WO2013191473A1 - Procédé permettant de cribler des microorganismes grands producteurs de l-tryptophane au moyen d'un riborégulateur - Google Patents

Procédé permettant de cribler des microorganismes grands producteurs de l-tryptophane au moyen d'un riborégulateur Download PDF

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WO2013191473A1
WO2013191473A1 PCT/KR2013/005423 KR2013005423W WO2013191473A1 WO 2013191473 A1 WO2013191473 A1 WO 2013191473A1 KR 2013005423 W KR2013005423 W KR 2013005423W WO 2013191473 A1 WO2013191473 A1 WO 2013191473A1
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tryptophan
seq
screening
riboswitch
present
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PCT/KR2013/005423
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English (en)
Korean (ko)
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정규열
양진아
장성호
서상우
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포항공과대학교 산학협력단
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Priority claimed from KR1020130069803A external-priority patent/KR101500839B1/ko
Application filed by 포항공과대학교 산학협력단 filed Critical 포항공과대학교 산학협력단
Priority to US14/406,422 priority Critical patent/US9493767B2/en
Publication of WO2013191473A1 publication Critical patent/WO2013191473A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • 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.
  • strains 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.
  • MSG monosodium glutamate
  • strain library 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.
  • PCR polymerase chain reaction
  • LC / gas chromatography 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.
  • 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.
  • a small amount of sample and using a multiplate a relatively large number of variant strains can be analyzed simultaneously.
  • the processing capacity is low to analyze a large strain library produced by the above-described preparation method.
  • 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 .
  • FACS fluorescence activated cell sorting
  • 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.
  • 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.
  • SELEX Systematic Evolution of Ligand by Exponential Enrichment
  • RNA devices 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.
  • 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.
  • 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.
  • 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.
  • the tryptophan aptamer is preferably described by SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 15.
  • 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.
  • 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.
  • the tryptophan aptamer is preferably described by SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 15.
  • 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.
  • 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 ⁇ lacI ⁇ T7 promoter plasmid in one embodiment of the present invention.
  • telomere 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 ⁇ lacI ⁇ T7 promoter plasmid ) Is the result of PCR.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • the tryptophan aptamer is preferably described by SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 15.
  • 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.
  • 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.
  • the tryptophan aptamer is preferably described by SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 15.
  • 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.
  • the selection marker gene may include, but is not limited to, the tetA gene.
  • 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.
  • 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.
  • a sequence in which the U of the L-tryptophan aptamer RNA sequence was substituted with T (SEQ ID NO: 2) was used.
  • 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).
  • 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 °C 30 seconds, (98 °C 10 seconds, 55 °C 15 seconds, 72 °C 1 minutes) ⁇ 3 cycles, 72 °C 3 minutes, and then stored at 4 °C.
  • 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.
  • 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 °C 30 seconds, (98 °C 10 seconds, 60 °C 30 seconds, 72 °C 2 minutes) ⁇ 3 cycles, 72 °C 5 minutes, 4 °C 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 °C 10 seconds, 55 °C 15 seconds, 72 °C 2 minutes) ⁇ 30 cycles, 72 °C 5 minutes PCR was performed, and then stored at 4 °C.
  • overlap PCR overlap PCR
  • 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.
  • 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.
  • 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.
  • the gene was recovered by gel-electrophoresis after digesting the pACYC Duet ⁇ lacI ⁇ T7 promoter plasmid with restriction enzymes KpnI and SacI.
  • T4 ligase T4 ligase
  • 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.
  • a reverse primer (SEQ ID NO: 11) complementary to the pACYC Duet ⁇ lacI ⁇ T7 promoter plasmid having an overhang of the J23100 promoter and a 10 bp random sequence. 12) using the PCR at 95 °C 30 seconds, (95 °C 30 seconds, 56 °C 30 seconds, 72 °C 5 minutes) ⁇ 30 cycles, 72 °C 7 minutes conditions, and stored at 4 °C, 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.
  • the riboSwitch library of the present invention was completed by transforming the product of 9 into MegaX DH ⁇ 10B cells.
  • RNA switch ribo switch
  • the characteristics of the tetA gene were used in the selection process.
  • the tetA gene 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.
  • the tetA gene if the tetA gene is overexpressed, the cells are killed by nickel ions.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g / ml) and incubated for 24 hours.
  • colonies 1 to 3 are cells selected from 40 ⁇ g / ml tetracycline
  • colonies 4 to 9 are cells selected from 100 ⁇ g / ml tetracycline.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention porte sur un procédé permettant de cribler des microorganismes grands producteurs de L-tryptophane au moyen d'un riborégulateur. L'invention se rapporte, en particulier, à un riborégulateur destiné au criblage de microorganismes grands producteurs de L-tryptophane, qui comprend des aptamères de tryptophane, une séquence d'ADN constituée de 1 à 20 nucléotides et un gène marqueur sélectionnable, et à un procédé de criblage faisant appel audit riborégulateur. Le riborégulateur et le procédé permettant de cribler des microorganismes grands producteurs de L-tryptophane au moyen d'un riborégulateur selon l'invention peuvent permettre de choisir, de façon relativement rapide et aisée, une souche produisant des concentrations élevées de L-tryptophane et, par conséquent, améliorer la compétitivité des prix de la production de tryptophane au moyen de microorganismes.
PCT/KR2013/005423 2012-06-20 2013-06-19 Procédé permettant de cribler des microorganismes grands producteurs de l-tryptophane au moyen d'un riborégulateur WO2013191473A1 (fr)

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US14/406,422 US9493767B2 (en) 2012-06-20 2013-06-19 Method for screening for high L-tryptophan producing microorganisms using riboswitch

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KR10-2012-0066009 2012-06-20
KR20120066009 2012-06-20
KR1020130069803A KR101500839B1 (ko) 2012-06-20 2013-06-18 리보스위치를 이용한 ｌ-트립토판 고생산균 스크리닝 방법
KR10-2013-0069803 2013-06-18

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20100017905A (ko) * 2007-05-29 2010-02-16 예일 유니버시티 리보스위치, 리보스위치의 사용 방법 및 리보스위치를 함유하는 조성물

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20100017905A (ko) * 2007-05-29 2010-02-16 예일 유니버시티 리보스위치, 리보스위치의 사용 방법 및 리보스위치를 함유하는 조성물

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JANG, SUNG HO ET AL.: "Engineering of L-tryptophan aptamer to produce RNA switch", KOREA SOCIETY FOR BIOTECHNOLOGY AND BIOENGINEERING SPRING MEETING ABSTRACTS, 14 April 2011 (2011-04-14), pages 189 *
MURANAKA, N. ET AL.: "An efficient platform for genetic selection and screening of gene switches in Escherichia coli", NUCLEIC ACIDS RES., vol. 37, no. 5, 3 February 2009 (2009-02-03), pages E39 *
WEIGAND, J. E. ET AL.: "Screening for engineered neomycin riboswitches that control translation initiation", RNA, vol. 14, 13 November 2007 (2007-11-13), pages 89 - 97 *

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