US20090197309A1 - Mutant Acetolactate Synthase and a Method for Producing Branched-Chain L-Amino Acids - Google Patents

Mutant Acetolactate Synthase and a Method for Producing Branched-Chain L-Amino Acids Download PDF

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US20090197309A1
US20090197309A1 US11/854,601 US85460107A US2009197309A1 US 20090197309 A1 US20090197309 A1 US 20090197309A1 US 85460107 A US85460107 A US 85460107A US 2009197309 A1 US2009197309 A1 US 2009197309A1
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acetolactate synthase
mutant
amino acid
small subunit
bacterium
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Elena Viktorovna Sycheva
Vsevolod Aleksandrovich Serebryanyy
Tatyana Abramovna Yampolskaya
Ekaterina Sergeevna Preobrazhenskaya
Natalia Viktorovna Stoynova
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Ajinomoto Co Inc
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Assigned to AJINOMOTO CO., INC. reassignment AJINOMOTO CO., INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: STOYNOVA, NATALIA VIKTOROVNA, YAMPOLSKAYA, TATYANA ABRAMOVNA, PREOBRAZHENSKAYA, EKATERINA SERGEEVNA, SEREBRYANYY, VSEVOLOD ALEKSANDROVICH, SYCHEVA, ELENA VIKTOROVNA
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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
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • 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.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
    • 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
    • C12N9/88—Lyases (4.)
    • 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
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
    • C12Y202/01—Transketolases and transaldolases (2.2.1)
    • C12Y202/01006—Acetolactate synthase (2.2.1.6)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Definitions

  • the present invention relates to biotechnology, and specifically to a method for producing branched-chain L-amino acids. More specifically, the present invention discloses the use of a new L-valine-resistant enzyme which is involved in the biosynthesis of branched-chain L-amino acids. More specifically, the present invention concerns a L-valine-resistant mutant acetolactate synthase (AHAS I) purified from E. coli , a bacteria from the Enterobacteriaceae family which contains this synthase enzyme, and a method for producing branched-chain L-amino acids by fermentation using the strains of this bacteria.
  • AHAS I L-valine-resistant mutant acetolactate synthase
  • L-amino acids have been industrially produced by fermentation utilizing strains of microorganisms obtained from natural sources, or mutants thereof which have been specifically modified to enhance L-amino acid productivity.
  • the biosynthesis of isoleucine, leucine, and valine occurs through a branched pathway in which three steps are common to each end product.
  • the AHAS reaction represents the first biosynthetic step common to the three products.
  • the reaction is catalyzed by isoenzymes which are the target of end-product inhibition by valine. This regulation plays a major role in the physiological control of the pathway in bacteria.
  • the reaction includes condensation of active acetaldehyde (derived from pyruvate) with either ⁇ -ketobutyrate or pyruvate to yield ⁇ -aceto- ⁇ -hydroxybutyrate (a precursor of isoleucine) or ⁇ -acetolactate (a precursor of leucine and valine), respectively.
  • valine and its keto-acid precursor ⁇ -ketoisovaleric acid inhibit the growth of E. coli K12, and that isoleucine counters this inhibition (Tatum, E. L., Fed. Proc. 8:511 (1946)). At present, it is commonly accepted that inhibition of valine primarily results from blocking ⁇ -aceto- ⁇ -hydroxybutyrate synthesis.
  • An analysis of E. coli K12 has revealed that this strain contains the structural genes for the three AHAS activities, which are designated as isoenzymes AHAS I, AHAS II, and AHAS III.
  • AHAS I and AHAS III are both inhibited by valine, whereas AHAS II is resistant to it; however, AHAS II is not normally expressed in E. coli K12 cells (Guardiola, J. et al, Mol. Gen. Genet. 156:17-25 (1977)).
  • All AHAS isozymes from enterobacteria are composed of a large and a small subunit in an ⁇ 2 ⁇ 2 structure, with the large subunits performing a catalytic function and the small subunits performing a regulatory function. The small subunits are absolutely required for sensitivity of the enzyme activity to the feedback inhibitor valine.
  • a study of the individual properties of the AHAS I and AHAS III subunits (Weinstock O. et al, J. Bacteriol. 174:5560-5566 (1992)) showed that the small subunits specifically induced a catalytically competent conformation of the whole enzyme and stabilized the transition state.
  • mutant bacterial acetolactate synthase (AHAS I) which is feedback resistant to valine and the use of such a mutant acetolactate synthase for improving branched-chain L-amino acid production in corresponding L-amino acid producing strains.
  • the present invention provides a new mutant bacterial acetolactate synthase for the purpose of developing branched-chain L-amino acid-producing strains with enhanced productivity of branched-chain L-amino acids, and to provide a method for producing branched-chain L-amino acids using these strains.
  • the present invention was achieved by constructing a new mutant acetolactate synthase from E. coli .
  • This mutant acetolactate synthase from E. coli has a mutation in the IlvN regulatory unit, specifically Asn-17, Ala-30, and/or Ile-44.
  • This mutant acetolactate synthase was shown to enhance branched-chain L-amino acid production when DNA encoding the mutant enzyme is introduced into the branched-chain L-amino acid-producing strain.
  • AHAS I mutant bacterial acetolactate synthase
  • AHAS I mutant small subunit of bacterial acetolactate synthase
  • AHAS I mutant small subunit of bacterial bacterial acetolactate synthase
  • AHAS I mutant small subunit of bacterial bacterial acetolactate synthase
  • AHAS I mutant small subunit of bacterial acetolactate synthase
  • branched-chain L-amino acids are selected from the group consisting of L-leucine, L-isoleucine, and L-valine.
  • It is a further aspect of the present invention to provide a method for producing branched-chain L-amino acids comprising cultivating the bacterium described above in a culture medium, and collecting the branched-chain L-amino acids from the culture medium.
  • branched-chain L-amino acid is selected from the group consisting of L-leucine, L-isoleucine, and L-valine.
  • FIG. 1 shows the construction of the plasmid pMIV-P ivbL -ilvBN.
  • FIG. 2 shows the strategy for sequencing the ilvBN33 operon.
  • FIG. 3 shows an alignment of the nucleotide and amino acid sequences of ilvN and ilvN33.
  • the nucleotide sequence is shown in small letters, the amino acids in capital.
  • a 34 bp direct repeat in ilvN33 is shown in bold and marked with arrows.
  • FIG. 4 shows the construction of the plasmid pM12.
  • FIG. 5 shows the construction of the plasmid pM12-ter(thr).
  • FIG. 6 shows the construction of the intJS integrative cassette.
  • FIG. 7 shows the construction of the plasmid pMIV-5JS.
  • FIG. 8 shows the construction of a chromosomal DNA fragment with the inactivated ilvBN gene.
  • bacterial acetolactate synthase means the wild-type, or endogenous, acetolactate synthase present in bacteria of the Enterobacteriaceae family, corynebacteria, bacteria belonging to the genus Bacillus etc.
  • the Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Erwinia, Providencia , and Serratia .
  • the genus Escherichia is preferred.
  • activity of acetolactate synthase means the activity which catalyzes the formation of 2-aceto-2-hydroxy-butyrate and CO 2 from pyruvate and 2-oxobutanoate, or the formation of 2-acetolactate and CO 2 from two molecules of pyruvate. This activity can be measured in bacterial extracts using the method of F. C. Stormer and H. E. Umbarger (Biochem. Biophys. Res. Commun., 17, 5, 587-592 (1964)).
  • Acetohydroxybutanoate synthase I also called acetolactate synthase I (AHAS I) is a heterotetramer protein which includes two catalytic and two regulatory domains (Weinstock, O. et al, J. Bacteriol., 174(17), 5560-5566 (1992)). It is generally accepted that the large (ca. 60-kDa) subunits are catalytic, while the small ones are regulatory. AHAS I is coded for by the ilvB and ilvN genes.
  • a typical example of such a mutant is IlvN33 (SEQ ID NO: 11).
  • the small subunit of the wild-type acetolactate synthase which has a substitution(s) at position 17, and/or position 30,, or has several amino acids replaced in the N-terminus portion downstream from position 44, may be referred to as the “mutant small subunit”.
  • the acetolactate synthase containing the mutant small subunit may be referred to as the “mutant acetolactate synthase”.
  • a DNA coding for the mutant small subunit may be referred to as the “mutant ilvN gene”.
  • the small subunit of acetolactate synthase without any substitutions may be referred to as “a wild-type small subunit”.
  • An acetolactate synthase which contains wild-type small subunits may be referred to as “a wild-type acetolactate synthase”.
  • a DNA encoding the mutant small subunit of the present invention and a large subunit of acetolactate synthase may be referred to as the “mutant acetolactate synthase gene”.
  • the ilvB gene (synonym-b3671) encodes the acetolactate synthase large subunit.
  • the ilvB gene (nucleotides complementary to nucleotide positions 3849119 to 3850807; GeneBank accession no. NC — 000913.2; gi:16129170) is located between the ilvN gene and ivbL gene on the chromosome of E. coli K-12.
  • the ilvN gene (synonym-b3670) encodes the acetolactate synthase small subunit.
  • the ilvN gene (nucleotides complementary to nucleotide positions 3848825 to 3849115; GeneBank accession no. NC — 000913.2; gi:49175990) is located between the uhpA and ilvB genes on the chromosome of E. coli K-12.
  • the nucleotide sequence of the ilvN gene and the amino acid sequence of the acetolactate synthase small subunit encoded by the ilvN gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the ilvB and ilvN genes forms the ilvBN operon.
  • the mutant small subunit is obtained by introducing mutations into a wild-type ilvN gene using known methods.
  • the ilvBN operon can be obtained by PCR (polymerase chain reaction; refer to White, T. J. et al., Trends Genet., 5, 185 (1989)) utilizing primers based on the nucleotide sequence of the operon.
  • Genes coding for acetolactate synthase from other microorganisms can be obtained in a similar manner.
  • the mutant small subunit may include deletions, substitutions, insertions, or additions of one or several amino acids at one or more positions other than 17, 30, and/or 44, provided that the activity of acetolactate synthase which contains the mutant subunits is maintained.
  • the number of “several” amino acids differs depending on the position in the three dimensional structure of the protein or the type of amino acid residues. This is because some amino acids are similar to one another in their structure and function within a protein, and interchanging of such amino acids does not greatly affect the three dimensional structure or the function of the protein.
  • the mutant acetolactate synthase of the present invention may be one which has homology of not less than 70%, preferably 80%, and more preferably 90%, and most preferably 95% with respect to the entire amino acid sequence for acetolactate synthase, and which maintains the acetolactate synthase activity.
  • the number of “several” amino acids may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5.
  • substitutions, deletions, insertions or additions of one or several amino acid residues is/are conservative mutation(s) so that the activity is maintained.
  • the representative conservative mutation is a conservative substitution.
  • conservative substitutions include substitution of Ser or Thr for Ala, substitution of Gln, His or Lys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn, substitution of Asn, Glu or Gln for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gln, substitution of Asn, Gln, Lys or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gln, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for Ile, substitution of Ile, Met, Val or Phe for Leu, substitution of Asn, Glu, Gln, His or Arg for Lys, substitution of Ile, Leu, Val or Phe for Met,
  • the large subunit of the acetolactate synthase encoded by the ilvB gene also may include deletions, substitutions, insertions, or additions of one or several amino acids at one or more positions.
  • L-amino acid at position 17, 30, and/or 44 means an amino acid residue corresponding to the amino acid residue at position 17, 30, and/or 44 in the amino acid sequence of SEQ ID NO: 2, which is the sequence of the E. coli wild-type small subunit of acetolactate synthase.
  • the amino acid residue in position 17 is asparagine
  • the amino acid residue in position 30 is alanine
  • the amino acid residue in position 44 is isoleucine.
  • the position of an amino acid residue may change.
  • amino acid residue at position 17, 30, and/or 44 becomes position 18, 31, and/or 45.
  • amino acid residue at the original position 17, 30, and/or 44 is designated as the amino acid residue at the position 17, 30 and/or 44 in the present invention.
  • the amino acid sequence of the small subunit of the acetolactate synthase from E. coli (SEQ ID NO: 2) is aligned with the amino acid sequence of the small subunit of the acetolactate synthase from the bacterium of interest, and the L-amino acids at positions 17, 30 and/or 44 in the small subunit of the acetolactate synthase from the bacterium of interest can be determined.
  • the DNA which codes for substantially the same protein as the mutant small subunit described above is obtained, for example, by modifying the nucleotide sequence by site-directed mutagenesis so that one or more amino acid residues at a specified site are deleted, substituted, inserted, or added. DNA modified as described above is obtained by conventionally known mutation treatments.
  • Such mutation treatments include treating a DNA containing the mutant ilvN gene in vitro, for example, with hydroxylamine, and treating a microorganism, for example, a bacterium belonging to the genus Escherichia harboring the mutant ilvN gene, with ultraviolet irradiation or a known mutating agent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid.
  • NTG N-methyl-N′-nitro-N-nitrosoguanidine
  • substitutions, deletions, insertions, or additions of nucleotides as described above also include naturally occurring mutations (mutant or variant), for example, on the basis of individual differences or differences in species or genus of the bacterium which contains acetolactate synthase.
  • the DNA which codes for substantially the same protein as the mutant small subunit can be obtained by isolating a DNA which hybridizes with DNA having a sequence complimentary to the known ilvN gene sequence or part of it under stringent conditions, and which codes for a protein which forms a whole enzyme having acetolactate synthase activity with a large subunit, from a cell which is has been subjected to mutation treatment.
  • Stringent conditions include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, preferably not less than 70%, more preferably not less than 80%, still more preferably not less than 90%, and most preferably not less than 95%, is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed.
  • stringent conditions are exemplified by washing one time or more, preferably two or three times at a salt concentration of 1 ⁇ SSC, 0.1% SDS, preferably 0.1 ⁇ SSC, 0.1% SDS at 60° C. Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer.
  • the recommended duration of washing for the HybondTM N+ nylon membrane (Amersham) under stringent conditions is 15 minutes.
  • washing may be performed 2 to 3 times.
  • the length of the probe may be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
  • BLAST search BLAST search
  • FASTA search FASTA search
  • ClustalW ClustalW
  • BLAST Basic Local Alignment Search Tool
  • blastp, blastn, blastx, megablast, tblastn, and tblastx are the programs ascribe significance to their findings using the statistical methods of Karlin, Samuel and Stephen F. Altschul (“Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes”. Proc. Natl. Acad. Sci. USA, 1990, 87:2264-68; “Applications and statistics for multiple high-scoring segments in molecular sequences”. Proc. Natl. Acad. Sci. USA, 1993, 90:5873-7).
  • the FASTA search method is described by W. R.
  • the gene which is able to hybridize under the conditions as described above includes genes which have a stop codon within the coding region, and genes which are inactive due to mutation of the active site.
  • Such inconveniences can be easily removed by ligating the gene with a commercially available expression vector, and investigating the acetolactate synthase activity of whole enzyme containing the expressed protein.
  • the bacterium of the present invention is a branched-chain L-amino acid-producing bacterium of the Enterobacteriaceae family which contains a DNA which codes for the mutant small subunit of acetolactate synthase. Furthermore, the bacterium of the present invention is a branched-chain L-amino acid-producing bacterium of the Enterobacteriaceae family which has increased mutant acetolactate synthase activity.
  • the bacterium of the present invention is a branched-chain L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein branched-chain L-amino acid production is increased due to the introduction into the bacterium of the mutant ilvN gene which encodes the mutant small subunit of the present invention.
  • the bacterium of the present invention is a branched-chain L-amino acid-producing bacterium belonging to the genus Escherichia , wherein branched-chain L-amino acid production is increased by enhancing the activity of acetolactate synthase, namely valine-resistant mutant acetolactate synthase, in the bacterium.
  • the bacterium of present invention contains the mutant ilvN gene on the bacterial chromosome or in a plasmid, wherein the gene is over-expressed, and this bacterium has an enhanced ability to produce branched-chain L-amino acids.
  • bacterium which has the ability to produce branched-chain L-amino acids indicates a bacterium which has the ability to cause accumulation of branched-chain L-amino acids in a medium, such as L-leucine, L-isoleucine and L-valine, when the bacterium of the present invention is cultured in the medium.
  • the branched-chain L-amino acid producing ability may be imparted or enhanced by breeding.
  • bacterium which has an ability to produce branched-chain L-amino acid also indicates a bacterium which is able to produce and cause accumulation in the culture medium of a larger amount of branched-chain L-amino acids than a wild-type or parental strain, and preferably means that the bacterium is able to produce and cause accumulation in the medium of not less than 0.5 g/L, more preferably not less than 1.0 g/L of the branched-chain L-amino acids.
  • Exemplary L-amino acids include L-leucine, L-isoleucine, and L-valine.
  • a bacterium belonging to the genus Escherichia means the bacterium classified as the genus Escherichia according to the classification known to a person skilled in the microbiology.
  • An example is Escherichia coli ( E. coli ).
  • the phrase “activity of the mutant acetolactate synthase is enhanced” means that the activity per cell is higher than that of a non-modified strain, for example, a wild-type strain.
  • Exemplary enhanced activities include when the number of the mutant acetolactate synthase molecules per cell increases, and when the specific activity per mutant acetolactate synthase molecule increases, and so forth.
  • an exemplary wild-type strain that may be used for comparison for example, is Escherichia coli K-12.
  • the mutant acetolactate synthase activity in a bacterial cell can be enhanced by increasing the expression of the gene coding for the mutant acetolactate synthase.
  • Any gene which codes for mutant acetolactate synthase derived or isolated from bacteria of the Enterobacteriaceae family or coryneform bacteria can be used. Among these, genes derived from bacteria belonging to the genus Escherichia are preferred.
  • Transforming a bacterium with a DNA coding for a protein means introducing the DNA into a bacterium cell, for example, by conventional methods, to increase the expression of the gene coding for the protein of present invention and to enhance the activity of the protein in the bacterial cell.
  • Methods for enhancing gene expression include increasing the gene copy number. Introducing a gene into a vector that is able to function in a bacterium belonging to the genus Escherichia increases the copy number of the gene.
  • multi-copy vectors can be preferably used, such as pBR322, pUC19, pBluescript KS + , pACYC177, pACYC184, pAYC32, pMW119, pET22b, or the like.
  • Gene expression can also be enhanced by introducing multiple copies of the gene into the bacterial chromosome by, for example, homologous recombination, or the like.
  • Gene expression can also be enhanced by placing the DNA of the present invention under the control of a promoter which is stronger than the native promoter.
  • the strength of a promoter is defined by the frequency of RNA synthesis initiation. Methods for evaluating the strength of a promoter and examples of potent promoters are described by Deuschle, U., Kammerer, W., Gentz, R., Bujard, H. (Promoters in Escherichia coli : a hierarchy of in vivo strength indicates alternate structures. EMBO J. 1986, 5, 2987-2994).
  • the P R promoter is known as a potent constitutive promoter.
  • Other known potent promoters are the P L promoter, lac promoter, trp promoter, trc promoter, of lambda phage, and the like.
  • SD sequence Shine-Dalgarno sequence
  • Using a potent promoter can be combined with using multiple gene copies.
  • Methods for preparing chromosomal DNA, hybridization, PCR, preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be typical methods which are well known to one skilled in the art. These methods are described in Sambrook, J., and Russell D., “Molecular Cloning A Laboratory Manual, Third Edition”, Cold Spring Harbor Laboratory Press (2001), and the like.
  • the bacterium of the present invention can be obtained by introducing the aforementioned DNAs into a bacterium which inherently is able to produce branched-chain L-amino acids.
  • the bacterium of present invention can be obtained by imparting the ability to produce branched-chain L-amino acid to a bacterium already containing the DNAs.
  • L-valine producing bacteria belonging to the genus Escherichia such as H-81 (VKPM B-8066), NRRL B-12287 and NRRL B-12288 (U.S. Pat. No. 4,391,907), VKPM B-4411 (U.S. Pat. No. 5,658,766), VKPM B-7707 (European patent application EP1016710A2), or the like is employed.
  • L-leucine producing bacteria belonging to the genus Escherichia may be used, such as H-9070 (FERM BP-4704) and H-9072 (FERM BP-4706) (U.S. Pat. No.
  • VKPM B-7386 and VKPM B-7388 (RU2140450), W1485atpA401/pMWdAR6, W1485lip2/pMWdAR6 and AJ12631/pMWdAR6 (EP0872547), or the like.
  • L-isoleucine producing bacteria belonging to the genus Escherichia may be used, such as strain (NZ10) TDH6/pVIC40, pMWD5 (Hashiguchi, K. et al, Biosci. Biotechnol. Biochem., 63(4), 672-679 (1999)), or strain AJ12919 described in European patent application EP 685555 A1, or the like.
  • the method of present invention includes producing a branched-chain L-amino acid, such as L-leucine, L-isoleucine, and L-valine, by cultivating the bacterium of the present invention in a culture medium, allowing the branched-chain L-amino acid to be produced in the culture medium, and collecting the branched-chain L-amino acid from the culture medium.
  • a branched-chain L-amino acid such as L-leucine, L-isoleucine, and L-valine
  • the cultivation, the collection and purification of branched-chain L-amino acids from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein an amino acid is produced using a microorganism.
  • the medium used for culture may be either synthetic or natural, so long as it includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the microorganism requires for growth.
  • the carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids.
  • alcohol including ethanol and glycerol may be used.
  • ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate and digested fermentative microorganism are used.
  • minerals potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like, are used. Additional nutrients can be added to the medium if necessary. For instance, if the microorganism requires proline for growth (proline auxotrophy) a sufficient amount of proline can be added to the medium.
  • the cultivation is performed preferably under aerobic conditions such as a shaking culture, and a stirring culture with aeration, at a temperature of 20 to 42° C., preferably 37 to 40° C.
  • the pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2.
  • the pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to the accumulation of the target L-amino acid in the liquid medium.
  • solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the target L-amino acid can be collected and purified by ion-exchange, concentration, and crystallization methods.
  • the ilvBN operon was cloned into the pMIV5JS vector as a part of a 2439 bp PCR product. Construction of the vector pMIV5JS is described below in Reference example 1.
  • the MG1655 chromosome was used as a template in the PCR reaction.
  • Synthetic oligonucleotides ilvBX60 (SEQ ID NO: 3) and ilvBR64 (SEQ ID NO: 4) were used as primers.
  • Primer ilvBX60 contains the XbaI-restriction site at the 5′-end
  • primer ilvBR64 contains the SalI-restriction site at the 5′-end.
  • the strain B7 ⁇ ilvBN ⁇ ilvGM ⁇ ilvIH/pMIV-P ivbL -ilvBN described in Example 1 contains only one operon encoding AHAS (ilvBN operon).
  • Spontaneous mutants resistant to valine were selected on plates with minimal medium and supplemented with 1 g/l of valine.
  • Acetolactate synthase activity and enzymes resistance to L-valine inhibition were determined in crude extracts by the method of F. C. Stomer and H. E. Umbarger (Biochem. Biophys. Res. Commun., 17, 5, 587-592 (1964)).
  • This plasmid contains an operon encoding AHAS I resistant to valine inhibition. Residual AHAS activity in the presence of 10 mM L-valine was measured. Residual AHAS activity is equal to the activity in the presence of L-valine (nmol/min mg)*100% /the activity in the absence of L-valine (nmol/min mg). AHAS activity was measured by the method of F. C. Stormer and H. E. Umbarger (Biochem. Biophys. Res. Commun., 17, 5, 587-592 (1964)). Results of AHAS activity measurements for this strain are presented in Table 1.
  • oligonucleotides were used to sequence the ilvBN DNA fragment cloned in pMIV-P ivbL -ilvBN33: ML74 (SEQ ID NO: 5), LattRS1 (SEQ ID NO: 6), ilvbS31 (SEQ ID NO: 7), ilvbS32 (SEQ ID NO: 8), and ilvbS33 (SEQ ID NO: 9).
  • the sequencing strategy is presented in FIG. 2
  • ilvBN33 SEQ ID NO: 10
  • ilvBN33 SEQ ID NO: 10
  • Comparison of this sequence with calculating programs revealed a direct repeat of 34 nucleotides in the region coding for the small subunit.
  • the mutant gene was named ilvN33 ( FIG. 3 ). This DNA rearrangement lead to earlier translation termination, resulting in replacement of the N-terminus portion downstream from the isoleucine at position 44 with arginine and phenylalanine, which forms the 45 amino acid truncated protein IlvN33 (SEQ ID NO: 11).
  • Chloramphenicol resistant (Cm R ) clones were selected at 30° C. on LB agar plates containing 20 mg/l chloramphenicol. After eliminating both plasmids by cultivation of these clones in LB, Cm R Km S Ap S clones were obtained which were able to grow on minimal medium without additions.
  • strain B7 ⁇ ilvBN ⁇ ilvGM ⁇ ilvIH mini-Mu::cat-P ivbL -ilvBN ValR33 containing the integrated cassette mini-Mu::cat-P ivbL -ilvBN ValR33 in a non-identified chromosomal locus was obtained.
  • mini-Mu::cat-P ivbL -ilvBN ValR33 cells were transformed with the plasmid pMW118-int-xis (AP R ) (WO2005/010175).
  • Ap R clones were grown on LB agar plates containing 150 mg/l ampicillin at 30° C. Several tens of Ap R clones were picked up and tested for chloramphenicol sensitivity. The plasmid pMW118-int-xis was eliminated from Cm S cells by incubation on LB agar plates at 42° C.
  • Modifying the regulator region of the ilvBN ValR33 operon namely replacing the native promoter region of the ilvBN operon with the P L promoter, was accomplished by the method first developed by Datsenko and Wanner (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called “Red-driven integration”. According to this procedure, the PCR primers ilvB-attR1 (SEQ ID NO: 12) and ilvB-PLSD (SEQ ID NO:13) were constructed.
  • Oligonucleotide ilvB-attR1 (SEQ ID NO: 12) is homologous to the region upstream of the ilvB gene and the region adjacent to the gene conferring antibiotic resistance present in the chromosomal DNA of BW25113 cat-P L -yddG.
  • Oligonucleotide ilvB-PLSD (SEQ ID NO: 13) is homologous to both the ilvB region and the region downstream to the PL promoter present in the chromosome of BW25113 cat-P L -yddG. Obtaining BW25113 cat-P L -yddG has been described in detail (EP1449918A1, Russian patent RU2222596).
  • the chromosomal DNA of strain BW25113 cat-P L -yddG was used as a template for PCR.
  • Conditions for PCR were the following: denaturation for 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 34° C., 40 sec at 72° C.; profile for the last 30 cycles: 30 sec at 95° C., 30 sec at 50° C., 40 sec at 72° C.; final step: 5 min at 72° C.
  • the PCR product was obtained (SEQ ID NO: 14), purified in agarose gel, and used for electroporation of the E.
  • the plasmid pKD46 (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) includes the 2,154 nt (31088-33241) DNA fragment of phage ⁇ (GenBank accession No. J02459), containing the genes of the ⁇ Red homologous recombination system ( ⁇ , ⁇ , exo genes) under the control of the arabinose-inducible P araB promoter.
  • the plasmid pKD46 is necessary for integration of the PCR product into the chromosome of the strain B7 ⁇ ilvBN ⁇ ilvGM ⁇ ilvIH mini-Mu::P ivbL -ilvBN ValR33 .
  • Electrocompetent cells were prepared as follows: E. coli strain B7 ⁇ ilvBN ⁇ ilvGM ⁇ ilvIH mini-Mu::P ivbL -ilvBN ValR33 was grown overnight at 30° C. in LB medium containing ampicillin (100 mg/l), and the culture was diluted in 100 times with 5 ml of SOB medium (Sambrook et al, “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989)) with ampicillin and L-arabinose (1 mM). The cells were grown with aeration at 30° C. to an OD 600 of ⁇ 0.6 and then made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H 2 O.
  • Electroporation was performed using 70 ⁇ l of cells and ⁇ 100 ng of PCR product. Following electroporation, the cells were incubated with 1 ml of SOC medium (Sambrook et al, “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989)) at 37° C. for 2.5 h, plated onto L-agar, and grown at 37° C. to select Cm R recombinants. Then, to eliminate the pKD46 plasmid, 2 passages on L-agar with Cm at 42° C. were performed and the resulting colonies were tested for sensitivity to ampicillin.
  • SOC medium Standardbrook et al, “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989)
  • the clone B7 ⁇ ilvBN ⁇ ilvGM ⁇ ilvIH mini-Mu::cat-P L -ilvBN ValR33 was obtained, which contains the cassette mini-Mu::cat-P L -ilvBN ValR33 with the mutant ilvBN ValR33 operon encoding the feedback-resistant AHAS I under the control of the phage lambda PL promoter, marked with Cm resistance gene.
  • Phage Mu right attachment-specific primer MR74 (SEQ ID NO: 15) and the primer specific to chloramphenicol acetyltransferase gene Cm-test2 (SEQ ID NO: 16) were used in PCR for the verification.
  • Conditions for PCR verification were the following: denaturation for 3 min at 94° C.; profile for the 30 cycles: 30 sec at 94° C., 30 sec at 55° C., 1 min at 72° C.; final step: 7 min at 72° C.
  • the PCR product obtained using the cells of B7 ⁇ ilvBN ⁇ ilvGM ⁇ ilvIH mini-Mu::cat-P ivbL -ilvBN ValR33 as a template was 586 nt in length (SEQ ID NO: 17).
  • the PCR product obtained using the cells of B7 ⁇ ilvBN ⁇ ilvGM ⁇ ilvIH mini-Mu::cat-P L -ilvBN ValR33 as a template was 879 nt in length (SEQ ID NO: 18). Residual AHAS activity in the B7 ⁇ ilvBN ⁇ ilvGM ⁇ ilvIH mini-Mu::cat-P L -ilvBN ValR33 strain in the presence of 10 mM L-valine was measured. Results of AHAS activity measurements for this strain are presented in Table 1.
  • Both E. coli strains B7 ⁇ ilvBN ⁇ ilvGM ⁇ ilvIH mini-Mu::P ivbL -ilvBN ValR33 and B7 ⁇ ilvBN ⁇ ilvGM ⁇ ilvIH mini-Mu::cat-P L -ilvBN ValR33 were grown for 18 hours at 37° C. on L-agar plates. Then, cells from about 0.5 cm 2 of the plate surface were introduced into the fermentation medium (2 ml) and cultivated in tubes with aeration for 72 hours at 32° C.
  • the fermentation medium was additionally supplemented with 100 ⁇ g/ml each of isoleucine and valine. The accumulated L-valine was measured by TLC. The results are presented in Table 2.
  • Glucose 60.0 (NH 4 ) 2 SO 4 18.0 KH 2 PO 4 3H 2 O 2.0 MgSO 4 7H 2 O 1.0 CaCO 3 25.0 Thiamin 0.02 Mameno 4.0
  • the native regulator region of the ilvBN operon was replaced with the phage lambda P L promoter by the same method as described in Example 5 in strain B7 ⁇ ilvIH ⁇ ilvGM (see Reference Example 2, section 5).
  • This strain has only AHAS I.
  • the resulting strain B7 ⁇ ilvIH ⁇ ilvGM cat-P L -ilvBN was sensitive to valine. New valine-resistant spontaneous mutants of AHAS I were obtained from this strain. Strains which grew better on 1 g/l of valine were characterized (Table 3).
  • Valine-resistant mutations which were resistant to isoleucine were obtained, as well. Variants with a specific activity higher than that of the wild-type were obtained. As seen in Table 4, expression of valine-resistant AHAS I resulted in the production of valine.
  • the fermentation medium contained 6% glucose.
  • IlvBN ValR1 contained one point mutation in IlvN: A30P Ala-Pro (Ala at position 30 is replaced with Pro; corresponding codon gcc was replaced with ccc), and IlvBN ValR4 also contained one point mutation in IlvN: N17K Asn-Lys (Asn at position 17 is replaced with Lys; corresponding codon aac was replaced with aag). In both cases, such substitutions were rare.
  • the cassette cat-P L -ilvBN ValR4 was introduced into L-leucine-producing E. coli strain 57 (USDn patent RU 2140450, VKPM B-7386).
  • the strain E. coli 57 was infected with phage P1 vir grown on the donor strain B7 ⁇ ilvIH ⁇ ilvGM cat-P L -ilvBNValR4.
  • the transductants were selected on L-agar plates supplemented with chloramphenicol (20 ⁇ g/ml).
  • the strain 57 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (USD, 117545 Moscow, 1 Dorozhny proezd, 1) on May 19, 1997 under accession number VKPM B-7386.
  • strain 57 cat-P L -ilvBNValR4 produced a larger amount of L-leucine.
  • PMIV-5JS was constructed according to the following scheme.
  • plasmid pM12 was constructed by integrating in vivo a Mu-derived integration cassette into plasmid pMW1, which is a derivative of pMW119 ( FIG. 4 ).
  • Two terminator oligonucleotide sequences complementary to each other were synthesized (SEQ ID NO: 19 and SEQ ID NO: 20).
  • Terminator thrL was obtained by annealing these synthetic oligonucleotides in the forward (SEQ ID NO: 19) and reverse directions (SEQ ID NO: 20). Terminator thrL was flanked with sites HindIII and PstI.
  • plasmid pM12-ter(thr) was constructed by inserting the synthetic terminator sequence Ter(thr) into pM12 which had been digested with HindIII and Mph1103I ( FIG. 5 ).
  • the intJS integrative cassette was constructed as following ( FIG. 6 ):
  • the fragment LattL-Cm R -LattR-MCS was digested by BgIII and HindIII and cloned into pM12-ter(thr) which had been digested with BamHI and HindIII to yield plasmid pMIV-5JS ( FIG. 7 ).
  • the ilvBN operon was deleted by the method firstly developed by Datsenko and Wanner (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called as a “Red-driven integration”. According to this procedure, the PCR primers ilvBN1 (SEQ ID NO: 28) and ilvBN2 (SEQ ID NO: 29) homologous to both the region adjacent to the ilvBN operon and the gene conferring chloramphenicol resistance in the template plasmid were constructed. The plasmid pMW-attL-Cm-attR (PCT application WO 05/010175) was used as a template in the PCR reaction.
  • Conditions for PCR were the following: denaturation step for 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 34° C., 40 sec at 72° C.; profile for the last 30 cycles: 30 sec at 95° C., 30 sec at 50° C., 40 sec at 72° C.; final step: 5 min at 72° C.
  • the resulting 1713 bp PCR product was purified in agarose gel and used for electroporation of the E. coli strain MG1655, which contains the plasmid pKD46 with a temperature-sensitive replication origin.
  • the plasmid pKD46 (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) includes a 2,154 nt (31088-33241) DNA fragment of phage ⁇ (GenBank accession No. J02459), which contains genes of the ⁇ Red homologous recombination system ( ⁇ , ⁇ , exo genes) under the control of the arabinose-inducible P araB promoter.
  • the plasmid pKD46 is necessary for integration of the PCR product into the chromosome of the E. coli strain MG1655.
  • Electrocompetent cells were prepared as follows: overnight culture of E. coli MG1655/pKD46 grown at 30° C. in LB medium, supplemented with ampicillin (100 mg/l), was diluted in 100 times with 5 ml of SOB medium (Sambrook et al, “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989)) with ampicillin and L-arabinose (1 mM). The culture was grown with aeration at 30° C. to an OD 600 of ⁇ 0.6 and then made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H 2 O. Electroporation was performed using 70 ⁇ l of cells and ⁇ 100 ng of PCR product.
  • the ilvIH operon was deleted by the same methods as the deletion of ilvBN operon described in Section 1. According to this procedure, the PCR primers ilvIH1 (SEQ ID NO: 32) and ilvIH2 (SEQ ID NO: 33) homologous to both the region adjacent to the ilvIH operon and gene conferring chloramphenicol resistance in the template plasmid were constructed.
  • the plasmid pMW-attL-Cm-attR (PCT application WO 05/010175) was used as a template in the PCR reaction.
  • Conditions for PCR were the following: denaturation step for 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 34° C., 40 sec at 72° C.; profile for the last 30 cycles: 30 sec at 95° C., 30 sec at 50° C., 40 sec at 72° C.; final step: 5 min at 72° C.
  • the 1713 bp PCR product was purified in an agarose gel and used for electroporation of the E. coli strain MG1655/pKD46. Chloramphenicol-resistant recombinants were selected after electroporation and verified by means of PCR with the locus-specific primers ilvIHC3 (SEQ ID NO: 34) and ilvIHC4 (SEQ ID NO: 35). Conditions for PCR verification were the following: denaturation step for 3 min at 94° C.; profile for the 30 cycles: 30 sec at 94° C., 30 sec at 53° C., 1 min 20 sec at 72° C.; final step: 7 min at 72° C.
  • the PCR product obtained in the reaction using the chromosomal DNA from parental IlvIH + strain MG1655B7 ⁇ ilvBN::cat as a template was 2491 nt in length.
  • the PCR product obtained in the reaction using the chromosomal DNA from mutant MG1655B7 ⁇ ilvBN::cat ⁇ ilvIH::cat strain as a template was 1823 nt in length.
  • the strain MG1655 ⁇ ilvIH::cat was obtained.
  • the ilvGM operon was deleted by the same methods as the deletion of ilvBN operon described in Section 1. According to this procedure, the PCR primers ilvGM1 (SEQ ID NO: 36) and ilvGM2 (SEQ ID NO: 37) homologous to both the region adjacent to the ilvGM operon and the gene conferring chloramphenicol-resistance in the template plasmid were constructed.
  • the plasmid pMW-attL-Cm-attR (PCT application WO 05/010175) was used as a template in the PCR reaction.
  • Conditions for PCR were the following: denaturation step for 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 34° C., 40 sec at 72° C.; profile for the last 30 cycles: 30 sec at 95° C., 30 sec at 50° C., 40 sec at 72° C.; final step: 5 min at 72° C.
  • the 1713 bp PCR product was purified in an agarose gel and used for electroporation of the E. coli strain MG1655/pKD46. Chloramphenicol-resistant recombinants were selected after electroporation and verified by PCR with the locus-specific primers ilvGMC3 (SEQ ID NO: 38) and ilvGMC4 (SEQ ID NO: 39). Conditions for PCR verification were the following: denaturation step for 3 min at 94° C.; profile for the 30 cycles: 30 sec at 94° C., 30 sec at 54° C., 1 min 30 sec at 72° C.; final step: 7 min at 72° C.
  • the PCR product obtained in the reaction using the chromosomal DNA from parental strain MG1655 as a template was 2209 nt in length.
  • the PCR product obtained in the reaction using the chromosomal DNA from mutant MG1655 ⁇ ilvGM::cat strain as a template was 1941 nt in length.
  • the strain MG1655 ⁇ ilvGM::cat was obtained.
  • the ilvIH genes ( ⁇ ilvIH::cat) were deleted in the wild-type strain E. coli K12 (VKPM B-7) by P1 transduction (Sambrook et al, “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989).
  • the E. coli strain MG1655 ⁇ ilvIH::cat described in Section 3 was used as a donor strain, and CmR transductants were selected.
  • the strain B7 ⁇ ilvIH::cat was obtained.
  • cells were transformed with the plasmid pMW118-int-xis (Ap R ) (WO2005/010175).
  • Ap R clones were grown on LB agar plates containing 150 mg/l ampicillin at 30° C. Several tens of Ap R clones were picked up and tested for chloramphenicol sensitivity. The plasmid pMW118-int-xis was eliminated from the Cm S cells by incubation on LB agar plates at 42° C. As a result, the strain B7 ⁇ ilvIH was obtained.
  • the ilvBN genes ( ⁇ ilvBN::cat) were deleted in the E. coli strain B7 ⁇ ilvIH by P1 transduction.
  • the strain E. coli MG1655 ⁇ ilvBN::cat described in Section 1 was used as a donor strain, and CmR transductants were selected.
  • the strain B7 ⁇ ilvIH ⁇ ilvBN::cat was obtained.
  • the chloramphenicol resistance marker was eliminated from B7 ⁇ ilvIH ⁇ ilvBN::cat as described above.
  • the strain B7 ⁇ ilvIH ⁇ ilvBN was obtained.
  • the ilvGM genes ( ⁇ ilvGM::cat) were deleted in the E. coli strains B7 ⁇ ilvIH by P1 transduction.
  • the strain E. coli MG1655 ⁇ ilvGM::cat described in Section 4 was used as a donor strain, and CmR transductants were selected.
  • the strain B7 ⁇ ilvIH ⁇ ilvGM::cat was obtained.
  • the chloramphenicol resistance marker was eliminated from B7 ⁇ ilvIH ⁇ ilvGM::cat as described above.
  • the strain B7 ⁇ ilvIH ⁇ ilvGM was obtained.
  • the strain B7 ⁇ ilvIH ⁇ ilvGM was prototrophic, therefore deletion of ilvGM genes did not prevent expression of the distal genes of the isoleucine-valine operon.
  • the ilvGM genes ( ⁇ ilvGM::cat) were deleted in the E. coli strains B7 ⁇ ilvIH ⁇ ilvBN by P1 transduction.
  • the strain E. coli MG1655 ⁇ ilvGM::cat described in Section 4 was used as a donor strain; CmR transductants were selected.
  • the strain B7 ⁇ ilvIH ⁇ ilvBN ⁇ ilvGM::cat was obtained.
  • the chloramphenicol resistance marker was eliminated from B7 ⁇ ilvIH ⁇ ilvBN ⁇ ilvGM::cat as described above.
  • the strain B7 ⁇ ilvIH ⁇ ilvBN ⁇ ilvGM was obtained.

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EP1942183B1 (fr) 2009-10-28
BRPI0703528B1 (pt) 2017-02-14
EP1942183A1 (fr) 2008-07-09
BRPI0703528A2 (pt) 2011-05-31
CN101386841A (zh) 2009-03-18
US20140335574A1 (en) 2014-11-13
DE602007002975D1 (de) 2009-12-10
RU2355763C2 (ru) 2009-05-20
JP5813907B2 (ja) 2015-11-17
CN101386841B (zh) 2012-06-13
US9279137B2 (en) 2016-03-08
ATE447014T1 (de) 2009-11-15

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