preferably a DNA sequence consisting of 10 bases, and may include those described in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, but are not limited thereto. Do not.

In the present invention, the selection marker gene may include, but is not limited to, the tetA gene.

The ribo switch of the present invention and a method for screening high L-tryptophan producing bacteria using the same can relatively quickly and easily select a strain producing L-tryptophan at a high concentration, and using this to produce tryptophan using microorganisms Can increase the price competitiveness.

Figure 1 shows the structure of L-tryptophan aptamer (Trp 70-727). The RNA sequence of L-tryptophan aptamer (Trp 70-727) is shown in SEQ ID NO: 1.

Figure 2 shows the design of the riboswitch library of the present invention.

Figure 3 shows a schematic diagram of a cloning process for the fabrication of riboswitch library of the present invention.

Figure 4 shows the electrophoresis results for confirming the TA cloning results of pMD20-tetA-linker-sGFP.

5 is a result of confirming the result of recovering the tetA-linker-sGFP gene by electrophoresis after cutting the inserted vector gene with a restriction enzyme.

Figure 6 shows the results of electrophoresis after ligation of the tetA-linker-sGFP gene and the pACYC Duet ΔlacI ΔT7 promoter plasmid in one embodiment of the present invention.

7 is a reverse primer that complementarily binds to a forward primer having a J23100 promoter and a 10 bp random sequence as an overhang and a pACYC Duet ΔlacI ΔT7 promoter plasmid ) Is the result of PCR.

8 is a schematic diagram showing a process for selecting L-tryptophan-specific riboswitch from the riboswitch library of the present invention.

Figure 9 shows the results of measuring the performance of each ribo switch using the sGFP gene.

10 is a schematic diagram showing a process of applying the technology proposed in the present invention to various target materials.

The present invention has developed an RNA switch that specifically recognizes L-tryptophan and regulates the expression level of a gene located downstream. First, a library capable of switching using a known L-tryptophan RNA aptamer was prepared. We only picked the switches that actually worked from the library.

The present invention provides riboswitches for screening L-tryptophan high-producing bacteria comprising tryptophan aptamers, DNA sequences consisting of 1 to 20 bases, and selection marker genes.

In the present invention, the tryptophan aptamer is preferably described by SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 15.

In the present invention, the DNA sequence consisting of 1 to 20 bases is preferably a DNA sequence consisting of 10 bases, and may include those described in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, but are not limited thereto. Do not.

In the present invention, the selection marker gene may include, but is not limited to, a tetA gene encoding a tetratracycline resistance protein.

The present invention also provides a method for screening L-tryptophan high-producing bacteria using a ribo switch comprising a tryptophan aptamer, a DNA sequence consisting of 1 to 20 bases, and a selection marker gene.

In the present invention, the tryptophan aptamer is preferably described by SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 15.

In the present invention, the DNA sequence consisting of 1 to 20 bases is preferably a DNA sequence consisting of 10 bases, and may include those described in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, but are not limited thereto. Do not.

In the present invention, the selection marker gene may include, but is not limited to, the tetA gene.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Taq polymerase, Phusion polymerase, and restriction enzymes used in the present invention were purchased from TaKaRa or New England Biolabs, pACYC_Duet and pCDF_Duet vectors were purchased from Novagen, and oligonucleotides were used. Synthesis by Genotech. All other materials for the preparation of the culture were purchased from Sigma.

Example 1. RNA device library construction

First, a library having various sequences was prepared to make a switch for controlling the expression level of a gene located downstream according to the concentration of L-tryptophan. This library contains 10 known L-tryptophan aptamers (Irene Majerfeld and Michael Yarus, Nucl.Acids.Res., 2005) and downstream selection marker genes (tetA-sGFP fusion: SEQ ID NO: 6). Created by inserting random sequences of base pairs (bp) length.

L-tryptophan aptamer (Trp 70-727) is shown in SEQ ID NO: 1 and FIG. 1 and provides a site to which L-tryptophan can bind. In the cloning step of the present invention, a sequence in which the U of the L-tryptophan aptamer RNA sequence was substituted with T (SEQ ID NO: 2) was used. Among the downstream selection marker genes, the tetA gene was used to select a sequence that acts as an RNA switch, and the sGFP linked thereto was used to investigate the performance of the riboselector selected using fluorescence. A 10 bp long random sequence was located upstream of the ribosomal binding site (RBS).

When L-tryptophan binds to aptamers, some of the random sequences can bind RBS inside the secondary or tertiary structure through complementary binding to the ribosomal binding site (RBS) or vice versa. This hidden or exposed RBS affects the binding of ribosomes, resulting in the regulation of the expression level of the downstream selection marker gene (tetA-sGFP fusion). The schematic diagram of the riboswitch library of the present invention is shown in FIG. 2.

Cloning of the following process was performed to make a library having the above structure, and each process is briefly shown in FIG. 3.

① First, a forward primer (SEQ ID NO: 7) having an overhang of a KpnI site and an SD sequence and a reverse primer having an overhang of a linker sequence (Gly-Gly-Gly-Ser) × 4 tetA gene was PCR using reverse primer: SEQ ID NO: 8). PCR conditions were 98 ℃ 30 seconds, (98 ℃ 10 seconds, 55 ℃ 15 seconds, 72 ℃ 1 minutes) × 3 cycles, 72 ℃ 3 minutes, and then stored at 4 ℃.

② Next, a forward primer having the linker sequence (Gly-Gly-Gly-Ser) × 4 as an overhang and a reverse primer having the SacI site as an overhang 10) was used to PCR the sGFP gene.

③ The PCR products were mixed to perform overlap PCR (overlap PCR) to obtain the tetA-linker-sGFP gene. Specifically, after mixing the PCR products obtained in ① and ② (98 ℃ 30 seconds, (98 ℃ 10 seconds, 60 ℃ 30 seconds, 72 ℃ 2 minutes) × 3 cycles, 72 ℃ 5 minutes, 4 ℃ After storage, add a forward primer (SEQ ID NO: 7) having an overhang of the KpnI site and the SD sequence and a reverse primer (SEQ ID NO: 10) having the SacI site overhanging at 98 ° C for 30 seconds, ( 98 ℃ 10 seconds, 55 ℃ 15 seconds, 72 ℃ 2 minutes) × 30 cycles, 72 ℃ 5 minutes PCR was performed, and then stored at 4 ℃.

④ The gene tetA-linker-sGFP obtained in the above ③ was inserted into the T-vector. 4 shows the results of PCR confirming the gene inserted into the T-vector through TA cloning. Specifically, the PCR products obtained in the above ① and ② are connected by performing an overlap PCR, followed by attaching A-tail at both ends of the structure using Taq polymerase. Subject to T-vector. As a final result, KpnI-SD-tetA-linker-sGFP-SacI is in the T-vector. Colony PCR was performed using a primer set that binds upstream and downstream of the inserted product in this state. When properly inserted, as shown in FIG. 4, it can be seen that a band of about 1959 bp appears.

Sequencing selected plasmids containing the correct constructs. Treatment of KpnI and SacI restriction enzymes to plasmids inserted into the T-vectors results in cleavage of restriction site recognition sites located at 5 'and 3' of the vector fragments (vector fragments). ) And a structure fragment are created. The gene was recovered by gel-electrophoresis, and as shown in FIG. 5, it can be seen that a band corresponding to 1959 bp is a structure we intend to recover.

⑥ Meanwhile, the gene was recovered by gel-electrophoresis after digesting the pACYC Duet ΔlacI ΔT7 promoter plasmid with restriction enzymes KpnI and SacI.

⑦ Next, using the T4 ligase (T4 ligase) was combined (ligation) the results of the above (⑤) and (⑥), and the results confirmed by electrophoresis is shown in FIG. Specifically, the plasmid from which the lacI gene and the T7 promoter were deleted from the pACYC Duet vector was digested with KpnI and SacI restriction enzymes, and then linked to the construct recovered in ⑤. The structures 5 'and 3' were cut with restriction enzymes KpnI and SacI, respectively, so that they could be bound to pACYC accurately, and the results can be seen in FIG. 6.

⑧ Next, a reverse primer (SEQ ID NO: 11) complementary to the pACYC Duet ΔlacI ΔT7 promoter plasmid having an overhang of the J23100 promoter and a 10 bp random sequence. 12) using the PCR at 95 ℃ 30 seconds, (95 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 5 minutes) × 30 cycles, 72 ℃ 7 minutes conditions, and stored at 4 ℃, the results Is shown in FIG. 7. A forward primer was designed to insert an aptamer and a random sequence upstream of tetA with tetA-linker-sGFP in pACYC. The reverse primer was designed to stick directly above tetA and PCR with the forward primer yielded the entire pACYC-aptamer-random sequence-tetA-linker-sGFP. This linear DNA sequence was blunt end ligation to make a plasmid, which was confirmed in FIG.

⑨ Next, attach both ends of the result obtained in ⑧ by the Blunt-end ligation method,

결과 The riboSwitch library of the present invention was completed by transforming the product of ⑨ into MegaX DHα 10B cells.

Example 2 Screening of L-Tryptophan Specific Riboswitches

Through two screening processes, it was possible to select a ribo switch (RNA switch) in response to L-tryptophan from the ribo switch library prepared in Example 1 (RNA switch library).

The characteristics of the tetA gene were used in the selection process. When the tetA gene is expressed, the tetA protein moves to the cell membrane and releases tetracycline inside the cell to the outside, thereby making the cell resistant to tetracycline. However, if the tetA gene is overexpressed, the cells are killed by nickel ions. Using the properties of the tetA gene as described above, it is possible to select a cell having a high tetA expression when the L- tryptophan concentration in the cell, and a plasmid that does not express tetA when the L- tryptophan concentration is low.

The process of selecting L-tryptophan-specific riboswitches is shown in FIG. 8. The cells that survive the addition of tetracycline to the medium at high intracellular L-tryptophan concentrations are cells that express much tetA. These cells can be harvested and changed to low L-tryptophan concentrations, followed by the addition of nickel ions to the medium, and cells that are still alive when grown (Adapted from Muranaka, N. et al) , Nucl.Acids Res., 37, e39, 2009).

Cells that survived the two selection processes as described above have a plasmid that expresses the tetA gene when the L-tryptophan concentration is high, but does not express the tetA gene when the L-tryptophan concentration is low. Harvesting and sequencing these plasmids can identify which of the 10 bp random sequences enables the operation of riboswitches.

Meanwhile, the performance of each ribo switch can be measured using the sGFP gene linked to each tetA. Gene expression control ability can be confirmed by measuring the intensity of sGFP fluorescence at high and low intracellular L-tryptophan concentrations.

Detailed experimental procedure is as follows.

① E. coli W3110 cells containing Riboswitch library plasmids were incubated for 8 hours in CM9 medium containing chloramphenicol in M9 medium, and the culture was transferred to CM9 containing 0.2 mM NiCl ₂ . Incubated for 24 hours.

② The resulting product of ① was transferred to CM9 to which 1 mM L-tryptophan was added, and then incubated for 8 hours. The culture was transferred to CM9 containing 1 mM L-tryptophan and tetracycline (40 or 100 μg / ml) and incubated for 24 hours.

③ The result of ② was grown in a CLB plate in which chloramphenicol was added to LB medium. Several colonies were selected and grown in medium with and without L-tryptophan, and fluorescence intensity was measured.

The experimental results are shown in FIG. 9. In FIG. 9, colonies 1 to 3 are cells selected from 40 μg / ml tetracycline, and colonies 4 to 9 are cells selected from 100 μg / ml tetracycline. As shown in Figure 9, colonies 2, 3, 6 showed good performance, their activation ratio (activation ratio) was about 2.3. The nucleotide sequences of colonies 2, 3 and 6 showed good performance as shown in SEQ ID NOs: 3, 4 and 5.

On the other hand, sequencing analysis of riboswitch colonies 2, 3, and 6, which showed good performance, showed that the sequences of tryptophan aptamers were different from each other. That is, in the case of colony 3, the tryptophan aptamer sequence had the sequence of SEQ ID NO: 2, whereas colonies 2 and 6 had the sequences of SEQ ID NO: 13 and 15, respectively, and it was confirmed that mutation occurred during selection.

As used herein, the term "activation ratio" refers to the OD of each culture after tryptophan is added to and without fluorescence of cell culture. It means the ratio of normalized value divided by and can be expressed as below. However, in the following equation, background fluorescence is a value of measuring fluorescence intensity of PBS (Phosphate Buffered Saline), and the reason for using it as a background is that it is measured by diluting with PBS when measuring fluorescence intensity of cell culture. Because I did.

The method for screening high production of L-tryptophan using the ribo switch prepared by the above embodiment of the present invention is briefly shown in FIG. 10.

10 describes the process of applying the technology of the present invention to various target materials. Through the SELEX process, RNA aptamers that bind to the target substance are generated, riboswitches are generated based on the generated aptamers, and then enriched in a strain library using the generated riboswitches. By performing enrichment, a superior strain can be obtained. The strain obtained in this way can be investigated in detail and used immediately for the production of the target substance, or a better strain can be obtained by repeating the enrichment step several times.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

L-Tryptophan High Production Bacteria Screening Method Using Riboswitch of the Invention Since a strain producing L-tryptophan at a high concentration can be selected relatively quickly and easily, it is possible to produce tryptophan at a low price.

Claims (8)

Riboswitch for L-tryptophan high producing bacteria screening, comprising tryptophan aptamer, DNA sequence consisting of 1 to 20 bases, and a selection marker gene. The method of claim 1, The tryptophan aptamer is described in SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 15, L-tryptophan (R-tryptophan) high production bacteria screening for riboswitch. The method of claim 1, The DNA sequence consisting of the 1 to 20 bases are those described in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, L-tryptophan (R-tryptophan) high production bacteria screening for riboswitch. The method of claim 1, The selection marker gene is a tetA gene, riboswitch for screening L-tryptophan high production bacteria. A method for screening L-tryptophan high producing bacteria using tryptophan aptamer, a DNA sequence consisting of 1 to 20 bases, and a riboswitch comprising a selection marker gene. The method of claim 5, The tryptophan aptamer is described in SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 15, a method for screening L-tryptophan high producing bacteria. The method of claim 5, The DNA sequence consisting of 1 to 20 bases are those described in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, a method for screening L-tryptophan high-producing bacteria. The method of claim 5, The selection marker gene is a tetA gene, a method for screening L-tryptophan high producing bacteria.

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