EP1421202A2 - Method for producing vitamin b12 - Google Patents

Method for producing vitamin b12

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Publication number
EP1421202A2
EP1421202A2 EP02796247A EP02796247A EP1421202A2 EP 1421202 A2 EP1421202 A2 EP 1421202A2 EP 02796247 A EP02796247 A EP 02796247A EP 02796247 A EP02796247 A EP 02796247A EP 1421202 A2 EP1421202 A2 EP 1421202A2
Authority
EP
European Patent Office
Prior art keywords
vitamin
megaterium
bacillus megaterium
nucleotide sequence
production
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Withdrawn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
EP02796247A
Other languages
German (de)
French (fr)
Inventor
Andreas KÜNKEL
Jan-Henning Martens
Dieter Jahn
Heiko Barg
Martin Warren
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.)
BASF SE
Original Assignee
BASF SE
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Filing date
Publication date
Priority claimed from DE10150359A external-priority patent/DE10150359A1/en
Application filed by BASF SE filed Critical BASF SE
Publication of EP1421202A2 publication Critical patent/EP1421202A2/en
Withdrawn legal-status Critical Current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
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes

Definitions

  • the present invention relates to a method for producing vitamin B12 using Bacillus megaterium.
  • vitamin B 12 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 B 12 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 B 12 . Nature 176, 325-328 and Nature 178, 64-70).
  • vitamin B 12 The naturally occurring end products of vitamin B 2 biosynthesis are 5 ' - deoxyadenosylcobalamin (coenzyme B 12 ) and methylcobalamin (MeCbl), while vitamin B 12 by definition stands for cyanocobalamin (CNCbl), which is mainly the one manufactured and traded by industry Represents form.
  • CNCbl cyanocobalamin
  • vitamin B 12 stands for the designation of all three analog molecules.
  • ß. megaterium was first described by De Bary over 100 years ago (1884). Although generally classified as a soil bacterium, ß. megaterium 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. ß. megaterium, like its close relative Bacillus subtilis, is a gram-positive bacterium and is characterized, among other things, by its relatively pronounced, eponymous size of 2x5 ⁇ m, a G + C content of approx. 38% and a very pronounced ability to sporulate.
  • ß. megaterium a species of ß. megaterium. It recycles a very large number of sugars and has been found, for example, in grain syrup, waste from the meat industry and even in petrochemical waste. In view of this ability to metabolize an extremely wide range of carbon sources, ß. megaterium can be equated with the pseudomonas without restriction (Vary, PS, 1994, Microbiology, 40, 1001-1013, Prime time for Bacillus megaterium).
  • megaterium products of commercial interest are effectively secreted, such as B. is used in the production of ⁇ - and ß-amylase. It is also with ß. megaterium possible due to its size to accumulate a high biomass until too high a population density leads to death. Of paramount importance in industrial production using ß. Megaterium continues to prove the favorable circumstance that this species is able to produce products of high value and highest quality from waste and inferior substances. This possibility of metabolizing an extremely wide range of substrates is also reflected in the use of ß. megaterium as a soil detoxifier that can break down cyanides, herbicides and persistent pesticides. Finally, the fact that ß. megaterium is completely apathogenic and also does not produce any toxins, especially of great importance in food and cosmetic production.
  • ß. megaterium is already used in a variety of industrial applications, such as the production of ⁇ - and ⁇ -amylase, penicillin amidase, the processing of toxic waste or aerobic vitamin Bi 2 production (summarized in Vary, PS, 1994, Microbiology, 40 , 1001-1013, Prime time for Bacillus megaterium).
  • Bacillus megaterium Due to its many advantages for use in the biotechnological production of various, industrially interesting products, the use of Bacillus megaterium is economically very interesting. In industrial fermentation of aerobic microorganisms, however, problems regularly occur on an industrial scale, particularly in the efficient oxygen aeration of the bacterial cultures, which are associated with considerable losses in the product yield.
  • the object of the present invention is to optimize the production of vitamin B12 with Bacillus megaterium.
  • This object is achieved by a process for the production of vitamin B12 by means of a culture containing Bacillus megaterium, in which the fermentation is carried out under anaerobic conditions.
  • B. megaterium strains which are suitable as vitamin B12 production strains can be used for the purposes of the present invention.
  • vitamin B12 production strains are to be understood as Bacillus megaterium strains or homologous microorganisms which are modified by classic and / or molecular genetic methods in such a way that their metabolic flow is increasingly in the direction of the biosynthesis of vitamin B12 or its derivatives ( 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 present invention includes all known vitamin B12 production strains, preferably of the Bacillus genus or homologous organisms.
  • the strains advantageous according to the invention include in particular the strains of B.
  • B. megaterium is capable of an anaerobic lifestyle and that 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 clearly shows that the anaerobic vitamin B12 production is increased by at least a factor of 3 to 4 in all strains compared to the aerobic vitamin B12 production. Further increases can be achieved by systematically optimizing the growth conditions and the composition of the culture medium and the bacterial strains used.
  • the production of vitamin B12 by means of Bacillus megaterium 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 according to the process of the invention.
  • the aforementioned compounds individually or their combinations in combination with cobalt can also be advantageous.
  • the present invention accordingly also relates to a method which is distinguished by the fact that at least cobalt is added to the culture medium. That Cobalt can be added, for example, individually or in combination with at least betaine, methionine, glutamate, dimethylbenzimidazole or choline or combinations of the last-mentioned compounds.
  • the vitamin B-12 content can be increased by adding about 200 to 750 ⁇ M, preferably 250 to 500 ⁇ M cobalt per liter of culture medium.
  • a method of the aforementioned type is included, in which B. megaterium is first fermented 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.
  • a method is preferred in which the transition from aerobic to anaerobic fermentation takes place as soon as the aerobic culture has reached its maximum optical density, but at least an optical density of approximately 2 to 3.
  • anaerobic conditions both in the one-stage and in the two-stage process according to the invention, are to be understood as those conditions which occur when the bacteria are transferred to anaerobic bottles after aerobic cultivation and are 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 after being transferred to the anaerobic bottles, the bacteria consume the oxygen present there and no more oxygen is added.
  • These conditions can also be called semi-anaerobic.
  • the corresponding procedures are common laboratory practice and known to the person skilled in the art.
  • Comparable conditions also exist if the bacteria are first cultivated aerobically in a fermenter and then the oxygen supply is successively reduced, so that semi-anaerobic conditions develop over time.
  • strictly anaerobic conditions can also be created, for example, by adding reducing agents to the culture medium.
  • anaerobic conditions whether semi-anaerobic or strictly anaerobic
  • an aerobic cultivation (preculture) of the bacteria is not absolutely necessary.
  • the bacteria can also be grown under anaerobic conditions and then fermented further 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.
  • the fermentation takes place under aerobic conditions with the addition of about 250 ⁇ M cobalt; Under anaerobic conditions it is advantageous to add about 500 ⁇ M cobalt.
  • 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 B-12, through which the metabolic flow can be specifically controlled in the direction of maximum vitamin B 2 production.
  • Targeted modifications of genes involved in the regulation of the metabolic flow also include investigations and changes in the regulatory areas before and after the structural genes, such as the optimization and / or the exchange of promoters, enhancers, terminators, ribosome binding sites, etc.
  • the improvement of stability the DNA, mRNA or the proteins encoded by them for example by reducing or Prevention of degradation by nucleases or proteases is included in the invention.
  • the present invention relates to the corresponding nucleotide sequences coding for the enzymes involved in the biosynthesis of vitamin B12.
  • the present invention also relates to an isolated nucleotide sequence coding for the enzymes involved in the biosynthesis of uroporphyrinogen III, organized in the hemAXCDBL operon from B. megaterium with a nucleotide sequence according to SEQ ID No.1 or its alleles.
  • Isoforms are to be understood as enzymes with the same or comparable substrate and activity specificity, but which have a different primary structure.
  • modified forms are understood to mean 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.
  • 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 functionally equivalent, i.e. H. to understand 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. 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 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 are conserved at the protein level, for example for the exchange Amino acids can lead, but which do not lead to a fundamental change in the activity of the protein and are therefore functionally 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 are obtained by modification of the nucleotide sequence, resulting in corresponding derivatives.
  • the aim of 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 sequences includes, according to the invention, substantially similar nucleotide sequences from the group of DNA or RNA, which enter into a specific interaction (binding) with the aforementioned nucleotide sequences under known stringent conditions.
  • 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 comprises a gene structure containing the isolated nucleotide sequence of the aforementioned type or parts thereof, as well as nucleotide sequences operatively linked thereto with a regulatory function.
  • 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.
  • These regulatory nucleotide sequences can be of natural origin or obtained by chemical synthesis.
  • any promoter which can control gene expression in the corresponding host organism is suitable as the promoter. According to the invention, 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. To connect the DNA fragments to one another, adapters or linkers can be attached to the fragments.
  • the invention also includes a vector containing an isolated nucleotide sequence of the aforementioned type or parts thereof or a gene structure of the aforementioned type, as well as additional nucleotide sequences for selection, replication in the host cell and / or integration into the host cell genome. Examples of such additional sequences are widely described in the literature and are not described further.
  • Suitable systems for the transformations and overexpression of the genes mentioned in B. megaterium are, for example, the plasmids pWH1510 and pWH1520 and the plasmid-free overexpression strain B. megaterium WH320, which were 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 plasmids are also commercially available (Q-biogene and MoBiTec). However, the systems mentioned are not limiting for the present invention.
  • the present invention also relates to a method which is characterized in that a Bacillus megaterium strain is fermented, the hemAXCDBL operon or parts thereof are / are increasingly expressed.
  • a variant of the present invention also includes a method in which a genetically modified Bacillus megaterium strain is fermented, the hemAXCDBL operon or parts thereof of which is / are present in the cell in an increased number of copies compared to the genetically unmodified Bacillus.
  • the number of copies can vary from two to several hundred.
  • the number of copies of the corresponding genes can be increased in order to achieve increased gene expression (overexpression). Furthermore, the promoter and / or regulatory region and / or the
  • Ribosome binding site which is located upstream of the structural gene, are changed accordingly 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 a different number of copies and / or integrated and / or amplified in the chromosome.
  • the activity of the enzyme itself can also be increased or increased by preventing the breakdown of the enzyme protein.
  • overexpression of the genes in question can also be achieved by changing the media composition and culture management.
  • the present invention furthermore relates to a transformed Bacillus megaterium strain for use in a method for producing vitamin B12 of the aforementioned type, which has an increased expression and / or increased copy number of the nucleotide sequence of the hemAXCDBL operon or parts thereof.
  • the invention also encompasses a transformed Bacillus megaterium strain which, in replicating form, contains a gene structure or a vector of the type mentioned above.
  • the present invention furthermore relates to the use of the isolated nucleotide sequence of the hemAXCDBL operon or parts thereof or the gene structure or a vector of the aforementioned type for producing a transformed Bacillus megaterium strain of the aforementioned type.
  • the present invention also includes the use of the transformed one Bacillus megaterium strain of the aforementioned type for the production of vitamin B12.
  • 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 ⁇ d 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:
  • 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 mepaterium 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 78 of one corresponds to a cell number of 1 ⁇ 10 9 cells.
  • the strains used are the strains B. megaterium DSMZ 32 (wild type) or the "production strains" DSMZ 509 and DSMZ 2894 which are suitable under aerobic conditions for vitamin B12 production.
  • the present invention is not limited to the use of these strains
  • Other strains suitable for the production of vitamin B12 are also conceivable, including genetically modified bacterial strains which can be produced by classic mutagenesis or targeted molecular biological techniques and corresponding selection processes.
  • B. megaterium In the investigation of the extent to which B. megaterium is capable of anaerobic growth, instead of the aerobic electron acceptor Oxygen, the alternative electron acceptors nitrate, nitrite and fumarate added to the culture medium ( Figures 4-6). In parallel, it was investigated whether B. megaterium is also capable of fermentation in a medium without the addition of these electron acceptors. It is clear from the results of the present invention that the electron acceptor nitrite has a toxic effect on the growth of the keratin. Fumarate also inhibits growth. None of the potential electron acceptors added can stimulate anaerobic growth beyond the level of fermentative growth.
  • 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 the ß resuspended in deionized, sterile water and boiled in a water bath for 15 min. Megater / wm samples were placed on the cooled plates and incubated for 18 h at 37 ° C.
  • the diameter of the grown salmonella colonies are then proportional to the content of vitamin B 12 in the applied ß. megater / ' i / m samples.
  • a calibration curve made from the addition of 0.01, 0.1, 1, 10 and 40 pmol of vitamin B1 2 , the content of vitamin B 12 in the examined samples was inferred. This standard method allows the detection of small amounts of vitamin B 2 in biological materials quickly and very reproducibly. 6.
  • LB medium 50 ml LB medium were mixed with 1 ml of an overnight culture of ⁇ . inoculated megaterium and incubated at 37 ° C. With an OD 5 ⁇ of 1, the cells were centrifuged at 15,000 rpm and 4 ° C. for 15 min (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 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 hardly visible sediment in 500 ⁇ l SMMP buffer resuspended. The suspension was incubated at 37 ° C. for 90 min with gentle shaking.
  • megaterium DNA a 3855 kb insert could be identified which codes for the hemAXCDBL operon sought.
  • the corresponding nucleotide sequence is in SEQ ID No. 1 and the amino acid sequences derived therefrom in SEQ ID No. 2-6 listed.
  • the sequences upstream and downstream of the DNA fragment obtained by functional complementation were obtained using the Vectorette TM system from Sigma Genosis. For this purpose, the Turbo Pfu DNA polymerase from Strategene was used, which has an extremely low error rate due to its proofreading function.
  • 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 Regulatory Elements for Transcription of the Bacillus megaterium Encoded Regulon for Xylose Utilization, Arch.
  • 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 approximately 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 education choose a transcription or translation fusion because the xylA reading frame upstream of the polylinker is still completely intact.
  • 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
  • Figure 4 shows anaerobic growth of ⁇ . Megaterium DSM32 (wild type) at 30 ° C with the addition of 10 mM nitrate (diamonds), 10 mM nitrite (triangles) and 10 mM fumarate (crosses). Fermentative growth (circles) took place in LB medium without additives. Samples were taken at the specified times and the optical density at 578 nm was determined.
  • Figure 5 shows anaerobic growth of ⁇ . Megaterium DSM509 at 30 ° C with the addition of 10 mM nitrate (diamonds), 10 mM nitrite (triangles) and 10 mM fumarate (crosses). Fermentative growth (circles) took place in LB medium without additives. Samples were taken at the specified times and the optical density at 578 nm was determined.
  • Figure 6 shows anaerobic growth of ⁇ . Megaterium DSM2894 at 30 ° C with the addition of 10 mM nitrate (diamonds), 10 mM nitrite (triangles) and 10 mM fumarate (crosses). Fermentative growth (circles) took place in LB medium without additives. Samples were taken at the specified times and the optical density at 578 nm was determined.
  • Figure 7 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 / ODs 8 for the wild-type strain ⁇ . megaterium DSM32 aerobically grown (1) and anaerobically grown (2), for ß. megaterium DSM509 aerobically grown (3) and anaerobically grown (4), as well as for ß. megaterium DSM2894 grown aerobically (5) and grown anaerobically (6).
  • Figure 8 shows the aerobic vitamin B- ⁇ 2 production of ß. megaterium with and without external addition of 50 ⁇ g / ml ALA.

Abstract

The invention relates to a method for producing vitamin B12 using Bacillus megaterium.

Description

Verfahren zur Herstellung von Vitamin B12 Process for the production of vitamin B12
Die vorliegende Erfindung betrifft ein Verfahren zur Herstellung von Vitamin B12 mittels Bacillus megaterium.The present invention relates to a method for producing vitamin B12 using Bacillus megaterium.
Bereits in den zwanziger Jahren unseres Jahrhunderts wurde Vitamin B12 indirekt durch seine Wirkung auf den menschlichen Körper durch George Minot und William Murphy entdeckt (Stryer, L, 1988, In Biochemie, vierte Auflage, pp. 528-531 , Spektrum Akademischer Verlag GmbH, Heidelberg, Berlin, New York). Im Jahre 1948 konnte Vitamin B12 erstmals gereinigt und isoliert werden, so daß bereits acht Jahre später, im Jahre 1956, die Aufklärung seiner komplexen dreidimensionalen Kristallstruktur durch Dorothy Hodgkin gelang (Hodgkin, D. C. et al., 1956, Structure of Vitamin B12. Nature 176, 325-328 und Nature 178, 64-70). Die in der Natur vorkommenden Endprodukte der Vitamin Bι2-Biosynthese sind 5'- Desoxyadenosylcobalamin (Coenzym B12) und Methylcobalamin (MeCbl), während Vitamin B12 per Definition für Cyanocobalamin (CNCbl) steht, das die hauptsächlich von der Industrie hergestellte und gehandelte Form darstellt. In der vorliegenden Erfindung steht, wenn nicht speziell angegeben, Vitamin B12 einheitlich für die Bezeichnung aller drei analogen Moleküle.Already in the twenties of our century, vitamin B 12 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). In 1948, vitamin B 12 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 B 12 . Nature 176, 325-328 and Nature 178, 64-70). The naturally occurring end products of vitamin B 2 biosynthesis are 5 ' - deoxyadenosylcobalamin (coenzyme B 12 ) and methylcobalamin (MeCbl), while vitamin B 12 by definition stands for cyanocobalamin (CNCbl), which is mainly the one manufactured and traded by industry Represents form. In the present invention, unless specifically stated, vitamin B 12 stands for the designation of all three analog molecules.
Die Spezies ß. megaterium wurde bereits vor über 100 Jahren (1884) das erste mal durch De Bary beschrieben. Obwohl generell als ein Bodenbakterium klassifiziert, läßt sich ß. megaterium auch in diversen anderen Habitaten wie Seewasser, Sedimenten, Reis, getrocknetem Fleisch, Milch oder Bienenhonig nachweisen. Er tritt dabei oft in Begleitung von Pseudomonaden und Actinomyceten auf. ß. megaterium ist, wie sein naher Verwandter Bacillus subtilis, ein Gram-postives Bakterium und zeichnet sich unter anderem aus durch seine relativ ausgeprägte, namengebende Größe von 2x5 μm, einem G+C-Gehalt von ca. 38 % und einer sehr ausgeprägten Fähigkeit zur Sporulation. Bereits geringste Mengen an Mangan im Wachstumsmedium genügen dieser Spezies, um eine vollständige Sporulation durchzuführen, eine Fähigkeit die nur noch vergleichbar ist mit der Sporulationseffizienz einiger thermophiler Bacillen. Aufgrund seiner Größe und seiner sehr effizienten Sporulation und Germination wurden vielfältige Untersuchungen der molekularen Grundlagen dieser Vorgänge an ß. megaterium durchgeführt, so daß mittlerweile mehr als 150 Gene in ß. megaterium beschrieben sind, die an seiner Sporulation und Germination beteiligt sind. Physiologische Untersuchungen an ß. megaterium (Priest, F. G. et al., 1988, A Numerical Classification of the Genus Bacillus, J. Gen. Microbiol. 134, 1847-1882) klassifizierten diese Spezies als obligat aerobes, Sporenbildendes Bakterium, das Urease-positiv und Voges-Proskauer-negativ ist und kein Nitrat reduzieren kann. Eine der herausragendsten Eigenschaften von ß. megaterium ist seine Fähigkeit, eine Vielzahl von Kohlenstoffquellen für sich zu nutzen. So verwertet er eine sehr große Zahl von Zuckern und wurde z.B. in Korn-Sirup, Abfällen aus der Fleischindustrie und sogar in petrochemischen Abfällen gefunden. In Hinsicht auf diese Fähigkeit zur Metabolisierung eines äußerst breiten Spektrums von Kohlenstoffquellen, kann ß. megaterium ohne Einschränkung mit den Pseudomonaden gleichgesetzt werden (Vary, P. S., 1994, Microbiology, 40, 1001-1013, Prime time for Bacillus megaterium).The species ß. megaterium was first described by De Bary over 100 years ago (1884). Although generally classified as a soil bacterium, ß. megaterium 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. ß. megaterium, like its close relative Bacillus subtilis, is a gram-positive bacterium and is characterized, among other things, by its relatively pronounced, eponymous size of 2x5 μm, a G + C content of approx. 38% and a very pronounced ability to sporulate. Even the smallest amounts of manganese in the growth medium are sufficient for this species to perform a complete sporulation, an ability that is only comparable to the sporulation efficiency of some thermophilic bacilli. Due to its size and its very efficient sporulation and germination, various studies of the molecular basis of these processes have been carried out. megaterium carried out, so that now more than 150 genes in ß. megaterium that are involved in its sporulation and germination. Physiological examinations at ß. megaterium (Priest, FG et al., 1988, A Numerical Classification of the Genus Bacillus, J. Gen. Microbiol. 134, 1847-1882) classified this species as an obligate aerobic, spore-forming bacterium that is urease-positive and Voges-Proskauer- is negative and cannot reduce nitrate. One of the most outstanding properties of ß. megaterium is its ability to use a variety of carbon sources. It recycles a very large number of sugars and has been found, for example, in grain syrup, waste from the meat industry and even in petrochemical waste. In view of this ability to metabolize an extremely wide range of carbon sources, ß. megaterium can be equated with the pseudomonas without restriction (Vary, PS, 1994, Microbiology, 40, 1001-1013, Prime time for Bacillus megaterium).
Die Vorteile der breiten Anwendung von ß. megaterium bei der industriellen Produktion verschiedenster Enzyme, Vitamine etc. sind vielfältig. Dazu gehört zum ersten sicherlich der Umstand, daß in ß. megaterium transformierte Plasmide sich als sehr stabil erweisen. Dies muß in direktem Zusammenhang gesehen werden mit der mittlerweile etablierten Möglichkeit, diese Spezies beispielsweise durch Polyethylenglykolbehandlung zu transformieren. Dies war bis vor wenigen Jahren noch ein Haupthindernis der Anwendung von ß. megaterium als Produktionsstamm. Parallel hierzu ist auch der Vorteil einer relativ gut entwickelten Genetik zu sehen, die innerhalb des ßac/7/us-Genus nur von ß. subtilis übertroffen wird. Zweitens weist ß. megaterium keine alkalischen Proteasen auf, so daß bei der Produktion heterologer Proteine kaum eine Degradation beobachtet wurde. Zudem ist bekannt, daß ß. megaterium Produkte von kommerziellem Interesse effekiv sezerniert, wie dies z. B. bei der Produktion von α- und ß-Amylase ausgenutzt wird. Außerdem ist es mit ß. megaterium durch seine Größe möglich, eine hohe Biomasse anzusammeln, bis eine zu hohe Populationsdichte zum Absterben führt. Von größter Bedeutung bei der industriellen Produktion mittels ß. megaterium erweist sich weiterhin der günstige Umstand, daß diese Spezies in der Lage ist, aus Abfällen und minderwertigen Stoffen Produkte hohen Wertes und höchster Qualität herzustellen. Diese Möglichkeit der Metabolisierung eines enorm breiten Substratspektrums spiegelt sich auch wieder in der Anwendung von ß. megaterium als Bodendetoxifizierer, der selbst Cyanide, Herbizide und persistente Pestizide abzubauen vermag. Schließlich ist die Tatsache, daß ß. megaterium vollkommen apathogen ist und auch keine Toxine produziert, insbesondere in der Lebensmittel- und Kosmetikproduktion von größter Wichtigkeit. Wegen dieser mannigfaltigen Vorzüge wird ß. megaterium bereits heute in einer Vielzahl von industriellen Anwendungen eingesetzt, wie der Produktion von α- und ß-Amylase, Penicillin-Amidase, dem Aufbereiten toxischer Abfälle oder der aeroben Vitamin Bi2-Produktion (zusammengefaßt in Vary, P. S., 1994, Microbiology, 40, 1001-1013, Prime time for Bacillus megaterium).The advantages of widespread use of ß. Megaterium in the industrial production of various enzymes, vitamins etc. are diverse. The fact that in ß. Plasmids transformed with megaterium have proven to be very stable. This has to be seen in direct connection with the meanwhile established possibility to transform this species for example by polyethylene glycol treatment. This was until a few years ago Years still a major obstacle to the use of ß. megaterium as a production master. In parallel, there is also the advantage of a relatively well developed genetics, which within the ßac / 7 / us genus only of ß. subtilis is surpassed. Second, ß. megaterium no alkaline proteases, so that hardly any degradation was observed in the production of heterologous proteins. It is also known that ß. megaterium products of commercial interest are effectively secreted, such as B. is used in the production of α- and ß-amylase. It is also with ß. megaterium possible due to its size to accumulate a high biomass until too high a population density leads to death. Of paramount importance in industrial production using ß. Megaterium continues to prove the favorable circumstance that this species is able to produce products of high value and highest quality from waste and inferior substances. This possibility of metabolizing an extremely wide range of substrates is also reflected in the use of ß. megaterium as a soil detoxifier that can break down cyanides, herbicides and persistent pesticides. Finally, the fact that ß. megaterium is completely apathogenic and also does not produce any toxins, especially of great importance in food and cosmetic production. Because of these diverse advantages, ß. megaterium is already used in a variety of industrial applications, such as the production of α- and β-amylase, penicillin amidase, the processing of toxic waste or aerobic vitamin Bi 2 production (summarized in Vary, PS, 1994, Microbiology, 40 , 1001-1013, Prime time for Bacillus megaterium).
Aufgrund seiner vielfältigen Vorteile zum Einsatz in der biotechnologischen Herstellung verschiedener, industriell interessanter Produkte ist der Einsatz von Bacillus megaterium wirtschaftlich sehr interessant. Bei der industriellen Fermentation aerober Mikroorganismen treten jedoch im großtechnischen Maßstab regelmäßig Probleme, insbesondere bei der effizienten Sauerstoffdurchlüftung der Bakterienkulturen auf, die mit erheblichen Verlusten in der Produktausbeute verbunden sind.Due to its many advantages for use in the biotechnological production of various, industrially interesting products, the use of Bacillus megaterium is economically very interesting. In industrial fermentation of aerobic microorganisms, however, problems regularly occur on an industrial scale, particularly in the efficient oxygen aeration of the bacterial cultures, which are associated with considerable losses in the product yield.
Aufgabe der vorliegenden Erfindung ist es, die Herstellung von Vitamin B12 mit Bacillus megaterium zu optimieren.The object of the present invention is to optimize the production of vitamin B12 with Bacillus megaterium.
Diese Aufgabe wird durch ein Verfahren zur Herstellung von Vitamin B12 mittels einer Kultur enthaltend Bacillus megaterium gelöst, bei welchem die Fermentation unter anaeroben Bedingungen durchgeführt wird.This object is achieved by a process for the production of vitamin B12 by means of a culture containing Bacillus megaterium, in which the fermentation is carried out under anaerobic conditions.
Grundsätzlich können für die Zwecke der vorliegenden Erfindung alle üblichen B. megaterium-Stämme eingesetzt werden, die als Vitamin B12- Produktionsstämme geeignet sind.In principle, all the usual B. megaterium strains which are suitable as vitamin B12 production strains can be used for the purposes of the present invention.
Unter Vitamin B12-Produktionsstämmen sind im Sinne der vorliegenden Erfindung Bacillus megaterium-Stämme oder homologe Mikroorganismen zu verstehen, die durch klassische und/oder molekulargenetische Methoden derart verändert sind, daß ihr Stoffwechselfluß verstärkt in die Richtung der Biosynthese von Vitamin B12 oder dessen Abkömmlingen verläuft (metabolic engineering). Beispielsweise sind bei diesen Produktionsstämmen ein oder mehrere Gen(e) und/oder die korrespondierenden Enzyme, die an entscheidenden und entsprechend komplex regulierten Schlüsselpositionen des Stoffwechselweges (Flaschenhals) stehen in ihrer Regulation verändert oder sogar dereguliert. Die vorliegende Erfindung umfaßt hierbei sämtliche bereits bekannte Vitamin B12-Produktionsstämme, bevorzugt der Gattung Bacillus oder homologer Organismen. Zu den erfindungsgemäß vorteilhaften Stämmen gehören insbesondere die Stämme von B. megaterium DSMZ 32, DSMZ 509 und DSMZ 2894. Erfindungsgemäß konnte gezeigt werden, daß B. megaterium zu einer anaeroben Lebensweise fähig ist und außerdem unter diesen Bedingungen die Vitamin B12 Produktion höher ist, als unter aeroben Bedingungen. Der Vergleich der Vitamin B12 Produktion von B. megaterium unter aeroben und anaeroben Wachstumsbedingungen zeigt deutlich, daß die anaerobe Vitamin B12 Produktion bei allen untersuchten Stämmen gegenüber der aeroben Vitamin B12 Produktion wenigstens um den Faktor 3 bis 4 gesteigert ist. Weitere Steigerungen lassen sich durch eine systematische Optimierung der Wachstumsbedingungen und der Zusammensetzung des Kulturmediums sowie der eingesetzten Bakterienstämme erreichen.For the purposes of the present invention, vitamin B12 production strains are to be understood as Bacillus megaterium strains or homologous microorganisms which are modified by classic and / or molecular genetic methods in such a way that their metabolic flow is increasingly in the direction of the biosynthesis of vitamin B12 or its derivatives ( metabolic engineering). For example, in 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 present invention includes all known vitamin B12 production strains, preferably of the Bacillus genus or homologous organisms. The strains advantageous according to the invention include in particular the strains of B. megaterium DSMZ 32, DSMZ 509 and DSMZ 2894. According to the invention, it was possible to show that B. megaterium is capable of an anaerobic lifestyle and that 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 clearly shows that the anaerobic vitamin B12 production is increased by at least a factor of 3 to 4 in all strains compared to the aerobic vitamin B12 production. Further increases can be achieved by systematically optimizing the growth conditions and the composition of the culture medium and the bacterial strains used.
Erfindungsgemäß kann die Herstellung von Vitamin B12 mittels Bacillus megaterium beispielsweise dadurch gesteigert werden, daß dem Kulturmedium wenigstens Cobalt zugesetzt wird. Auch der Zusatz von beispielsweise Betain, Methionin, Glutamat, Dimethylbenzimidazol oder Cholin oder deren Kombinationen wirkt sich vorteilhaft auf die Vitamin B12 Produktion gemäß dem erfindungsgemäßen Verfahren aus. Vorteilhaft können auch die zuvor genannten Verbindungen einzeln oder deren Kombinationen in Kombination mit Cobalt sein.According to the invention, the production of vitamin B12 by means of Bacillus megaterium 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 according to the process of the invention. The aforementioned compounds individually or their combinations in combination with cobalt can also be advantageous.
Gegenstand der vorliegenden Erfindung ist demgemäß auch ein Verfahren, das sich dadurch auszeichnet, das dem Kulturmedium wenigstens Cobalt zugesetzt wird. D.h. Cobalt kann beispielsweise einzeln oder in Kombination mit wenigstens Betain, Methionin, Glutamat, Dimethylbenzimidazol oder Cholin oder Kombinationen der zuletzt genannten Verbindungen zugesetzt werden.The present invention accordingly also relates to a method which is distinguished by the fact that at least cobalt is added to the culture medium. That Cobalt can be added, for example, individually or in combination with at least betaine, methionine, glutamate, dimethylbenzimidazole or choline or combinations of the last-mentioned compounds.
In einer Variante des erfindungsgemäßen Verfahrens kann der Vitamin B- 12 Gehalt durch die Zugabe von etwa 200 bis 750 μM, vorzugsweise 250 bis 500 μM Cobalt pro Liter Kulturmedium angehoben werden. In einer weiteren Variante der vorliegenden Erfindung ist ein Verfahren der zuvor genannten Art umfasst, bei dem B. megaterium zunächst aerob und anschließend anaerob fermentiert wird. In einer vorteilhaften Variante erfolgt der Übergang von der aeroben zur anaeroben Fermentation in der exponentiellen Wachstumsphase der aerob fermentierten Zellen. Hierbei ist es vorteilhaft, wenn der Übergang von der aeroben zur anaeroben Fermentation in der Mitte oder am Ende, bevorzugt am Ende, der exponentiellen Wachstumsphase der aerob wachsenden Zellen erfolgt. Erfindungsgemäß bevorzugt ist hierbei ein Verfahren, bei dem der Übergang von der aeroben zur anaeroben Fermentierung erfolgt, sobald die aerobe Kultur ihre maximale optische Dichte, wenigstens jedoch eine optische Dichte von etwa 2 bis 3 erreicht hat.In a variant of the method according to the invention, the vitamin B-12 content can be increased by adding about 200 to 750 μM, preferably 250 to 500 μM cobalt per liter of culture medium. In a further variant of the present invention, a method of the aforementioned type is included, in which B. megaterium is first fermented aerobically and then anaerobically. In an advantageous variant, 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. According to the invention, a method is preferred in which the transition from aerobic to anaerobic fermentation takes place as soon as the aerobic culture has reached its maximum optical density, but at least an optical density of approximately 2 to 3.
Unter anaeroben Bedingungen, sowohl in dem einstufigen, als auch in dem zweistufigen erfindungsgemäßen Verfahren, sind im Sinne der vorliegenden Erfindung diejenigen Bedingungen zu verstehen, die eintreten, wenn die Bakterien nach aerober Anzucht in Anaerobierflaschen überführt und dort fermentiert werden. Der Zeitpunkt der Überführung in die Anaerobierflaschen erfolgt insbesondere bei dem zweistufigen Verfahren, sobald sich die aerob angezüchteten Bakterienzellen in der exponentiellen Wachstumsphase befinden. D.h. nach der Überführung in die Anaerobierflaschen verbrauchen die Bakterien den dort vorliegenden Sauerstoff und es wird kein Sauerstoff mehr zugeführt. Diese Bedingungen können auch mit semi-anaerob bezeichnet werden. Die entsprechenden Vorgehensweisen sind gängige Laborpraxis und dem Fachmann bekannt.For the purposes of the present invention, anaerobic conditions, both in the one-stage and in the two-stage process according to the invention, are to be understood as those conditions which occur when the bacteria are transferred to anaerobic bottles after aerobic cultivation and are 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 after being transferred to the anaerobic bottles, the bacteria consume the oxygen present there and no more oxygen is added. These conditions can also be called semi-anaerobic. The corresponding procedures are common laboratory practice and known to the person skilled in the art.
Vergleichbare Bedingungen herrschen auch vor, wenn die Bakterien in einem Fermenter zunächst aerob kultiviert werden und dann die Sauerstoffzufuhr sukzessive reduziert wird, so daß sich mit der Zeit semi- anaerobe Bedingungen einstellen. In einer besonderen Variante der vorliegenden Erfindung können auch beispielsweise durch die Zugabe von Reduktionsmitteln zum Kulturmedium strikt anaerobe Bedingungen geschaffen werden. Generell ist für eine erfindungsgemäße Fermentation unter anaeroben Bedingungen (ob nun semi-anaerob oder strikt anaerob), eine aerobe Anzucht (Vorkultur) der Bakterien nicht zwingend erforderlich. D.h. die Bakterien können auch unter anaeroben Bedinungen angezüchtet werden und anschließend unter semi-anaeroben oder strikt anaeroben Bedingungen weiter fermentiert werden. Denkbar ist auch, daß das Inocculum direkt aus der Stammhaltung entnommen und zur Herstellung von Vitamin B12 unter anaeroben Bedingungen eingesetzt wird.Comparable conditions also exist if the bacteria are first cultivated aerobically in a fermenter and then the oxygen supply is successively reduced, so that semi-anaerobic conditions develop over time. In a special variant of the present invention, strictly anaerobic conditions can also be created, for example, by adding reducing agents to the culture medium. Generally, for a fermentation according to the invention under anaerobic conditions (whether semi-anaerobic or strictly anaerobic), an aerobic cultivation (preculture) of the bacteria is not absolutely necessary. This means that the bacteria can also be grown under anaerobic conditions and then fermented further 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.
In einer Variante der vorliegenden Erfindung erfolgt die Fermentierung unter aeroben Bedingungen unter Zusatz von etwa 250 μM Cobalt; unter anaeroben Bedingungen ist ein Zusatz von etwa 500 μM Cobalt vorteilhaft.In a variant of the present invention, the fermentation takes place under aerobic conditions with the addition of about 250 μM cobalt; Under anaerobic conditions it is advantageous to add about 500 μM cobalt.
Eingeschlossen sind erfindungsgemäß auch genetisch veränderte Bakterienstämme, die durch klassische Mutagenese oder gezielte molekularbiologische Techniken und entsprechende Selektionsverfahren hergestellt werden können. Interessante Ansatzpunkte zur gezielten gentechnischen Manipulation sind u.a. Verzweigungsstellen der zum Vitamin B-12 führenden Biosynthesewege, durch die der Stoffwechselfluß gezielt in Richtung einer maximalen Vitamin Bι2-Produktion gesteuert werden kann. Gezielte Modifikationen von an der Regulation des Stoffwechesflusses beteiligten Genen schließt auch Untersuchungen und Veränderungen der regulatorischen Bereiche vor und hinter den Strukturgenen ein, wie z.B. die Optimierung und/oder den Austausch von Promotoren, Enhancem, Terminatoren, Ribosomenbindungsstellen etc.. Auch die Verbesserung der Stabilität der DNA, mRNA oder der durch sie kodierten Proteine, beispielsweise durch die Verringerung oder Verhinderung des Abbaus durch Nukleasen bzw. Proteasen ist erfindungsgemäß umfaßt.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 B-12, through which the metabolic flow can be specifically controlled in the direction of maximum vitamin B 2 production. Targeted modifications of genes involved in the regulation of the metabolic flow also include investigations and changes in the regulatory areas before and after the structural genes, such as the optimization and / or the exchange of promoters, enhancers, terminators, ribosome binding sites, etc. Also the improvement of stability the DNA, mRNA or the proteins encoded by them, for example by reducing or Prevention of degradation by nucleases or proteases is included in the invention.
Gegenstand der vorliegenden Erfindung sind die entsprechenden Nukleotidsequenzen, kodierend für die an der Biosynthese von Vitamin B12 beteiligten Enzyme. Insbesondere ist Gegenstand der vorliegenden Erfindung auch eine isolierte Nukleotidsequenz kodierend für die an der Biosynthese von Uroporphyrinogen III beteiligten Enzyme, organisiert in dem hemAXCDBL-Operon aus B. megaterium mit einer Nukleotidsequenz gemäß SEQ ID No.1 oder deren Allele. Erfindungsgemäß sind auch die durch das hemAXCDBL-Operon aus B. megaterium kodierten Enzyme mit den Aminosäuresequenzen gemäß SEQ ID No. 2-6 sowie Isoenzyme oder modifizierte Formen davon umfaßt.The present invention relates to the corresponding nucleotide sequences coding for the enzymes involved in the biosynthesis of vitamin B12. In particular, the present invention also relates to an isolated nucleotide sequence coding for the enzymes involved in the biosynthesis of uroporphyrinogen III, organized in the hemAXCDBL operon from B. megaterium with a nucleotide sequence according to SEQ ID No.1 or its alleles. According to the invention, the enzymes encoded by the hemAXCDBL operon from B. megaterium with the amino acid sequences according to SEQ ID No. 2-6 and isoenzymes or modified forms thereof.
Unter Isoformen sind Enzyme mit gleicher oder vergleichbarer Substrat- und Wirkungsspezifität zu verstehen, die jedoch eine unterschiedliche Primärstruktur aufweisen.Isoforms are to be understood as enzymes with the same or comparable substrate and activity specificity, but which have a different primary structure.
Unter modifizierten Formen sind erfindungsgemäß Enzyme zu verstehen, bei denen Änderungen in der Sequenz, beispielsweise am N- und/oder C- Teminus des Polypeptids oder im Bereich konservierter Aminosäuren vorliegen, ohne jedoch die Funktion des Enzyms zu beeinträchtigen. Diese Veränderungen können in Form von Aminosäureaustauschen nach an sich bekannten Methoden vorgenommen werden.According to the invention, modified forms are understood to mean 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.
Eine besondere Ausführungsvariante der vorliegenden Erfindung umfaßt Varianten der erfindungsgemäßen Polypeptide, deren Aktivität beispielsweise durch Aminosäureaustausche, verglichen mit dem jeweiligen Ausgangsprotein, abgeschwächt oder verstärkt ist. Gleiches gilt für die Stabilität der erfindungsgemäßen Enzyme in den Zellen, die beispielsweise gegenüber dem Abbau durch Proteasen verstärkt oder vermindert anfällig sind.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. The same applies to the stability of the enzymes according to the invention in the cells which for example, are more or less susceptible to degradation by proteases.
Unter einer isolierten Nukleinsäure oder einem isolierten Nukleinsäurefragment ist erfindungsgemäß ein Polymer aus RNA oder DNA zu verstehen, das einzel- oder doppelsträngig sein kann und optional natürliche, chemisch synthetisierte, modifizierte oder artifizielle Nukleotide enthalten kann. Der Begriff DNA-Polymer schließt hierbei auch genomische DNA, cDNA oder Mischungen davon ein.According to the invention, 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. The term DNA polymer also includes genomic DNA, cDNA or mixtures thereof.
Unter Allelen sind erfindungsgemäß funktioneil äquivalente, d. h. im wesentlichen gleichwirkende Nukleotidsequenzen zu verstehen. Funktioneil äquivalente Sequenzen sind solche Sequenzen, welche trotz abweichender Nukleotidsequenz, beispielsweise durch die Degeneriertheit des genetischen Codes noch die gewünschten Funktionen besitzen. Funktionelle Äquivalente umfassen somit natürlich vorkommende Varianten der hierin beschriebenen Sequenzen sowie künstliche, z. B. durch chemische Synthese erhaltene und gegebenenfalls an den Kodon- Gebrauch des Wirtsorganismus angepaßte Nukleotid-Sequenzen. Darüber hinaus umfassen funktionell äquivalente Sequenzen solche, die eine veränderte Nukleotidsequenz aufweisen, welche dem Enzym beispielsweise eine Desensitivität oder Resistenz gegenüber Inhibitoren verleiht.According to the invention, alleles are functionally equivalent, i.e. H. to understand 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. obtained by chemical synthesis and possibly adapted to the codon use of the host organism nucleotide sequences. In addition, functionally equivalent sequences include those which have a modified nucleotide sequence which gives the enzyme, for example, a desensitivity or resistance to inhibitors.
Unter einem funktioneilen Äquivalent versteht man insbesondere auch natürliche oder künstliche Mutationen einer ursprünglich isolierten Sequenz, welche weiterhin die gewünschte Funktion zeigen. Mutationen umfassen Substitutionen, Additionen, Deletionen, Vertauschungen oder Insertionen eines oder mehrerer Nukleotidreste. Inbegriffen sind hier auch sogenannte Sinnmutationen (sense mutations), die auf Proteinebene beispielsweise zum Austausch konservierter Aminosäuren führen können, welche aber zu keiner grundsätzlichen Veränderung der Aktivität des Proteins führen und somit funktionsneutral sind. Dies beinhaltet auch Veränderungen der Nukleotidsequenz, die auf Proteinebene den N- oder C-Terminus eines Proteins betreffen, ohne jedoch die Funktion des Proteins wesentlich zu beeinträchtigen. Diese Veränderungen können sogar stabilisierenden Einfluß auf die Proteinstruktur ausüben.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 are conserved at the protein level, for example for the exchange Amino acids can lead, but which do not lead to a fundamental change in the activity of the protein and are therefore functionally 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.
Ferner werden beispielsweise auch solche Nukleotidsequenzen durch die vorliegende Erfindung mit umfaßt, welche man durch Modifikation der Nukleotidsequenz, resultierend in entsprechenden Derivaten, erhält. Ziel einer solchen Modifikation kann z. B. die weitere Eingrenzung der darin enthaltenen kodierenden Sequenz oder z. B. auch die Einfügung weiterer Restriktionsenzym-Schnittstellen sein. Funktionelle Äquivalente sind auch solche Varianten, deren Funktion, verglichen mit dem Ausgangsgen bzw. Genfragment, abgeschwächt oder verstärkt ist.Furthermore, the present invention also encompasses, for example, nucleotide sequences which are obtained by modification of the nucleotide sequence, resulting in corresponding derivatives. The aim of 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.
Außerdem sind artifizielle DNA-Sequenzen Gegenstand der vorliegenden Erfindung, solange sie, wie oben beschrieben, die gewünschten Eigenschaften vermitteln. Solche artifiziellen DNA-Sequenzen können beispielsweise durch Rückübersetzung von mittels computergestützten Programmen (molecular modelling) erstellten Proteinen oder durch in- vitro-Selektion ermittelt werden. Besonders geeignet sind kodierende DNA-Sequenzen, die durch Rückübersetzung einer Polypeptidsequenz gemäß der für den Wirtsorganismus spezifischen Kodon-Nutzung erhalten wurden. Die spezifische Kodon-Nutzung kann ein mit molekulargenetischen Methoden vertrauter Fachmann durch Computerauswertungen anderer, bereits bekannter Gene des zu transformierenden Organismus leicht ermitteln. Unter homologen Sequenzen sind erfindungsgemäß solche zu verstehen, die zu den erfindungsgemäßen Nukleotidsequenzen komplementär sind und/oder mit diesen hybridisieren. Der Begriff hybridisierende Sequenzen schließt erfindungsgemäß substanziell ähnliche Nukleotidsequenzen aus der Gruppe von DNA oder RNA ein, die unter an sich bekannten stringenten Bedingungen eine spezifische Wechselwirkung (Bindung) mit den zuvor genannten Nukleotidsequenzen eingehen. Hierzu zählen auch kurze Nukleotidsequenzen mit einer Länge von beispielsweise 10 bis 30, bevorzugt 12 bis 15 Nukleotiden. Dies umfaßt erfindungsgemäß u. a. auch sogenannte Primer oder Sonden.In addition, 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. According to the invention, 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, according to the invention, substantially similar nucleotide sequences from the group of DNA or RNA, which enter into a specific interaction (binding) with the aforementioned nucleotide sequences under known stringent conditions. 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.
Erfindungsgemäß sind auch die den kodierenden Bereichen (Strukturgenen) vorausgehenden (5'- oder upstream) und/oder nachfolgenden (3'- oder downstream) Sequenzbereiche eingeschlossen. Insbesondere sind hierin Sequenzbereiche mit regulatorischer Funktion inbegriffen. Sie können die Transkription, die RNA-Stabilität oder das RNA processing sowie die Translation beeinflussen. Beispiele für regulatorische Sequenzen sind u. a. Promotoren, Enhancer, Operatoren, Terminatoren oder Translationsverstärker.According to the invention, the (5 'or upstream) and / or subsequent (3' or downstream) sequence regions preceding the coding regions (structural genes) are also included. In particular, 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.
Die vorliegende Erfindung umfaßt ferner eine Genstruktur enthaltend die isolierte Nukleotidsequenz der zuvor genannten Art oder Teile davon, sowie operativ damit verknüpfte Nukleotidsequenzen mit regulatorischer Funktion.The present invention further comprises a gene structure containing the isolated nucleotide sequence of the aforementioned type or parts thereof, as well as nucleotide sequences operatively linked thereto with a regulatory function.
Unter einer operativen Verknüpfung versteht man die sequenzielle Anordnung beispielsweise von Promotor, kodierender Sequenz, Terminator und ggf. weiterer regulatorischer Elemente derart, daß jedes der regulatorischen Elemente seine Funktion bei der Expression der kodierenden Sequenz bestimmungsgemäß erfüllen kann. Diese regulatorischen Nukleotidsequenzen können natürlichen Ursprungs sein oder durch chemische Synthese erhalten werden. Als Promotor ist grundsätzlich jeder Promotor geeignet, der die Genexpression in dem entsprechenden Wirtsorganismus steuern kann. Hierbei kann es sich erfindungsgemäß auch um einen chemisch induzierbaren Promotor handeln, durch den die Expression der ihm unterliegenden Gene in der Wirtszelle zu einem bestimmten Zeitpunkt gesteuert werden kann. Beispielhaft sei hier das ß-Galakosidase- oder Arabinose-System genannt.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. These regulatory nucleotide sequences can be of natural origin or obtained by chemical synthesis. In principle, any promoter which can control gene expression in the corresponding host organism is suitable as the promoter. According to the invention, 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 β-galacosidase or arabinose system may be mentioned here as an example.
Die Herstellung einer Genstruktur erfolgt durch Fusion eines geeigneten Promotors mit wenigstens einer erfindungsgemäßen Nukleotidsequenz nach gängigen Rekombinations- und Klonierungstechniken, wie sie beispielsweise in Sambrook, J. et al., 1989, In Molecular cloning; a laboratory manual. 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York beschrieben sind. Für die Verbindung der DNA-Fragmente miteinander können an die Fragmente Adaptoren oder Linker angesetzt werden.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. To connect the DNA fragments to one another, adapters or linkers can be attached to the fragments.
Erfindungsgemäß umfasst ist auch ein Vektor enthaltend eine isolierte Nukleotidsequenz der zuvor genannten Art oder Teile davon oder eine Genstruktur der zuvor genannten Art, sowie zusätzliche Nukleotidsequenzen zur Selektion, Replikation in der Wirtszelle und/oder Integration in das Wirtszellgenom. Beispiele für solche zusätzlichen Sequenzen sind in der Literatur zahlreich beschrieben und werden nicht weiter ausgeführt. Geeignete Systeme für die Transformationen und Überexpressionen der erwähnten Gene in B. megaterium sind beispielsweise die Plasmide pWH1510 und pWH1520 sowie der plasmidfreie Überexpressionsstamm B. megaterium WH320, die bei Rygus, T. et al. (1991 , Inducible high level expression of heterologous genes in Bacillus megaterium, Appl. Microbiol. And Biotechnol., 35, 5: 594-599) beschrieben sind. Die Plasmide sind auch kommerziell erhältlich (Q-biogene und MoBiTec). Die genannten Systeme sind jedoch nicht limitierend für die vorliegende Erfindung.The invention also includes a vector containing an isolated nucleotide sequence of the aforementioned type or parts thereof or a gene structure of the aforementioned type, as well as additional nucleotide sequences for selection, replication in the host cell and / or integration into the host cell genome. Examples of such additional sequences are widely described in the literature and are not described further. Suitable systems for the transformations and overexpression of the genes mentioned in B. megaterium are, for example, the plasmids pWH1510 and pWH1520 and the plasmid-free overexpression strain B. megaterium WH320, which were 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 plasmids are also commercially available (Q-biogene and MoBiTec). However, the systems mentioned are not limiting for the present invention.
Gegenstand der vorliegenden Erfindung ist auch ein Verfahren, das sich dadurch auszeichnet, daß ein Bacillus megaterium-Stamm fermentiert wird, dessen hemAXCDBL-Operon oder Teile davon verstärkt exprimiert wird/werden.The present invention also relates to a method which is characterized in that a Bacillus megaterium strain is fermented, the hemAXCDBL operon or parts thereof are / are increasingly expressed.
In einer Variante der vorliegenden Erfindung ist auch ein Verfahren umfasst, bei dem ein genetisch veränderter Bacillus megaterium-Stamm fermentiert wird, dessen hemAXCDBL-Operon oder Teile davon gegenüber dem genetisch nicht veränderten Bacillus in erhöhter Kopienzahl in der Zelle vorliegt/vorliegen. Die Anzahl der Kopien kann zwischen 2 bis mehrere hundert variieren.A variant of the present invention also includes a method in which a genetically modified Bacillus megaterium strain is fermented, the hemAXCDBL operon or parts thereof of which is / are present in the cell in an increased number of copies compared to the genetically unmodified Bacillus. The number of copies can vary from two to several hundred.
Zur Erzielung einer erhöhten Genexpression (Überexpression) kann die Kopienzahl der entsprechenden Gene erhöht werden. Ferner kann die Promotor- und/oder Regulationsregion und/oder dieThe number of copies of the corresponding genes can be increased in order to achieve increased gene expression (overexpression). Furthermore, the promoter and / or regulatory region and / or the
Ribosomenbindungsstelle, die sich stromaufwärts des Strukturgens befindet, entsprechend so verändert werden, daß die Expression mit erhöhter Rate erfolgt. In gleicher Weise wirken Expressionskassetten, die stromaufwärts des Strukturgens eingebaut werden. Durch induzierbare Promotoren ist es zusätzlich möglich die Expression im Verlaufe der Vitamin B12-Produktion zu steigern.Ribosome binding site, which is located upstream of the structural gene, are changed accordingly 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.
Durch Maßnahmen zur Verlängerung der Lebensdauer der mRNA wird ebenfalls die Expression verbessert. Die Gene oder Genkonstrukte können entweder in Plasmiden mit unterschiedlicher Kopienzahl vorliegen und/oder im Chromosom integriert und/oder amplifiziert sein.Expression is also improved by measures to extend the life of the mRNA. The genes or gene constructs can either be present in plasmids with a different number of copies and / or integrated and / or amplified in the chromosome.
Weiterhin kann auch die Aktivität des Enzyms selbst erhöht sein oder durch die Verhinderung des Abbaus des Enzymproteins verstärkt werden. Alternativ kann ferner eine Überexpression der betreffenden Gene durch Veränderung der Medienzusammensetzung und Kulturführung erreicht werden.Furthermore, the activity of the enzyme itself can also be increased or increased by preventing the breakdown of the enzyme protein. Alternatively, overexpression of the genes in question can also be achieved by changing the media composition and culture management.
Gegenstand der vorliegenden Erfindung ist ferner ein transformierter Bacillus megaterium-Stamm zum Einsatz in ein Verfahren zur Vitamin- B12-Herstellung der zuvor genannten Art, der eine verstärkte Expression und/oder erhöhte Kopienzahl der Nukleotidsequenz des hemAXCDBL- Operons oder Teile davon aufweist. Erfindungsgemäß umfaßt ist auch ein transformierter Bacillus megaterium- Stamm, der in replizierender Form eine Genstruktur oder einen Vektor der zuvor erwähnten Art enthält.The present invention furthermore relates to a transformed Bacillus megaterium strain for use in a method for producing vitamin B12 of the aforementioned type, which has an increased expression and / or increased copy number of the nucleotide sequence of the hemAXCDBL operon or parts thereof. The invention also encompasses a transformed Bacillus megaterium strain which, in replicating form, contains a gene structure or a vector of the type mentioned above.
Gegenstand der vorliegenden Erfindung ist ferner die Verwendung der isolierten Nukleotidsequenz des hemAXCDBL-Operons oder Teile davon oder der Genstruktur oder eines Vektors der zuvor genannten Art zur Herstellung eines transformierten Bacillus megaterium-Stammes der zuvor erwähnten Art. Die vorliegende Erfindung schießt auch die Verwendung des transformierten Bacillus megaterium-Stammes der zuvor genannte Art zur Herstellung von Vitamin B12 ein. The present invention furthermore relates to the use of the isolated nucleotide sequence of the hemAXCDBL operon or parts thereof or the gene structure or a vector of the aforementioned type for producing a transformed Bacillus megaterium strain of the aforementioned type. The present invention also includes the use of the transformed one Bacillus megaterium strain of the aforementioned type for the production of vitamin B12.
Die nachfolgenden Beispiele dienen der Erläuterung der vorliegenden Erfindung. Sie wirken sich jedoch nicht limitierend auf die Erfindung aus.The following examples serve to illustrate the present invention. However, they have no limiting effect on the invention.
1. Chemikalien und molekularbioloqische Agenzien1. Chemicals and molecular biological agents
DNA-Isolations-Kit QiagenQiagen DNA isolation kit
Fast-Link Ligation Kit BiozymFast-Link Ligation Kit Biozym
Molekularbiologische Enzyme Amersham-Pharmacia, NEN-LifeScienceMolecular biological enzymes Amersham-Pharmacia, NEN-LifeScience
Wachstumsmedien DifcoGrowth media Difco
2. Bakterienstämme und Plasmide2. Bacterial strains and plasmids
Alle in dieser Arbeit verwendeten Bakterienstämme und Plasmide sind in den Tabellen 1 und 2 aufgeführt.All bacterial strains and plasmids used in this work are listed in Tables 1 and 2.
3. Puffer und Lösungen 3.1. Minimalmedium E. coli-Minimalmedium mM3. Buffers and solutions 3.1. Minimal medium E. coli minimal medium mM
KH2PO4 33.1 mMKH 2 PO 4 33.1 mM
(NH )2SO4 7.6 mM(NH) 2 SO 4 7.6 mM
Natriumeitrat 1.7 mMSodium citrate 1.7 mM
MgSO4 1.0 mMMgSO 4 1.0 mM
D-Glucose 10.1 mMD-glucose 10.1 mM
Thiamin 3.0 μMThiamine 3.0 µM
Casaminoacids 0.025Casaminoacids 0.025
% (w/v)% (w / v)
Für feste Medien wurden 15 g/l Agar-Agar zugesetzt.15 g / l agar agar was added for solid media.
Mopso-MinimalmediumMopso minimal medium
Mopso (pH 7.0) 50.0 mMMopso (pH 7.0) 50.0 mM
Tricin (pH 7.0) 5.0 mM MgCI2 520.0 μMTricin (pH 7.0) 5.0 mM MgCI 2 520.0 µM
K2SO4 276.0 μM FeSO4 50.0 μMK 2 SO 4 276.0 μM FeSO 4 50.0 μM
CaCI2 1.0 mMCaCI 2 1.0 mM
MnCI2 100.0 μMMnCI 2 100.0 μM
NaCI 50.0 mMNaCI 50.0 mM
KCI 10.0 mMKCI 10.0 mM
nM nM
C0CI2 300.0 pMC0CI2 300.0 pM
CuSO4 100.0 pM ZnSO4 100.0 pMCuSO 4 100.0 pM ZnSO 4 100.0 pM
D-Glucose 20.2 mMD-glucose 20.2 mM
NH4CI 37.4 mMNH 4 CI 37.4 mM
Titrationsreagenz war KOH-Lösung.Titration reagent was KOH solution.
S. typhimurium-MinimalmediumS. typhimurium minimal medium
NaCI 8.6 mMNaCI 8.6 mM
Na2HPO4 33.7 mMNa 2 HPO 4 33.7 mM
KH2PO4 22.0 mM NH CI 18.7 mMKH 2 PO 4 22.0 mM NH CI 18.7 mM
D-Glucose 20.2 mMD-glucose 20.2 mM
MgSO 2.0 mM CaCI2 0.1 mMMgSO 2.0 mM CaCl 2 0.1 mM
Für feste Medien wurden 15 g/l Agar-Agar zugesetzt.15 g / l agar agar was added for solid media.
3.2. Lösungen zur Protoplastentransformation von ß. megaterium SMMP-Puffer Antibiotic Medium No. 3 (Difco) 17.5 g/ι3.2. Protoplast transformation solutions for ß. megaterium SMMP buffer Antibiotic Medium No. 3 (Difco) 17.5 g / ι
Saccharose 500.0 mMSucrose 500.0 mM
Na-Maleinat (pH 6.5) 20.0 mMNa maleinate (pH 6.5) 20.0 mM
MgCI2 20.0 mM Titrationsreagenz war NaOH-Lösung.MgCI 2 20.0 mM titration reagent was NaOH solution.
PEG-P-LösungPEG-P solution
PEG 6000 40.0PEG 6000 40.0
% (w/v) Saccharose 500.0 mM% (w / v) sucrose 500.0 mM
Na-Maleinat (pH 6.5) 20.0 mMNa maleinate (pH 6.5) 20.0 mM
MgCI2 20.0 mMMgCl 2 20.0 mM
Titrationsreagenz war NaOH-Lösung.The titration reagent was NaOH solution.
cR5 Top-AgarcR5 top agar
Saccharose 300.0 mMSucrose 300.0 mM
Mops (pH 7.3) 31.1 mMPug (pH 7.3) 31.1 mM
NaOH 15.0 mM L-Prolin 52.1 mMNaOH 15.0 mM L-Proline 52.1 mM
D-Glucose 50.5 mMD-glucose 50.5 mM
K2SO4 1.3 mM MgCI2 x 6 H2O 45.3 mMK 2 SO 4 1.3 mM MgCl 2 x 6 H 2 O 45.3 mM
KH2PO4 313.0 μM CaCI2 13.8 mM Agar-Agar 4.0 g/ιKH 2 PO 4 313.0 μM CaCI 2 13.8 mM agar agar 4.0 g / ι
Casaminoacids 0.2 g/lCasaminoacids 0.2 g / l
Hefe-Extrakt 10.0 g/ιYeast extract 10.0 g / ι
Titrationsreagenz war NaOH-Lösung.The titration reagent was NaOH solution.
4. Medien und Medienzusätze 4.1 Medien4. Media and media additions 4.1 Media
Falls nicht gesondert angegeben, wurde mit Vollmedium Luria-Bertani Broth (LB) wie bei Sambrook, J. et al. (1989, In Molecular cloning; a laboratory manual. 2πd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) beschrieben, gearbeitet.Unless otherwise specified, Luria-Bertani Broth (LB) was used with complete medium as in Sambrook, J. et al. (1989, Molecular cloning; a laboratory manual. 2 πd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
4.2. Zusätze4.2. additions
Zusätze wie Kohlenstoffquellen, Aminosäuren oder Antibiotika wurden entweder den Medien zugefügt und zusammen autoklaviert oder als konzentrierte Stammlösungen in Wasser angesetzt und sterilisiert oder sterilfiltriert. Die Substanzen wurden den autoklavierten und auf unter 50°C abgekühlten Medien zugesetzt. Bei lichtempfindlichen Substanzen, wie Tetracyclin wurde auf Inkubation im Dunkeln geachtet. Die üblicherweise verwendeten Endkonzentrationen waren folgende: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:
Ampicillin 296 μMAmpicillin 296 µM
Tetracyclin 21 μM ALA 298 μMTetracycline 21 µM ALA 298 µM
Häm 153 μM X-Gal 98 μMHeme 153 µM X-Gal 98 µM
Methionin 335 μMMethionine 335 µM
Cystein 285 μMCysteine 285 µM
Natriumnitrat 10 mMSodium nitrate 10 mM
Natriumnitrit 10 mM Dinatriumfumarat 10 mMSodium nitrite 10 mM disodium fumarate 10 mM
Glucose 10 mMGlucose 10 mM
Ammoniumchlorid 37 mMAmmonium chloride 37 mM
Xylose 33 mMXylose 33 mM
Lysozym 10 μg/ml Casaminoacids 0.025Lysozyme 10 µg / ml casaminoacids 0.025
% (w/v)% (w / v)
5. Mikrobiologische Techniken 5.1. Sterilisation Soweit nicht anders angegeben wurden sämtliche Medien und Puffer für 20 min bei 120°C und 1 bar Überdruck dampfsterilisiert. Temperaturempfindliche Substanzen wurden sterilfiltriert, Glaswaren mindestens 3 h bei 180°C hitzesterilisiert.5. Microbiological techniques 5.1. Sterilization Unless otherwise stated, all media and buffers were steam sterilized for 20 min at 120 ° C and 1 bar overpressure. Temperature-sensitive substances were sterile filtered, glassware was heat sterilized at 180 ° C for at least 3 h.
5.2.Allgemeine Wachstumsbedingungen5.2.General growth conditions
Aerobe Bakterienkulturen wurden in Schikanekolben bei 37°C und einer minimalen Drehzahl von 180 rpm inkubiert. Die Inkubationszeiten wurden entsprechend den erwünschten optischen Dichten der Bakterienkulturen variiert. 5.3.Bedingungen für Bacillus mepaterium Wachstum Aerobe Kulturen wurden für eine bestmögliche Durchlüftung in Schikanekolben bei 250 rpm und, falls nicht anders angegeben, bei 30 °C inkubiert. Anaerobe Kulturen wurden in einem Volumen von 100 ml in kleinen Anaerobenflaschen bei 30 °C und 100 rpm fermentiert. In beiden Fällen wurde auf die Verwendung qualitativ konstanter Medien, Animpfung im Verhältnis 1 :100 aus Übernachtkulturen, sowie Verwendung gleichbleibender Bedingungen für die Übernachtkulturen geachtet.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 mepaterium 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.
5.4.Bestimmunq der Zelldichte5.4. Determination of cell density
Die Zelldichte einer Bakterienkultur wurde durch Messung der optischen Dichte bei 578 nm bestimmt, wobei davon ausgegangen wurde, daß einer OD 78 von eins eine Zellzahl von 1x109 Zellen entspricht.The cell density of a bacterial culture was determined by measuring the optical density at 578 nm, it being assumed that an OD 78 of one corresponds to a cell number of 1 × 10 9 cells.
5.5. Vergleichende Wachstumsuntersuchungen5.5. Comparative growth studies
Es wurden vergleichende Untersuchungen zum aeroben und anaroben Wachstumsverhalten verschiedener Bacillus megaterium-Stämme durchgeführt, die in den Figuren 1 , 2 und 3 dargestellt sind. Bei den eingesetzten Stämmen handelt es sich um die Stämme B. megaterium DSMZ 32 (Wildtyp) oder die unter aeroben Bedingungen zur Vitamin B12- Produktion geeigneten „Produktionsstämme" DSMZ 509 und DSMZ 2894. Die vorliegende Erfindung ist jedoch nicht auf den Einsatz dieser Stämme limitiert. Denkbar sind auch andere zur Herstellung von Vitamin B12 geeignete Stämme, eingeschlossen genetisch veränderte Bakterienstämme, die durch klassische Mutagenesen oder gezielte molekularbiologische Techniken und entsprechende Selektionsverfahren hergestellt werden können.Comparative studies on the aerobic and anarobic growth behavior of various Bacillus megaterium strains were carried out, which are shown in FIGS. 1, 2 and 3. The strains used are the strains B. megaterium DSMZ 32 (wild type) or the "production strains" DSMZ 509 and DSMZ 2894 which are suitable under aerobic conditions for vitamin B12 production. However, the present invention is not limited to the use of these strains Other strains suitable for the production of vitamin B12 are also conceivable, including genetically modified bacterial strains which can be produced by classic mutagenesis or targeted molecular biological techniques and corresponding selection processes.
Bei den Untersuchungen, inwieweit B. megaterium zum anaeroben Wachstum befähigt ist, wurden anstelle des aeroben Elektronenakzeptors Sauerstoff, die alternativen Elektronenakzeptoren Nitrat, Nitrit und Fumarat zum Kulturmedium zugesetzt (Figuren 4-6). Parallel hierzu wurde untersucht, ob B. megaterium auch zur Fermentation in einem Medium ohne Zusatz dieser Elektronenakzeptoren fähig ist. Aus den Ergebnissen der vorliegenden Erfindung wird dabei deutlich, daß der Elektronenakzeptor Nitrit eine toxische Wirkung auf das Bekterienwachstum ausübt. Auch Fumarat wirkt sich wachstumshemmend aus. Keiner der zugesetzten potentiellen Elektronenakzeptoren kann ein anaerobes Wachstum über das Maß des fermentativen Wachstums hinaus stimulieren.In the investigation of the extent to which B. megaterium is capable of anaerobic growth, instead of the aerobic electron acceptor Oxygen, the alternative electron acceptors nitrate, nitrite and fumarate added to the culture medium (Figures 4-6). In parallel, it was investigated whether B. megaterium is also capable of fermentation in a medium without the addition of these electron acceptors. It is clear from the results of the present invention that the electron acceptor nitrite has a toxic effect on the growth of the keratin. Fumarate also inhibits growth. None of the potential electron acceptors added can stimulate anaerobic growth beyond the level of fermentative growth.
5.6. Vergleichende Untersuchungen zur Vitamin B12 Produktion5.6. Comparative studies on vitamin B12 production
Ferner wurden Analysen zur Vitamin B12 Produktion unter aeroben und anaeroben Wachstumsbedingungen von Bacillus megaterium durchgeführt. Erfindungsgemäß wurden beispielhaft die Stämme Bacillus megaterium DSMZ 32, DSMZ 509 und DSMZ 2894 unter anaeroben Bedingungen in Glukose-haltigem (LB-Voll)-Medium herangezogen und am Ende der exponentiellen Wachstumsphase der Vitamin B12 Gehalt in pmol/OD5 8 ermittelt. Parallel dazu wurde die Vitamin B12 Produktion bei aerober Lebensweise in der Mitte der exponentiellen Wachstumsphase untersucht. Die Ergebnisse sind in Figur 7 wiedergegeben.Analyzes of vitamin B12 production under aerobic and anaerobic growth conditions of Bacillus megaterium were also carried out. According to the invention, the strains Bacillus megaterium DSMZ 32, DSMZ 509 and DSMZ 2894 were used 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 5 8 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. 7.
5.7. Einfluß von 5-Amino-Lävulinsäure (ALA) auf die Vitamin B12 Produktion Da ein zentraler Regulationspunkt im Tetrapyrrolbiosysntheseweg zur Synthese von Vitamin B12 die Bildung von 5-Amino-Lävulinsäure (ALA) ist, wurde erfindungsgemäß der Einfluß von ALA auf die Vitamin B-12 Produktion im Bacillus megaterium untersucht. Die aerobe Vitamin B-12 Produktion von B. megaterium mit und ohne externe Zugabe von ALA ist in Figur 8 dargestellt. Dabei wurden ALA-Konzentrationen von etwa 50 μg/ml Kulturmedium eingesetzt. Erfindungsgemäß konnte gezeigt werden, daß sowohl bei aerober als auch bei anaerober Fermentation des Bakteriums die Zugabe von ALA keinen Effekt auf die Vitamin B-12 Produktion hat. Aufgrund dessen scheint die Regulation der Vitamin B-12 Biosynthese nicht alleine durch die Bildung von ALA limitiert zu sein.5.7. Influence of 5-amino-levulinic acid (ALA) on vitamin B12 production Since a central regulation point in the tetrapyrrole biosynthetic pathway for the synthesis of vitamin B12 is the formation of 5-amino-levulinic acid (ALA), the influence of ALA on vitamin B- 12 Production in the Bacillus megaterium examined. The aerobic vitamin B-12 production of B. megaterium with and without external addition of ALA is shown in FIG. 8. ALA concentrations of approximately 50 μg / ml culture medium were used. According to the invention, it was possible to show that the addition of ALA had no effect on vitamin B-12 production in both aerobic and anaerobic fermentation of the bacterium. Because of this, the regulation of vitamin B-12 biosynthesis does not appear to be limited solely by the formation of ALA.
5.8.Quantitative Vitamin Bip-Analyse5.8.Quantitative Vitamin Bip Analysis
Aerobe ß. megater/um-Kulturen wurden in der Mitte der exponentiellenAerobic ß. Megater / um cultures were in the middle of the exponential
Wachstumsphase, anaerobe Kulturen am Ende der exponentiellen Wachstumsphase nach Bestimmung der OD578 durch 15-minütiges Abzentrifugieren bei 5000 rpm geerntet (Centrifuge 5403, Eppendorf). Nach Waschen mit 40 ml Salzlösung wurde erneut 15 min bei 5000 rpm zentrifugiert (Centrifuge 5403, Eppendorf). Die erhaltenen Zellsedimente wurden abschließend lyophilisiert. S. typhimurium metE cysG Doppelmutanten (Raux, E. et al., 1996, J. Bacteriol., 178: 753-767) wurden über Nacht auf Methionin und Cystein enthaltendem Minimalmedium bei 37 °C inkubiert, von der Platte gekratzt und mit 40 ml isotonischer Salzlösung gewaschen. Nach Abzentrifugieren wurde das Zellsediment wieder in isotonischer Salzlösung resuspendiert. Die gewaschene Bakterienkultur wurde sorgfältig mit 400 ml 47-48 °C warmem, Cystein enthaltendem Minimalmedium-Agar gemischt. 10 μl der in deionisiertem, sterilem Wasser resuspendierten und 15 min im Wasserbad gekochten ß. megater/wm-Proben wurden auf die abgekühlten Platten gegeben und für 18 h bei 37 °C inkubiert. Die Durchmesser der gewachsenen Salmonellenkolonien sind dann proportional dem Gehalt an Vitamin B12 der aufgetragenen ß. megater/'i/m-Proben. Durch Vergleich mit einer Eichkurve, angefertigt aus der Zugabe von 0.01 , 0.1 , 1 , 10 und 40 pmol Vitamin B12, wurde auf den Gehalt an Vitamin B12 in den untersuchten Proben zurückgeschlossen. Diese Standardmethode erlaubt schnell und sehr reproduzierbar den Nachweis geringer Vitamin Bι2- Mengen in biologischen Materialien. 6. Molekularbiologische TechnikenGrowth phase, anaerobic cultures at the end of the exponential growth phase after determining the OD 578 by centrifuging at 5000 rpm for 15 minutes (Centrifuge 5403, Eppendorf). After washing with 40 ml of saline, the mixture was centrifuged again at 5000 rpm for 15 min (Centrifuge 5403, Eppendorf). The cell sediments obtained were finally lyophilized. S. typhimurium metE cysG double mutants (Raux, E. et al., 1996, J. 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 μl of the ß resuspended in deionized, sterile water and boiled in a water bath for 15 min. Megater / wm samples were placed on the cooled plates and incubated for 18 h at 37 ° C. The diameter of the grown salmonella colonies are then proportional to the content of vitamin B 12 in the applied ß. megater / ' i / m samples. By comparison with a calibration curve, made from the addition of 0.01, 0.1, 1, 10 and 40 pmol of vitamin B1 2 , the content of vitamin B 12 in the examined samples was inferred. This standard method allows the detection of small amounts of vitamin B 2 in biological materials quickly and very reproducibly. 6. Molecular biological techniques
Allgemeine Techniken, wie DNA-Präparation, Restriktion von DNA, Ligation, PCR, Sequenzierung, funktioneile Komplementation, Proteinexpression etc gehören zur gängigen Laborpraxis und sind bei Sambrook, J. et al. (1989, In Molecular cloning; a laboratory manual. 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) beschrieben.General techniques such as DNA preparation, restriction of DNA, ligation, PCR, sequencing, functional complementation, protein expression etc. are part of common laboratory practice and are described by Sambrook, J. et al. (1989, In Molecular cloning;.. A laboratory manual 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
6.1.Protoplastentransformation von Bacillus megaterium Protoplastenpreparation:6.1.Protoplast transformation of Bacillus megaterium protoplast preparation:
50 ml LB-Medium wurden mit 1 ml einer Übernachtkultur von ß. megaterium angeimpft und bei 37 °C inkubiert. Bei einer OD5 β von 1 wurden die Zellen bei 15,000 rpm und 4 °C 15 min abzentrifugiert (RC 5B Plus, Sorvall) und in 5 ml SMMP-Puffer resuspendiert. Nach Zugabe von Lysozym in SMMP-Puffer wurde die Suspension 60 min bei 37 °C inkubiert und die Protoplastenbildung unter dem Mikroskop kontrolliert. Nach Ernten der Zellen durch Zentrifugation bei 3000 rpm und Rt (Centrifuge 5403, Eppendorf) wurde das Zellsediment vorsichtig in 5 ml SMMP-Puffer resuspendiert und der Zentrifugations- und Waschschritt ein zweites mal durchgeführt. Die Protoplastensuspension konnte nun, nach Zugabe von 10 % (v/v) Glycerin, portioniert und bei -80 °C eingefroren werden.50 ml LB medium were mixed with 1 ml of an overnight culture of β. inoculated megaterium and incubated at 37 ° C. With an OD 5 β of 1, the cells were centrifuged at 15,000 rpm and 4 ° C. for 15 min (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. After harvesting the cells by centrifugation at 3000 rpm and Rt (Centrifuge 5403, Eppendorf), the cell sediment was carefully resuspended in 5 ml of SMMP buffer and the centrifugation and washing step was carried out a second time. After adding 10% (v / v) glycerol, the protoplast suspension could now be portioned and frozen at -80 ° C.
Transformation:Transformation:
500 μl der Protoplastensuspension wurden mit 0.5 bis 1 μg DNA in SMMP-Puffer versetzt und 1.5 ml PEG-P-Lösung zugegeben. Nach Inkubation bei Rt für 2 min wurden 5 ml SMMP-Puffer hinzugefügt, vorsichtig gemischt und die Suspension bei 3000 rpm und Rt für 10 min zentrifugiert (Centrifuge 5403, Eppendorf). Sofort danach wurde der Überstand abgenommen und das kaum sichtbare Sediment in 500 μl SMMP-Puffer resuspendiert. Die Suspension wurde 90 min bei 37 °C unter leichtem Schütteln inkubiert. Danach wurden 50 - 200 μl der transformierten Zellen mit 2.5 ml cR5-Topagar gemischt und auf LB-Agar- Platten gegeben, die die für die Selektion geeigneten Antibiotika enthielten. Transformierte Kolonien wurden nach zweitägiger Inkubation bei 37 °C sichtbar.500 μl of the protoplast suspension were mixed with 0.5 to 1 μg 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 hardly visible sediment in 500 μl SMMP buffer resuspended. The suspension was incubated at 37 ° C. for 90 min with gentle shaking. 50-200 μl of the transformed cells were then mixed with 2.5 ml of cR5 top agar and placed on LB agar plates which contained the antibiotics suitable for the selection. Transformed colonies became visible after two days of incubation at 37 ° C.
6.2. Identifizierung und Seguenzierung des hemAXCDBL-Operons aus B. megaterium Zur Klonierung des hemAXCDBL-Operons wurde die Strategie der funktioneilen Komplementation häm-auxotropher E. coli-Mutanten gewählt. Dazu wurde eine B. megaterium-Genbank nach gängigen Methoden hergestellt. Durch funktioneile Komplementation der E. coli hemB-Mutante RP523 mit dieser genomischen B. megaterium Plasmidgenbank konnten Kolonien isoliert werden, die wieder zur vollkommenen Tetrapyrrolbiosynthese befähigt waren, also phänotypisch den hämprototrophen Wildtyp darstellten. Nach Plasmid-DNA-Präparation und DNA-Sequenzierung der Vektor-Iokalisierten B. megaterium-DNA konnte ein 3855 kb großes Insert identifiziert werden, das für das gesuchte hemAXCDBL Operon codiert. Die entsprechende Nukleotidsequenz ist in SEQ ID No. 1 und die davon abgeleiteten Aminosäuresequenzen in SEQ ID No. 2-6 aufgeführt. Die Sequenzen stromauf- und stromabwärts des durch funktioneile Komplementation erhaltenen DNA-Fragments wurden mit Hilfe des VectoretteTM-Systems der Firma Sigma Genosis erhalten. Hierzu wurde die Turbo-Pfu-DNA-Polymerase der Firma Strategene verwendet, die aufgrund ihrer proofreading Funktion eine äußerst geringe Fehlerquote aufweist.6.2. Identification and sequencing of the hemAXCDBL operon from B. megaterium. The strategy of functional complementation of heme auxotrophic E. coli mutants was chosen to clone the hemAXCDBL operon. For this purpose, a B. megaterium gene bank was produced using common methods. By functional complementation of the E. coli hemB mutant RP523 with this genomic B. megaterium plasmid library, it was possible to isolate colonies which were again capable of complete tetrapyrrole biosynthesis, that is to say phenotypically represented the hematoprotrophic wild type. After plasmid DNA preparation and DNA sequencing of the vector-localized B. megaterium DNA, a 3855 kb insert could be identified which codes for the hemAXCDBL operon sought. The corresponding nucleotide sequence is in SEQ ID No. 1 and the amino acid sequences derived therefrom in SEQ ID No. 2-6 listed. The sequences upstream and downstream of the DNA fragment obtained by functional complementation were obtained using the Vectorette ™ system from Sigma Genosis. For this purpose, the Turbo Pfu DNA polymerase from Strategene was used, which has an extremely low error rate due to its proofreading function.
6.3. Transformations- und Expressionssysteme Geeignete Plasmide zur Transformation und Überexpression von Genen in B. megaterium sind pWH1510 und PWH1520 sowie der plasmidfreie Überexpressionsstamm 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).6.3. Transformation and expression systems 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).
Das Kontrollplasmid pWH1510 enthält im unterbrochenen xylA-Leseraster eine spoVG-lacZ Fusion. SpoVG-lacZ bezeichnet dabei die Fusion aus einer sehr starken Ribosomen-Bindesequenz eines Sporulationsproteins aus B. subtilis (spoVG) mit dem für die ß-Galaktosidase codierenden Gen (lacZ) aus E. coli. Dieses Plasmid ist damit hervorragend geeignet zur Untersuchung von Transformationseffizienzen undThe 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 β-galactosidase (lacZ) from E. coli. This plasmid is therefore ideally suited for the study of transformation efficiencies and
Überexpressionsbedingungen bei B. megaterium. Das Plasmid pWH1520 fungiert als eigentlicher Klonierungs- und Expressionsvektor. Beide Vektoren weisen ein Tetracyclin- und eine Ampicillinresistenz sowie die für die Replikation in E. coli und Bacillus spp wichtigen Elemente auf. Damit sind sie zugänglich für alle in E. coli etablierten Techniken für Abkömmlinge des Plasmids pBR322. Beiden Vektoren enthalten die B. megaterium xylA- und xylR- Gene des xyl Operons mit ihren regulatorischen Sequenzen (Rygus, T. et al., 1991 , Molecular Cloning, Structure; Promoters and Regulatory Elements for Transcription of the Bacillus megaterium Encoded Regulon for Xylose Utilization, Arch. Microbiol. 155, 535-542). XylA codiert für die Xyloseisomerase, während xylR für ein Regulatorprotein codiert, das eine starke transkriptionelle Kontrolle auf xylA ausübt. XylA wird reprimiert in Abwesenheit von Xylose. Bei Zugabe von Xylose kommt es zu einer ca. 200-fachen Induktion durch Derepression von xylA. Mit Hilfe eines Polylinkers im xylA-Leseraster wird eine Fusion von Genen mit xylA möglich, die dann ebenfalls unter der starken transkriptioneilen Kontrolle von XylR stehen. Dabei kann man zwischen den Alternativen zur Bildung einer Transkriptions- oder Translationsfusion wählen, da das xylA- Leseraster stromaufwärts des Polylinkers noch vollkommen intakt ist. Overexpression conditions in B. megaterium. 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 Regulatory Elements for Transcription of the Bacillus megaterium Encoded Regulon for Xylose Utilization, Arch. Microbiol. 155, 535-542). XylA encodes xylose isomerase, while xylR encodes a regulatory protein that exerts strong transcriptional control over xylA. XylA is repressed in the absence of xylose. When xylose is added, there is an approximately 200-fold induction due to the depression of xylA. 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 education choose a transcription or translation fusion because the xylA reading frame upstream of the polylinker is still completely intact.
Legende zu den FigurenLegend to the figures
Es wurden Bakterienstämme und Plasmide gemäß den Tabellen 1 und 2 eingesetzt. Tabelle 1 : Verwendete Bakterienstämme Tabelle 2: Verwendete PlasmideBacterial strains and plasmids according to Tables 1 and 2 were used. Table 1: Bacterial strains used Table 2: Plasmids used
Im folgenden wird die vorliegende Erfindung anhand der Figuren noch erläutert.In the following, the present invention will be explained with reference to the figures.
Figur 1 zeigt einen Vergleich des Wachstums von ß. megaterium DSMZ32 (Wildtyp) bei 30 °C unter aeroben sowie anaeroben Bedingungen. Anaerobes Wachstum wurde unter Zugabe von 10 mM Nitrat (leere Rauten), 10 mM Nitrit (leere Dreiecke) und 10 mM Fumarat (Kreuze) gemessen. Fermentatives (leere Kreise) und aerobes Wachstum (gefüllte Rauten) fanden in LB-Medium ohne Zusätze statt. Zu angegebenen Zeitpunkten wurden Proben genommen und die optische Dichte bei 578 nm bestimmt.Figure 1 shows a comparison of the growth of β. 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.
Figur 2 zeigt einen Vergleich des Wachstums von ß. megaterium DSM509 bei 30 °C unter aeroben sowie anaeroben Bedingungen. Anaerobes Wachstum wurde unter Zugabe von 10 mM Nitrat (leere Rauten), 10 mM Nitrit (leere Dreiecke) und 10 mM Fumarat (Kreuze) gemessen. Fermentatives (leere Kreise) und aerobes Wachstum (gefüllte Rauten) fanden in LB-Medium ohne Zusätze statt. Zu angegebenen Zeitpunkten wurden Proben genommen und die optische Dichte bei 578 nm bestimmt.Figure 2 shows a comparison of the growth of β. 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.
Figur 3 zeigt einen Vergleich des Wachstums von ß. megateriumFigure 3 shows a comparison of the growth of β. megaterium
DSM2894 bei 30 °C unter aeroben sowie anaeroben Bedingungen. Anaerobes Wachstum wurde unter Zugabe von 10 mM Nitrat (leereDSM2894 at 30 ° C under aerobic and anaerobic conditions. Anaerobic growth was carried out with the addition of 10 mM nitrate (empty
Rauten), 10 mM Nitrit (leere Dreiecke) und 10 mM Fumarat (Kreuze) gemessen. Fermentatives (leere Kreise) und aerobes Wachstum (gefüllte Rauten) fanden in LB-Medium ohne Zusätze statt. Zu angegebenen Zeitpunkten wurden Proben genommen und die optische Dichte bei 578 nm bestimmt.Diamonds), 10 mM nitrite (empty triangles) and 10 mM fumarate (crosses) measured. 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.
Figur 4 zeigt anaerobes Wachstum von ß. megaterium DSM32 (Wildtyp) bei 30 °C unter Zusatz von 10 mM Nitrat (Rauten), 10 mM Nitrit (Dreiecke) und 10 mM Fumarat (Kreuze). Fermentatives Wachstum (Kreise) fand in LB-Medium ohne Zusätze statt. Zu angegebenen Zeitpunkten wurden Proben genommen und die optische Dichte bei 578 nm bestimmt.Figure 4 shows anaerobic growth of β. Megaterium DSM32 (wild type) at 30 ° C with the addition of 10 mM nitrate (diamonds), 10 mM nitrite (triangles) and 10 mM fumarate (crosses). Fermentative growth (circles) took place in LB medium without additives. Samples were taken at the specified times and the optical density at 578 nm was determined.
Figur 5 zeigt anaerobes Wachstum von ß. megaterium DSM509 bei 30 °C unter Zusatz von 10 mM Nitrat (Rauten), 10 mM Nitrit (Dreiecke) und 10 mM Fumarat (Kreuze). Fermentatives Wachstum (Kreise) fand in LB- Medium ohne Zusätze statt. Zu angegebenen Zeitpunkten wurden Proben genommen und die optische Dichte bei 578 nm bestimmt.Figure 5 shows anaerobic growth of β. Megaterium DSM509 at 30 ° C with the addition of 10 mM nitrate (diamonds), 10 mM nitrite (triangles) and 10 mM fumarate (crosses). Fermentative growth (circles) took place in LB medium without additives. Samples were taken at the specified times and the optical density at 578 nm was determined.
Figur 6 zeigt anaerobes Wachstum von ß. megaterium DSM2894 bei 30 °C unter Zusatz von 10 mM Nitrat (Rauten), 10 mM Nitrit (Dreiecke) und 10 mM Fumarat (Kreuze). Fermentatives Wachstum (Kreise) fand in LB- Medium ohne Zusätze statt. Zu angegebenen Zeitpunkten wurden Proben genommen und die optische Dichte bei 578 nm bestimmt.Figure 6 shows anaerobic growth of β. Megaterium DSM2894 at 30 ° C with the addition of 10 mM nitrate (diamonds), 10 mM nitrite (triangles) and 10 mM fumarate (crosses). Fermentative growth (circles) took place in LB medium without additives. Samples were taken at the specified times and the optical density at 578 nm was determined.
Figur 7 zeigt die Vitamin B-ι2-Produktion von ß. megaterium unter aeroben und anaeroben Wachstumsbedingungen. Es wurde der Gehalt an Vitamin B12 pro Zellmasse angegeben in pmol/ODs 8 für den Wildtypstamm ß. megaterium DSM32 aerob gewachsen (1) und anaerob gewachsen (2), für ß. megaterium DSM509 aerob gewachsen (3) und anaerob gewachsen (4), sowie für ß. megaterium DSM2894 aerob gewachsen (5) und anaerob gewachsen (6), bestimmt. Figur 8 zeigt die aerobe Vitamin B-ι2-Produktion von ß. megaterium mit und ohne externe Zugabe von 50 μg/ml ALA. Es wurde der Gehalt an Vitamin B12 pro Zellmasse, gemessen in pmol/OD578, für den Wildtypstamm ß. megaterium DSM32 ohne ALA-Zugabe (1), mit ALA- Zugabe (2), für ß. megaterium DSM509 ohne ALA-Zugabe (3), mit ALA- Zugabe (4), sowie für ß. megaterium DSM2894 ohne ALA-Zugabe (5) und mit ALA-Zugabe (6), bestimmt. Figure 7 shows the vitamin B-ι 2 production of ß. megaterium under aerobic and anaerobic growth conditions. The content of vitamin B 12 per cell mass was given in pmol / ODs 8 for the wild-type strain β. megaterium DSM32 aerobically grown (1) and anaerobically grown (2), for ß. megaterium DSM509 aerobically grown (3) and anaerobically grown (4), as well as for ß. megaterium DSM2894 grown aerobically (5) and grown anaerobically (6). Figure 8 shows the aerobic vitamin B-ι 2 production of ß. megaterium with and without external addition of 50 μg / ml ALA. The content of vitamin B 12 per cell mass, measured in pmol / OD 5 78, was determined for the wild-type strain β. megaterium DSM32 without ALA addition (1), with ALA addition (2), for ß. megaterium DSM509 without ALA addition (3), with ALA addition (4), and for ß. megaterium DSM2894 without ALA addition (5) and with ALA addition (6).
Tabelle 1 :Table 1 :
Stamm Beschreibung Referenz/QuelleStrain description reference / source
Escherichia coliEscherichia coli
DH5α Fλ'supE44Δ(argF- Sambrook er a/., 1989 lac)U169φ89dlacZΔM15hsdRDH5α Fλ ' supE44Δ (argF-Sambrook er a /., 1989 lac) U169φ89dlacZΔM15hsdR
11recA 1endA 1gyrA96thi-11recA 1endA 1gyrA96thi-
1relA11relA1
RP523 Fλ'supE44thr-1leuB6thi- Li et al., 1lacY1tonA21hemBRP523 Fλ ' supE44thr-1leuB6thi- Li et al., 1lacY1tonA21hemB
Bacillus megateriumBacillus megaterium
DSMZ32 Wildtyp DSMZ * DSMZ32 wild type DSMZ *
DSMZ509 Vitamin Bi2-Produzent DSMZ *DSMZ509 Vitamin Bi 2 producer DSMZ *
DSMZ2894 Riboflavin- und Vitamin B-ι2- DSMZ * ProduzentDSMZ2894 Riboflavin and Vitamin B-ι 2 - DSMZ * producer
WH320 DSMZ319 lac Rygus eWH320 DSMZ319 lac Rygus e
Sαlmonellα typhimuriumSαlmonellα typhimurium
AR 3612 Leu+ cysG metE, Smr Ra x et al., 1996AR 3612 Leu + cysG metE, Sm r Rax et al., 1996
* DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig Tabelle 2 * DSMZ: German collection of microorganisms and cell cultures, Braunschweig Table 2
Plasmid Beschreibung Referenz/QuellePlasmid Description Reference / Source
pBlueskript II SK+ Phagemid Klonierungs- und StratagenepBluescript II SK + Phagemid cloning and stratagens
Expressionsvektor mit sehr hoher Kopienzahl, Apr pWH1510 xylA-lacZ, Apr, Tcr Rygus et al.,Very high copy number expression vector, Ap r pWH1510 xylA-lacZ, Ap r , Tc r Rygus et al.,
1991 pWH1520 Klonierungs- und Expressionsvektor für Rygus et al., Bacillus spp., Apr, Tcr 1991 1991 pWH1520 cloning and expression vector for Rygus et al., Bacillus spp., Ap r , Tc r 1991

Claims

Ansprüche: Expectations:
1. Verfahren zur Herstellung von Vitamin B12 mittels einer Kultur enthaltend Bacillus megaterium, dadurch gekennzeichnet, daß die Fermentation unter anaeroben Bedingungen durchgeführt wird.1. A process for the preparation of vitamin B12 by means of a culture containing Bacillus megaterium, characterized in that the fermentation is carried out under anaerobic conditions.
2. Verfahren gemäß Anspruch 1 , dadurch gekennzeichnet, daß B. megaterium zunächst aerob und anschließend anaerob fermentiert wird.2. The method according to claim 1, characterized in that B. megaterium is fermented first aerobically and then anaerobically.
Verfahren gemäß einem der Ansprüche 1 oder 2, dadurch gekennzeichnet, daß der Übergang von der aeroben zur anaeroben Fermentation in der exponentiellen Wachstumsphase der aerob fermentierten Zellen erfolgt.Method according to one of claims 1 or 2, characterized in that the transition from aerobic to anaerobic fermentation takes place in the exponential growth phase of the aerobically fermented cells.
4. Verfahren gemäß einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß der Übergang von der aeroben zur anaeroben Fermentation in der Mitte oder am Ende, bevorzugt am Ende der exponentiellen Wachstumsphase der aerob wachsenden Zellen erfolgt.4. The method according to any one of claims 1 to 3, characterized in that 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.
5. Verfahren gemäß einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, daß dem Kulturmedium wenigstens Cobalt zugesetzt wird.5. The method according to any one of claims 1 to 4, characterized in that at least cobalt is added to the culture medium.
Verfahren gemäß einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, daß ein Bacillus megaterium-Stamm fermentiert wird, dessen hemAXCDBL-Operon oder Teile davon verstärkt exprimiert wird. Method according to one of claims 1 to 5, characterized in that a Bacillus megaterium strain is fermented, the hemAXCDBL operon or parts thereof is expressed more intensely.
7. Verfahren gemäß einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß ein Bacillus megaterium-Stamm fermentiert wird, dessen hemAXCDBL-Operon in erhöhter Kopienzahl in der Zelle vorliegt.7. The method according to any one of claims 1 to 6, characterized in that a Bacillus megaterium strain is fermented, the hemAXCDBL operon is present in the cell in an increased number of copies.
8. Isolierte Nukleotidsequenz kodierend für die an der Biosynthese von Uroporphyrinogen III beteiligten Enzyme gemäß SEQ ID No. ID No. 2-6 oder deren Isoformen organisiert in dem hemAXCDBL- Operon mit einer Nukleotidsequenz gemäß SEQ ID No. 1 oder deren Allele.8. Isolated nucleotide sequence coding for the enzymes involved in the biosynthesis of uroporphyrinogen III according to SEQ ID No. ID No. 2-6 or their isoforms organized in the hemAXCDBL operon with a nucleotide sequence according to SEQ ID No. 1 or their alleles.
9. Genstruktur enthaltend die isolierte Nukleotidsequenz gemäß Anspruch 8 oder Teile davon sowie operativ damit verknüpfte Nukleotidsequenzen mit regulatorischer Funktion.9. gene structure containing the isolated nucleotide sequence according to claim 8 or parts thereof and operatively linked nucleotide sequences with regulatory function.
10. Vektor enthaltend eine isolierte Nukleotidsequenz gemäß Anspruch 8 oder Teile davon oder eine Genstruktur gemäß Anspruch 9 sowie zusätzliche Nukleotidsequenzen zur Selektion, Replikation in der Wirtszelle und/oder Integration in das Wirtszell-Genom.10. Vector containing an isolated nucleotide sequence according to claim 8 or parts thereof or a gene structure according to claim 9 and additional nucleotide sequences for selection, replication in the host cell and / or integration into the host cell genome.
,.
11. Transformierter Bacillus megaterium-Stamm zum Einsatz in ein11. Transformed Bacillus megaterium strain for use in a
Verfahren zur Vitamin-B12-Herstellung gemäß einem derProcess for the production of vitamin B12 according to one of the
Ansprüche 1 bis 7, dadurch gekennzeichnet, daß er eine verstärkteClaims 1 to 7, characterized in that it is a reinforced
Expression und/oder erhöhte Kopienzahl der Nukleotidsequenz gemäß Anspruch 8 oder Teile davon aufweist.Expression and / or increased copy number of the nucleotide sequence according to claim 8 or parts thereof.
12. Transformierter Bacillus megaterium-Stamm gemäß Anspruch 11 , dadurch gekennzeichnet, daß er in replizierender Form eine Genstruktur gemäß Anspruch 9 oder einen Vektor gemäß Anspruch 10 enthält. 3712. Transformed Bacillus megaterium strain according to claim 11, characterized in that it contains in replicating form a gene structure according to claim 9 or a vector according to claim 10. 37
13. Verwendung der isolierten Nukleotidsequenz gemäß Anspruch 8 oder Teile davon oder der Genstruktur gemäß Anspruch 9 oder eines Vektors gemäß Anspruch 10 zur Herstellung eines transformierten Bacillus megaterium-Stammes gemäß einem der Ansprüche 11 oder 12.13. Use of the isolated nucleotide sequence according to claim 8 or parts thereof or the gene structure according to claim 9 or a vector according to claim 10 for the production of a transformed Bacillus megaterium strain according to one of claims 11 or 12.
14. Verwendung des transformierten Bacillus megaterium-Stammes gemäß einem der Ansprüche 11 oder 12 zur Herstellung von Vitamin B12. 14. Use of the transformed Bacillus megaterium strain according to one of claims 11 or 12 for the production of vitamin B12.
EP02796247A 2001-08-22 2002-08-20 Method for producing vitamin b12 Withdrawn EP1421202A2 (en)

Applications Claiming Priority (5)

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DE10141132 2001-08-22
DE10141132 2001-08-22
DE10150359A DE10150359A1 (en) 2001-08-22 2001-10-11 Process for the production of vitamin B12
DE10150359 2001-10-11
PCT/EP2002/009272 WO2003018826A2 (en) 2001-08-22 2002-08-20 Method for producing vitamin b12

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DE10300719A1 (en) * 2003-01-11 2004-07-22 Basf Ag Improved process for the production of vitamin B12
WO2005105999A1 (en) * 2004-04-01 2005-11-10 Basf Aktiengesellschaft Improved method for the production of vitamin b12
CN101748177B (en) * 2008-12-09 2013-06-12 华东理工大学 Optimized method for producing vitamin B12 through pseuomonas denitrifican fermentation and synthetic medium
CN103103235B (en) * 2010-02-05 2014-07-30 冯胜利 Preparation method for vitamin B12
CN107365718B (en) * 2017-04-27 2021-02-02 延边大学 Bacillus megaterium MYB3 and application thereof in straw fermented feed

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US2576932A (en) * 1950-02-01 1951-12-04 John A Garibaldi Fermentation process for production of vitamin b12
FR2780733A1 (en) * 1998-06-25 2000-01-07 Gist Brocades Bv POLYNUCLEOTIDE, VECTOR AND HOST CELL CONTAINING SAME, CORRESPONDING POLYPEPTIDE, PROCESS FOR PRODUCTION THEREOF AND USE THEREOF

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