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WO2000003009A2 - Genes pour la biosynthese d'anticapsine et de bacilysine, leur isolement et leur utilisation - Google Patents

Genes pour la biosynthese d'anticapsine et de bacilysine, leur isolement et leur utilisation Download PDF

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Publication number
WO2000003009A2
WO2000003009A2 PCT/DE1999/002179 DE9902179W WO0003009A2 WO 2000003009 A2 WO2000003009 A2 WO 2000003009A2 DE 9902179 W DE9902179 W DE 9902179W WO 0003009 A2 WO0003009 A2 WO 0003009A2
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bacilysin
plasmid
genes
bacillus subtilis
bacillus
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English (en)
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WO2000003009A3 (fr
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Gerhard Steinborn
Jürgen HOFEMEISTER
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Institut fuer Pflanzengenetik und Kulturpflanzenforschung
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Institut fuer Pflanzengenetik und Kulturpflanzenforschung
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Priority to CA002334079A priority Critical patent/CA2334079A1/fr
Priority to EP99947209A priority patent/EP1097214A2/fr
Priority to AU60774/99A priority patent/AU6077499A/en
Publication of WO2000003009A2 publication Critical patent/WO2000003009A2/fr
Publication of WO2000003009A3 publication Critical patent/WO2000003009A3/fr
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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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)

Definitions

  • the invention relates to genes for the biosynthesis of anticapsin and bacilysin, their isolation and use, in particular in medicine and agriculture.
  • Bacilysin is a well-known dipeptide that has been described as an antibiotic agent (Zuber and Marahiel, 1992). It is species-specific from Bacillus subtle and also from individual strains of other Bacillus species from the Bacillus subtilis group (Loeffler et al., 1986) and is secreted from the cell into the surrounding culture medium.
  • Bacilysin consists of the N-terminal of L-alanine and the C-terminal of the non-proteinogenic epoxy amino acid L-anticapsin (Rogers et al., 1965; Walker and Abraham, 1970a; Neuss et al., 1970) and was chemically described as L-alanyl- L- ⁇ - (2,3-epoxycyclohexanono-4) alanine (Walker and Abraham, 1970b; Chmara et al., 1981). After absorption into sensitive cells, bacilysin is proteolytically cleaved, releasing the antibiotic anticapsin.
  • Anticapsin inhibits glucosamine synthetase in bacteria and fungi and thereby the biosynthesis of the aminosugars glucosamine and N-acetylglucosamine, which are required for cell wall synthesis. This inhibits cell wall synthesis and causes cell lysis (Whitney and Funderburk, 1970; Kenig et al., 1976; Milewski, 1993). Bacilysin and anticapsin are competitively inhibited by other di- and tripeptides (Perry and Abraham, 1979) as well as by glucosamine and N-acetylglucosamine (Walton and Rickes, 1962; Chmara, 1985). a. thereby be distinguished from other antibiotic agents.
  • Another way of identifying anticapsin and bacilysin is to prevent their biosynthesis by mutations in ⁇ roGenes for the biosynthesis of the aromatic amino acids phenylalanine and tyrosine (Hilton et al., 1988a).
  • Bacillus subtilis AI 4 (Walker and Abraham, 1970a, b) and a strain of Streptomyces griseoplanus (Whitney et al., 1970) also immediately produce extracellular anticapsin, but such findings are limited to these exceptional cases. It is also known that bacilysin has a relatively broad, nonspecific activity against Gram-positive and negative bacteria as well as against shoot and filamentous fungi and from sensitive cells via a dipeptide and oligopeptide permease system (Diddens et al., 1979; Perry and Abraham , 1979; Chmara et al., 1981) is effectively included. Defect mutations in this permease system lead to resistance to bacylisin.
  • Extracellular anticapsin is usually not incorporated by bacteria and therefore has a relatively low antibacterial activity, except against Streptococcus pyogenes and Salmonella gallinarum (Neuss et al., 1970). In contrast, anticapsin has a strong antifungal Effect, inter alia against Candida albicans and Botrytis an ea and other filamentous fungi (Neuss et al, 1970, Kenig et al, 1976, Chmara et al, 1980)
  • Bacillus subtilis 168 is known to branch off from the aromatic amino acid synthesis pathway in the prephenate, which apparently serves as a metaphoric precursor for the synthesis of anticapsin (Hilton et al, 1988a).
  • the enzymatic reactions to this and also to the following compound from anticapsin with L-alanm to bacilysin are still unknown
  • the excretion of the bacilysm formed from the cell is presumably carried out by means of a peptide transport system (Diddens et al, 1979)
  • halogenated Bacilysmde ⁇ vate chlorotetam, bromotetain
  • halogenated Bacilysmde ⁇ vate chlorotetam, bromotetain
  • these halogen derivatives are of interest insofar as they have some differences in their mode of action and spectrum compared to bacilysin of their producers (Loeffler et al, 1990, Katzer, 1991)
  • the object is achieved in that genes or a gene group for the biosynthesis of anticapsin and bacilysin are first genetically engineered using a suitable bacterial host-vector system is cloned and amphfected and thereby accessible to a structural and functional characterization due to the gene amphfication and the resulting gene dose effect and possibly using other bacterial host vector systems with increased gene amphfication and Increased and / or producible gene expression achieves an economically usable biosynthesis of the active substances, which are then used against microbial pathogens and putrefactive agents.
  • An excretion of the active substances is advantageous, so that they can be isolated more cost-effectively from the culture supernatants.
  • Such organisms are available as host strains to the naturally anticapsin or bacilysin from the cell is also advantageous to suppress the end product inhibition, as in bacilysin biosynthesis by accumulated bacilysin, so that the overproduction of the antibiotics is not caused by a high concentration of the ant ibiotics itself is inhibited
  • the present invention can also be used for the biotechnological production of chlorotetam and bromotetain or of new, stronger or more specific effective Bacilysin and Anticapsindenvaten
  • the recombined active substance formers can be used as cellular antagonists by applying them in the environment of microbial putrefaction and pathogens, for example in the soil, in culture fluids or on the surface of other organisms, and by activating the secreted antibiotics
  • moniated bacilysin genes Another possibility of using the moniated bacilysin genes is to insert them into the organisms to be protected against antibiotics, so that they themselves form the antibiotic active ingredient and thus achieve self-protection against microbial pathogens and putrefactive organisms.
  • the genes that are directly used for the biosynthesis of Anticapsin or bacilysin are required, transferred and useful in crops and thus an increased tolerance or resistance to a broad spectrum of different types of phytopathogenic pathogens can be achieved by transgenic synthesis of anticapsin or bacilysin
  • the object of the invention is achieved in particular in that corresponding genes according to the invention are isolated from a bacterial strain of the Bacillus subtilis group, preferably from Bacillus subtilis 168, using methods customary in genetic engineering
  • the invention relates to genes for the m-vitro and m-vivo biosynthesis of anticapsin and bacilysin and their fragments, variants and mutants which are characterized by base exchanges or other mutations, and also RNA nucleic acid sequences in which T is replaced by U, and nuclear acid sequences that are completely or partially complementary or homologous
  • the genes and their products themselves represent diagnostic and / or therapeutic substances or serve as targets for the search for new therapeutically active substances.
  • these are preferably bacilysin genes from Bacillus subtilis 168, hereinafter referred to as “Z> ⁇ c” genes, within a 7471 bps DNA sequence according to SEQ ID No. 1 (Appendix 1), their mutants, variants and their fully or partially complementary DNA sequences.
  • positive-selective cloning vectors with high cloning capacity and stability are preferably used and gene banks with large chromosomal DNA inserts are isolated, so that not only individual genes but also gene groups with sufficient probability can be isolated.
  • the palindromic, simply positive selective cloning vector pPS15 has proven to be an advantageous cloning vector (Steinborn, 1996).
  • This palindrome plasmid vector and also the non-palindrome plasmid vector pSB595 (see below) used for subsequent clonings belong to the incompatibility group inc ⁇ 8, and their particularly high cloning capacity and structural and segregative plasmid stability are due to their theta replication or a partition gene parS.
  • Bacterial bacilysin-negative strains preferably of Bacillus subtilis 168, are used as the host strain for the primary cloning of the genes according to the invention. Because of a chromosomal ⁇ ⁇ c mutation, they have no chromosomally encoded bacilysin formation, and the cloning of the ⁇ c genes can be demonstrated due to the complementation of the chromosomal mutation.
  • a mutant NG79 bac- (Hilton et al, 1988b) or derivatives of NG79 from the Bacillus subtilis 168 family are preferably used as Bacilysin-negative host strains.
  • a stabilizing strain Bacillus subtilis GSB298 hip-X bac-X or a comparable strain as a bacilysin-negative host strain is used, which has an increased transformability and stabilizing effect on recombinant plasmid derivatives with a large insert, this GSB298 as spontaneous hip (host increased plasmid stability) mutant derived from NG79 bac-X and isolated due to its stabilizing effect on palindromic, double positive selective cloning vectors (Steinborn, 1996).
  • Gene banks with large chromosomal DNA inserts are obtained preferably by partial restriction cleavage of the donor DNA and insertion of preselected, large (> 10 kb) DNA fragments.
  • a gene bank is isolated from which a recombinant strain Bacillus subtilis GSB298 (pSB657) with t ⁇ c genes from Bacillus subtilis 168 is isolated. This excretes plasmid-encoded bacilysin, which is detected by the inhibition of a bacilysin-sensitive indicator strain, preferably Escherichia coli B or Proteus vulgaris, and the inhibition of bacilysin by N-acetylglucosamine.
  • pSB657 does not contain it a host strain Bacillus subtilis GSB285 aroI906 (Steinborn, 1996) because the aro ⁇ 906 mutation inhibits the biosynthesis of prephenate as a precursor for anticapsin.
  • Optimal formation and antibiotic effectiveness of bacilysin is achieved by using a minimal PA medium (Perry and Abraham, 1979) and cultivation at 30 ° C. Agar diffusion tests are used for the semi-quantitative determination of bacilysin activity using zones of inhibition around growing colonies or punch holes that are loaded with culture supernatants.
  • the bac genes are contained in the recombinant plasmid pSB657 generated by primary cloning within a 21.5 kb BamHI insert.
  • a further recombinant strain Bacillus subtilis GSB298 (pSB660) is derived from the 21.5 kb BamHI insert of pSB657 by subcloning, the bac genes contained in a 7.5 kb PstI insert in the recombinant plasmid pSB660 thus obtained are (embodiment 2).
  • the PstI insert of pSB660 is localized in the region from bp 3867455 to bp 3 874 925 of the genomic DNA sequence on the basis of DNA homologies and corresponding restriction data (Fig. 1) as a 7471 bps DNA sequence (Fig. 2) (SubtiList database, Institut Pasteur, Moszer et al., 1995; Moszer, 1998) of the genome of Bacillus subtilis 168, identified (embodiment 3). This position lies within the 90 kb region in which the ⁇ c-1 locus has been mapped by transduction (Hilton, H, et al., 1988b); this confirms the location and function of the bac genes.
  • the recombinant plasmid pSB660 in Bacillus subtilis GSB298 encodes an increased or at least comparable bacilysin formation (Fig. 3) with the gene donor strain Bacillus subtilis 168 (Fig. 3), so that the bac-X mutation (Hilton et al., 1988b) caused this Defect in the ligase reaction in GSB298 is fully complemented by pSB660.
  • a different course of bacilysin formation is observed, which begins immediately with the start of growth in the recombinant strain GSB298 (pSB660).
  • the chromosomally coded bacilysin formation takes place with a clear lag phase and only adapts to the level of the plasmid-coded bacilysin formation in the stationary phase. This approximation of the maximum values is interpreted as an expression of the end product inhibition by accumulated bacilysin.
  • the Pstl insert contains 6 open reading frames (ORF 's) with previously unknown function and the original name (see SubtiList database) ywfk to w F, which due to the described complementation and further findings (see below) on their function in the Bacilysin biosynthesis renamed back to bacY (Fig. 1) and claimed according to the invention.
  • a deletion derivative pSB674 pSB672 AbacE even causes a complete failure of the formation of extracellular bacilysin and a complete inhibition of growth and cell lysis in GSB298, correlated with conspicuous bloating of the naturally rod-shaped cells to protoplast-like cell forms, such as those for the antibiotic effect of anticapsin are typical.
  • bacE encodes an enzyme required for the ligase reaction of bacilysin formation and in GSB298 (pSB674) with inactive bacE gene in both pSB674 and in GSB298 bac-X (as demonstrated for the bac-X mutation; Hilton et al., 1988b) intracellularly, a lethal concentration of anticapsin for the host cell.
  • an additional gene is mutated in GSB298, e.g. B. a transport gene so that the excretion of bacilysin and / or anticapsin is inhibited.
  • Fig. 1 Restriction map of the PstI insert of pSB660 corresponding to the DNA sequence in Appendix 1 with the genes back, bacB, bacC, bacD, bacE and bacF from Bacillus subtilis 168. Only those restriction sites that are required for the construction of Plasmid derivatives can be used.
  • Fig. 2 Growth and bacilysin formation in the recombinant strain Bacillus subtilis GSB298 (pSB672) compared to the gene donor strain Bacillus subtilis 168 and the bacilysin-negative host strain GSB298 bac-X at 30 ° C in PA minimal medium
  • Fig. 3 Growth and bacilysin formation in the recombinant strains Bacillus subtilis GSB298 (pSB672), GSB298 (pSB674) and GSB298 (pSB676) at 30 ° C in PA minimal medium
  • Bacillus subtilis bacterial strain GSB285 h ⁇ X (Steinborn, 1996/98) as recipient strain for subcloning of bac genes; derived as a spontaneous, plasmid-stabilizing (for recombinant plasmids with large insert) mutant of GSB26 .s ⁇ cA321 ⁇ roI906 metB5 amyE strX from the strain line of Bacillus subtilis 168.
  • the ⁇ roI906 mutation prevents the biosynthesis of anticapsin and bacilysin, so that these do not alter the plasmid transformation in protoplasts)
  • Plasmid vector pPS15 (15.4 kb; MLS 1 ) as a palindromic, positive selective cloning vector with high cloning capacity and stability (Steinbom, 1996/98), so that when cloned almost all plasmid transformants contain a recombinant plasmid and also recombinant plasmids with a large insert remain stable
  • Plasmid vector pSB595 (7 kb; MLS r ) as a non-palindromic cloning vector with high cloning capacity and stability, derived from a plasmid pSB472 (Steinborn, 1996/98) by introducing a multiple cloning site
  • Both plasmid vectors belong to the incompatibility group / «cl8 and their high structural and segregative stability is due to the theta type of their replication or to a partition gene parS
  • Insertion mutagenesis (Niaudet et al., 1982) for the inactivation of chromosomal bac genes of the gene donor Bacillus subtilis 168 by chromosomal insertion.
  • Exemplary embodiment 1 Primary cloning of the Bacillus subtilis 168 bac genes.
  • the primary cloning is carried out by “shot gun” after partial fragmentation cleavage of the chromosomal donor DNA and preselection of large DNA fragments (> 10 kb) for ligation.
  • a positively selective cloning vector pPS15 used, transformed into a stabilizing, Bacilysin-negative (Bac) recipient strain Bacillus subtilis GSB298 bac-1 and a screening for Bacilysin-positive (Bac ⁇ ) clones on PA agar plates with Escherichia coli B designed as a bacilysin-sensitive indicator organism.
  • GSB298 pSB657
  • a 21.5 kb BamHI insert is determined in pSB657 and the plasmid-encoded formation of bacilysin confirmed by all (see above) biological and biochemical evidence.
  • pSB657 complements the bac-X mutation in GSB298, so that GSB298 (pSB657) compared to the donor strain Bacillus subtilis 168 gives at least comparable or increased bacilysin formation ((as for GSB298 (pSB672) in Fig. 3)) With comparable growth, a strongly delayed formation of bacilysin is observed for the donor strain.
  • Exemplary embodiment 2 Localization of the bac genes in the primary insert of pSB657
  • subcloning is carried out after cleavage of the insert of pSB657 by various restrictases and pPS15 and GSB298 are again used as the host vector system.
  • Highly expressing clones comparable to GSB298 (pSB657), are obtained after Pstl cleavage and these are referred to as "GSB298 (pSB660)".
  • PSB660 contains a 7.5 kb Pstl insert which, after recloning in pPS15 and retransformation in GSB298 high bacilysin formation (Fig. 3) is reproducibly encoded.
  • Exemplary embodiment 3 Localization of the bac genes in the genomic DNA sequence of Bacillus subtilis 168
  • the 7.5 kb-Pstl insert contained in pSB660 (exemplary embodiment 2) is cloned in pUC18 and the recombinant strain Escherichia coh XL1 (pSB661) is thereby obtained. Sequencing is then carried out from both ends of the Pstl insert and a homology comparison with the genomic DNA sequence (SubtiList database) of the gene donor Bacillus subtilis 168 is carried out with the DNA sequences obtained (429 bps or 226 bps). Complete DNA homology is found for the region from bp 3 867 455 to bp 3 874 925 of the genomic DNA sequence so that the PstI insert contains 7 471 bps. Restriction cleavage sites (FIG.
  • the PstI fragment 6 contains complete, functionally still unknown ORF's ywfk to ywfi, which are renamed back to bacF because of their function in bacilysin biosynthesis (Fig. 1).
  • First indications of their function are obtained by a homology comparison of the proteins BacA to BacF, which are putatively encoded by the genes back to bacF, to known proteins of other organisms using the “SWISS- PROT database "(ExPASY Molecular Biology Server, Fasta 3): High homologies for BacB to a prephenate dehydratase from Streptomyces coelicolor (28%), for BacE to D-alanine-D-alanine ligases from Haemophüus influenzae (29%) and from Bacillus subtilis (21.5%) and for BacF to a macrolide efflux protein (24.8%) indicate functions in the synthesis from prephenate to Antikapsin, ligation of Antikapsin with L-Alanine to Bacily
  • promoter motifs are determined upstream to bacB (from bp 3874425 to bp 3874360 of the genomic DNA sequence), while a transcription terminator is probably only present downstream from back (from bp 3873120 to bp 3873061).
  • Embodiment 4 mutants of pSB660
  • PSB672 also complements the bac-X mutation in GSB298 and encodes a level of bacilysin comparable to that of pSB657 and pSB660 (Fig. 3).
  • pSB679 pSB660 A (back bacD bacE bacF) is generated by cloning a subfragment from PvuII (bp 1680) to Sacl (bp 3558) into the plasmid vector pSB595.
  • GSB298 (pSB674) forms no bacilysin
  • PA medium ie under conditions of bacilysin formation
  • PA medium does not grow, forms atypical, bloated cell forms and finally lyses.
  • Example 5 Insertion mutagenesis of chromosomal bac genes in Bacillus subtilis 168 by double crossover
  • chromosomal bac genes of the gene donor are deactivated by insertion in order to determine their function in bacilysin biosynthesis.
  • the insertion is carried out by general recombination via double cross-over and is carried out using integration plasmids.
  • integration plasmids a selection marker is inserted within the bac gene to be inactivated and is thus flanked by fragments of the bac gene which in a homologous host strain have the double cross - over effect.
  • Chloramphenicol resistance introduced as a selection marker in the mutated site.
  • pSB682 pSB672 bacB * .cmlH
  • pSB683 pSB672 AbacC: .cmlR
  • pSB684 pSB672 bacDv.cmlW
  • pSB680 pSB672 AbacEv.cmlR
  • pSB685 is derived from pSB660 (see Fig. 1) after deletion from PvuII (bp 1680) to EcoRV (bp 5681).
  • the integration plasmids are linearized (as a prerequisite for double cross-over) and transformed into competent cells from Bacillus subtilis 168.
  • the selection is made via chloramphenicol resistance, and double cross-over (to exclude single cross-over and mutations) is demonstrated by the absence of plasmid-coded MLS antibiotic resistance (coded by the plasmid-specific selection marker of the integration plasmid) and by free plasmid DNA of the integration vector, by Southern hybridization with the cmRI gene as a labeled sample and by cloning and restriction analysis of the relevant chromosomal region.
  • the insertion mutants are streaked on PA minimal agar and identified as prototrophic for the amino acids phenylalanine and tyrosine, whose biosynthesis (as well as for anticapsin) branches off from prephenate in the biosynthetic pathway of the aromatic amino acids.
  • Exemplary embodiment 6 Function of the bac genes for protecting the cells of Bacillus subtilis 168 against extracellular bacilysin
  • insertion mutants are tested for bacylis insensitivity. All insertion mutants, including GSB322 with deletion in back to bacF, show no Zones of inhibition and (as GSB298 and Bacillus subtilis 168) prove to be unchanged bacilysin-resistant.
  • Embodiment 7 Effect of deletion plasmids on chromosomal insertion mutants
  • # GSB322 shows a plasmid-encoded bacilysin formation comparable to GSB298 (pSB660) or GSB298 (pSB672), so that bacB to bacE can be rated as essential or sufficient, while back and bacF can be assessed as insignificant or not relevant for high bacilysin formation
  • # GSB322 (pSB674) (but not plasmid transformants of pSB674 with other chromosomal insertion mutants) behaves with respect to defects in bacilysin formation, aberrant cell forms and cell lysis like GSB298 (pSB674) when cultivated in PA minimal medium, so that this mutant phenotype (as also with NG79 or GSB298) cannot be traced back to the defect in only one & ac structure gene.
  • Exemplary embodiment 8 plasmid-coded bacilysin formation in heterologous Bacillus cells pSB660 and various deletion plasmids (see exemplary embodiment 4) are tested in Bacillus amyloliquefaciens GSB272, Bacillus licheniformis ATCC9789, Bacillus megaterium PV361, and Bacillus plasmid plasmid-transformed 404 pilbumid .
  • Plasmid transformants of Bacillus coagulans, Bacillus licheniformis ATCC9789 and Bacillus megaterium ATCC 12140 do not form extracellular bacilysin, while in Bacillus pumilus ATCC 12140 (as in Bacillus subtilis GSB298) pSB672 with bacB to bacE as active genes already encodes a high Bacilysin.
  • a particularly high plasmid-coded bacilysin formation up to 5-fold compared to GSB298 (pSB660) or GSB298 (pSB672), is achieved in Bacillus amyloliquefaciens GSB272, for which the deletion plasmid pSB679 (exemplary embodiment 4) with bacB and bacC is sufficient.

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Abstract

L'invention concerne des gènes pour la biosynthèse de l'anticapsine et de la bacilysine, leur isolement et leur utilisation, notamment dans le domaine de la médecine et de l'agriculture. Il s'agit de préférence de produits géniques qui s'utilisent généralement comme antagonistes de la synthèse de la glucosamine et peuvent être utilisés comme moyens pour l'antibiose contre des agents pathogènes et saprogènes microbiens.
PCT/DE1999/002179 1998-07-10 1999-07-12 Genes pour la biosynthese d'anticapsine et de bacilysine, leur isolement et leur utilisation Ceased WO2000003009A2 (fr)

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CA002334079A CA2334079A1 (fr) 1998-07-10 1999-07-12 Genes pour la biosynthese d'anticapsine et de bacilysine, leur isolement et leur utilisation
EP99947209A EP1097214A2 (fr) 1998-07-10 1999-07-12 Genes pour la biosynthese d'anticapsine et de bacilysine, leur isolement et leur utilisation
AU60774/99A AU6077499A (en) 1998-07-10 1999-07-12 Genes for the biosynthesis of anticapsin and bacilysine, their isolation and their use

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DE19830912 1998-07-10
DE19830912.0 1998-07-10
DE19921807.2 1999-05-11
DE19921807 1999-05-11

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WO2004058960A1 (fr) * 2002-12-26 2004-07-15 Kyowa Hakko Kogyo Co., Ltd. Procede de production d'un dipeptide
WO2006001382A1 (fr) * 2004-06-25 2006-01-05 Kyowa Hakko Kogyo Co., Ltd. Procédé de fabrication d'un dipeptide ou d'un dérivé de dipeptide
WO2006001381A1 (fr) * 2004-06-25 2006-01-05 Kyowa Hakko Kogyo Co., Ltd. Methode de production de dipeptide ou de derive de dipeptide
US7514243B2 (en) 2004-06-25 2009-04-07 Kyowa Hakko Kogyo Co., Ltd. Process for producing dipeptides
EP2179652A1 (fr) 2008-09-10 2010-04-28 ABiTEP GmbH Gesellschaft für AgroBioTechnische Entwicklung und Produktion Produit antibactérien destiné au traitement d'un incendie dans des vergers et d'autres maladies bactériennes des plantes
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EP1983059A2 (fr) 2002-12-26 2008-10-22 Kyowa Hakko Kogyo Co., Ltd. Procédé de production de dipeptides
US7514242B2 (en) 2002-12-26 2009-04-07 Kyowa Hakko Kogyo Co., Ltd. Process for producing dipeptides
US8039236B2 (en) 2002-12-26 2011-10-18 Kyowa Hakko Bio Co., Ltd. Process for producing dipeptides
KR101043562B1 (ko) * 2002-12-26 2011-06-22 교와 핫꼬 바이오 가부시키가이샤 디펩티드의 제조법
CN100400660C (zh) * 2002-12-26 2008-07-09 协和发酵工业株式会社 生产二肽的方法
EP1908773A2 (fr) 2002-12-26 2008-04-09 Kyowa Hakko Kogyo Co., Ltd. Procédé de production de dipeptides
EP1908773A3 (fr) * 2002-12-26 2008-04-16 Kyowa Hakko Kogyo Co., Ltd. Procédé de production de dipeptides
WO2004058960A1 (fr) * 2002-12-26 2004-07-15 Kyowa Hakko Kogyo Co., Ltd. Procede de production d'un dipeptide
EP1433791A3 (fr) * 2002-12-26 2005-03-30 Kyowa Hakko Kogyo Co., Ltd. Procédé de préparation de dipeptides
US8709752B2 (en) 2004-06-25 2014-04-29 Kyowa Hakko Bio Co., Ltd. Process for producing dipeptides or dipeptide derivatives
JPWO2006001382A1 (ja) * 2004-06-25 2008-04-17 協和醗酵工業株式会社 ジペプチドまたはジペプチド誘導体の製造法
JPWO2006001381A1 (ja) * 2004-06-25 2008-04-17 協和醗酵工業株式会社 ジペプチドまたはジペプチド誘導体の製造法
EP1616963A3 (fr) * 2004-06-25 2006-05-31 Kyowa Hakko Kogyo Co., Ltd. Procédé de préparation de dipeptides ou dérivés dipeptidiques.
WO2006001382A1 (fr) * 2004-06-25 2006-01-05 Kyowa Hakko Kogyo Co., Ltd. Procédé de fabrication d'un dipeptide ou d'un dérivé de dipeptide
US7514243B2 (en) 2004-06-25 2009-04-07 Kyowa Hakko Kogyo Co., Ltd. Process for producing dipeptides
CN1973037B (zh) * 2004-06-25 2011-10-19 协和发酵生化株式会社 二肽或二肽衍生物的制造方法
US7939302B2 (en) * 2004-06-25 2011-05-10 Kyowa Hakko Bio Co., Ltd. Process for producing dipeptides
WO2006001381A1 (fr) * 2004-06-25 2006-01-05 Kyowa Hakko Kogyo Co., Ltd. Methode de production de dipeptide ou de derive de dipeptide
EP2179652A1 (fr) 2008-09-10 2010-04-28 ABiTEP GmbH Gesellschaft für AgroBioTechnische Entwicklung und Produktion Produit antibactérien destiné au traitement d'un incendie dans des vergers et d'autres maladies bactériennes des plantes
EP2179652B1 (fr) 2008-09-10 2019-05-08 ABiTEP GmbH Gesellschaft für AgroBioTechnische Entwicklung und Produktion Utilisation d'un agent antibactérien pour le traitement des infections bactériennes chez les plantes cultivées
CN112226437A (zh) * 2020-10-26 2021-01-15 湖北大学 梯度调控芽胞杆菌启动子启动效率的序列组合及应用
CN112226437B (zh) * 2020-10-26 2022-02-15 湖北大学 梯度调控芽胞杆菌启动子启动效率的序列组合及应用

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