EP1322764A2 - Procede de fabrication amelioree de cyanophycine et de ses produits secondaires - Google Patents

Procede de fabrication amelioree de cyanophycine et de ses produits secondaires

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Publication number
EP1322764A2
EP1322764A2 EP01962878A EP01962878A EP1322764A2 EP 1322764 A2 EP1322764 A2 EP 1322764A2 EP 01962878 A EP01962878 A EP 01962878A EP 01962878 A EP01962878 A EP 01962878A EP 1322764 A2 EP1322764 A2 EP 1322764A2
Authority
EP
European Patent Office
Prior art keywords
cyanophycin
synthetase
nucleotide sequence
production
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Withdrawn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
EP01962878A
Other languages
German (de)
English (en)
Inventor
Winfried Joentgen
Alexander Steinbüchel
Fred Bernd Oppermann-Sanio
Elsayed Aboulmagd
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 EP1322764A2 publication Critical patent/EP1322764A2/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Withdrawn legal-status Critical Current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 thermostable cyanophycin synthetases, transformed organisms containing such an enzyme and a process 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 an approximately equimolar ratio.
  • the chemical 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.
  • a disadvantage of the production of cyanophycin according to the processes known hitherto is that a relatively narrow temperature range, generally below 35 ° C., must not be exceeded for optimum product yield. This represents a considerable restriction of the degrees of freedom within the process control for the production of cyanophycin and / or its secondary products.
  • the present invention relates to cyanophycin synthetases with increased stability and activity in the temperature range of> 35 ° C., characterized in that they
  • the cyanophycin synthetases according to the invention have an optimum temperature in the range from 35 ° C. to 55 ° C., preferably in the range from 35 ° C. to 50 ° C.
  • the cyanophycin synthetases according to the invention are thermostable enzymes.
  • the present invention also relates to isoenzymes of the invention
  • Cyanophycin synthetases 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 the cyanophycin synthetases. 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 enzymes. 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 synthetases according to the invention, the substrate specificity of which is weakened or enhanced, for example by amino acid exchange, compared to the respective starting protein.
  • the present invention furthermore relates to polypeptides with the function of a cyanopyhcin synthetase, the amino acid sequence of which has been modified in such a way that they are resistant to compounds having a regulatory action, for example those which they contain their activity-regulating metabolic end products are desensitive (feedback-desensitive).
  • an isolated nucleotide sequence or an isolated nucleic acid fragment is to be understood as a polymer from RNA or DNA which can be single or double-stranded and optionally can contain natural, chemically synthesized, modified or artificial nucleotides.
  • DNA polymer also includes genomic DNA, cDNA or mixtures thereof.
  • alleles are functionally equivalent, i.e. H. to understand essentially equivalent nucleotide sequences.
  • Function-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, e.g. B. obtained by chemical synthesis and possibly adapted to the codon use of the host organism nucleotide sequences.
  • functionally equivalent sequences include those which have a modified nucleotide sequence which, for example, imparts a desensitivity or resistance to inhibitors to the enzyme.
  • a functional equivalent is also understood to mean, in particular, natural or artificial mutations in an originally isolated sequence which continue to show the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
  • 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 are at the protein level the N- or C-terminus of a protein affect, but without significantly affecting the function of the protein. These changes can even have a stabilizing influence on the protein structure.
  • nucleotide sequences are also, for example, by the present invention.
  • 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 are particularly suitable which are obtained by back-translating a polypeptide sequence in accordance with the codon sequence specific for the host organism.
  • 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.
  • the term 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, before moves 12 to 15 nucleotides. According to the invention, this also includes so-called nucleotide primers or probes.
  • the preceding (5'- or upstream) and / or subsequent (3'- or downstream) coding regions are also also present.
  • the present invention furthermore relates to a gene structure comprising at least one of the previously described nucleotide sequences coding for a cyanophycin synthetase and to regulatory sequences which are operatively linked thereto and which control the expression of the coding sequences in the host cell.
  • An operational 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 is its own
  • These regulatory nucleotide sequences can be of natural origin or can be obtained by chemical synthesis.
  • 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- ⁇ -thiogalactopyranoside) 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 in the literature. To connect the DNA fragments to one another, adapters or linkers can be attached to the fragments.
  • the present invention relates to a vector containing at least one nucleotide sequence of the type described above coding for a cyanophycin synthetase specific for the production of cyanophycin, regulative nucleotide sequences operatively linked thereto and 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.
  • vectors are those that are in microorganisms, such as. B. bacteria,
  • Mushrooms and / or plants are replicated.
  • Known vectors are, for example, pBluescript (Stratagene, 11099 North Torney Pines Red., La Jolla, CA 92 037, USA) or pEKO (Eikmanns, BJ. Et al., Gene, 1991, 102: 93-98).
  • pBluescript Stratagene, 11099 North Torney Pines Red., La Jolla, CA 92 037, USA
  • pEKO Eikmanns, BJ. Et al., Gene, 1991, 102: 93-98.
  • this list is not limiting for the present invention.
  • probes or nucleotide primers can be synthesized and used to amplify and isolate, for example, genes from other single or multicellular organisms, preferably bacteria, fungi, algae or plants, using the PCR technique.
  • 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 suitable Contains marker.
  • the probe can be a partial section of the sequence according to the invention, for example from a conserved area which, for. B. 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 a single-cell or multi-cell system
  • bacteria are preferred, particularly preferred the enterobacterium genus and in particular the Escherichia coli species or coryneform bacteria, in particular the genus Corynebacterium or Brevibacterium, especially Corynebacterium glutamicum.
  • 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 or agrobacterial-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 nucleic acid transfer thus differs from that corresponding non-transformed organism in that it contains additional nucleic acids of the type according to the invention and can express them accordingly.
  • the present invention thus also relates to a transformed single or multicellular organism containing a cyanophycin synthase according to the invention and / or a vector containing a cyanophycin synthetase of the type described above.
  • the present invention further relates to a method for providing a cyanophycin synthetase according to the invention of the type described above, the nucleotide sequence coding for the enzyme being isolated from a single- or multicellular organism, optionally operatively linked to regulatory structures and or in one for heterologous expression suitable vector is cloned, possibly transferred to a heterologous host system, expressed there and then isolated from this host system and, if necessary, purified and / or enriched.
  • the enzymes of the cyanophycin synthetases of the type described above can then be used, for example, in an in vitro system for the synthesis of cyanophycin and / or its secondary products.
  • polyaspartic acids can be modified with the aid of a cyanophycin synthetase of the type described above in such a way that graft copolymers are formed when the polyaspartic acids are used as primers in the enzymatic polymerization reaction (Table 1, Table 2).
  • a cyanophycin synthetase of the type described above in such a way that graft copolymers are formed when the polyaspartic acids are used as primers in the enzymatic polymerization reaction (Table 1, Table 2).
  • the amount of arginine incorporated is determined after overnight incubation using the method described below (cyanophycin synthetase enzyme test, see below).
  • the polymeric primers are added to the reaction mixture at a final concentration of 0.87 mg / ml.
  • the polyaspartic acids are produced by chemical synthesis from maleic acid and ammonia or from aspartic acid.
  • Table 2 Influence of aspartic acid-containing polymers and N-acetylglucosamine on the incorporation of arginine by the purified cyanophycin synthetase from Synechococcus sp. MA 19.
  • the amount of arginine incorporated is determined after overnight incubation using the method described below (cyanophycin synthetase enzyme test).
  • the substances used as primers are used in a final concentration of 1.0 mg / ml.
  • 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 includes not only the production of cyanophycin and / or its secondary products in a living host system, but also the in vitro synthesis of cyanophycin using 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 20 ° C. to 60 ° C., preferably in a range from 35 ° C. to 55 ° C.
  • the influence of temperature on the activity and stability of the enzymes Synechocystis sp. PCC 6308 and from Synechococcus sp. MA 19 is shown in FIGS. 2 to 5. As can be seen in FIG. 5, the stability of the enzyme from Synechococcus sp. MA 19 can also be increased.
  • 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). This requires both monomers, ATP, Mg 2+ , K + , a sulfhydryl reagent and small amounts of cyanophycin as a primer.
  • the dependence of the cyanophycin synthetases according to the invention on their substrates, cosubstrates, arginine-analogous compounds and other substances of the different enzyme preparations are shown in Table 3, Table 4 and Table 5.
  • FIG. 7 shows in particular the influence of arginine and aspartic acid-like compounds on the by the purified cyanophycin synthetase from Synechocystis sp.
  • PCC 6308 catalyzed incorporation of arginine and aspartic acid into
  • Cyanophycin shown This clearly shows that on the one hand canavanine and lysine instead of arginine and on the other hand aspartic acid- ⁇ -methyl ester and asparagine instead of aspartic acid can be incorporated into the polymer.
  • Table 3 Dependence of the cyanophycin synthetase activity in the soluble cell fraction on substrates, cosubstrates and the arginine analogue canavanine d .
  • the activities were determined as the rate of incorporation of the amino acid monomers in cyanophycin within 15 min after the start of the reaction in the soluble cell fraction of Synechocystis PCC6308 and of IPTG-induced E. coli TOP 10 ⁇ pSK :: cphA co ).
  • the activities which corresponded to 100% in each case, were 9.1 nmol arginine min "1 (g protein) _1 for Synechocystis PCC6308 and 26.6 nmol arginine min " 1 (g protein) _1 for E. coli.
  • the soluble cell fraction was incubated at 95 ° C for 2 min. f.
  • the activity was determined using 0.1 mM L [U- 1 C] aspartic acid as substrate.
  • the activity was determined using 0.01 mM L [U- 1 C] glutamic acid as substrate.
  • the final concentrations of the radiolabeled compounds are as follows: L- [U- 14 C] arginine, 0.5 mM; L- [U- 14 C] aspartic acid; 5 mM; L- [U- 3 H] - canavanine, 1 mM; L- [4,5- 3 H] lysine, IMM; L- [U- 14 C] -glutamic acid, 0.5 mM (if used instead of L-arginine), 5 mM (used instead of L-aspartic acid).
  • the present invention further relates to an improved cyanophycin synthetase enzyme test combined with an improved separation of the enzymatically formed cyanophycin from the reaction mixture.
  • the cyanophycin formed is first washed after its precipitation from the reaction mixture and the precipitate (pellet) is then resuspended in an acidic aqueous medium.
  • the cyanophycin goes into solution and can be removed from the supernatant and used directly for determining the concentration in a scintillation counter.
  • This procedure is distinguished from the known methods in that a large number of washing steps are dispensed with, no transfer of insoluble cyanophycin is required, the amount of radioactive waste is drastically reduced, and the cyanophycin by solubilization under acidic conditions of others Impurities in the precipitate are cleaned and a homogeneous cyanophycin solution is available, from which a much more precise and reproducible determination can be made by means of scintillation counting than from a dispersion, as was previously customary.
  • a Comparative comparison of the isolation of cyanophycin from the reaction mixture is shown in flow diagrams in FIG. 6.
  • the present invention further relates to the use of a vector containing a cyanophycin synthetase according to the invention of the aforementioned type for producing 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.
  • cyanophycin for the production of food supplements and / or agents in the field of agriculture and / or crop protection is covered in the present invention. Further areas of application of cyanophycin and / or its secondary products can be seen in the paper, textile, pigment, lacquer, ceramic, building material or detergent industry and in the areas of water and wastewater treatment.
  • Areas of application of the secondary products of cyanophycin are agents in the field of agriculture and / or crop protection, in the paper, textile, pigment, varnish, ceramic, building material or detergent industry as well as in the areas of water and waste water treatment.
  • PCC6308 cphB gene and primer P6 (5-TGGCGGCGGTGTATGAAAAC-3 '), the sequence of which part of the Synechocystis sp. PCC6308 represents cphA gene.
  • primer P6 5-TGGCGGCGGTGTATGAAAAC-3 '
  • PCC6308 represents cphA gene.
  • a genomic region comprising the 3 'end of cphB, the intergenic region between cphB and cphA and the complete cphA gene was created using primer P3 (see above) and primer P7 (5'-
  • TTCAGAATTCACTACTCACTATTC-3 ' the sequence of which is complementary to the 3' end of cphA, amplified.
  • the primers were obtained from MWG-Biotech AG (Ebersberg). Vent DNA polymerase (New England Biolabs, Schwalbach, Taunus) was used for the PCR according to the information in the PCR application manual (Biochemica 1995, Boehringer Mannheim). After electrophoretic separation, the PCR products were isolated from agarose gels, ligated with EcoRV-linearized vector pBluescript SK- (Stratagene Cloning Systems, San Diego, Calif, USA) and according to E. coli TOP 10 (Invitrogen, San Diego, Calif, USA) transferred.
  • the method is a modification of the method described by Ziegler et al. in Eur. J. Biochem. 254, 154 (1998).
  • the reaction mixture contained in a total volume of 0.125 ml 50 mM Tris, 20 mM MgCl 2 , 20 mM KC1, 4 mM ATP,
  • the insoluble cyanophycin under these conditions was separated by centrifugation (15 min, 14000 x g) and washed twice with 1 ml each of 50 mM Tris-HCl, 2 mM EDTA (pH 8.2). The pellet was resuspended in 1.0 ml of 1.5 M HC1. The suspension was centrifuged again.
  • SEQUENCE PROTOCOL 1 of the amino acid sequence of the thermostable cyanophycin synthetase according to the invention derived from the corresponding cyanophycin synthetase gene of the Synechocystis PCC 6308.
  • Fig. 1 Time course of the incorporation of the amino acid monomers in cyanophycin.
  • the built-in content of L-arginine was determined at certain times (min) in a reaction mixture totaling 125 ⁇ l, which contains the following components: 1.25 nmol radioactive L [U- 14 C] arginine, 12.5 nmol unlabeled L-aspartate, 500 nmol ATP, 0.38 mg soluble cell protein of the in
  • Fig. 3b Time course of the incorporation of arginine in cyanophycin at 28 ° C (-D-) and at 50 ° C (- • -). The amount of arginine incorporated was determined at the times indicated in 125 ⁇ l reaction mixture.
  • Fig. 4 Temperature profile (- • -) and heat stability (- ⁇ -) of the purified cyanophycin synthetase from Synechococcus sp. MA 19. For the temperature profile, the enzyme activities were determined at a time interval of 15 min after the start of the reaction at the specified temperature. The heat stability of the enzyme was determined as the activity (measured at 28 ° C.) after every 30 min incubation of the enzyme solution at the temperatures indicated.
  • Fig. 6 Flow diagrams of the isolation of cyanophycin from the reaction mixture in comparison (A) according to Simon, R.D. (Biochim Biophys Acta, 1976, 422: 407-418), (B) according to Ziegler, K. et al. (Eur. J. Biochem., 1998, 254: 154-159) and (C) by the method according to the invention.
  • the insoluble cyanophycing granules are shown as spheres; Transfer steps are shown by curved arrows.
  • Fig. 7a incorporation of aspartic acid (Hatched columns) and arginine (shaded columns) after adding 5 mM of the specified compound to the otherwise complete reaction mixture.
  • Fig. 7b Influence of the specified compounds when these in a concentration of 5 mM instead of aspartic acid (hatched columns) or in a concentration of 0.5 mM instead of arginine in the

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Abstract

La présente invention concerne des cyanophycine synthétases thermostables ainsi qu'un procédé de fabrication améliorée de cyanophycine et/ou de ses produits secondaires.
EP01962878A 2000-08-09 2001-07-27 Procede de fabrication amelioree de cyanophycine et de ses produits secondaires Withdrawn EP1322764A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10038776 2000-08-09
DE10038776 2000-08-09
PCT/EP2001/008687 WO2002012508A2 (fr) 2000-08-09 2001-07-27 Procede de fabrication amelioree de cyanophycine et de ses produits secondaires

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EP1322764A2 true EP1322764A2 (fr) 2003-07-02

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PL2133419T3 (pl) * 2008-06-13 2019-02-28 Westfälische Wilhelms-Universität Münster Zastosowania beta-dipeptydów cyjanoficyny

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