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WO2025101028A1 - Procédé de synthèse d'une banque de régions non traduites à l'aide de désoxynucléotidyl transférase terminale (tdt) et de dntp - Google Patents

Procédé de synthèse d'une banque de régions non traduites à l'aide de désoxynucléotidyl transférase terminale (tdt) et de dntp Download PDF

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WO2025101028A1
WO2025101028A1 PCT/KR2024/017762 KR2024017762W WO2025101028A1 WO 2025101028 A1 WO2025101028 A1 WO 2025101028A1 KR 2024017762 W KR2024017762 W KR 2024017762W WO 2025101028 A1 WO2025101028 A1 WO 2025101028A1
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prut
dntp
library
tdt
strain
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조병관
김의기
유어진
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Korea Advanced Institute of Science and Technology KAIST
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Definitions

  • the present invention relates to a method for synthesizing a PRUT (promoter, RBS-containing 5'-UTR, transcription terminator) random sequence using TdT (terminal deoxynucleotidyl transferase) and dNTP.
  • PRUT promoter, RBS-containing 5'-UTR, transcription terminator
  • TdT terminal deoxynucleotidyl transferase
  • a chemical synthesis method using phosphoramidites is commercially available for synthesizing short ( ⁇ 250 nt) ssDNA (oligonucleotides) with specific base sequences, and is also used for synthesizing random sequence oligonucleotide libraries by setting some or all of the base sequences as random sequences.
  • Non-patent Document 1 Tong, Y., Zhou, J., Zhang, L. and Xu, P. (2021) A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica. ACS Synth Biol, 10, 115-124.
  • Non-patent Document 2 Choe, D., Kim, K., Kang, M., Lee, S.G., Cho, S., Palsson, B. and Cho, B.K. (2022) Synthetic 3'-UTR valves for optimal metabolic flux control in Escherichia coli. Nucleic Acids Res, 50, 4171-4186.
  • the present invention overcomes the limitations described above and is a technology for easily producing various types of random sequence libraries in a laboratory.
  • the present invention has been completed by developing a technology for controlling the base composition (ratio between A, T, G, and C) of a random sequence in a TdT enzyme reaction using dNTP as a substrate according to the user's needs.
  • An object of the present invention is to provide a method for synthesizing a PRUT (PRUT: promoter, RBS-containing 5'-UTR, Terminator) random sequence library using TdT (Terminal deoxynucleotidyl transferase) and dNTP, characterized in that it includes a step of controlling the ratio of a specific deoxyribonucleotide in a dNTP mixture.
  • PRUT Physical deoxynucleotidyl transferase
  • Another object of the present invention is to provide a strain comprising PRUT synthesized through the above method.
  • Another object of the present invention is to provide a method for producing a strain including PRUT synthesized through the above method and a method for producing a useful product including a step of culturing the strain.
  • the synthetic method of the present invention has the advantage that the synthesis is carried out under aqueous conditions simulating biological conditions, so there is no toxic organic solvent waste generated from the phosphoramidite chemical synthesis method, there is no base damage, and even a length longer than 400 nt can be synthesized, and synthesis can be easily performed within 1 hour using only an incubator without complex mechanical equipment under laboratory conditions.
  • the base composition, random sequence length, etc. can be easily adjusted according to the user's convenience.
  • Figure 1 shows the results of equalizing the preference of the oligo library by adjusting the dNTP and cofactor concentrations.
  • Figure 1A is the base composition in various cofactor types and concentrations.
  • Figure 1B confirms the uniformity of the base composition of the oligo library when the initial concentration of the substrate is adjusted.
  • Figure 1C plots the correlation of the variables, where A, T, G, and C are the ratios of each base in the TdT synthetic library; [dATP], [dTTP], [dGTP], and [dCTP] are the concentrations of each dNTP under the reaction conditions; and H is the Shannon entropy, a measure of the library base uniformity.
  • Figure 1D is a correlation modeling between the fractional ratio of dNTPs with respect to the total mixture concentration and the N ratio of the resulting library (wherein N is A, C, G, or T).
  • Figure 2 is a schematic diagram of the experimental procedure and the results of the PRUT library selection.
  • Figure 2A shows the process and results of inserting the TdT synthetic library into the regulatory region.
  • Figure 2B shows the construction of the promoter library and the profiling of its fluorescence distribution.
  • Figure 2C shows the construction of the 5'UTR library according to three base compositions and the profiling of its fluorescence distribution.
  • Figure 2D shows the results of confirming that three different transcription terminator libraries have different termination efficiencies.
  • the rightmost table shows the Pearson R 2 value and the tangent value.
  • Figure 3 is about the individual sequence discovery process from each PRUT library
  • Figure 3A is a NGS data normalization method
  • Figure 3B is a summary of the number of candidate sequences finally obtained through FACS-sorting and NGS.
  • Figure 3C is data that confirmed the expression level more precisely by introducing some of the discovered promoter sequences into E. coli colonies
  • Figure 3D is data that confirmed the expression level by introducing some of the discovered 5'UTR sequences into E. coli colonies.
  • Figure 4 shows the results of optimizing the biosynthetic pathway for high-purity production of violacein.
  • Figure 4A shows the violacein biosynthetic pathway
  • Figure 4B shows the design of the violacein pathway using the classified PRUT parts
  • Figure 4C shows the results of confirming the productivity of violacein in E. coli including the designed violacein biosynthetic system.
  • Fig. 5 shows the results of optimizing the biosynthetic pathway for high-purity lycopene production.
  • Fig. 5A shows a vector including a promoter sequence that is partially randomized using the TdT random synthesis library technology of the present invention in the promoter sequence that controls the expression level of an operon composed of lycopene synthesis genes.
  • Fig. 5B shows the results of confirming the growth and lycopene production level of E. coli DH5 ⁇ strain including the vector of Fig. 5A, and shows the lycopene production levels of high-producing strains selected through image analysis.
  • the positive control is the BBa_J23119 promoter, which is the promoter before the random sequence synthesized by TdT is inserted, and the negative control (N.C.) is the promoter sequence removed.
  • the light gray bar (left) indicates the optical density at 600 nm wavelength (OD 600 nm), which is an indicator of the final cell density of the culture medium, and the black bar (right) indicates the lycopene production (titer, in g/l).
  • One aspect of the present invention is a method for synthesizing a PRUT random sequence library using TdT (Terminal deoxynucleotidyl transferase) and dNTP.
  • TdT Terminal deoxynucleotidyl transferase
  • a PRUT random sequence library synthesis method is characterized by including a step of controlling the ratio of a specific deoxyribonucleotide in a dNTP mixture.
  • a PRUT random sequence library synthesis method according to the above specific example, characterized in that the TdT is of murine origin.
  • a PRUT random sequence library synthesis method according to any one of the preceding specific examples, wherein the PRUT comprises at least one of a promoter, a 5' UTR including an RBS, and a transcription terminator.
  • a PRUT random sequence library synthesis method according to any one of the preceding specific examples, characterized in that the method comprises a step of calculating the amount of dNTP input to control the base composition constituting the library according to any one of the following formulas 1 to 4:
  • P(dNTP) represents the ratio of a specific dNTP in the mixture.
  • the value of P(dNTP) can be calculated as follows.
  • the method may further include a step of calculating a corrected P n (N) value by substituting the P (N) value calculated according to any one of Equations 1 to 4 into Equations 5 and 6 below:
  • P n (N) is a normalized value of P (N), which adjusts the ratio of each base so that the sum of the ratios of the bases becomes exactly 1.
  • the method is characterized in that the amount of dGTP input is calculated according to the following Equation 4 to control the composition of guanine (G) constituting the library:
  • a PRUT random sequence library synthesis method according to any one of the preceding specific examples, characterized in that the PRUT is a PRUT for expression in E. coli.
  • a method for synthesizing a PRUT random sequence library according to any one of the preceding specific examples, wherein the PRUT is a 5'UTR including an RBS, and the method is characterized by including a step of controlling the ratio of dATP and dGTP so that the content of A and G exceeds 50% within the entire synthesized polynucleotide sequence.
  • a method for synthesizing a PRUT random sequence library according to any one of the preceding specific examples, wherein the PRUT is a 5'UTR including an RBS, and the method comprises a step of controlling the ratio of dATP and dGTP so that the content of A and G in the entire synthesized polynucleotide sequence exceeds 50%, and is characterized in that the average expression level of the synthesized 5' UTR library is higher than that in a case where the content of A and G in the entire synthesized polynucleotide sequence is 50% or less.
  • a method for synthesizing a PRUT random sequence library according to any one of the preceding specific examples, wherein the PRUT is a transcription terminator, and the method comprises a step of controlling a ratio so that the content of C is higher than that of G in the entire synthesized polynucleotide sequence, and the termination efficiency of the synthesized transcription terminator library is higher than that in a case where the content of G is equal to or lower than the content of C in the entire synthesized polynucleotide sequence.
  • a PRUT random sequence library synthesis method according to any one of the preceding specific examples, wherein the method is characterized in that at least one metal cation selected from Mg 2+ , Zn 2+ , Co 2+ and Mn 2+ is used as a cofactor in the dNTP and TdT reaction step.
  • a PRUT random sequence library synthesis method according to any one of the preceding specific examples, wherein the method is characterized in that Co 2+ is used as a cofactor in the dNTP and TdT reaction steps.
  • a PRUT random sequence library synthesis method according to any one of the preceding specific examples, wherein the method is characterized in that the concentration of the cofactor used in the dNTP and TdT reaction steps is 0.5 mM to 1.5 mM.
  • Another aspect of the present invention is a strain comprising PRUT synthesized by the above method.
  • the strain is E. coli.
  • a strain according to any one of the preceding specific examples wherein the strain is characterized in that it comprises at least one of a promoter, a 5' UTR including an RBS, and a transcription terminator synthesized through the random sequence library synthesis method.
  • the strain is characterized in that it comprises the synthesized PRUT in a gene expression control region of the violacein biosynthetic pathway.
  • a strain according to any one of the preceding specific examples characterized in that the strain comprises the synthesized PRUT in a gene expression control region of a lycopene biosynthetic pathway.
  • a strain according to any one of the preceding specific examples characterized in that the strain comprises a promoter synthesized by the above method upstream of vioA and/or vioD of the violacein biosynthetic pathway.
  • Another aspect of the present invention is a method for producing a strain comprising PRUT synthesized by the above method.
  • Another aspect of the present invention is a method for producing a useful product, comprising the step of culturing a strain containing PRUT synthesized through the above method in a medium.
  • the strain is characterized in that it is E. coli.
  • a method for producing a useful product according to the above specific example is characterized in that the useful product is violacein.
  • Another aspect of the present invention is a method for producing a strain having increased target product production ability, comprising: a step of producing a strain including PRUT synthesized through the above method; and a step of comparing the target product production ability of the produced strain with a control group to select a strain having increased target product production ability compared to the control group.
  • PRUT refers to a promoter, an RBS-containing 5'UTR, and a transcription terminator
  • a sequence synthesized by the PRUT random sequence library synthesis method provided in the present invention is characterized by being at least one selected from a promoter sequence, an RBS-containing 5'UTR sequence, and a transcription terminator sequence.
  • promoter means a polynucleotide sequence that controls the transcription of an operably linked polynucleotide sequence.
  • a promoter in prokaryotes is defined as a region around a transcription initiation site where RNA polymerase binds, and is generally composed of two short base sequences located one base pair apart from the transcription initiation site in the -10 region and the -35 region (also referred to as the -10 and -35 elements).
  • the -10 region and the -35 region of the promoter are fixed as sequences capable of inducing expression, and the remaining region can be synthesized by replacing it with a random sequence.
  • the "5'-untranslated region (5'UTR)" refers to a region that exists at the 5'-end of an mRNA transcript but is not translated into an amino acid.
  • the process of translating mRNA into a protein begins with the binding of the 30S subunit of the ribosome to the 5'UTR.
  • the 16S rRNA (16S ribosomal RNA) in the 30S subunit of the ribosome binds to the RBS in the 5'UTR and tRNA recognizes and binds to the initiation codon (AUG) of the mRNA, translation into a protein begins.
  • the 5'-UTR of the present invention includes a ribosome binding site (RBS).
  • the "ribosome binding site (RBS)” means a region (structure) existing inside an mRNA chain, to which a ribosome directly binds and can also initiate translation, or a region (structure) of a DNA chain that causes the region by being transcribed.
  • Most prokaryotes possess an RBS as a short sequence that enables the ribosome to easily recognize and bind to mRNA, located close to the 5' upstream side from the start codon (usually 'AUG') on the mRNA containing the open reading frame (ORF).
  • the RBS contains a sequence complementary to the sequence at the 3' end of 16S rRNA, and this is called the Shine-Dalgarno sequence.
  • transcription terminator means a polynucleotide sequence located downstream of a protein coding region and involved in transcription termination when a polynucleotide is transcribed into mRNA.
  • TdT Terminal deoxynucleotidyl transferase
  • DNTT DNA nucleotidylexotransferase
  • dNTP refers to deoxyribonucleoside triphosphate
  • dATP deoxyribonucleoside triphosphate
  • dGTP deoxyribonucleoside triphosphate
  • dCTP deoxynucleoside triphosphate
  • dNTP When referred to as dNTP in the present invention, it may be interpreted as a mixture of the above four types of deoxyribonucleoside triphosphates or any one of the above four types of deoxyribonucleoside triphosphates depending on the context.
  • P(dATP), P(dCTP), P(dTTP), and P(dGTP) represent the ratio of specific dNTPs in a mixture of four types of deoxyribonucleoside triphosphates.
  • P(dATP) represents P(dATP), P(dCTP), P(dTTP), P(dGTP), i.e., the ratio of dATP in the entire dNTP mixture.
  • P n (A), P n (C), P n (T), and P n (G) represent the proportion of each base (A, C, T, G) included in the entire synthesized polynucleotide, respectively.
  • P n (A) represents the proportion of the adenine (A) base included in the entire generated polynucleotide, and accordingly, the sum of P n (A), P n (C), P n (T), and P n (G) becomes 1.
  • Equations 1 to 6 capable of calculating the content of a specific base included in the generated library based on the ratio of a specific dNTP in a mixture of four types of deoxyribonucleoside triphosphates were derived to enable more accurate and precise composition prediction and control, and a method capable of controlling the base composition of the library by controlling the ratio of dNTPs was developed.
  • the ratio of dATP and dGTP is adjusted so that the total content of A and G in the entire 5'UTR sequence polynucleotide generated by the random sequence library synthesis method exceeds 50%, it was confirmed that the average expression level of the synthesized 5' UTR library is higher compared to when the total content of A and G is 50% or less.
  • a step of controlling the ratio of C to be higher than G within the entire polynucleotide sequence of a transcription terminator sequence library generated by the random sequence library synthesis method is included, and it was confirmed that the termination efficiency of the transcription terminator library is higher compared to a case where the G content is equal to or lower than the C content.
  • E. coli K-12 MG1655 E. coli DH5 ⁇ (Enzynomics, Daejeon, Korea), and Endura TM electrocompetent cells (Lucigen, Middleton, WI, USA) were cultured in Difco TM LB Broth - Miller (BD Biosciences, San Jose, CA, USA) in broth or agar plate format.
  • E. coli MG1655 harboring pDRA2_CI and other pDRA2_CI-driven plasmids were cultured in M9 glucose minimal medium (47.75 mM Na2HPO4 , 22.04 mM KH2PO4 , 18.70 mM NH4Cl , 8.56 mM NaCl , 2 mM MgSO4 , 0.1 mM CaCl2 , and 2 g/L glucose) supplemented with 100 ⁇ g/mL ampicillin to an initial OD600 value of 0.05.
  • M9 glucose minimal medium 47.75 mM Na2HPO4 , 22.04 mM KH2PO4 , 18.70 mM NH4Cl , 8.56 mM NaCl , 2 mM MgSO4 , 0.1 mM CaCl2 , and 2 g/L glucose
  • 100 ⁇ g/mL ampicillin 100 ⁇ g/mL ampicillin to an initial OD600 value
  • Murine TdT was expressed and purified from E. coli BL21(DE) pET28a murineTdT strain. All TdT-synthetic libraries were first synthesized by elongation of the initiator oligo using a TdT reaction (2 ⁇ l 10x reaction buffer, 4 ⁇ l 10 ⁇ M initiator, 1 ⁇ l 20 mM CoCl2, 2 ⁇ l 10x dNTP mix, final 0.05 mg/ml murine TdT, and nuclease-free purified water to a final volume of 20 ⁇ l). The reaction was initiated by incubating the TdT reaction without TdT at 37 °C for 1 min and adding TdT and mixing by pipetting.
  • TdT reaction 2 ⁇ l 10x reaction buffer, 4 ⁇ l 10 ⁇ M initiator, 1 ⁇ l 20 mM CoCl2, 2 ⁇ l 10x dNTP mix, final 0.05 mg/ml murine TdT, and nuclease-free pur
  • the reaction was quenched after the desired time by adding 4 ⁇ l of 0.5 M EDTA.
  • the TdT reaction mixture was purified using the Oligo Clean & Concentration Kit (Zymo Research) and eluted with 20 ⁇ l of nuclease-free distilled water. 5 ⁇ l ( ⁇ 10 pmol) of the purified TdT-synthetic library was analyzed by electrophoresis on a 15% urea-PAGE gel (Invitrogen) at 180 V for 70 min. PAGE-gel purification was performed when necessary. Prior to amplification, 3'-adapter ligation was performed for reverse primer coupling.
  • Dual reporter assay system pDRA2_CI was derived from pDRA2 vector by inserting a strong synthetic transcription terminator L3S2P21 between lacI and egfp to minimize leaky expression of lacI. All PRUT libraries were inserted into the appropriate sites of the egfp-mrfp operon of pDRA2_CI.
  • pDRA2_CI was linearized by High Fidelity Phusion polymerase, and the replaced region was removed, leaving 20-bp homology arms. The purified PCR products were treated with FastDigest DpnI for ⁇ 12 h to remove the template plasmid, and only the major band of the desired size was gel purified.
  • 1st sorting Library samples were sorted into 4-6 regions and target events were set to a coverage of at least 15 times the expected colony count, i.e. (library size in CFU) X 15 X (region ratio).
  • 2nd sorting The entire volume of the 1st sorted cell population was sorted into the corresponding gate. Optional centrifugation was performed (3,134 rcf, 4°C, 10 min) to reduce the time required for 2nd sorting. The supernatant was removed, leaving 2-3 ml, and the cell pellet was resuspended. After 2nd sorting, the sorted cell population was concentrated by centrifugation (3,134 rcf, 4°C for 10 min or 16,000 rcf, 4°C for 10 min) to a final volume of ⁇ 10 ⁇ l.
  • High-throughput screening was performed using image analysis equipment and software for E. coli colonies on solid media obtained through the above plasmid construction method. The redness of the colonies was quantified to select the colonies that were expected to produce the most red lycopene. All plates were photographed in the same photographing environment to ensure consistency in the data. Afterwards, the obtained images were batch-processed using CellProfiler software (CellProfilerTM: Lamprecht, M.R., Sabatini, D.M. and Carpenter, A.E. (2007) Free, Versatile Software for Automated Biological Image Analysis. BioTechniques, 42(1), 71-75) and its examples, and the location (coordinates), size, shape, and redness of individual colonies were confirmed. The 1-2 colonies that appeared the reddest on each plate were identified, and the lycopene production was measured under LB medium liquid culture (batched culture, 3 ml) conditions.
  • the enzymatic reaction with a uniformly composed dNTP mixture does not actually mean the generation of a uniform oligonucleotide library (each of 25% of A, C, G, and T).
  • a uniform oligonucleotide library (each of 25% of A, C, G, and T).
  • the relationship between the base composition of the dNTP mixture and the base composition of the generated oligonucleotide library has not yet been elucidated, despite its necessity for controlling the base composition of the oligonucleotide library. Therefore, the present inventors analyzed the relationship between the base composition of the dNTP mixture and the base composition of the generated oligonucleotide library.
  • Fig. 1A several options for divalent cation cofactors were investigated when a homogeneous composition of dNTP mixtures was provided (Fig. 1A). All conditions indicated that murine TdT most commonly prefers G. In addition, among the three concentrations of Mg 2+ , Zn 2+ , Co 2+ , and Mn 2+ known to activate TdT, 1 mM Co 2+ was selected as an additional reaction condition because it generated the most homogeneous oligonucleotide library (Fig. 1A).
  • dNTP mixing conditions 81 different dNTP mixing conditions were screened (Fig. 1B).
  • the final concentrations of each of the four dNTPs were varied to 0.67, 1.0, and 1.5 mM.
  • the case with the minimum dGTP ratio 1.5 mM of dATP, dCTP, and dTTP; 0.67 mM of dGTP
  • the base-homogeneous oligonucleotide library with the highest uniformity with an error of less than 2%p, thereby showing the highest Shannon entropy value.
  • Equations 1 to 4 were derived to calculate the amount of dNTP input according to base composition.
  • the dNTP ratio was calculated according to the above formula, and the dNTP concentration was determined so that the sum of the dNTP concentrations was in the range of 4 to 6 mM. Then, a random library was synthesized and an experiment was performed to confirm the actual base composition.
  • Target base composition Sample name A C G T Experimental Example 1 Uniform (A, T 50%) 25% 25% 25% 25% A, T 90% 45% 5% 5% 45% Experimental example 2 Uniform (C 25%) 25% 25% 25% 25% C 30% 25% 30% 20% 25% C 35% 25% 35% 15% 25% Experimental Example 3 A, G 60% 30% 20% 30% 20% A, G 70% 35% 15% 35% 15%
  • the actual base composition was confirmed by sequencing as follows. For the sequencing process, at least 10 sequences were sequenced (at least 1000 bp analysis) using the next generation sequencing (NGS) kit and devices such as Mi-Seq and Next-Seq provided by Illumina or the Sanger sequencing service provided by Macrogen.
  • NGS next generation sequencing
  • the random sequence productivity of TdT was exploited to explore the unnatural sequence space to determine a fine-tuned PRUT set. After extending the initiator oligonucleotide to the desired length by TdT reaction, the length of each product was confirmed by electrophoresis. A random sequence oligonucleotide library of appropriate length was amplified and inserted into various sites of the reporter operon (Fig. 2A).
  • the base compositions of random sequences in the PRUT library were also varied.
  • the Cb library had UP elements with uniform or AT-rich (45% A and 45% T); 5'-UTR libraries with uniform or 60% purine (30% A and 30% G) or % purine (35% A and 35% G) base compositions; and transcription terminators with uniform or 30% C and 20% G, or 35% C and 15% G base compositions (Fig. 2B).
  • each TdT-synthetic PRUT sequence Based on the read count of each TdT-synthetic PRUT sequence, we were able to roughly estimate its intensity by assigning the PRUT sequence to one of the gates. After normalizing for the total read count per sample, each sample was again normalized to account for the actual cluster size (Fig. 3B). For a particular sequence, by comparing the read count across expression levels, the sequence was assigned to the most enriched region, generating a variety of P-values as a significance metric. We were able to sort the PRUT sequences across all expression levels and determine a PRUT set for further validation. The top 20 most significant sequences were synthesized as oligonucleotides and cloned into pDRA2_CI to obtain single colonies, which were then profiled for expression levels. This resulted in a fine-tuned PRUT toolkit set of approximately 100 sequences with a significant dynamic range (Fig. 3E).
  • Random sequence synthesis using TdT is suitable for library customization and the subsequent process is suitable for profiling using conventional cloning methods and flow cytometry.
  • the PRUT library was tested for metabolic engineering of biosynthetic pathways.
  • the promoter, 5'-UTR and transcription terminator libraries were cloned into violacein and lycopene production vectors in a combinatorial manner after a simple FACS sorting step to ensure significant expression levels.
  • Violacein is a representative high value-added bioactive compound known for its antibacterial function and vivid purple color. For decades, many studies have been conducted to produce violacein with higher titer by utilizing various bacterial and yeast platforms. However, in addition to titer, the purity of violacein with respect to the inevitable byproduct, deoxyviolacein, is another key factor due to the nature of the violacein biosynthetic pathway. Specifically, at the end of the pathway, VioD reduces protodeoxyviolaceinic acid (PDVA) to protoviolaceinic acid (PVA), while VioC acts on both PDVA and PVA to produce deoxyviolacein (DV) and violacein (V), respectively.
  • PDVA protodeoxyviolaceinic acid
  • PVA protoviolaceinic acid
  • VioC acts on both PDVA and PVA to produce deoxyviolacein (DV) and violacein (V), respectively.
  • the final strain produced a DV/V ratio of about 7.5% (Tong, Y., Zhou, J., Zhang, L. and Xu, P. (2021) A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica. ACS Synth Biol, 10, 115-124.). These characteristics provide room for optimizing V production by optimizing the level of vioC relative to vioD to redirect the waste material from DV to V. In particular, if the violacein production gene found in nature is directly introduced into the production strain E. coli, the synthetic efficiency may decrease because the gene expression deviates from the optimal point.
  • the TdT-synthetic transcription terminator was utilized as a metabolic valve to provide various distributions of vioC and vioD flux (Choe, D., Kim, K., Kang, M., Lee, SG, Cho, S., Palsson, B. and Cho, BK (2022) Synthetic 3'-UTR valves for optimal metabolic flux control in Escherichia coli. Nucleic Acids Res , 50 , 4171-4186).
  • the strong promoters were inserted upstream of vioA and vioD, and the TdT-synthetic terminator synthesized from the random library was inserted between vioD and vioC to explore various gene expression levels.
  • the constructed strains exhibited various productivities, which could be confirmed through color and absorbance in the range of 565-585 nm using a BioTek instrument (Fig. 4).
  • Lycopene is a representative red carotenoid compound produced from red crops such as tomatoes and carrots. It has antioxidant effects, removes active oxygen in the body to prevent additional cell damage, and has been proven to have anticancer and anti-inflammatory effects. Extraction of lycopene from crops is difficult due to the difficult purification process, and chemical synthesis incurs high costs, so attempts have been made to produce lycopene from bacteria such as E. coli to improve this.
  • four genes ipiHP1, crtE, crtB, and crtI, required for lycopene synthesis were introduced into an E.
  • coli strain and their expression levels are controlled by a single promoter (Yoon, SH, Lee, YM, Kim, JE, Lee, SH, Lee, JH, Kim, JY, Jung, KH, Shin, YC, Keasling, JD, and Kim, SW (2006) Enhanced Lycopene Production in Escherichia coli Engineered to Synthesize Isopentenyl Diphosphate and Dimethylallyl Diphosphate From Mevalonate. Biotechnol Bioeng, 94 (6), 1025-1032).
  • the synthesized random sequence was used to replace part of the promoter, as shown in Fig. 5A, and the produced strain was selected.
  • the selection process consisted of an image analysis process to screen thousands of colony colors to identify those likely to be high-producing, and a process to actually grow candidate strains in liquid media and measure lycopene production.

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Abstract

La présente invention concerne un procédé de synthèse de banque de séquences aléatoires PRUT (PRUT : promoteur, RBS contenant 5'-UTR, terminateur) par l'utilisation d'une désoxynucléotidyl transférase terminale (TdT) et de dNTP.
PCT/KR2024/017762 2023-11-10 2024-11-11 Procédé de synthèse d'une banque de régions non traduites à l'aide de désoxynucléotidyl transférase terminale (tdt) et de dntp Pending WO2025101028A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160138581A (ko) * 2014-04-17 2016-12-05 디엔에이 스크립트 핵산, 특히 긴 핵산을 합성하는 방법, 상기 방법의 용도 및 상기 방법을 수행하기 위한 키트
KR20180038401A (ko) * 2016-10-06 2018-04-16 포항공과대학교 산학협력단 라이코펜을 생산하는 재조합 미생물 및 이를 이용한 라이코펜의 생산방법
KR20200026874A (ko) * 2017-06-06 2020-03-11 지머젠 인코포레이티드 대장균 개량을 위한 htp 게놈 공학 플랫폼
WO2022003116A1 (fr) * 2020-07-01 2022-01-06 Danmarks Tekniske Universitet Banques d'aptamères de taille variable
KR20220114026A (ko) * 2019-12-12 2022-08-17 디엔에이 스크립트 폴리뉴클레오타이드의 주형-부재 효소 합성을 위한 키메라 말단 데옥시뉴클레오타이드 전달효소

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160138581A (ko) * 2014-04-17 2016-12-05 디엔에이 스크립트 핵산, 특히 긴 핵산을 합성하는 방법, 상기 방법의 용도 및 상기 방법을 수행하기 위한 키트
KR20180038401A (ko) * 2016-10-06 2018-04-16 포항공과대학교 산학협력단 라이코펜을 생산하는 재조합 미생물 및 이를 이용한 라이코펜의 생산방법
KR20200026874A (ko) * 2017-06-06 2020-03-11 지머젠 인코포레이티드 대장균 개량을 위한 htp 게놈 공학 플랫폼
KR20220114026A (ko) * 2019-12-12 2022-08-17 디엔에이 스크립트 폴리뉴클레오타이드의 주형-부재 효소 합성을 위한 키메라 말단 데옥시뉴클레오타이드 전달효소
WO2022003116A1 (fr) * 2020-07-01 2022-01-06 Danmarks Tekniske Universitet Banques d'aptamères de taille variable

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