EP1309694A2 - Procede de fabrication amelioree de cyanophycine et de ses produits secondaires - Google Patents

Procede de fabrication amelioree de cyanophycine et de ses produits secondaires

Info

Publication number
EP1309694A2
EP1309694A2 EP01955376A EP01955376A EP1309694A2 EP 1309694 A2 EP1309694 A2 EP 1309694A2 EP 01955376 A EP01955376 A EP 01955376A EP 01955376 A EP01955376 A EP 01955376A EP 1309694 A2 EP1309694 A2 EP 1309694A2
Authority
EP
European Patent Office
Prior art keywords
cyanophycin
synthetase
cyanophycin synthetase
asp
nucleotide sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01955376A
Other languages
German (de)
English (en)
Inventor
Karl Ziegler
Wolfgang Lockau
Jan Ebert
Kirill Piotukh
Holger Berg
Rudolf Volkmer-Engert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Chemicals AG
Original Assignee
Bayer AG
Bayer Chemicals AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer AG, Bayer Chemicals AG filed Critical Bayer AG
Publication of EP1309694A2 publication Critical patent/EP1309694A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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

Definitions

  • the present invention relates to a thermostable cyanophycin synthase, transformed organisms containing such an enzyme, and a method for the improved production of cyanophycin and / or its secondary products, for example polyaspartic acid or arginine.
  • Multi-L-Arginyl-Poly-L-aspartate is a branched polypeptide that contains aspartic acid and arginine in a ratio of 1: 1.
  • Structure corresponds to a poly- ⁇ -aspartate backbone with arginine side residues, which are linked to almost all ⁇ -carboxyl groups of the backbone via peptide bonds.
  • DE-A 197 09 024 discloses the extraction and purification of cyanophycin from Aphanocapsa PCC 6308, the synthesis being carried out at 20 ° C.
  • FEMS Microbiology Letters 181 (1999) 229-236 describes the production of cyanophycin from Synechococcus sp. MA 19 known.
  • a disadvantage of the large-scale production of cyanophycin according to the known processes is that a relatively narrow temperature range, generally below 35 ° C., must not be exceeded for optimum product yield.
  • the present invention relates to a cyanophycin synthetase which is characterized in that it codes a temperature optimum in the range of> 35 ° C. and an amino acid sequence according to SEQUENCE PROTOCOL 1 by an isolated nucleotide sequence according to SEQUENCE PROTOCOL 2, an allele, homologue or derivative of this nucleotide sequence or with this has a hybridizing nucleotide sequence.
  • the cyanophycin synthetase according to the invention has an optimum temperature in the range from 35 ° C. to 55 ° C., preferably in the range from 35 to 50 ° C.
  • the cyanophycin synthetase according to the invention is distinguished by the fact that it originates from Synechococcus elongatus.
  • the cyanophycin syn- thetase is a thermostable enzyme.
  • the present invention also relates to isoenzymes of the cyanophycin synthetase according to the invention.
  • This includes enzymes with the same or comparable substrate and activity specificity, but which have a different primary structure.
  • the present invention also includes modified forms of cyanophycin synthetase. According to the invention, this includes enzymes in which there are changes in the sequence, for example at the N- and / or C-terminus of the polypeptide or in the region of conserved amino acids, but without impairing the function of the enzyme. These changes can be made by exchanging one or more amino acids according to known methods.
  • a special embodiment variant of the present invention comprises variants of the cyanophycin synthetase according to the invention, the substrate specificity of which has been changed, for example, by amino acid exchange compared with the respective starting protein, for example with regard to the production of polyaspartic acid.
  • the present invention furthermore relates to polypeptides with the function of a cyanophycin synthetase, the amino acid sequence of which is changed in such a way that they are desensitive to regulatory compounds, for example the end products of metabolism regulating their activity (feedback-desensitive).
  • an isolated nucleotide sequence or an isolated nucleic acid fragment is to be understood as a polymer made from RNA or DNA, which can be single or double-stranded and optionally contain natural, chemically synthesized, modified or artificial nucleotides.
  • DNA polymer also includes genomic DNA, cDNA or mixtures thereof.
  • alleles are to be understood as functionally equivalent, ie essentially equivalent nucleotide sequences. Functionally equivalent sequences are those sequences which, despite a different nucleotide sequence, for example due to the degeneracy of the genetic code, still have the desired functions.
  • Functional equivalents thus include naturally occurring variants of the sequences described here as well as artificial nucleotide sequences, for example those obtained by chemical synthesis and possibly adapted to the codon use of the host organism.
  • functionally equivalent sequences include those which have an altered nucleotide sequence which corresponds to the
  • Enzyme for example, gives a desensitivity or resistance to inhibitors.
  • a functional equivalent is also understood to mean, in particular, natural or artificial mutations in an originally isolated sequence which continues to show the desired function. Mutations include substitutions, additions,
  • nucleotide residues Deletions, exchanges or insertions of one or more nucleotide residues. Also included here are so-called sense mutations, which can lead to the exchange of conserved amino acids at the protein level, but which do not lead to a fundamental change in the activity of the protein and are therefore function-neutral. This also includes changes in the nucleotide sequence that affect the N- or C-terminus of a protein at the protein level, but without significantly impairing the function of the protein. These changes can even have a stabilizing influence on the protein structure.
  • the present invention also encompasses, for example, those nucleotide sequences which are obtained by modifying the nucleotide sequence, resulting in corresponding derivatives.
  • the aim of such a modification can, for example, be to further narrow down the coding sequence contained therein or, for example, also to insert further recognition sites for restriction enzymes.
  • Functional equivalents are also those variants whose function is weakened or enhanced compared to the original gene or gene fragment.
  • artificial DNA sequences are the subject of the present invention as long as they impart the desired properties as described above and can be incorporated or appended into the gene of the cyanophycin synthetase according to the invention.
  • Such artificial DNA sequences can be determined, for example, by back-translating proteins created using computer-aided programs (molecular modeling) or by in-vitro selection. Coding DNA sequences which are obtained by back-translating a polypeptide sequence according to the codon usage specific for the host organism are particularly suitable. The specific codon usage can easily be determined by a person familiar with molecular genetic methods by computer evaluations of other, already known genes of the organism to be transformed.
  • homologous sequences are to be understood as those which are complementary to and / or hybridize with the nucleotide sequences according to the invention.
  • hybridizing sequences includes substantially similar nucleotide sequences from the group of DNA or RNA which, under known stringent conditions, enter into a specific interaction (binding) with the aforementioned nucleotide sequences. This also includes short nucleotide sequences with a length of, for example, 10 to 30, preferably
  • nucleotide primers or probes 12 to 15 nucleotides. According to the invention, this includes also so-called nucleotide primers or probes.
  • the preceding (5'- or upstream) and / or subsequent (3'- or downstream) coding regions are also also present.
  • regulatory sequences include promoters, enhancers, operators, terminators or translation enhancers.
  • An operative link is understood to mean the sequential arrangement of, for example, the promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence.
  • These regulatory nucleotide sequences can be of natural origin or can be obtained by chemical synthesis. In principle, any promoter which can control gene expression in the corresponding host organism is suitable as the promoter.
  • this can also be a chemically inducible promoter by means of which the expression of the genes underlying it in the host cell can be controlled at a specific point in time.
  • a promoter that can be induced by IPTG (isopropyl- ⁇ -thiogalactoside) is mentioned here as an example.
  • a gene structure is produced by fusing a suitable promoter with the nucleotide sequence according to the invention using common recombination and cloning techniques as are known from the literature.
  • DNA fragments with one another can be attached to the fragments adapters or linkers.
  • the present invention relates to a vector comprising at least one nucleotide sequence of the type described above coding for a cyanophycin synthetase specific for the production of cyanophycin, to regulative nucleotide sequences operatively linked thereto and to additional nucleotide sequences for selecting transformed host cells, for replication within the host cell or for Integration into the corresponding host cell genome.
  • the vector according to the invention can contain a gene structure of the aforementioned type.
  • Suitable vectors are those which are replicated in microorganisms, such as bacteria, fungi and / or plants.
  • Known vectors are, for example, pBluescript (Stratagene, 11099 North Torney Pines Rd., La Jolla, CA 92 037, USA) or pET (Novagen, 601 Science Drive, Madison, WJ 53 711, USA).
  • pBluescript Stratagene, 11099 North Torney Pines Rd., La Jolla, CA 92 037, USA
  • pET Novagen, 601 Science Drive, Madison, WJ 53 711, USA.
  • this list is not limiting for the present invention.
  • corresponding probes or nucleotide primers can be synthesized and used to amplify and isolate analog genes from other single or multicellular organisms, preferably bacteria, fungi, algae or plants, for example with the aid of PCR technology.
  • the present invention thus also relates to a probe for identifying and / or isolating genes coding for proteins involved in the biosynthesis of cyanophycin, preferably further thermostable cyanophycin synthetases, this probe being produced on the basis of the nucleic acid sequences of the type described above and one for detection contains suitable marking.
  • the probe can be a partial section of the sequence according to the invention, for example from a conserved area, which e.g. has a length of 10 to 30 or preferably 12 to 15 nucleotides and can hybridize specifically with homologous nucleotide sequences under stringent conditions. Suitable markings are well known from the literature.
  • the present invention further relates to the transfer of the nucleic acid sequence according to the invention or a part thereof coding for a cyanophycin synthetase, an allele, homolog or derivative thereof or a nucleotide sequence hybridizing with these sequences into a heterologous host system.
  • This also includes the transfer of a gene construct or vector according to the invention into a heterologous host system.
  • a heterologous host system is understood to mean a single-cell or multi-cell organism. Examples of these are microorganisms, fungi, lower or higher plants, tissues or cells thereof. Bacteria are preferred according to the invention, particularly preferred the genus of enterobacteria and in particular the type Escherichia coli. Furthermore, useful plants, such as potatoes or
  • the nucleotide sequence coding for a thermostable cyanophycin synthetase according to the invention is transferred into one of the host systems mentioned above by known methods. Examples of methods for DNA transfer into suitable host systems are transformation, electroporation, conjugation and agrobacterium-mediated DNA transfer or “particle bombardment”. These lists are only used to explain the present invention and are not limiting.
  • a transformed single or multicellular organism resulting from a successfully carried out nuclear acid transfer thus differs from the correspondingly not transformed organism in that it contains additional nucleic acids of the type according to the invention and can accordingly express them.
  • the present invention thus also relates to a transformed single or multicellular organism containing a cyanophycin synthetase according to the invention and / or a vector containing a cyanophycin synthetase of the type described above.
  • the present invention relates to a method for providing a cyanophycin synthetase according to the invention of the type described above, wherein the nucleotide sequence coding for the enzyme is isolated from a thermophilic single or multicellular organism, if necessary operatively linked to regulatory structures and / or into one suitable for heterologous expression Vector is cloned, optionally transferred to a heterologous host system, expressed there and then isolated from this host system and optionally purified and / or enriched.
  • cyanophycin synthetase it is also conceivable to directly isolate an amount of cyanophycin synthetase sufficient for the synthesis of cyanophycm from a thermophilic organism. mus, without the previous enrichment in a heterologous system. Furthermore, the enzyme cyanophycin synthetase according to the invention can then be used, for example, in an in vitro system for the synthesis of cyanophycin and / or its secondary products.
  • the present invention also relates to a process for the preparation of cyanophycin and / or its secondary products, a cyanophycin synthetase and / or a vector and / or a transformed single or multicellular organism of the type described above being used.
  • the present invention comprises not only the production of cyanophycin and / or its secondary products in a living host system, but also the in vitro synthesis of cyanophycin with the aid of an isolated cyanophycin synthetase of the type described above.
  • the process according to the invention for the production of cyanophycin is characterized in that the enzyme-catalyzed synthesis takes place in a temperature range from 35 ° C. to 55 ° C., preferably in a range from 35 ° C. to 50 ° C.
  • the process according to the invention is advantageously characterized in that the process is less susceptible to faults due to the wide temperature range, in particular above 28 ° C., allows greater variability in the process control and thus delivers an improved product yield.
  • the production of cyanophycin and / or its secondary products according to the invention is thus substantially more reproducible and more economical than the previously known processes.
  • the cyanophycin synthetase according to the invention catalyzes an ATP-dependent chain extension reaction (elongation). Surprisingly, the enzyme has two active (catalytic) centers.
  • the cyanophycin synthetase according to the invention gradually and alternately (sequentially) incorporates a molecule of aspartic acid and then a molecule of arginine into a cyanophyne precursor (peptide primer). Without a primer, the enzyme-catalyzed
  • the present invention further relates to the use of a vector containing a cyanophycin synthetase according to the invention of the aforementioned type for position of a transformed single or multicellular organism as described above.
  • a transformed single or multi-cell organism for the production of a cyanophycin synthetase according to the invention and / or for the production of cyanophycin and / or its secondary products is also included in the present invention.
  • a cyanophycin synthetase isolated according to the invention can also be used for the in vitro production of cyanophycin and / or its secondary products.
  • the use of cyanophycin and / or its derivatives for the production of food supplements and / or agents in the field of agriculture and / or crop protection is covered in the present invention.
  • cyanophycin and / or its secondary products can be seen in the paper, textile, pigment, lacquer, ceramic, building material or detergent industries as well as in the areas of water and waste water treatment.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • the peptide primers are linked by the following reaction: (i) coupling of Fmoc-Asp-OtBu and subsequently (ii) twice attaching the building block Fmoc-Asp- [Arg- (Pmc) -OtBu] -OH. Furthermore, the N-terminally blocked peptide primer ⁇ -Ahx 2 - (b-Asp-Arg) 3 was produced by attaching Fmoc- ⁇ -aminohexanoic acid (Novabiochem) twice to the peptide primer bound to the resin described above. The finished peptides were made by
  • the peptide primer ( ⁇ -Asp-Arg) 3 -Asp was synthesized on a TentaGel-S-PHB resin (Rapp polymers) loaded with Fmoc-Asp (OtBu) by attaching the corresponding building block three times.
  • the peptide was also cleaved from the resin and deprotected as previously described. Here, the peptide was obtained as a free acid.
  • All peptide primers were purified on a C-18 column (Vydac 201SP54) and analyzed using RP-HPLC and MALDI-MS.
  • the dipeptide ⁇ -Asp-Arg was also produced on a TentaGel-S-PHB phase, which had previously been loaded with Fmoc-Arg (Pmc).
  • Fmoc-Arg Pmc
  • the resin was treated with 4 eq (equivalents) of Boc-Asp-OtBu (Bachern Chemicals), 4 eq O- (benzotriazol-l-yl) - N , N, N ', N'-tetramethyluronium tetrafluoroborate and 8 eq diisopropyl-emylamine treated in DMF.
  • the peptide was cleaved from the resin with trifluoroacetic acid containing 1% phenol, 2% water and 3% triisobutylsilane, then precipitated with cold t-butylmethyl and finally purified on a C-18 column (Vydac 201SP54) and with RP-HPLC and MALDI-MS analyzed.
  • the reaction batches for product analysis by mass spectrometry contain the following components in a volume of 125 ⁇ l: 100 mM NEUHCO; ⁇ (pH 8.0), 4 mM ATP disodium salt, 20 mM MgCl 2 , 8 mM KC1, 2 mM DTT, 0.2 mM L-aspartic acid, 0.2 mM L-arginine, ⁇ 10 ⁇ M synthetic primers and 3 ⁇ g cyanophycin synthetase.
  • the reaction mixture of 125 ⁇ l contains the following: 50 mM Tris-HCl (pH 8.0), 4 mM ATP disodium salt, 20 mM MgCl 2 , 20 mM KC1, 1 mM DTT, 0.8 mM L Aspartic acid, 0.4 mM L-arginine,> 10 ⁇ M synthetic primer and 3 ⁇ g cyanophycin synthetase.
  • Various primers (Fig. 1) are added to the reaction mixture. After incubation of the reaction batches for 24 hours at room temperature
  • Lane 5 like lane 4 but with about 160 ⁇ M primer. " The diffuse bands below 29 kDa in lanes 1, 2 and 3 represent cyanophycin-like material.

Abstract

La présente invention concerne une cyanophycine synthétase thermostable provenant de Synechococcus elongatus, ainsi qu'un procédé de fabrication améliorée de cyanophycine et/ou de ses produits secondaires.
EP01955376A 2000-08-09 2001-07-27 Procede de fabrication amelioree de cyanophycine et de ses produits secondaires Withdrawn EP1309694A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10038775 2000-08-09
DE10038775 2000-08-09
PCT/EP2001/008690 WO2002012459A2 (fr) 2000-08-09 2001-07-27 Procede de fabrication amelioree de cyanophycine et de ses produits secondaires

Publications (1)

Publication Number Publication Date
EP1309694A2 true EP1309694A2 (fr) 2003-05-14

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EP01955376A Withdrawn EP1309694A2 (fr) 2000-08-09 2001-07-27 Procede de fabrication amelioree de cyanophycine et de ses produits secondaires

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US (1) US20020115141A1 (fr)
EP (1) EP1309694A2 (fr)
AU (1) AU2001277555A1 (fr)
WO (1) WO2002012459A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2008268150A1 (en) 2007-06-27 2008-12-31 Arizona Board Of Regents, A Body Corporate Acting On Behalf Of Arizona State University Reagents and methods for cyanobacterial production of bioplastics and biomaterials
JP2009171908A (ja) * 2008-01-26 2009-08-06 Univ Waseda シアノフィシンの製造方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19709024A1 (de) * 1997-03-06 1998-09-10 Bayer Ag Polyasparaginsäure Homo- und Copolymere, ihre biotechnologische Herstellung und Verwendung
DE19813692A1 (de) * 1998-03-27 1999-09-30 Norddeutsche Pflanzenzucht Han Cyanophycinsynthetasegene zur Erzeugung von Cyanophycin oder Cyanophycinderivaten, und ihre Verwendung
DE19825509A1 (de) * 1998-06-02 1999-12-09 Bayer Ag Biotechnische Herstellung von Polyasparaginsäure und deren Modifikation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0212459A2 *

Also Published As

Publication number Publication date
AU2001277555A1 (en) 2002-02-18
WO2002012459A2 (fr) 2002-02-14
WO2002012459A3 (fr) 2002-06-20
US20020115141A1 (en) 2002-08-22

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