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EP1421202A2 - Procede pour produire de la vitamine b12 - Google Patents

Procede pour produire de la vitamine 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Withdrawn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)
English (en)
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
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE10150359A external-priority patent/DE10150359A1/de
Application filed by BASF SE filed Critical BASF SE
Publication of EP1421202A2 publication Critical patent/EP1421202A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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.

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Abstract

La présente invention concerne un procédé pour produire de la vitamine B12 au moyen de Bacillus megaterium.
EP02796247A 2001-08-22 2002-08-20 Procede pour produire de la vitamine b12 Withdrawn EP1421202A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE10141132 2001-08-22
DE10141132 2001-08-22
DE10150359A DE10150359A1 (de) 2001-08-22 2001-10-11 Verfahren zur Herstellung von Vitamin B12
DE10150359 2001-10-11
PCT/EP2002/009272 WO2003018826A2 (fr) 2001-08-22 2002-08-20 Procede pour produire de la vitamine b12

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EP1421202A2 true EP1421202A2 (fr) 2004-05-26

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US (1) US20040235120A1 (fr)
EP (1) EP1421202A2 (fr)
JP (1) JP2005500852A (fr)
CN (1) CN1636066A (fr)
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DE10300719A1 (de) * 2003-01-11 2004-07-22 Basf Ag Verbessertes Verfahren zur Herstellung von Vitamin B12
WO2005105999A1 (fr) * 2004-04-01 2005-11-10 Basf Aktiengesellschaft Procede ameliore de production de vitamine b12
CN101748177B (zh) * 2008-12-09 2013-06-12 华东理工大学 优化的脱氮假单胞菌发酵生产维生素b12方法与合成培养基
CN103103235B (zh) * 2010-02-05 2014-07-30 冯胜利 一种维生素b12的制备方法
CN107365718B (zh) * 2017-04-27 2021-02-02 延边大学 巨大芽孢杆菌myb3及其在秸秆发酵饲料中的应用

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US2576932A (en) * 1950-02-01 1951-12-04 John A Garibaldi Fermentation process for production of vitamin b12
FR2780733A1 (fr) * 1998-06-25 2000-01-07 Gist Brocades Bv Polynucleotide, vecteur et cellule hote le contenant, polypeptide correspondant, procede pour leur production et leur utilisation

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JP2005500852A (ja) 2005-01-13
CA2458187A1 (fr) 2003-03-06
US20040235120A1 (en) 2004-11-25
WO2003018826A2 (fr) 2003-03-06
WO2003018826A3 (fr) 2003-11-13

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