WO2022055056A1 - Micro-organisme recombinant pour produire de la s-méthylméthionine et procédé de préparation de la s-méthylméthionine l'utilisant - Google Patents

Micro-organisme recombinant pour produire de la s-méthylméthionine et procédé de préparation de la s-méthylméthionine l'utilisant Download PDF

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WO2022055056A1
WO2022055056A1 PCT/KR2021/002981 KR2021002981W WO2022055056A1 WO 2022055056 A1 WO2022055056 A1 WO 2022055056A1 KR 2021002981 W KR2021002981 W KR 2021002981W WO 2022055056 A1 WO2022055056 A1 WO 2022055056A1
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methylmethionine
mmt
gene
recombinant microorganism
seq
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오민규
이준민
박부수
박민호
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고려대학교 산학협력단
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    • C12Y205/01006—Methionine adenosyltransferase (2.5.1.6), i.e. adenosylmethionine synthetase

Definitions

  • a gene encoding methionine S-methylmethionine transferase is overexpressed in a microorganism having glycolysis and S-adenosylmethionine (SAM) biosynthetic pathway process, S It relates to a recombinant microorganism having the ability to produce -methylmethionine (S-Methylmethionine, SMM) and a method for producing SMM using the recombinant microorganism.
  • SMM S-Methylmethionine
  • the present inventors were intensively researching on the production method of SMM through microbial fermentation, methionine S-methyl in microorganisms having glycolysis and S-adenosylmethionine (S-Adenosylmethionine, SAM) biosynthetic pathway process
  • S-Adenosylmethionine, SAM S-adenosylmethionine
  • SMM S-methylmethionine
  • Another object of the present invention is to provide a method for producing S-methylmethionine using the recombinant microorganism.
  • the present invention encodes methionine S-methylmethionine transferase (MMT) in a microorganism having a glycolysis process and a S-adenosylmethionine (S-adenosylmethionine, SAM) biosynthetic pathway process
  • MMT methionine S-methylmethionine transferase
  • SAM S-adenosylmethionine
  • the present invention also comprises the steps of (a) culturing a recombinant microorganism having the S-methylmethionine (SMM) producing ability to produce S-methylmethionine; and (b) obtaining the produced S-methylmethionine; provides a method for producing S-methylmethionine comprising.
  • SMM S-methylmethionine
  • S-methylmethionine can be produced in excellent yield through microbial biosynthesis, unlike conventional plant extraction or chemical synthesis, and it is expected that it can be used as a core technology for S-methylmethionine production.
  • FIG. 1 shows a pathway for biosynthesis of SMM by introducing an MMT gene in Escherichia coli having a glycolysis process and a SAM biosynthetic pathway process according to the present invention.
  • Figure 2 shows the results of measuring the SMM production of the recombinant microorganism (E. coli) according to Example 1 of the present invention
  • dJ ⁇ metJ
  • dJMP ⁇ metJ
  • ⁇ mmuP ⁇ mmuM
  • aK pZA (metK)
  • aKF pZA(metK, metF)
  • cM pCDFDuet(A.MMT)
  • sA * : pZS(metA * fbr
  • cBM pCDFDuet(B.MMT)
  • csfM pCDFDuet(Sf.MMT)
  • Figure 3 shows the results of measuring the SMM production for each MMT gene origin of the recombinant microorganism (E. coli) according to Example 1 of the present invention ( Arabidopsis thaliana (dJMP + aKcMsA * ), Barley (dJMP + aKcBMsA * ), Sunflower (dJMP+aKcsfMsA * )).
  • Figure 4 shows the results of measuring the SMM production of the recombinant microorganism (Saccharomyces cerevisiae) according to Example 2 of the present invention (Y1: BY4742 + pSH47 (MMT::URA). Y2: K6-1 + p425(MMT::Leu))
  • Figure 5 shows the results of measuring the SMM production of the recombinant microorganism (Bacillus subtilis) according to Example 3 of the present invention (BS + M (wtBacillus + pBE-S (MMT), BS + MK (wtBacillus + pBE) -S(metK, MMT))
  • Example 6 shows the results of measuring the SMM production of the recombinant microorganism (Streptomyces Venezuela) according to Example 4 of the present invention.
  • a recombinant microorganism capable of producing S-methylmethionine in excellent yield through microbial biosynthesis was prepared, and the prepared recombinant microorganism exhibits excellent S-methylmethionine yield and productivity.
  • the present invention is a glycolysis process and S-adenosylmethionine (S-adenosylmethionine, SAM) in a microorganism having a biosynthetic pathway process, methionine S-methylmethionine transferase (methionine S-methylmethionine transferase, MMT) encoding
  • SAM S-adenosylmethionine
  • MMT methionine S-methylmethionine transferase
  • the gene is overexpressed, and relates to a recombinant microorganism having S-methylmethionine (SMM) producing ability (FIG. 1).
  • Escherichia coli or Saccharomyces cerevisiae as a microorganism having the above glycolysis process and S-adenosylmethionine (SAM) biosynthetic pathway process was used, In order to receive a methyl group from SAM and generate S-methylmethionine from methionine, a gene encoding methionine S-methylmethionine transferase (MMT) was introduced and overexpressed.
  • SAM S-adenosylmethionine
  • the S-methylmethionine may be S-methyl-L-methionine (S-Methyl-L-methionine).
  • the gene encoding the MMT may be represented by any one of SEQ ID NOs: 1 to 3, but is not limited thereto.
  • the genes of SEQ ID NOs: 1 to 3 may be derived from Arabidopsis thaliana, barley, and sunflower, respectively.
  • the MMT-encoding gene may be any if the amino acid sequence corresponding to the MMT-encoding gene has at least 60% sequence homology with the amino acid sequence of SEQ ID NO: 47, which is the amino acid sequence corresponding to SEQ ID NO: 1.
  • the gene encoding the MMT is grape (scientific name: Vitis vinifera), coyote tobacco (scientific name: Nicotiana attenuata), mulberry (scientific name: Arabidopsis lyrata), corn (scientific name: Zea mays), tulip (scientific name: Anthurium amnicola) may be derived from, and their amino acid sequences may be those represented by SEQ ID NOs: 50 to 54, respectively.
  • the gene metJ (methionine repressor), which encodes a repressor of a methionine biosynthesis pathway present in wild-type E. coli, was further deleted.
  • a transport of S-methylmethionine (mmuP) gene and a homocysteine S-methyltransferase (mmuM) gene which is a gene for synthesizing methionine from SMM, are additionally added. deleted it
  • metK S-adenosylmethionine synthetase
  • SAM S-adenosylmethionine synthetase
  • methionine a precursor of SMM
  • metF gene encoding 5,10-methylenetetrahydrofolate reductase, which helps in production, was further overexpressed.
  • the metA gene encoding homoserine O-succinyltransferase involved in methionine biosynthesis was mutated.
  • the mutant gene of metA may be one in which cytosine, which is the 190th base, is substituted with guanine in the nucleotide sequence of the metA gene, and is represented by SEQ ID NO: 4.
  • the microorganism can be used without limitation as long as it has a glycolytic process and a S-adenosylmethionine (SAM) biosynthetic pathway process, for example, Escherichia coli ), yeast, Bacillus subtilis, or actinomycetes, wherein the yeast is Saccharomyces cerevisiae , the Bacillus subtilis is Bacillus subtilis , and the actinomycetes are Streptomyces venezuelae ( Streptomyces venezuelae ).
  • SAM S-adenosylmethionine
  • the present invention comprises the steps of (a) culturing the recombinant microorganism to produce S-methylmethionine; And (b) obtaining the produced S-methylmethionine; relates to a method for producing S-methylmethionine comprising a.
  • the method of culturing the recombinant microorganism may be performed using a method widely known in the art.
  • the culture is not particularly limited as long as SMM can be produced from the recombinant microorganism, but may be continuously cultured in a batch process or injection batch or repeated fed batch process.
  • the medium used for culture may contain a carbon source, a nitrogen source, amino acids, vitamins, etc., and must meet the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc.
  • the carbon source that can be used is sucrose, lactose, maltose, trehalose, turanose, cellobiose, raffinose, melechtose, malotriose, acarbose, stachyose, glucose, amylose, cellulose, fructose, anose, or It may be one or more selected from the group consisting of galactose.
  • nitrogen source examples include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, defatted soybean cake or its degradation products, etc. can These nitrogen sources may be used alone or in combination.
  • the medium may contain monopotassium phosphate, dipotassium phosphate and the corresponding sodium-containing salt as phosphorus.
  • the phosphorus that may be used includes potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt.
  • potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used.
  • essential growth substances such as amino acids and vitamins can be used.
  • precursors suitable for the culture medium may be used.
  • the above-mentioned raw materials may be added in a batch, fed-batch or continuous manner by an appropriate method to the culture during the culturing process, but is not particularly limited thereto.
  • MG1655 K-12 strain was used as E. coli
  • S. cerevisiae K6-1 was used as yeast.
  • Frozen stocks of E. coli and S. cerevisiae were prepared by adding 30% (v/v) glycerol to exponentially grow the cells.
  • E. coli cells were cultured using two different media.
  • LB (Luria-Bertani) medium was used for E. coli cell growth.
  • M9 medium was used as a minimal medium, and the composition of M9 is as follows: Na 2 HPO 4 7H 2 O (6g) , KH 2 PO 4 (3g), NaCl (0.5g), NH 4 Cl (1g), 1M MgSO 4 2mL, 1M CaCl 2 10uL and 50g/L glucose (40mL) in 1L bottle.
  • YPD medium and SD medium were used for yeast culture.
  • a minimal medium, SD medium was prepared using yeast nitrogenous base w/o amino acids (6.7 g), H 2 O (865 mL), AA-Mix 10x concentrate (10 mL) without L-leucine.
  • E. coli cultures were grown with each required antibiotic (kanamycin, chloramphenicol, spectinomycin (25 mg/mL)).
  • Agar plates were prepared with 20 g/L agar.
  • E. coli DH5a was used for all plasmid cloning and propagation. All plasmids used were constructed using Gibson Asembly (New England BioLab.). The genes used were derived from E. coli MG1655 K-12 (metF, metH, metA) and Arabidopsis thaliana (MMT), Sunflower (MMT) and Barley (MMT). All MMT genes were integrated into the pCDFDuet vector. For yeast (K6-1), a pESC vector with a bidirectional promoter was used. Successful genomic integration of overexpression was confirmed by diagnostic PCR from Bionics.
  • E. coli cells were cultured at 37 °C while shaking at 30 °C.
  • the antibiotic required for recombinant microorganisms for 1 day was inoculated into 5 mL LB.
  • each was transferred to 5 mL of M9 medium (minimum medium) for acclimatization in the medium required for antibiotics.
  • M9 medium minimum medium
  • the seeds were scaled up in 25 mL M9 medium to an initial OD (0.125) in unbaffled flasks for culture.
  • E. coli was cultured to a maximum OD (4.2) for 48 h.
  • S. cerevisiae K6 (Sake yeast kyokai No.6) cells were cultured at 30° C. while shaking at 30 rpm.
  • Yeast cells (S. cerevisiae K6-1 with pESC vector (MMT, SAM2)) were first inoculated into 5 mL YPD without additional amino acids for 36 hours, and then transferred to 5 mL SD medium (minimum medium) to adapt to the medium. Next, the seeds were scaled up in 25 mL SD medium, to an initial OD (0.125) in baffled flasks for cultivation.
  • Yeast (S. cerevisiae K6-1) carrying a pESC vector (MMT, SAM2) was cultured at a maximum OD (5.0) for 72 hours.
  • S-methylmethionine and L-methionine were determined by high performance liquid chromatography using UV spectroscopy. Two mobile phases were used for the analysis (1: Sodium phosphate di-basic 8.51 g with 3 mL phosphoric acid in 3 L DW, 2: Acetonitrile (45), Methanol (45), DW (10) in 3 L bottle). In addition, Agilent's amino acid analysis column was used for all samples, and OPA reagent (Sigma-Aldrich) was used for amino acid derivatization.
  • Escherichia as a microorganism having glycolysis and S-adenosylmethionine (SAM) biosynthetic pathway process coli K-12 MG1655) was used, and the names of strains or plasmids according to genetic manipulation, etc. are shown in Table 1 below.
  • SAM S-adenosylmethionine
  • strains or plasmids Description source strains dJ Escherichia coli K-12 MG1655 ⁇ metJ this study dJMP Escherichia coli K-12 MG1655 ⁇ metJ, ⁇ mmuM, ⁇ mmuP this study Plasmids pRedET ⁇ phage red ⁇ , ⁇ , ⁇ -producing vector, pBAD_promoter; ori101 Tetr Gene Bridges 707-FLP Flp recombinase-producing vectors; Psc101 ori cI1578 Tetr Gene Bridges pKD4 FRT-flanked resistance cassette-involved vector; oriR ⁇ Kmr Datsenko KA and Wanner BL, 2000 aK pZA (metK) this study aKF pZA (metK, metF) this study cM pCDFDuet (MMT) this study cBM pCDFDuet (Barley MMT) this study csfM p
  • a lambda red recombination method was used to remove the metJ gene of K-12 MG1655.
  • an enzyme expression vector for preventing degradation of the linear gene introduced into the cell and increasing the efficiency of homologous recombination was introduced into the wild type E. coli K-12 MG1655 strain prepared as competent cells.
  • a pRedET vector having a BAD promoter and resistant to tetracycline was used in the present invention, and transformation was confirmed.
  • a linear gene for homologous recombination was prepared.
  • a pKD4 vector having an FRT-kanamycin-FRT cassette was used as a template. Homologous 50 base pairs on both sides of the metJ gene of wild type E.
  • coli K-12 MG1655 the site at which homologous recombination occurs, and 20 base pairs for polymerization of the FRT-kanamycin-FRT cassette of pKD4 used as a template, a total of 70 base pairs was prepared as a primer, and a linear gene for removing the metJ gene was prepared using polymerase chain reaction. Kanamycin is used as a selection marker to confirm successful homologous recombination, and the FRT region serves to remove a selection marker for removal of other genes after the target gene is removed.
  • the MG1655+pRedET strain confirmed to be transformed with the pRedET vector was cultured at 30°C.
  • the pRedET vector is a temperature-sensitive vector and loses its activity when it is over 37°C, so it was cultured at 30°C.
  • the expression of pRedET's red lambda recombinase was induced with 10% arabinose (L-arabinose).
  • the cells were transformed into competent cells for electroporation and then the prepared linear gene. After 1 hour incubation at 37° C. in LB medium, it was spread on LB solid medium to which 12.5 mg/ml of kanamycin was added and cultured for 12 hours.
  • a lambda red recombination method was used.
  • an enzyme expression vector for preventing degradation of the linear gene introduced into the cell and increasing the efficiency of homologous recombination was introduced into the dJ strain prepared as competent cells.
  • a pRedET vector having a BAD promoter and resistant to tetracycline was used in the present invention, and transformation was confirmed.
  • a linear gene for homologous recombination was prepared.
  • a pKD4 vector having an FRT-kanamycin-FRT cassette was used as a template. Homologous 50 base pairs on both sides of the metJ gene of wild type E.
  • coli K-12 MG1655 the site where homologous recombination occurs, and 20 base pairs for polymerization of the FRT-kanamycin-FRT cassette of pKD4 used as a template, a total of 70 A base pair was prepared as a primer, and a linear gene for removing the metJ gene was prepared using polymerase chain reaction. Kanamycin is used as a selection marker to confirm successful homologous recombination, and the FRT region serves to remove a selection marker for removal of other genes after the target gene is removed.
  • the dJ+pRedET strain confirmed to be transformed with the pRedET vector was cultured at 30°C.
  • the pRedET vector is a temperature-sensitive vector and loses its activity when it exceeds 37°C, so it was incubated at 30°C.
  • the expression of pRedET red lambda recombinase was induced with 10% arabinose (L-arabinose).
  • the cells were transformed into competent cells for electroporation, and then the prepared linear gene was transformed.
  • 1 hour incubation at 37° C. in LB medium it was spread on LB solid medium to which 12.5 mg/ml of kanamycin was added and cultured for 12 hours.
  • All vectors used for gene overexpression according to the present invention were prepared through gibson assembly.
  • the primers used to overexpress the metK gene, which produces a lot of S-adenosylmethionine, are shown in Table 2 below. After cloning using the corresponding primer, re-cloning was performed to match the gibson primer, and each fragment was combined through gibson assembly.
  • a pZA (metK) vector was constructed in this way.
  • the primers used to overexpress the MMT gene, a gene that makes S-methylmethionine, are shown in Table 2 below. After cloning using the corresponding primer, re-cloning was performed according to the gibson primer, and each fragment was combined through gibson assembly. In this way, a pCDFDuet (MMT) vector was constructed.
  • the primers used to overexpress the mutant gene (SEQ ID NO: 4) of metA which is a gene for synthesizing O-succinyl homoserine in the methionine biosynthesis pathway in E. coli, are shown in Table 2 below. After cloning using the corresponding primer, re-cloning was performed to match the gibson primer, and each fragment was combined through gibson assembly. In this way, a pZS(metA*) vector was constructed.
  • the primers used to overexpress metK, a gene that generates SAM, which transfers a methyl group to methionine during SMM generation, and metF, a gene that enhances the activity of the tetrahydrofolate cycle in E. coli, are shown in Table 2 below. After cloning using the corresponding primer, re-cloning was performed to match the gibson primer, and each fragment was combined through gibson assembly. A pZA (metK, metF) vector was constructed in this way.
  • Recombinant E. coli according to the present invention was prepared through transformation using the vectors prepared as above. Transformation was performed according to a method generally known in the art. Briefly, the strain to be transformed was cultured in a 5mL falcon tube, and after 24 hours, cell down was performed using a centrifuge, washed twice, and glycerol After releasing the cells with a 10% solution, the prepared vectors were added thereto, and then electricity of 1950V was applied.
  • Saccharomyces cerevisiae (BY4742, Sake koykai No.6) was used as a microorganism having glycolysis and S-adenosylmethionine (SAM) biosynthetic pathway process, and according to genetic manipulation, etc.
  • SAM S-adenosylmethionine
  • Table 3 The strain or plasmid names are shown in Table 3 below.
  • all vectors used for gene overexpression according to the present invention were prepared through gibson assembly.
  • the primers used to express the gene making S-methylmethionine are shown in Table 4 below. After cloning using the corresponding primer, re-cloning was performed to match the gibson primer, and each fragment was combined through gibson assembly. In this way, p425 (MMT) and pSH47 (MMT) were prepared. Thereafter, a recombinant yeast in which the MMT gene was overexpressed was prepared through the same method as the MMT gene overexpression method described in Example 1.
  • Bacillus subtilis ATCC6633 was used as a microorganism having glycolysis process and S-adenosylmethionine (SAM) biosynthetic pathway process, and the names of strains or plasmids according to genetic manipulation, etc. are shown in Table 5 below.
  • SAM S-adenosylmethionine
  • Table 5 all vectors used for gene overexpression according to the present invention were prepared through gibson assembly.
  • Primers used to overexpress the metK gene are shown in Table 6 below. After cloning using the corresponding primer, re-cloning was performed to match the gibson primer, and each fragment was combined through gibson assembly. In this way, a pBE-S (metK) vector was constructed.
  • the primers used to overexpress the MMT gene are shown in Table 6 below. After cloning using the corresponding primer, re-cloning was performed to match the gibson primer, and each fragment was combined through gibson assembly. In this way, a pBE
  • strains and plasmids Description source strains BS wild type Bacillus subtilis this study Plasmid pBE-S(M) pBE-S (MMT) this study pBE-S(K,M) pBE-S (metK, MMT) this study
  • Streptomyces as a microorganism with glycolysis and S-adenosylmethionine (SAM) biosynthetic pathway process venezuelae ATCC 15439) was used, and the names of strains or plasmids according to genetic manipulation, etc. are shown in Table 7 below.
  • all vectors used for gene overexpression according to the present invention were prepared through gibson assembly. Primers used to overexpress the MMT gene are shown in Table 8 below.
  • MMT gene fragment was created using the corresponding primer, and backbone vector fragments cut with XbaI and NdeI were combined through gibson assembly. A pSET(M) vector was constructed in this way.
  • Recombinant E. coli according to Example 1 of the present invention was cultured in M9 (minimal media) medium supplemented with 10 g/L glucose for 24 hours, and SMM production was measured using HPLC.
  • Agilent's Amino Acid Analysis column was used for the column, and A: Sodium phosphate diabasic, Phosphoric acid, DW / B: DW, Acetonitrile, and Methanol were used as mobile phases for HPLC.
  • Figure 2 shows the results of measuring the SMM production of the recombinant microorganism (E. coli) according to Example 1 of the present invention
  • J ⁇ metJ, dJMP: ⁇ metJ, ⁇ mmuP, ⁇ mmuM
  • sA * :pZS(metA * fbr) cBM:pCDFDuet(B.MMT)
  • csfM:pCDFDuet(Sf.MMT) shows the results of measuring the SMM production of the recombinant microorganism (E. coli) according to Example 1 of the present invention
  • SMM when recombinant E. coli according to the present invention is used, SMM can be produced in excellent yield without adding methionine, a precursor of SMM (0.063 g/L and 0.063 g/L and 0.07 g/L production) was confirmed.
  • Figure 3 shows the results of measuring the SMM production for each MMT gene origin of the recombinant microorganism (E. coli) according to Example 1 of the present invention ( Arabidopsis thaliana (dJMP + aKcMsA * ), Barley (dJMP + aKcBMsA * ), Sunflower (dJMP+aKcsfMsA * )).
  • the culture conditions and the method of measuring the amount of SMM production are the same as those described in Evaluation Example 1 above.
  • the recombinant yeast according to Example 2 of the present invention was cultured for 24 hours in SD (minimal media) medium supplemented with 10 g/L glucose and 0.5 g/L methionine, and SMM production was measured using HPLC.
  • SD minimal media
  • Agilent's Amino Acid Analysis column was used for the column, and A: Sodium phosphate diabasic, Phosphoric acid, DW / B: DW, Acetonitrile, and Methanol were used as mobile phases for HPLC.
  • Example 4 shows the results of measuring the SMM production of the recombinant microorganism (Saccharomyces cerevisiae) according to Example 2 of the present invention (Y1: BY4742 + pSH47 (MMT::URA). Y2: K6-1 + p425(MMT::Leu))
  • Recombinant Bacillus subtilis according to Example 3 of the present invention was cultured for 24 hours in LB (rich media) medium supplemented with 10 g/L glucose and 0.5 g/L methionine, and SMM production was measured using HPLC.
  • Agilent's Amino Acid Analysis column was used for the column, and A: Sodium phosphate diabasic, Phosphoric acid, DW / B: DW, Acetonitrile, and Methanol were used as mobile phases for HPLC.
  • Figure 5 shows the results of measuring the SMM production of the recombinant microorganism (Bacillus subtilis) according to Example 3 of the present invention (BS + M (wtBacillus + pBE-S (MMT), BS + MK (wtBacillus + pBE) -S(metK, MMT))
  • Recombinant actinomycetes according to Example 4 of the present invention were cultured for 96 hours in GYM medium supplemented with 0.5 g/L methionine, and SMM production was measured using HPLC.
  • Agilent's Amino Acid Analysis column was used for the column, and A: Sodium phosphate diabasic, Phosphoric acid, DW / B: DW, Acetonitrile, and Methanol were used as mobile phases for HPLC.
  • Example 6 shows the results of measuring the SMM production of the recombinant microorganism (Streptomyces Venezuela) according to Example 4 of the present invention.
  • the multiple sequence alignment was performed through the ClustalX program, and as a result, unlike the amino acid sequence (SEQ ID NOs: 55 and 56) corresponding to the MMT gene possessed by orchids (Apostasia shenzhenica) and stink bug (Lygus hesperus), grapes (Vitis vinifera) , Coyote tobacco (Nicotiana attenuata), Arabidopsis lyrata, corn (Zea mays), the amino acid sequence corresponding to the MMT gene (SEQ ID NOs: 50 to 54) derived from tulips (Anthurium amnicola) Arabidopsis thaliana ), it was confirmed that the amino acid sequence (SEQ ID NO: 47) corresponding to the MMT gene (SEQ ID NO: 1) and the similarity and identity (similarity) and identity (identity) are at least 60%, and the MMT gene derived from them is also It was confirmed that the same can be applied to the invention.
  • S-methylmethionine can be produced in excellent yield through microbial biosynthesis, unlike conventional plant extraction or chemical synthesis, and thus can be usefully used in various fields requiring S-methylmethionine production.

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Abstract

La présente invention concerne un micro-organisme recombinant ayant la capacité de produire de la S-méthylméthionine (SMM) par surexpression d'un gène qui code la méthionine S-méthylméthionine transférase (MMT) dans un micro-organisme comprenant des procédés de voie de biosynthèse de la glycolyse et de la S-adénosylméthionine (SAM), et un procédé de production de la SMM à l'aide du micro-organisme recombinant. Selon la présente invention, la S-méthylméthionine peut être produite selon un excellent rendement par le biais de la biosynthèse microbienne, contrairement à une extraction de plante ou à une synthèse chimique classique, et la présente invention est prévue pour pouvoir être utilisée en tant que technologie principale pour la production de S-méthylméthionine.
PCT/KR2021/002981 2020-09-11 2021-03-10 Micro-organisme recombinant pour produire de la s-méthylméthionine et procédé de préparation de la s-méthylméthionine l'utilisant WO2022055056A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049157A2 (fr) * 1999-02-22 2000-08-24 University Of Florida Compositions et procedes pour modifier la teneur en soufre de plantes
KR101784633B1 (ko) * 2016-10-28 2017-10-17 심진선 S-메틸 메티오닌이 함유된 양배추 추출방법
KR20180093981A (ko) * 2016-01-08 2018-08-22 에보니크 데구사 게엠베하 발효적 생산에 의해 l-메티오닌을 생산하는 방법

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049157A2 (fr) * 1999-02-22 2000-08-24 University Of Florida Compositions et procedes pour modifier la teneur en soufre de plantes
KR20180093981A (ko) * 2016-01-08 2018-08-22 에보니크 데구사 게엠베하 발효적 생산에 의해 l-메티오닌을 생산하는 방법
KR101784633B1 (ko) * 2016-10-28 2017-10-17 심진선 S-메틸 메티오닌이 함유된 양배추 추출방법

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Title
KOCSIS MICHAEL G., RANOCHA PHILIPPE, GAGE DOUGLAS A., SIMON ERIC S., RHODES DAVID, PEEL GREGORY J., MELLEMA STEFAN, SAITO KAZUKI, : "Insertional Inactivation of the Methionine S -Methyltransferase Gene Eliminates the S -Methylmethionine Cycle and Increases the Methylation Ratio", PLANT PHYSIOLOGY, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, USA, vol. 131, no. 4, 1 April 2003 (2003-04-01), Rockville, Md, USA , pages 1808 - 1815, XP055782658, ISSN: 0032-0889, DOI: 10.1104/pp.102.018846 *
THANBICHLER MARTIN, NEUHIERL BERNHARD, BÖCK AUGUST: "S -Methylmethionine Metabolism in Escherichia coli", JOURNAL OF BACTERIOLOGY (PRINT), AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 181, no. 2, 15 January 1999 (1999-01-15), US , pages 662 - 665, XP055909981, ISSN: 0021-9193, DOI: 10.1128/JB.181.2.662-665.1999 *

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