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WO2012008809A2 - Micro-organisme ayant une productivité de l-ornithine améliorée, et procédé pour produire de la l-ornithine en utilisant celui-ci - Google Patents

Micro-organisme ayant une productivité de l-ornithine améliorée, et procédé pour produire de la l-ornithine en utilisant celui-ci Download PDF

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WO2012008809A2
WO2012008809A2 PCT/KR2011/005251 KR2011005251W WO2012008809A2 WO 2012008809 A2 WO2012008809 A2 WO 2012008809A2 KR 2011005251 W KR2011005251 W KR 2011005251W WO 2012008809 A2 WO2012008809 A2 WO 2012008809A2
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ornithine
microorganism
activity
gene
gluconate kinase
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WO2012008809A3 (fr
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조재용
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Industry Academy Cooperation Foundation of Sangji University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Definitions

  • the present invention relates to L-ornithine producing microorganisms in which gluconate kinase (GntK) activity is attenuated compared to endogenous activity.
  • the present invention also relates to a method for producing L-ornithine comprising culturing the L-ornithine producing microorganism to obtain a culture and recovering L-ornithine from the culture or microorganism.
  • Ornithine is one of the amino acids present on the urea cycle in vivo. High levels of ammonia in the blood cause liver damage. Ornithine and citrulline combine with ammonia in the blood to form citrulline and arginine, respectively, to reduce the level of ammonia in the blood. L-ornithine has a detoxifying function against ammonia, such as citrulline and proline, and is used not only as a therapeutic agent for liver disease but also as a food material for preventing obesity. have.
  • Corynebacterium strain in particular Corynebacterium glutamicum ( Corynebacterium glutamicum ) is a Gram-positive microorganism that is widely used for L-amino acid production.
  • Corynebacterium strain is important, many attempts have been made to improve the production method thereof.
  • microorganisms used for the production of ornithine include micrococcus glutamicus , which is a citrulline or arginine nutritionally demanding mutant strain by ultraviolet light treatment, etc., Kyowahako Kogyo Co., Japan, US Patent No. 2,988,489.
  • Brevibacterium lactofermentum (Brevibacterium lactofermentum), Brevibacterium Kawasaki (. B kawasaki), Brevibacterium Plastic boom (B flavum;. Ajinomoto, Japan Patent Publication No. 68-8712, No. 68-8714 .) and (No. 69-26910), Brevibacterium keto glutamicum (B ketoglutamicum), bakteo para pinewooseu by AAS (Arthrobacter paraffineus), Corynebacterium hydrocarbyl carbonyl class tooth (Corynebacterium hydrocarboclastus; T Waha twisted industry ( Note), Japan, US Patent No. 3,574,061) and the like are known.
  • bakteo sheet Reus to the Ars-arginine and citrulline auxotrophy strain A citreus.
  • Brevibacterium lactofermentum B Lactofermentum.
  • Corynebacterium glutamicum C glutamicum;
  • Ajinomoto European Patent No. 0393708
  • US Patent No. 3,574,061 discloses a method for producing L-ornithine by culturing the nutritionally demanding mutant Brevibacterium ketoglutacum ATCC 21092 in a medium containing hydrocarbons, for example, paraffin as the main carbon source.
  • L-ornithine is biosynthesized from L-glutamic acid via cyclic pathway in Corynebacterium glutamicum (Cunin et al., Microbiol Rev 50: 314-352, 1986), and during the biosynthesis process
  • the third step is catalyzed by NADPH dependent reductase encoded by the argC gene.
  • a direct link between the regeneration of NADPH and the biosynthesis of certain metabolites was revealed through the biosynthesis of L-lysine in Corynebacterium glutamicum (Wittmann and Heinzle, Microbiol 68: 5843-5849, 2002; Marx et al.
  • NADPH is an enzyme that catalyzes the pentose phosphate pathway during the production of lysine in Corynebacterium glutamicum (glucose-6-phosphate dehydrogenase (G6PDH) and 6- It is mainly produced during oxidation by 6-phosphogluconate dehydrogenase (6PGD) (Marx et al., J Biotechnol 104: 185-197, 2003; Wittmann and Heinzle, Microbiol 68: 5843-5849 , 2002).
  • G6PDH Corynebacterium glutamicum
  • 6- It is mainly produced during oxidation by 6-phosphogluconate dehydrogenase (6PGD) (Marx et al., J Biotechnol 104: 185-197, 2003; Wittmann and Heinzle, Microbiol 68: 5843-5849 , 2002).
  • the inventors have made diligent efforts to find a method for obtaining L-ornithine with high efficiency and high yield.
  • the present inventors have genetically modified the metabolic activity of the oxidative pentane phosphorylation pathway to produce NADPH production through the pentane phosphorylation pathway.
  • the L-ornithine production could be increased, thereby completing the present invention.
  • Another object of the present invention is to provide a method for producing L-ornithine comprising culturing the microorganism to obtain a culture, and recovering L-ornithine from the culture or microorganism.
  • the present invention is capable of producing L-ornithine with high yield and high efficiency as the gluconate kinase activity provides L-ornithine producing microorganisms inferior to the intrinsic activity, and thus the L-ornithine is produced in medicine and pharmaceuticals. It can be usefully used in industry, food industry, feed industry and chemical industry.
  • 1 is a schematic diagram of the metabolism of the pentose phosphorylation pathway of Corynebacterium glutamicum.
  • FIG. 2 is a diagram showing a cleavage map of the marker-free deletion vector pSJ1034 of the NCgl2399 gene in the Corynebacterium chromosome.
  • FIG. 3 is a diagram showing a cleavage map of the marker-free deletion vector pSJ1035 of the NCgl2905 gene in the Corynebacterium chromosome.
  • the present invention provides an L-ornithine producing microorganism in which the gluconate kinase (GntK) activity is weakened compared to the intrinsic activity.
  • L-ornithine is a basic amino acid present in the urea circuit of a higher animal, and serves to protect the liver by converting ammonia, which is highly toxic to the liver, into a non-toxic urea and releasing it with urine. Refers to amino acids. L-ornithine increases muscle synthesis by secreting growth hormone, promotes basal metabolism, and is widely used as a food material for preventing obesity. Recently, it has been found that there are new functions such as skin beauty action such as wrinkle improvement.
  • L-ornithine is in the form of L-ornithine alpha ketoglutarate (OKG: L-ornithine and alpha ketoglutaric acid in a 2: 1 ratio) for muscle enhancement, prevention of obesity and immunity. It is widely used as a material. L-ornithine is used as a medicine for improving liver disorders in Europe, in the form of L-ornithine salt in Japan, and as a food supplement in the United States.
  • glucose kinase As used herein, the term “gluconate kinase (GntK)” refers to an enzyme involved in the GntK pathway for biosynthesis of 6-phosphogluconic acid, which is an intermediate of the oxidation process of the pentose phosphorylation pathway of microorganisms.
  • GntK glycose kinase
  • Microorganisms of Corynebacterium have two metabolic pathways, 6 G6PDH pathway and GntK pathway, for the biosynthesis of 6-phosphogluconic acid, an intermediate of the oxidation of the pentose phosphorylation pathway (Fig. 1). It means that it can metabolize at least part of glucose.
  • the present inventors further block the carbon flow to 6-phosphogluconic acid through the blocking of the GntK pathway, and thus more carbon flow proceeds through oxidation of the pentose phosphorylation pathway, resulting in 6-phosphogluconate dehydrogena. It was predicted that NADPH regeneration was increased due to the specific increase in the activity of (6PGD), and as a result, a mechanism for increasing the production of L-ornithine was proposed. Thus, it was intended to weaken the activity of enzymes that are believed to have gluconate kinase (GntK) activity in microorganisms.
  • GntK gluconate kinase
  • the enzyme having gluconate kinase (GntK) activity in the present invention may be NCgl2399 represented by the amino acid sequence of SEQ ID NO: 1 or NCgl2905 represented by the amino acid sequence of SEQ ID NO: 2.
  • intrinsic activity refers to an active state of an enzyme possessed by a microorganism in a natural state, and in the present invention, refers to an active state of GntK naturally possessed by a microorganism.
  • "impairment of intrinsic activity” can be achieved by mutation, by deletion, substitution or insertion of all or part of the nucleotide sequence of a gene on a chromosome encoding a protein having gluconate kinase activity.
  • the gluconate kinase activity can be made by mutating by deletion, substitution or insertion of all or part of the expression control sequence of the gene on the chromosome encoding the protein.
  • the expression control sequence may be located above or below the chromosome, and may be a promoter, an enhancer, or the like.
  • GntK activity is weakened compared to the intrinsic activity means that GntK is not normally acted by the deletion or mutation of the gene on the chromosome encoding GntK, so that the GntK activity in which the microorganism has a natural state is weakened. do.
  • GntK activity is destroyed to improve L- ornithine production capacity.
  • the gene encoding NCgl2399 or NCgl2905, or all of the genes are mutated by deletion, substitution or insertion, or its expression control sequence is mutated by deletion, substitution or insertion, so that GntK does not function normally and thus is in a natural state.
  • GntK activity means a weakened state.
  • microorganisms with reduced GntK endogenous activity can be prepared by introducing a recombinant vector comprising a part of a gene encoding a gluconate kinase into a microorganism and deleting or mutating the endogenous gluconate kinase gene. Insertion of the gene into the chromosome can be by any method known in the art, for example homologous recombination.
  • the term "recombinant vector” refers to a vector that cannot be replicated separately from a chromosome of a host cell, and that the gene construct comprises an essential component capable of homologous recombination with a specific gene on the chromosome of the host cell.
  • it may include a selection gene such as an antibiotic resistance gene and levansucrase (levansucrase, sacB) gene.
  • a recombinant vector represented by the cleavage map of FIG. 2 or 3 may be used.
  • any microorganism capable of producing L-ornithine may be used without limitation, and preferably, a microorganism of the genus Corynebacterium or Brevibacterium may be used.
  • Corynebacterium hydrocarbyl carbonyl class tooth Corynebacterium hydrocarboclastus
  • Corynebacterium glutamicum C.
  • Corynebacterium glutamicum ATCC13032 Corynebacterium thermoaminogenes amino to Ness (thermoaminogenes) FERM BP-1539, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869, and L-amino acid producing mutants or strains prepared therefrom such as Corey Nebacterium glutamicum KFCC10881, Corynebacterium glutamicum KFCC11001, Corynebacterium glutamicum KCCM10770P, and preferably, Corynebacterium glutamicum SJC8039 ( C. glutamicum ATCC13032, argF ⁇ , argR ⁇ ).
  • Corynebacterium glutamicum SJC8039 C. glutamicum ATCC13032, argF ⁇ , argR ⁇
  • Corynebacterium glutamicum SJC8039 C. glutamicum ATCC13032, argF ⁇ , argR ⁇
  • gene deletion was performed by using a pK18mobsacB vector that does not replicate in Corynebacterium glutamicum but has the function of performing marker-free deletion of the target gene. That is, a recombinant vector is prepared to include a portion of a gene encoding NCgl2399 or NCgl2905 in the pK18mobsacB vector and an antibiotic marker and a Levansucrease gene ( SacB ), and then the recombinant vector is transferred to a microorganism by any method known in the art. By transformation and homologous recombination in chromosomes, Corynebacterium microorganisms for L-ornithine production with reduced GntK activity were produced.
  • a pK18mobsacB derivative including a part of the gene encoding NCgl2399 in the pK18mobsacB vector was prepared and named pSJ1034, and transformed to produce a recombinant strain in which the gene encoding NCgl2399 was destroyed, which was named SJC8245.
  • a pK18mobsacB derivative including a part of the gene encoding NCgl2905 in the pK18mobsacB vector was prepared and named as pSJ1035, which was transformed into the SJC8245 strain to prepare a recombinant strain in which the gene encoding NCgl2905 was further destroyed. Named SJC8399.
  • culture of SJC8245 a Corynebacterium glutamicum strain lacking the NCgl2399 gene, resulted in a 41% specific activity of GntK and a 23.5% increase in NADPH concentration. -The production of ornithine was found to increase by 24.8%.
  • a Corynebacterium glutamicum strain that additionally destroyed the NCgl2905 gene which is assumed to be another gene of the enzyme having gluconate kinase activity in the SJC8245 strain, the activity of the GntK pathway compared to SJC8245.
  • the microorganism of the present invention attenuates the activity of GntK, thereby increasing the activity of the pentose phosphorylation pathway by specifically increasing the activity of 6PGD, and significantly increased the concentration of NADPH in the cells (Table 4). ). Since the biosynthetic pathway of L-ornithine in the microorganism of the genus Corynebacterium is catalyzed by NADPH dependent reductase, it was found that the microorganism of the present invention increased L-ornithine production.
  • the activity of known enzymes related to the biosynthesis of L-ornithine can be further controlled.
  • the present invention may further increase the L-ornithine production by overexpressing the argCJBD gene by regulating argCJBD operon activity (Hwang et al., J Microbiol Biotechnol 18: 704-710, 2008).
  • the present invention provides a method for obtaining L-ornithine, comprising the steps of: 1) culturing a microorganism according to the present invention, and 2) recovering L-ornithine from the culture or microorganism. It provides a production method of
  • culture in the present invention means to grow microorganisms under environmental conditions that are appropriately artificially controlled.
  • the method of culturing L-ornithine using Corynebacteria may be performed using a method well known in the art. Specifically, the culture may be continuously cultured in a batch process, an injection batch or a repeated fed batch process, but is not limited thereto.
  • the medium used for cultivation must meet the requirements of the particular strain in an appropriate manner.
  • Culture media for Corynebacteria strains are known (eg, Manual of Methods for General Bacteriology. American Society for Bacteriology.Washington D.C., USA, 1981).
  • Sugar sources that can be used include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid Fatty acids such as linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials can be used individually or as a mixture.
  • Nitrogen sources that can be used include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources can also be used individually or as a mixture. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts.
  • the culture medium should contain metal salts such as magnesium sulfate or iron sulfate required for growth.
  • essential growth substances such as amino acids and vitamins can be used.
  • suitable precursors to the culture medium may be used. The above-mentioned raw materials may be added batchwise or continuously in a manner appropriate to the culture during the culturing process.
  • Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.
  • antifoaming agents such as fatty acid polyglycol esters can be used to inhibit bubble generation.
  • the temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. Incubation is continued until the maximum amount of L-ornithine production is obtained. For this purpose it is usually achieved in 10 to 160 hours.
  • L-ornithine may be excreted in culture medium or contained in cells.
  • the method for producing L-ornithine of the present invention includes recovering ornithine from cells or culture medium. Methods of recovering L-ornithine from cells or culture media are well known in the art. Filtration, anion exchange chromatography, crystallization and HPLC may be used for the L-ornithine recovery method, but is not limited to these examples.
  • the parent strain Corynebacterium glutamicum SJC8039 ( C. glutamicum ATCC13032, argF ⁇ , argR ⁇ ; Hwang et al., J Microbiol Biotechnol 18: 704-710, 2008) was used. Primers were prepared to prepare recombinant strains SJC8399 and SJC8245 of the present invention from the parent strain.
  • 2905R2 was produced (Table 1). Underlined sequences indicate restriction positions for restriction enzymes as shown in parentheses, and uppercase letters indicate sequences of microbial genes.
  • PK18mobsacB vector (Schafer et al.) With the ability to perform marker-free deletion of the target gene without replication in Corynebacterium glutamicum for site specific gene disruption al., Gene 145: 69-73, 1994), in which the open reading frames of NCgl2399 and NCgl2905 were internally deleted to create gene-disrupted mutant strains.
  • pK18mobsacB derivatives were made.
  • the pK18mobsacB derivatives were named pSJ1034 and pSJ1035, respectively.
  • pSJ1034 is a pK18mobsacB derivative containing the Xba I terminus of 2,267 bp NCgl2399, which includes the internal gene loss of NCgl2399.
  • the NCgl2399 internal gene loss was generated by cross-PCR using 2399F1-2399R1 primer and 2399F2-2399R2 primer pair as a template of Corynebacterium glutamicum SJC8039 genomic DNA.
  • pSJ1035 is a pK18mobsacB derivative with the Xba I terminus of 2,819 bp NCgl2905, which includes the internal gene loss of NCgl2905.
  • the NCgl2905 internal gene loss was generated by cross PCR with 2905F1-2905R1 primer pair and 2905F2-2905R2 primer pair as a template of Corynebacterium glutamicum SJC8039 genomic DNA.
  • the recombinant plasmid was transformed into wild Corynebacterium glutamicum by electroporation, and the plasmid was introduced into the chromosome by primary recombination (crossover). The plasmid was then excised from the chromosome via secondary recombination (crossing) in a solid plate medium containing 10% sucrose.
  • NCgl2905 and NCgl2399 genes were confirmed.
  • Genbank accession numbers of the open reading frame DNA sequences of NCgl2905 and NCgl2399 are NC_003450.
  • Example 2 Culture of Corynebacterium glutamicum with deletion of NCgl2905 and / or NCgl2399 gene
  • Corynebacterium glutamicum SJC8399 both NCgl2399 and NCgl2905 inactivated strains
  • Corynebacterium SJC8245 NCgl2399 inactivated strains
  • L-ornithine producing strains were prepared using the following method for L-ornithine production. Incubated with. The seed medium was incubated for 24 hours using Recovery Glucose medium (80 g BHI, 20 g glucose, 60 g sorbitol / L).
  • the production medium was MMY medium [0.8 g KH 2 PO 4 , 10 g (NH 4 ) 2 SO 4, 1 g MgSO 4 ⁇ 7H 2 O, 1.2 g Na 2 HPO 4 , 20 mg MnSO 4 ⁇ H 2 O, 20 mg FeSO 4 7H 2 O, 10 mg, ZnSO 4 7H 2 O, 10 g yeast extract, and 60 g glucose / L, pH 7.0] were used.
  • a 100 ml baffle flask containing a seed medium was inoculated with Corynebacterium glutamicum parent strain SJC8039 and the recombinant strain Corynebacterium glutamicum SJC8399 and Corynebacterium glutamicum SJC8245, respectively, and incubated with shaking ( 200 rpm).
  • the glutamicum cultured in the seed medium was separated and harvested, and then resuspended in a 100 ml baffle flask containing 10 ml of the production medium until the absorbance was 0.4-0.5 at 600 nm.
  • kanamycin was added to a final concentration of 50 ⁇ g / ml if necessary.
  • the culture was carried out in a 200 rpm rotary stirrer at 30 °C.
  • Corynebacterium glutamicum incubated in the production medium was harvested by centrifugation during the expontial phase and resuspended in 100 mM Tris / HCl buffer (pH 7.5). The cell wall was broken using glass beads and centrifuged to obtain cell extracts. All these treatments were performed at 4 ° C., and the supernatants were harvested for specific enzyme activity analysis. The amount of protein for enzymatic analysis was determined by the Bradford method (Bradford 1976). The activity of G6PDH and 6PGD was measured by NADPH spectroscopic formation at 340 nm (Frunzke et al., Mol Microbiol 67: 305-322, 2008).
  • Gluconate kinase (GntK) activity was determined by coupled enzymatic assay of 6PGD (Frunzke et al., Mol Microbiol 67: 305-322, 2008). Cell growth rate was determined spectroscopically at 600 nm, and L-ornithine concentration was determined by colorimetric method with ninhydrin (Chinard et al., J Biol Chem 199: 91-95, 1952). Dry cell mass was measured based on the absorbance value 1 at 600 nm corresponds to 0.3 g dry weight per 1 L. The concentration of NADPH was determined by an enzymatic cycling reaction using the EnzyChrom TM NADP + / NADPH Assay Kit (BioAssay Systems, CA, USA).
  • Tables 2 and 3 show the growth rate of Corynebacterium glutamicum and the accumulation concentrations of NADPH and L-ornithine, respectively, and Table 4 shows the specific activities of the core enzymes of the core carbon metabolism process.
  • SJC8245 strain with NCgl2399 inactivated
  • SJC8399 strains with both NCgl2399 and NCgl2905 inactivated
  • SJC8039 parent strain
  • intracellular NADPH concentration and L -The production of ornithine has increased significantly (Tables 2 and 3).
  • SJC8245 a Corynebacterium glutamicum strain lacking the gene encoding NCgl2399 of the present invention, retained about 41% of GntK specific activity (0.51 vs 0.21 U mg / protein), and in the SJC8245 strain.

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Abstract

La présente invention concerne un micro-organisme producteur de L-ornithine, dans lequel l'activité gluconate kinase (GntK) est affaiblie par rapport à l'activité intrinsèque de celle-ci, et un procédé pour produire de la L-ornithine à partir d'un micro-organisme producteur de L-ornithine. Selon la présente invention, la L-ornithine peut être produite à un rendement élevé et avec une efficacité élevée, et la L-ornithine produite ainsi peut être efficacement utilisée dans l'industrie médicale et pharmaceutique, l'industrie alimentaire, l'industrie de l'alimentation animale, l'industrie chimique, etc.
PCT/KR2011/005251 2010-07-15 2011-07-15 Micro-organisme ayant une productivité de l-ornithine améliorée, et procédé pour produire de la l-ornithine en utilisant celui-ci Ceased WO2012008809A2 (fr)

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EP2430152B1 (fr) * 2010-07-15 2016-08-24 CJ CheilJedang Corporation Micro-organisme ayant une productivité de l-lysine améliorée et procédé de production de l-lysine l'utilisant
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