EP1492877A2 - Procede ameliore de production de la vitamine b12 - Google Patents

Procede ameliore de production de la vitamine b12

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Publication number
EP1492877A2
EP1492877A2 EP03745269A EP03745269A EP1492877A2 EP 1492877 A2 EP1492877 A2 EP 1492877A2 EP 03745269 A EP03745269 A EP 03745269A EP 03745269 A EP03745269 A EP 03745269A EP 1492877 A2 EP1492877 A2 EP 1492877A2
Authority
EP
European Patent Office
Prior art keywords
seq
precorrin
chelatase
sirohydrochlorin
vitamin
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.)
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Application number
EP03745269A
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German (de)
English (en)
Inventor
Martin J. Warren
Evelyne Deery
Helen Leech
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BASF SE
Original Assignee
BASF SE
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Publication date
Application filed by BASF SE filed Critical BASF SE
Publication of EP1492877A2 publication Critical patent/EP1492877A2/fr
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    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • 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.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/42—Cobalamins, i.e. vitamin B12, LLD factor

Definitions

  • the present invention relates to an improved process for the production of vitamin B12, in which at least one enzyme of sirohaem synthesis is used.
  • vitamin B12 was indirectly discovered by its effects on the human body by George Minot and William Murphy (Stryer, L, 1988, In Biochemie, fourth edition, pp. 528-531, Spektrum Akademischer Verlag GmbH, Heidelberg , Berlin, New York).
  • vitamin B12 could be purified and isolated for the first time, so that eight years later, in 1956, Dorothy Hodgkin's complex three-dimensional crystal structure was elucidated (Hodgkin, DC et al., 1956, Structure of Vitamin B12. Nature 176 , 325-328 and Nature 178, 64-70).
  • vitamin B12 The naturally occurring end products of vitamin B12 biosynthesis are 5 - deoxyadenosylcobalamin (coenzyme B12) and methylcobalamin (MeCbl), while vitamin B12 by definition stands for cyanocobalamin (CNCbl), which is the form that is primarily manufactured and traded by industry.
  • CNCbl cyanocobalamin
  • vitamin B12 is the same for the designation of all three analog molecules.
  • B. megaterium was first described by De Bary over 100 years ago (1884). Although generally classified as a soil bacterium, B. megaterium can also be found in various other habitats such as sea water, sediments, rice, dried meat, milk or honey. It often occurs in the company of pseudomonas and actinomycetes. B. megaterium, like its close relative Bacillus subtilis, is a gram-positive bacterium and is characterized, among other things, by its relative nature pronounced, eponymous size of 2x5 ⁇ m, a G + C content of approx. 38% and a very pronounced ability to sporulate.
  • B. megaterium a group of microorganisms that are suitable for the biotechnological production of vitamin B12, such as. B. microorganisms of the genus Bacillus, Pseudomonas or Propionibacterium.
  • the object of the present invention is to further optimize the biotechnological production of vitamin B12 with microorganisms.
  • This object is achieved by a process for the improved production of vitamin B12, in which a culture is fermented, containing a microorganism in which at least the enzymes involved in the synthesis of sirohaem have uroporphyrinogen-Ill-methyltransferase and precorrin-2-dehydrogenase in their activity the corresponding endogenous enzyme activities are increased.
  • the invention also relates to a process for the improved production of vitamin B12, in which a culture is fermented, comprising a microorganism in which at least the enzymes involved in the sirohaem synthesis are uroporphyrinogen-ill-methyltransferase, sirohydrochlorin-iron chelatase and precorrin-2 - Dehydrogenase are increased in their activity compared to the corresponding endogenous enzyme activities.
  • the invention further relates to a process for the improved production of vitamin B12, in which a culture is fermented, comprising a microorganism in which at least the enzymes involved in the synthesis of sirohaem uroporphyrinogen-ill-methyltransferase, precorrin-2-dehydrogenase and the other Cobalamin synthesis involved enzyme Sirohydrochlorin-Cobalt-Chelatase in their activity compared to the corresponding endogenous enzyme activities are increased.
  • the vitamin B12 formed can be processed from the fermentation medium. Measures to do this are part of common laboratory practice and will not be discussed further here.
  • it is a method in which at least one uroporphyrinogen III methyltransferase according to SEQ ID No. 2 and a precorrin-2 dehydrogenase according to SEQ ID No. 6 or their isoenzymes are increased compared to the corresponding endogenous enzyme activities.
  • a method is included in which at least one uroporphyrinogen III methyl transferase according to SEQ ID No. 2 and a sirohydrochlorine iron chelatase according to SEQ ID No. 4 and a precorrin-2-dehydrogenase according to SEQ ID No. 6 or their isoenzymes are increased compared to the corresponding endogenous enzyme activities.
  • the invention comprises a variant of a method in which at least one uroporphyrinogen-III-methyltransferase according to SEQ ID No. 2, a precorrin-2-dehydrogenase according to SEQ ID No. 6 and the Sirohydrochlorin-Cobalt-Chelatase (CbiX) or their isoenzymes are increased compared to the corresponding endogenous enzyme activities.
  • the present invention thus relates to variants of a process for the improved production of vitamin B12, in which a culture is fermented, comprising a microorganism in which at least the enzymes involved in sirohaem synthesis, uroporphyrinogen-Ill-methyltransferase (sirA), precorrin-2 dehydrogenase (sirC), sirohydrochlorin-iron chelatase (sirB) and the enzyme sirohydrochlorin-cobalt chelatase (cbiX) involved in the synthesis of cobalamin are individually or in combination increased in their activity compared to the corresponding endogenous enzyme activities.
  • uroporphyrinogen-Ill-methyltransferase sirinogen-Ill-methyltransferase
  • precorrin-2 dehydrogenase precorrin-2 dehydrogenase
  • sirohydrochlorin-iron chelatase siro
  • Endogenous enzyme activity is to be understood as one that arises from the system (the basic genetic makeup) of the cell or organism (exists naturally) and is not caused by external influences or supply from outside, i.e. the level of the respective enzyme activity before genetic manipulation of the organism.
  • Isoforms are to be understood as enzymes with the same or comparable substrate and activity specificity, but which have a different primary structure.
  • the present invention further relates to modified forms of the enzymes mentioned.
  • Modified forms which are also encompassed according to the invention, are to be understood as 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 in the form of amino acid exchanges according to methods known per se.
  • a special embodiment variant of the present invention comprises variants of the polypeptides according to the invention, the activity of which is weakened or enhanced, for example, by amino acid exchanges compared to the respective starting protein.
  • Endogenous enzyme activity in the sense of the present invention is understood to mean the activity of an enzyme of the sirohaem or cobalamin synthesis that occurs naturally in a microorganism.
  • the synthesis of the sirohaem is based on the precursor uroporphyrinogen-III.
  • the reaction steps required for the conversion to the sirohaem and the biocatalysts involved are known to the person skilled in the art. In principle, these are biocatalysts with the following activities: Uroporphyrinogen-III- methyltransferase, sirohydrochlorin-iron chelatase or precorrin-2-dehydrogenase.
  • the enzyme with the activity of a sirohydrochlorine cobalt chelatase is an important biocatalyst.
  • the cbiX gene codes for the enzyme of the Sirohydrochlorin-Cobalt-Chelatase.
  • the activities of uroporphyrinogen-Ill-methyltransferase are less than 0.25 units / mg raw cell extract, the precorrin-2-dehydrogenase is approx. 0.002 units / mg raw cell extract and for the chelating agents approx. 0.01 unit / mg raw cell extract.
  • One Umit is 1 nM of Precarrin-2 produced per hour, 1 nM of Sirohydrochlorin per minute and for the chelatases the consumption (i.e. disappearance) of 1 nM Sirohydrochlorin per minute.
  • Each of the enzymes mentioned can be encoded by a gene or, for example, encode a gene for a multifunctional gene product which combines the activity of two or all three of the aforementioned enzymes or any other conceivable combination.
  • the genes are already coding for the enzymes involved in the synthesis of sirohaem and cobalamin known.
  • the nomenclature of these genes and the corresponding gene products is varied.
  • sirohaem synthesis in Escherichia coli is catalyzed by the multifunctional protein sirohaem synthase, the gene of which is designated cysG.
  • two enzymes are specifically involved in sirohaem synthesis (uroporphyrinogen III methyl transferase and a dehydrogenase / chelatase), the genes of which are designated metl and met ⁇ .
  • Bacillus subtilis in turn, has three genes, namely ylnD, ylnE and ylnF, which code for the above-mentioned enzymes for sirohaem synthesis.
  • a variety of the enzymes of cobalamin synthesis are also known. Examples include sirohydrochlorine chelatase, which is an enzyme at the branching point of sirohaem and cobalamin synthesis.
  • sirohydrochlorine chelatase which is an enzyme at the branching point of sirohaem and cobalamin synthesis.
  • Salmonella it is called CbiK or CbiL.
  • CbiX Rhohydrochlorine chelatase
  • Bacillus megaterium it is known under the name CbiX (Raux, E. et al., 1998, Biochem. J., 335: 159-166).
  • sirohaem genes and the enzymes of sirohaem synthesis encoded by them are in the process according to the invention for the production of vitamin B12 suitable.
  • the present invention further relates to an isolated nucleotide sequence according to SEQ ID No. 1 containing the sirABC operon from Bacillus megaterium coding for enzymes of sirohaem synthesis.
  • nucleotide sequence according to SEQ ID No. 1 coding for the entire operon sIRABC or parts of it Nucleotide sequence according to SEQ ID No. 1 includes. Below the parts are the individual coding regions of the sequence according to SEQ ID No. 1 meant that can be used according to the invention individually or in all conceivable combinations, such as, for example, sirA, sirB, sirC, sirAB, sirAC, sirBC or sirABC. All possible combinations with genes of cobalamin synthesis are also conceivable, advantageously with a gene coding for sirohydrochlorine chelatase (cbiX) or parts thereof.
  • sirA, sirB, sirC, sirAB, sirAC, sirBC or sirABC All possible combinations with genes of cobalamin synthesis are also conceivable, advantageously with a gene coding for sirohydrochlorine chelatase (cbiX) or parts thereof.
  • an isolated nucleotide sequence according to SEQ ID No. 1 or their alleles, coding from nucleotide 1-780 for a uroporphyrinogen III methyl transferase (sirA) according to SEQ ID No. 2 includes.
  • any combination of the said genes of sirohaem synthesis with the gene cbiX encoding a sirohydrochlorine chelatase and corresponding alleles is included.
  • an isolated nucleic acid 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 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, e.g. B.
  • functionally equivalent sequences include those which have a modified nucleotide sequence which gives the enzyme, for example, 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 continue to show the desired function. Mutations include substitutions, additions, 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, nucleotide sequences which can be obtained by modifying the present invention
  • Nucleotide sequence resulting in corresponding derivatives. aim such a modification can e.g. B. the further limitation of the coding sequence contained therein or z. B. also the insertion of further restriction enzyme interfaces. 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.
  • 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 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 nucleotide sequences in the sense of the invention are understood to mean oligonucleotides or polynucleotides which bind under standard hybridization conditions to the corresponding nucleotide sequence according to the invention.
  • standard hybridization conditions to the corresponding nucleotide sequence according to the invention.
  • Hybridization conditions are to be understood broadly and mean stringent and less stringent hybridization conditions. Such conditions are among others in Sambrook et al. (1989, Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press,).
  • hybridizing sequences essentially includes according to the invention similar nucleotide sequences from the group of DNA or RNA, which enter into a specific interaction (binding) with the nucleotide sequences according to the invention under standard hybridization conditions known per se. This also includes short nucleotide sequences with a length of, for example, 10 to 30, preferably 12 to 15 nucleotides. According to the invention, this also includes so-called primers or probes.
  • the (5 'or upstream) and / or subsequent (3' or downstream) sequence regions preceding the coding regions are also included.
  • this includes sequence areas with a regulatory function. You can influence the transcription, the RNA stability or the RNA processing as well as the translation. Examples of regulatory sequences include a. Promoters, enhancers, operators, terminators or translation enhancers.
  • the present invention further relates to a method of the aforementioned type, which is characterized in that the genes sirA coding for the enzymes of the sirohaem synthesis of nucleotide 1-780 according to SEQ ID No. 1 and sirC of nucleotide 1542-2150 according to SEQ ID No. 1, 3 or 5 are increasingly expressed and / or are present in an increased number of copies.
  • the genes sirA coding for the enzymes of the sirohaem synthesis of nucleotide 1-780 according to SEQ ID No. 1, sirB of nucleotide 761-1561 according to SEQ ID No. 1 or 3 and sirC of nucleotide 1542-2150 according to SEQ ID No. 1, 3 or 5 are increasingly expressed and / or are present in an increased number of copies.
  • a method is included in which the genes sirA coding for the enzymes of the sirohaem synthesis of nucleotide 1-780 according to SEQ ID No. 1, sirC of nucleotide 1542-2150 according to SEQ ID No. 1, 3 or 5 and the cbiX gene coding for the enzyme of cobalamin synthesis are expressed in an increased manner and / or are present in an increased number of copies.
  • the above variants are carried out by fermentation of a culture containing Bacillus megaterium.
  • the present invention also relates to the enzymes uroporphyrinogen III methyl transferase according to SEQ ID No. 2, sirohydrochlorine iron chelatase according to SEQ ID No. 4 and precorrin-2-dehydrogenase according to SEQ ID No. 6, which are encoded by the sIRABC operon or parts thereof from Bacillus megaterium.
  • the present invention further relates to modified forms of the enzymes mentioned.
  • Modified forms which are also encompassed according to the invention, are to be understood as enzymes in which there are changes in the sequence, for example at the N- and / or C-terminus of the polypeptide or in the range of conserved amino acids, but without impairing the function of the enzyme. These changes can be made in the form of amino acid exchanges according to methods known per se.
  • a special embodiment variant of the present invention comprises variants of the polypeptides according to the invention, the activity of which is weakened or enhanced, for example, by amino acid exchanges compared to the respective starting protein.
  • sirohydrochlorine iron chelatase according to the invention is distinguished according to SEQ ID No. 4 further characterized in that it additionally has a cobalt chelatase activity.
  • the enzyme according to the invention according to SEQ ID No. 4 with chelatase activity also accepts nickel, zinc, and copper ions.
  • vitamin B12 For the production of vitamin B12 according to the invention by biotechnological processes, at least the use of a sirohydrochlorin iron chelatase and / or a sirohydrochlorin cobalt chelatase in combination with other enzymes involved in sirohaem or cobalamin synthesis is conceivable in a suitable organism .
  • the metabolic flow can be controlled in favor of cobalamin by suitable fermentation conditions.
  • the fermentation conditions can be cultivation of the microorganism under aerobic, semi-anaerobic or anaerobic conditions. Furthermore, all conceivable variations of a shift from aerobic to anerobic conditions or vice versa are included.
  • a culture containing a microorganism of the genus Bacillus, Salmonella, Pseudomonas, Escherichia or Propionibacterium or a mixture of these microorganisms is fermented in the process according to the invention.
  • the Propionibacterium shermanii species can be used as the Propionibacterium.
  • a culture containing Salmonella typhimurium or Escherichia coli or Bacillus megaterium is preferably fermented in a method of the aforementioned type for producing vitamin B12.
  • Bacillus megaterium is economically very interesting because it has a number of advantages for use in the biotechnological production of various, industrially interesting products.
  • B. megaterium has no alkaline proteases, so that hardly any degradation was observed in the production of heterologous proteins.
  • B. megaterium effectively secretes products of commercial interest, such as e.g. B. is used in the production of ⁇ - and ß-amylase.
  • B. megaterium's size it is possible to accumulate high biomass before too high a population density leads to death.
  • B. megaterium is the fact that this species is able to produce products of high value and highest quality from waste and inferior substances. This possibility of metabolizing an enormously wide range of substrates is also reflected in the use of B.
  • B. megaterium as a soil detoxifier, which itself can break down cyanides, herbicides and persistent pesticides.
  • B. megaterium is completely non-pathogenic and does not produce toxins is of paramount importance, especially in food and cosmetic production. Because of these diverse advantages, B. megaterium is already used in a variety of industrial applications, such as the production of - and ß-amylase, penicillin amidase, the processing of toxic waste or the aerobic production of vitamin B12 (summarized in Vary, PS, 1994, Microbiology, 40, 1001-1013, Prime time for Bacillus megaterium).
  • strains of the genus Bacillus, Salmonella, Propionibacterium, Escherichia or Pseudomonas or mixtures thereof can be used for the purposes of the present invention, in which at least the activities of the enzymes of sirohaem synthesis and optionally cobalamin synthesis are increased compared to the corresponding wild types .
  • These include, in particular, genetically modified microorganisms which have at least increased expression and / or increased number of copies of the nucleotide sequences coding for enzymes of sirohaem synthesis and possibly cobalamin synthesis compared to the corresponding wild types.
  • vitamin B12 production strains The use of vitamin B12 production strains is preferred.
  • Salmonella strain is the (genetically modified) strain AR3612 (Raux et al, 1996; J. Bacteriol., 178, (3): 753-767).
  • Organisms of the genus Propionibacteria have become the species Propionibacterium shermanii was found to be particularly suitable.
  • the Bacillus megaterium strain DSMZ 509 and all the usual B. megaterium strains which are suitable as vitamin B12 production strains are very particularly preferred.
  • vitamin B12 production strains are preferably to be understood as microorganism strains which have been modified by classic and / or molecular genetic methods in such a way that their metabolic flow increasingly runs in the direction of the biosynthesis of vitamin B12 or its progeny (metabolic engineering).
  • these production strains one or more genes and / or the corresponding enzymes, which are at crucial and correspondingly complex regulated key positions in the metabolic pathway (bottleneck), are changed or even deregulated.
  • the microorganisms which can be used according to the invention include all the known vitamin B12
  • strains preferably of the genus Bacillus or homologous organisms.
  • the strains advantageous according to the invention include in particular the strains of B. megaterium DSMZ 509 and DSMZ 2894.
  • strains can also be used for the production of vitamin B12 which, without the invention, would not be able to produce vitamin B12, e.g. B. Escherichia coli strains ER 185 or ER 171 (see Table 1 below).
  • E. coli is not able to synthesize vitamin B12 de novo because it lacks some enzymes, e.g. B. are present in Salmonella or Bacillus and have been lost in the course of evolution. However, it is possible to reintroduce these missing enzymes by transformation using a suitable plasmid comprising these genes. If you also use a cysG Strain, then the enzymes for converting uroporphyrinogen III to cobinamide are also present in E. coli.
  • Genetically modified bacterial strains which can be produced by classic mutagenesis or targeted molecular biological techniques and corresponding selection processes are also included according to the invention.
  • interesting starting points for targeted genetic engineering manipulation include Branches of the biosynthetic pathways leading to vitamin B12, through which the metabolic flow can be controlled in the direction of maximum vitamin B12 production.
  • Targeted modifications of genes involved in the regulation of the metabolism also include studies and changes in the regulatory areas before and after the structural genes, such as. B. the optimization and / or the exchange of promoters, enhancers, terminators, ribosome binding sites etc.
  • Also improving the stability of the DNA, mRNA or the proteins encoded by them, for example by reducing or preventing the degradation by nucleases or proteases is according to the invention includes.
  • the present invention further comprises a gene structure containing an isolated nucleotide sequence of the sir operon of the aforementioned type or parts thereof, and nucleotide sequences operatively linked thereto with a regulatory function.
  • a nucleotide sequence coding for a sirohydrochlorin coblate chelatase (cbiX) or parts thereof can additionally be contained in a gene structure of the aforementioned type.
  • 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 perform its function as intended in the expression of the coding sequence.
  • 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.
  • the ⁇ -galacosidase or arabinose system may be mentioned here as an example.
  • a gene structure is produced by fusing a suitable promoter with at least one nucleotide sequence according to the invention using common recombination and cloning techniques, as described, for example, in Sambrook, J. et al., 1989, In Molecular cloning; a laboratory manual. 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York described.
  • adapters or linkers can be attached to the fragments.
  • the invention also includes a vector containing an isolated nucleotide sequence of the sir operon of the aforementioned type or parts thereof or a gene structure of the aforementioned type, as well as additional nucleotide sequences for selection, for replication in the host cell and / or for integration into the host cell genome. Examples of such additional sequences are described in the literature in large numbers and will not be further elaborated.
  • this also includes a vector which, in addition to the nucleotide sequences of the sir operon or parts thereof, contains a nucleotide sequence coding for a sirohydrochlorine coblate chelatase or parts thereof.
  • Suitable systems for the transformations and overexpression of the genes mentioned are, for example, the plasmids pACYC184 (New England Biolabs), pKK8668 (Pharmacia) and pWH1510 and pWH1520 as well as the plasmid-free overexpression strain B. megaterium WH320, which was described by Rygus, T. et al. (1991, Inducible high level expression of heterologous genes in Bacillus megaterium, Appl. Microbiol. And Biotechnol., 35, 5: 594-599).
  • the systems mentioned are not limiting for the present invention.
  • the present invention further relates to a transgenic microorganism for use in a process for the production of vitamin B12 of the aforementioned type, which is characterized in that it encodes an increased expression and / or increased number of copies of a nucleotide sequence coding for a uroporphyrinogen ill-methyltransferase and Has precorrin-2-dehydrogenase.
  • transgenic microorganism for use in a process for the preparation of vitamin B12 of the aforementioned type, which is characterized in that it encodes an increased expression and / or increased number of copies of a nucleotide sequence coding for a uroporphyrinogen III-methyltransferase, sirohydrochlorin iron -chelatase and precorrin-2 dehydrogenase.
  • the present invention relates to a transgenic microorganism for use in a method for producing vitamin B12 of the aforementioned type, which it encodes an increased expression and / or increased number of copies of a nucleotide sequence coding for a uroporphyrinogen III methyl transferase, sirohydrochlorine cobalt chelatase and Has precorrin-2-dehydrogenase.
  • nucleotide sequence which encodes a multifunctional enzyme.
  • a nucleotide sequence can be expressed more strongly and / or be present in the transgenic microorganism in increased copy number, which codes for several enzymes of sirohaem synthesis, since it contains, for example, an operon or a gene cluster.
  • the transgenic microorganism according to the invention preferably contains at least one nucleotide sequence according to SEQ ID No. 1 containing the sirABC operon from Bacillus megaterium or parts thereof. Also advantageous is a transgenic microorganism which has an increased expression and / or increased number of copies of at least one isolated nucleotide sequence according to SEQ ID No. 1 or their alleles coding from nucleotide 1-780 for a uroporphyrinogen-III-methyltransferase (sirA) according to SEQ ID No. 2, coding from nucleotide 761-1561 for a sirohydrochlorine iron chelatase (sirB) according to SEQ ID No.
  • sirA uroporphyrinogen-III-methyltransferase
  • sirB sirohydrochlorine iron chelatase
  • a transgenic microorganism according to the invention which has an increased expression and / or increased number of copies of at least one isolated nucleotide sequence according to SEQ ID No. 1 or parts or alleles thereof and additionally an increased expression and / or increased number of copies of at least one nucleotide sequence coding for an enzyme the cobalamin biosynthesis, advantageously the sirohydrochlorine cobalt chelatase.
  • transgenic microorganism which parts of the nucleotide sequence according to SEQ ID No. 1, wherein these parts are preferably expressed more and / or are present in an increased number of copies. These are preferably subregions of the type defined in more detail above, which have a coding function (sirA, sirB or sirC).
  • the partial sequences can be present individually or in any combination, as well as with regulatory sequences, such as Promoters, enhancers, terminators or the like can be operatively linked.
  • Combinations of the said sequences of the sir operon with sequences coding for enzymes of the cobalamin synthesis path, e.g. of the cbiX gene or parts thereof are also included according to the invention.
  • the present invention furthermore relates to a transgenic microorganism which, in replicating form, contains a gene structure according to the invention or a vector according to the invention.
  • a transgenic microorganism of the genus Bacillus, Salmonella, Pseudomonas, Escherichia or Propionibacterium is advantageous.
  • the transgenic microorganism according to the invention is preferably the species Bacillus megaterium, Escherichia coli or Salmonella typhimurium.
  • a Bacillus megaterium or Escherichia coli strain is fermented, the genes sirA, sirB or sirC or parts thereof are expressed individually or in combination and / or the gene cbiX or parts thereof in any conceivable combination, being opposite to the endogenous (naturally) existing activity of the enzymes of the sirohaem or cobalamin synthesis an increased enzyme activity is achieved.
  • the combination of the genes sIRAB, sirAC or sirABC or sirABcbiX, sirACcbiX or sirABCcbiX is advantageous.
  • the combination of the sirACcbiX genes is particularly preferred.
  • An equally advantageous variant of the present invention comprises a method in which a genetically modified Bacillus megaterium or Escherichia coli strain is fermented, the genes sirA, sirB or sirC or parts thereof individually or in combination and / or the gene cbiX or Parts of it are present in every conceivable combination compared to the genetically unmodified organism in an increased number of copies in the cell.
  • the number of copies can vary between 2 and several hundred and is achieved by introducing and replicating vectors or plasmids containing the aforementioned genes of sirohaem and possibly cobalamin synthesis in the transgenic cells.
  • the introduction of additional copies of the genes mentioned for sirohaem and possibly cobalamin synthesis into the genome of the transgenic organisms by homologous recombination is also included in the invention.
  • a combination of increasing the natural (endogenous) enzyme activity, increasing gene expression, increasing the number of gene copies, introducing the genes according to the invention or parts thereof, individually or in combination, into a microorganism, preferably a vitamin, is advantageous for increasing vitamin B12 productivity B12 production strain, particularly preferably of the species Bacillus megaterium.
  • the number of copies of the corresponding genes can be increased in order to achieve increased gene expression. Furthermore, the promoter and / or the regulatory region and / or the ribosome binding site, which is located upstream of the structural gene, are correspondingly changed so that expression takes place at an increased rate. Expression cassettes which are installed upstream of the structural gene act in the same way. With inducible promoters it is also possible to increase expression in the course of vitamin B12 production.
  • genes or gene constructs can either be present in plasmids with different copy numbers or can be integrated and amplified in the chromosome. Furthermore, the activity of the enzyme itself can also be increased or increased by preventing the breakdown of the enzyme protein.
  • an increased metabolic flow towards vitamin B12 can be achieved by changing the media composition and culture management.
  • vitamin B12 can be improved according to the invention by increasing the metabolic flow at key positions in the direction of vitamin B12 or corresponding precursors.
  • This can be done by fermentation engineering variants of the present method, such as fermentation under anaerobic conditions. Or fermentation takes place first under aerobic and then under anaerobic conditions.
  • a microorganism of the genus Bacillus, Salmonella, Pseudomonas, Escherichia or Propionibacterium is preferably used.
  • Bacillus megaterium, Escherichia coli or Salmonella typhimurium is particularly preferred.
  • B. megaterium is capable of an anaerobic lifestyle.
  • vitamin B12 production is higher under these conditions than under aerobic conditions.
  • the comparison of the vitamin B12 production of B. megaterium under aerobic and anaerobic growth conditions shows that the anaerobic vitamin B12 production in all strains investigated is at least 3 to 4 times higher than the aerobic vitamin B12 production (see FIGS. 1 to 3) and in particular 4). Further increases can be achieved by systematically optimizing the growth conditions and the composition of the culture medium and the bacterial strains used.
  • B. megaterium is fermented first aerobically and then anaerobically.
  • the transition from aerobic to anaerobic fermentation takes place in the exponential growth phase of the aerobically fermented cells. It is advantageous here if the transition from aerobic to anaerobic fermentation takes place in the middle or at the end, preferably at the end, of the exponential growth phase of the aerobically growing cells.
  • Anaerobic conditions both in the one-stage and in the two-stage fermentation of B. megaterium, are to be understood as those conditions which occur when the bacteria are transferred to anaerobic bottles after fermentation and fermented there.
  • the transfer to the anaerobic bottles takes place in particular in the two-stage process as soon as the aerobically grown bacterial cells are in the exponential growth phase. That is, after transfer to the anaerobic bottles, the bacteria consume the oxygen present there and no more oxygen is added.
  • These conditions can also be met with can be called semi-anaerobic.
  • the corresponding procedures are common laboratory practice and known to the person skilled in the art.
  • Oxygen supply is successively reduced so that semi-anaerobic conditions develop over time.
  • an aerobic cultivation pre-culture of the bacteria is not absolutely necessary.
  • the bacteria can also be grown under anaerobic conditions and then further fermented under semi-anaerobic or strictly anaerobic conditions. It is also conceivable that the inoculum is taken directly from the stock and used to produce vitamin B12 under anaerobic conditions.
  • vitamin B12 by means of Bacillus megaterium or Escherichia coli can be increased, for example, by adding at least cobalt to the culture medium.
  • the addition of, for example, betaine, methionine, glutamate, dimethylbenzimidazole or choline or their combinations also has an advantageous effect on vitamin B12 production.
  • the aforementioned compounds individually or their combinations in combination with cobalt can also be advantageous.
  • cobalt is strengthened, for example opposite Iron or magnesium ions, is built into common precursors of the biosynthetic pathway and thus, for example, the synthesis of sirohaem, hemoglobin or chlorophyll increases in favor of cobalamin (vitamin B12).
  • fermentation can also take place in a medium containing glucose as the C source.
  • a variant of the method according to the invention is also conceivable, in which the fermentation takes place in a medium containing glycerol as the C source.
  • This improved vitamin B12 production can be increased even further by converting the fermented Bacillus megaterium cells from aerobic growth conditions to anaerobic.
  • the use of a culture medium containing glycerol, cobalt and 5-aminolevulinic acid is advantageous.
  • By transferring the cultures from aerobic to anaerobic growth conditions a combination of high vitamin B 12 contents with high cell densities is possible.
  • the cultivation is advantageously carried out in a batch approach. Variants in which the fermentation takes place in a fed-batch approach or in continuous culture are also included according to the invention.
  • the present invention relates to the use of nucleotide sequences coding for the enzymes uroporphyrinogen-Ill-methyltransferase, sirohydrochlorin-iron chelatase, sirohydrochlorin-cobalt chelatase and precorrin-2-dehydrogenase individually or in combination for the production of vitamin B12.
  • the present invention furthermore relates to the use of the nucleotide sequence according to SEQ ID No. 1 or parts thereof coding for the enzymes of sirohaem synthesis for the production of vitamin B12.
  • Object of the present invention. also includes the use of a gene structure according to the invention or a vector according to the invention for the production of vitamin B12.
  • the invention also relates to the use of the nucleotide sequence according to SEQ ID No. 1 or parts thereof individually or in combination with the nucleotide sequence coding for the enzyme of the Sirohydrochlorin-Cobalt-Chelatase or parts thereof or a gene structure of the kind according to the invention or a vector of the kind according to the invention for the production of a transgenic microorganism as previously explained.
  • This also includes the use of the nucleotide sequences according to the invention, a gene structure or a vector for the production of a vitamin B12 production strain, preferably of the genus Bacillus or Escherichia, particularly preferably of the species Bacillus megaterium or Escherichia. Equally included is the use of a transgenic microorganism, preferably of the Bacillus genus and particularly preferably of the Bacillus megaterium species, for the production of vitamin B12.
  • Chemicals and molecular biological agents Chemicals and molecular biological agents Chemicals, reagents, antibiotics and DEAE sephacels were obtained from Amersham-Pharmacia, growth media from Oxoid. Enzymes were obtained from Promega, Bioline or Invitrogen Life Technologies.
  • Bacterial strains, plasmids and primers All bacterial strains, plasmids and primers used in this work are listed in Tables 1, 2 and 3.
  • Titration reagent was KOH solution.
  • MgCI 2 20.0 mM titration reagent was NaOH solution.
  • the titration reagent was NaOH solution.
  • the titration reagent was NaOH solution.
  • Luria-Bertani Broth was used with complete medium as in Sambrook, J. et al. (1989, Molecular cloning; a laboratory manual. 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
  • Additives such as carbon sources, amino acids or antibiotics were either added to the media and autoclaved together or prepared as concentrated stock solutions in water and sterilized or sterile filtered. The substances were added to the autoclaved and cooled to below 50 ° C media. In the case of light-sensitive substances such as tetracycline, care was taken to incubate in the dark. The final concentrations commonly used were as follows:
  • Temperature-sensitive substances were sterile filtered, glassware was heat sterilized at 180 ° C for at least 3 h.
  • Aerobic bacterial cultures were incubated in baffled flasks at 37 ° C and a minimum speed of 180 rpm. The incubation times were varied according to the desired optical densities of the bacterial cultures. 5.3. Conditions for Bacillus megaterium growth Aerobic cultures were incubated in baffled flasks at 250 rpm and, unless otherwise stated, at 30 ° C for the best possible aeration. Anaerobic cultures were fermented in a volume of 100 ml in small anaerobic bottles at 30 ° C. and 100 rpm. In both cases, attention was paid to the use of qualitatively constant media, inoculation in a ratio of 1: 100 from overnight cultures, and the use of constant conditions for the overnight cultures.
  • the cell density of a bacterial culture was determined by measuring the optical density at 578 nm, it being assumed that an OD 578 of one corresponds to a cell number of 1 ⁇ 10 9 cells.
  • the strains Bacillus megaterium DSMZ 32, DSMZ 509 and DSMZ 2894 were used, for example, under anaerobic conditions in glucose-containing (LB full) medium and, at the end of the exponential growth phase, the vitamin B12 content in pmol / OD 578 was determined. At the same time, vitamin B12 production in the aerobic lifestyle was examined in the middle of the exponential growth phase. The results are shown in FIG. 4.
  • Vitamin B12 can be isolated in a manner known to those skilled in the art.
  • Aerobic ß. megafer / ⁇ / m cultures were in the middle of the exponential
  • Bacteriol., 178: 753-767) were incubated overnight on minimal medium containing methionine and cysteine at 37 ° C., scratched from the plate and at 40 ml of isotonic saline. After centrifugation, the cell sediment was resuspended in isotonic saline. The washed bacterial culture was carefully mixed with 400 ml of 47-48 ° C minimal medium agar containing cysteine. 10 ⁇ l of ß resuspended in deionized, sterile water and boiled in a water bath for 15 min. Megaterium samples were placed on the cooled plates and incubated at 37 ° C for 18 h.
  • the diameter of the grown salmonella colonies are then proportional to the content of vitamin B 12 in the applied ß. megaterium samples.
  • the content was determined by comparison with a calibration curve, made from the addition of 0.01, 0.1, 1, 10 and 40 pmol of vitamin B 12 . of vitamin B 2 in the examined samples. This standard method allows the detection of small amounts of vitamin B 12 in biological materials quickly and very reproducibly.
  • the determination of uroporphyrinogen III methyltransferase activity was determined on the basis of the conversion of uroporphinogen III to precorrin-2.
  • the production of Precorrin-2 was monitored by monitoring the fluorescence and Precorrin-2 after excitation at 380 nm and emission at 610 nm using a commercially available fluorometer.
  • One unit of enzyme activity is defined as 1 nM of Precorrin-2 produced per hour.
  • the activity of the chelating reaction for the cobalt insertion in sirohydrochlorin was determined by measuring the rate of decrease of the syrohydrochlorin fluorescence at ⁇ max of 376 nm using an extinction coefficient of 2.4 x 10 5 M “1 cm " 1 .
  • One unit of enzyme activity was determined as a decrease of 1 nM sirohydrochlorine per minute.
  • raw cell extracts containing between about 50 and 200 ⁇ g protein were reacted with sirohydrochlorin (50 ⁇ M) and cobalt (10 ⁇ M) with 50 mM Tris / HCl buffer at pH 8.
  • the dehydrogenase reaction was determined by forming syrohydrochlorin at ⁇ max of 376 nmm using an extinction coefficient of 2.4 ⁇ 10 5 M “1 cm ” 1 .
  • One unit of enzyme activity is defined as the production of 1 nM sirohydrochlorin per minute.
  • LB medium 50 ml LB medium were inoculated with 1 ml of an overnight culture of B. megaterium and incubated at 37 ° C. With an OD 578 of 1, the cells were centrifuged at 15,000 rpm and 4 ° C. for 15 minutes (RC 5B Plus, Sorvall) and resuspended in 5 ml of SMMP buffer. After adding lysozyme in SMMP buffer, the suspension was incubated at 37 ° C. for 60 min and protoplast formation was checked under a microscope.
  • the cell sediment was carefully resuspended in 5 ml of SMMP buffer and the centrifugation and washing step was carried out performed a second time. After adding 10% (v / v) glycerol, the protoplast suspension could now be portioned and frozen at -80 ° C.
  • 500 ⁇ l of the protoplast suspension were mixed with 0.5 to 1 ⁇ g DNA in SMMP buffer and 1.5 ml PEG-P solution was added. After incubation at Rt for 2 min, 5 ml of SMMP buffer were added, mixed gently and the suspension was centrifuged at 3000 rpm and Rt for 10 min (Centrifuge 5403, Eppendorf). Immediately afterwards, the supernatant was removed and the barely visible sediment was resuspended in 500 ⁇ l SMMP buffer. The suspension was incubated at 37 ° C. for 90 min with gentle shaking.
  • the sirABC operon was cloned by means of PCR. Genomic DNA from B. megaterium was prepared according to Sambrook et al., 1989 and the genes sirA, sirB and sirC were isolated from the genomic DNA using the primers according to Table 3 as follows: using the known sequence of the cobA gene coding for a methyltransferase, converting uroporphyrinogen-III to precorrin-2 (Robin et al., 1991, J. Bacteriol., 172 (15): 4893-6), primers were made complementary to the 3 'end of the gene sequence. These primers were in a vectorette system from Sigma-Genosys according to the method of Lilleberg et al.
  • primers were synthesized, with the help of which the three genes could be cloned individually and in combination in two different expression vectors.
  • PCR products from sirA, sirAB, sirABC and sirAC were produced and cloned into the EcoRI-BamHI site of pKK8668 (modification of plasmid pKK223.3) under the control of the tac promoter.
  • SirC was cloned as a BamHI-PstI fragment in pKK8668.
  • SirA, sirB and sirC were individually cloned as Ndel-BamHI fragments in pET14b (a T7 expression vector). This allows the corresponding proteins to be overexpressed with an N-terminal Histag.
  • Suitable plasmids for the transformation and overexpression of genes in B. megaterium are pWH1510 and pWH1520 and the plasmid-free overexpression strain B. megaterium WH320 (Rygus, T. et al., 1991, Inducible high level expression of heterologous genes in Bacillus megaterium, Appl. Microbiol. And Biotechnol., 35, 5: 594-599).
  • the control plasmid pWH1510 contains a spoVG-lacZ fusion in the interrupted xylA reading frame. SpoVG-lacZ denotes the fusion of a very strong ribosome binding sequence of a sporulation protein from B. subtilis (spoVG) with the gene coding for ⁇ -galactosidase (lacZ) from E. coli.
  • This plasmid is therefore ideally suited for the study of transformation efficiencies and
  • the plasmid pWH1520 acts as the actual cloning and expression vector. Both vectors have a tetracycline and an ampicillin resistance as well as the elements important for replication in E. coli and Bacillus spp. This makes them accessible for all techniques established in E. coli for descendants of the plasmid pBR322. Both vectors contain the B. megaterium xylA and xylR genes of the xyl operon with their regulatory sequences (Rygus, T. et al., 1991, Molecular Cloning, Structure; Promoters and Regulator / Elements for Transcription of the Bacillus megaterium Encoded Regulon for Xylose Utilization, Arch. Microbiol.
  • XylA encodes xylose isomerase
  • xylR encodes a regulatory protein that exerts strong transcriptional control over xylA.
  • XylA is repressed in the absence of xylose.
  • xylose is added, there is an approx. 200-fold induction due to the depression of xylA.
  • a polylinker in the xylA reading frame With the help of a polylinker in the xylA reading frame, a fusion of genes with xylA is possible, which are then also under the strong transcriptional control of XylR. You can choose between the alternatives to form a transcription or translation fusion, since the xylA reading frame upstream of the polylinker is still completely intact.
  • SirB can act as sirohydrochlorin cobalt chelatase because it can restore vitamin B12 biosynthesis in the absence of cbiK.
  • the level of vitamin B12 formed is increased when SirC is also present.
  • Escherichia coli transformed according to the invention are therefore suitable for the production of vitamin B12.
  • E. coli 302 ⁇ a cysG-deficient strain without functional sirohaem synthase, which only grows on minimal medium when exogenous cysteine is added
  • plasmid pAR8766 pKK223.3 derivative containing cbiX from Bacillus megaterium plus cysG from E. coli ; Pharmacia; Raux, E. et al., 1998, Biochem. J., 1998, 335: 167-173
  • the plasmid pER179 pKK223.3 derivative, containing cobA gene from Pseudomonas denitrificans and cbiX gene from Bacillus megaterium; Pharmacia; Raux, E.
  • the genes were cloned separately into the vector pET14b under the control of the T7-inducible promoter T7. After transforming this
  • the proteins from the supernatant were purified according to standard methods and manufacturer's information using chromatography (metal
  • the Bacillus megaterium cbiX gene was derived from plasmid pAR8766, which contains the entire cop operon (Raux, E. et al., 1998, Biochem. J., 1998, 335: 167-173), as Sspl / SnaBI- Fraction isolated and cloned into the plasmid pKK223.3 (Pharmacia), resulting in the plasmid pAR8882. After transformation in E. coli 302 ⁇ a and addition of IPTG, the expression and purification took place analogously to the purification for SirA, SirB or SirC.
  • Escherichia coli is not able to synthesize vitamin B12 de novo because it lacks some enzymes that are required to convert precorrin-2 to cobinamide.
  • this ability can be restored in E. coli by inserting the missing cobalamin biosynthetic genes using a suitable plasmid.
  • a cysG strain is also used, all the enzymes for the conversion of uroporphyrinogen III to cobinamide are present again in the corresponding E. coli strain.
  • the need for enzymes for the synthesis of precorrin-2 and its oxidized product, sirohydrochlorin, in cobalamin biosynthesis was investigated. The following results show that vitamin B12 production is improved according to the invention in the presence of a precorrin-2 dehydrogenase.
  • CbiK is the cobalt chelatase from Salmonella enterica.
  • the E. coli cysG strain was transformed with a plasmid pER 185K D , which is a plasmid which contains all Salmonelle enterica cbi genes but has a deletion in the cbiK gene.
  • the E. coli were further cotransformed with another plasmid, which allows the investigation of the necessity of the genes coding for precorrin-II dehydrogenase. The following results were obtained:
  • CobA (representative of SirA), HemC and HemD were developed according to Raux et al. (1999, Bioorganic.Chem., 27: 100-118).
  • the mixture was mixed in 1 ml of 50 mM Tris / HCl pH8.
  • 0.1 mg SirB, 0.1 mg SirC, 0.5 mg NAD + and 21 ⁇ M CoCI 2 x6H 2 0 were added. This mixture was then incubated at 37 ° C. for 30 minutes and then analyzed by photospectrometry. As a control, spectra were run without SirB and SirC.
  • FIG. 7a shows that the synthesis of precorrin-2 in the coupled mixture containing PBG, HemC, HemD, CobA and SAM, after 20 minutes of incubation both in the presence and in the absence of NAD +, shows a spectrum with the specific spectrum for Precorrin-2 is comparable.
  • SirC a spectrum was obtained which corresponds to that of sirohydrochlorin with a maximum at 378 nm, comparable to the spectrum of the crude extract of E. coli pKK8668sirAC in FIG. 6.
  • a spectrum with an absorption maximum at 378 nm was also obtained by adding SirB and SirC (FIG. 7b). However, was When cobalt was added, a spectrum was obtained which had absorption maxima at 410 and 590 nm, which are equivalent to a spectrum of cobalt sirohydrochlorine. This means that SirB acts as a cobalt chelase.
  • E. coli ER 171 After transformation of E. coli ER 171 with the plasmid pKK8668 containing sirA (pKK8668sirA), the microorganism thus transformed was cultivated according to Example 5.3. As a control, the correspondingly non-transformed E. coli strain was cultivated under the same conditions. Gene expression was induced with IPTG and ALA at a concentration of 10 mg / l. The specific activity of the urophorphyrinogen III methyl transferase (sirA) was then determined by determining the precorrin-2 formed.
  • sirA urophorphyrinogen III methyl transferase
  • Precorrin-2 fluoresces and after excitation of the batch at 340 nm a clear emission of the fluorescent precorrin-2 can be measured at 600 nm (extinction coefficient: 8x10 5 M "1 L " 1 ).
  • the substrate Uroporphyrinogen-III does not fluoresce.
  • the urophorphyrinogen III methyl transferase activity was hardly measurable in the non-transformed E. coli strain.
  • an excess of the sirA-encoded enzyme Urophorphyrinogen-III-methyltransferase of at least 10 mg protein per liter of culture medium, with an enzyme activity of about 15,000 units per liter of culture medium, can be measured.
  • 1 unit corresponds to the production of 1 nmole Precorrin-2 per hour. Protein determination was carried out using the Protein Detection Kit (BioRad, Germany) based on the Bradford method (Anal. Biochem., 1989, 178, 263.268).
  • Bacillus megaterium strain DSMZ 509 was also transformed with the plasmid pKK8668 containing sirA (pKK8668sirA) and cultivated as described in Example 5.3.
  • the protein encoded by sirA is increasingly produced in the transformed B. megaterium strain, which is shown qualitatively by a red fluorescence of the cells as soon as the gene is expressed. Gene expression was induced with IPTG and ALA at a concentration of 10 mg / l.
  • B. megaterium strain had a specific activity of about 15,000
  • Urophorphyrinogen III methyl transferase measured (Bradford; Anal. Biochem., 1989, 178, 263.268). This resulted in an excess of SirA of 10 mg protein per liter of culture medium.
  • a clear emission of the fluorescent precorrin-2 can be measured at 600 nm (extinction coefficient: 8x10 5 M " V 1 ).
  • the substrate uroporphinogen-III does not fluoresce. This resulted in the non-transformed E. coli In the transformed E. coli strain, a specific enzyme activity of uroporphyrinogen-III-methyltransferase (SirA) of 2,500 units of precorrin-2 per liter of culture medium and a specific enzyme activity of precorrin-2-dehydrogenase (SirC) of 4,000 Units of Sirohydrochlorin per liter of culture medium can be determined.
  • SirA uroporphyrinogen-III-methyltransferase
  • SirC precorrin-2-dehydrogenase
  • sirohydrochlorin as an intermediate substance serves both as a substrate for the sirohaem-synthesizing enzyme encoded by sirB (sirohydrochlorin iron chelatase) and for cobalamin chelatase encoded by cbiX.
  • Figure 1 shows a comparison of the growth of ⁇ . megaterium DSMZ32 (wild type) at 30 ° C under aerobic and anaerobic conditions. Anaerobic growth was measured with the addition of 10 mM nitrate (empty diamonds), 10 mM nitrite (empty triangles) and 10 mM fumarate (crosses). Fermentatives (empty circles) and aerobic growth (filled diamonds) took place in LB medium without additives. Samples were taken at the specified times and the optical density at 578 nm was determined.
  • Figure 2 shows a comparison of the growth of ⁇ . megaterium DSM509 at 30 ° C under aerobic and anaerobic conditions. Anaerobic growth was measured with the addition of 10 mM nitrate (empty diamonds), 10 mM nitrite (empty triangles) and 10 mM fumarate (crosses). Fermentatives (empty circles) and aerobic growth (filled diamonds) took place in LB medium without additives. Samples were taken at the specified times and the optical density at 578 nm was determined.
  • Figure 3 shows a comparison of the growth of ⁇ . megaterium DSM2894 at 30 ° C under aerobic and anaerobic conditions. Anaerobic growth was measured with the addition of 10 mM nitrate (empty diamonds), 10 mM nitrite (empty triangles) and 10 mM fumarate (crosses). Fermentatives (empty circles) and aerobic growth (filled diamonds) took place in LB medium without additives. Samples were taken at the specified times and the optical density at 578 nm was determined.
  • Figure 4 shows the vitamin B ⁇ 2 production of ß. megaterium under aerobic and anaerobic growth conditions. The content of vitamin B 12 per cell mass was given in pmol / OD 578 for the wild-type strain ⁇ .
  • megaterium DSM32 aerobically grown (1) and anaerobically grown (2), for ß.
  • megaterium DSM2894 grown aerobically (5) and grown anaerobically (6).
  • Figure 5 shows an SDS-polyacrylamide gel from His-tagged, purified Sir A, SirB and SirC.
  • Lane 1 shows a molecular weight standard and lanes 2, 3 and 4 purified His-tagged protein SirA, SirB and SirC, respectively.
  • FIG. 6 shows a UV-Vis spectrum of accumulated pigments isolated from pKK8668SirA, pKK8668SirAB, pKK8668SirABC and pKK8668SirAC overexpressing bacterial strains of E. coli ER171, which were cultivated in the presence of ALA.
  • FIG. 7 shows UV-Vis spectra of in vitro accumulated pigments of a coupled Precorrin-2 enzyme batch with SirB and SirC.
  • FIG. 8 shows UV-Vis spectra of pigments of a coupled precorrin-2 enzyme batch accumulated in vitro with SirB and SirC with the addition of metals other than cobalt, namely Ni 2+ , Zn 2+ , Cu 2+ and Fe 2+ .
  • the Absorption maxima of Ni 2+ , Zn 2+ , Cu 2+ and Fe 2+ are 395 nm, 405 and 402 nm.

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Abstract

La présente invention concerne un procédé amélioré de production de la vitamine B12. Selon ce procédé, une culture contenant un micro-organisme est fermentée. Au moins les enzymes uroporphyrinogen-III-méthyltransférase et précorrin-2-déhydrogénase qui sont impliquées dans la synthèse de sirohaem, ont une activité accrue contrairement aux activités enzymatiques endogènes correspondantes.
EP03745269A 2002-03-28 2003-03-24 Procede ameliore de production de la vitamine b12 Withdrawn EP1492877A2 (fr)

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