Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/42—Cobalamins, i.e. vitamin B12, LLD factor

Definitions

• the present invention a method for the production of vitamin B12 using a genetically modified Bacillus megaterium strain and vectors for the production of genetically modified bacteria of the genus Bacillus.
• 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 ⁇ 2 could be cleaned 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 ⁇ 2 The naturally occurring end products of vitamin B ⁇ 2 biosynthesis are 5 ⁇ -deoxyadenosyIcobalamin (coenzyme B- ⁇ 2 ) and methylcobalamin (MeCbl), while vitamin B 12 by definition stands for cyanocobalamin (CNCbl), which is the one mainly manufactured by industry and represents traded form.
• CNCbl cyanocobalamin
• vitamin B 2 is the same for the designation of all three analog molecules.
• B. megaterium The species ß. megaterium was first described by De Bary over 100 years ago (1884). Although generally classified as a soil bacterium, B. megaterium can also be found in various other habitats such as sea water, sediments, rice, dried meat, milk or honey. It often occurs in the company of pseudomonas and actinomycetes. B. megaterium, like its close relative Bacillus subtilis, is a gram-positive bacterium and is characterized, among other things, by its relatively pronounced, eponymous size of 2x5 ⁇ m, a G + C content of approx. 38% and a very pronounced one Ability to sporulate.
• ß. megaterium can be equated with the pseudomonas without restriction (Vary, PS, 1994, Microbiology, 40, 1001-1013, Prime time for Bacillus megaterium).
• 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 ⁇ - and ß-amylase. It is also with ß. megaterium because of its size possible to accumulate a high biomass up to a too high one Population density leads to death. Of paramount importance in industrial production using ß.
• megaterium is already used today in a variety of industrial applications, such as the production of ⁇ - and ß-amylase, penicillin amidase, the processing of toxic waste or the aerobic vitamin B- ⁇ 2 production (summarized in Vary, PS, 1994, Microbiology , 40, 1001-1013, Prime time for Bacillus megaterium).
• the object of the present invention is to provide genetically modified Bacillus megaterium strains with which the production of vitamin B12 can be further improved.
• This also requires the provision of suitable vectors which allow overexpression of the enzymes for the formation of uroporphyinogen-III from glutamyl tRNA and advantageously a repression of the heme biosynthetic pathway combined with an increased metabolic flow towards vitamin B12.
• the vectors according to the invention should make it possible to stably integrate the desired genetic changes into the chromosome of the bacterial strain.
• an induction of the gene expression of the chromosomally encoded hemAXCDBL operon and / or the repression of the heme biosynthetic pathway should be controllable in the course of the fermentation.
• the problem is solved by providing a genetically modified Bacillus megaterium strain containing a gene hemA [KK] according to SEQ ID No. 4 coding for a feedback-resistant glutamyl tRNA reductase and / or part of the nucleotide sequence according to SEQ ID No. 1 (hemZ) as antisense RNA (ashemZ).
• a genetically modified Bacillus megaterium strain which has a gene hemA [KK] according to SEQ ID No. 4 coding for a feedback-resistant glutamyl tRNA synthase, organized in a hemA [KK] XCDBL operon and / or an antisense RNA (ashemZ) according to SEQ ID No. 3 contains.
• the present invention also includes a nucleotide sequence as shown in SEQ ID No. 1 coding for a coproporphyrinogen III oxidase.
• This nucleotide sequence according to the invention is further distinguished by the fact that it contains upstream (5 'or upstream) and / or downstream (3' or downstream) sequences with a regulatory function for the region of the hemZ gene coding for a coproporphyrinogen III oxidase ,
• sequences with a regulatory function are understood to be those sequences which transcribe, RNA stability or that can affect RNA processing and translation.
• regulatory sequences include promoters, enhancers, operators, terminators or translation enhancers. However, this list is not limiting for the present invention.
• the nucleotide sequence according to the invention preferably comes from SEQ ID No. 1 from Bacillus megaterium.
• the present invention also relates to so-called isolated nucleic acids.
• 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 can contain natural, chemically synthesized, modified or artificial nucleotides.
• the term DNA polymer also includes genomic DNA, cDNA or mixtures thereof.
• a coproporphyrinogen III oxidase according to SEQ ID No. 2 Subject of the present invention.
• the amino acid sequence according to SEQ ID No. is preferred. 2 by a nucleotide sequence according to SEQ ID No. 1 encoded.
• alleles are to be understood as functionally equivalent, ie essentially equivalent nucleotide sequences.
• Function-equivalent sequences are those sequences which, despite a different nucleotide sequence, for example due to the degeneracy of the genetic code, still have the desired functions.
• Functional equivalents thus include naturally occurring variants of the sequences described here, as well as artificial, e.g. B. obtained by chemical synthesis and possibly adapted to the codon use of the host organism nucleotide sequences.
• functionally equivalent sequences include those which have a modified nucleotide sequence which gives the enzyme, for example, a desensitivity or resistance to inhibitors.
• all the usual B. megaterium strains which are suitable as vitamin B12 production strains can be used.
• vitamin B12 production strains are to be understood as Bacillus megaterium strains or homologous microorganisms which have been modified by classic and / or molecular genetic methods in such a way that their metabolic flow increasingly runs in the direction of the biosynthesis of vitamin B12 or its derivatives ( metabolic engineering).
• these production strains one or more genes and / or the corresponding enzymes, which are at key and correspondingly complexly regulated key positions in the metabolic pathway (bottleneck), are changed or even deregulated.
• the present invention encompasses all known vitamin B12 production strains of the genus Bacillus or homologous organisms.
• the strains advantageous according to the invention include, in particular, the strains of B. megaterium DSMZ32, DSMZ 509 and DSMZ 2894.
• Bacterial strains genetically modified according to the invention can in principle be produced by classical mutagenesis and preferably by targeted molecular biological techniques and corresponding selection processes.
• 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 controlled in the direction of maximum vitamin B 12 production.
• Targeted modifications of genes involved in the regulation of the metabolic flow also include studies 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 of the DNA, mRNA or the proteins encoded by them for example by reducing or preventing degradation by nucleases or proteases, is included according to the invention.
• the hemA [KK] gene according to SEQ ID No. is in the genetically modified Bacillus megaterium strain. 4 integrated into the bacterial chromosome.
• Another variant of a genetically modified Bacillus megaterium strain is characterized in that part of the hemZ gene is plasmid-encoded as antisense RNA (ashemZ) in this bacterium and is present in an increased number of copies.
• part of the hemZ gene is to be understood to mean that, starting from the nucleotide sequence of the hemZ gene according to SEQ ID No. 1, the production of various antisense RNAs possible.
• Procedures for making antisense RNA e.g. via PCR, are known to the person skilled in the art and belong to common laboratory practice. The differences can result, for example, from the length of the antisense RNAs produced or from the selection of the regions of the hemZ nucleotide sequence from which the antisense RNA (s) are derived.
• the length of the antisense mRNA sequences can vary, for example, between a few nucleotides and the entire sequence section of the coding region.
• An antisense RNA (ashemZ) according to SEQ ID No. is preferred according to the invention.
• the increased number of copies may be due to an increased replication of a corresponding vector, resulting in an increased number of copies.
• an increased number of copies can also be achieved by multiple integration of a gene or parts thereof into the bacterial chromosome.
• the invention also includes a genetically modified Bacillus megaterium strain in which the hemA [KK] gene is incorporated into the bacterial Chromosome is integrated and part of the hemZ gene is present as an antisense RNA (ashemZ) in an increased number of copies.
• the present invention also relates to a genetically modified Bacillus megaterium strain in which the hemA [KK] gene, organized in the hemA [KK] XCDBL operon, and / or the part of the hemZ-
• promoter stands. Examples of inducible promoters are the xylose inducible XylA promoter or the beta-galactosidase inducible promoter (Miller, J.H., 1972, Experiments in Molecular Genetics, Cold Spring
• the xylose-inducible promoter is preferably the xylA promoter of the xylose operon from pWH1520 (Rygus, T. et al., 1991,
• xylose to the culture medium can increase the initiation of the transcription of the genes which are under the control of the xylA promoter, that is to say here the gene expression of hemA [KK] XCDBL and / or ashemZ.
• Suitable vectors according to the invention are constructed for the production of the genetically modified Bacillus megaterium strains described above, which are also the subject of the present invention.
• the present invention thus comprises an integrative vector containing a gene hemA [KK] coding for a feedback-resistant glutamyl-tRNA reductase according to SEQ ID No. 4 and sequences operatively linked thereto for induced gene expression, selection, replication and / or integration into the chromosome of the host cell.
• An integrative vector is to be understood as a vector which, by site-specific recombination at a defined point in the
• Host cell chromosome is integrated and there together with the chromosome replicated. In a variant according to the invention, this site-specific recombination takes place via the homologous sequences of the hemA gene.
• 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 stringent conditions known per se.
• homologous sequences can be isolated from other organisms on the basis of the DNA sequence described in SEQ ID NO: 4 or parts of these sequences, for example using conventional hybridization methods or the PCR technique. These DNA sequences hybridize to the sequences mentioned under standard conditions. Short oligonucleotides, for example the conserved regions, which can be determined by comparing them with other hemA genes in a manner known to the person skilled in the art, are advantageously used for hybridization. However, longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization. These standard conditions vary depending on the nucleic acid used: oligonucleotide, longer fragment or complete sequence or depending on the type of nucleic acid DNA or RNA used for the hybridization. For example, the melting temperatures for DNA: DNA hybrids are approx. 10 ° C lower than those of DNA: RNA hybrids of the same length.
• DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 30 ° C. to 45 ° C.
• DNA: RNA hybrids the hybridization conditions are advantageously 0.1 ⁇ SSC and temperatures between approximately 30 ° C.
• These specified temperatures for the hybridization are, for example, calculated melting temperature values for a nucleic acid with a length of approx. 100 nucleotides and a G + C content of 50% in the absence of formamide.
• the experimental conditions for DNA hybridization are described in relevant textbooks of genetics, such as Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be made according to formulas known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G + C content. The person skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al.
• homologous sequences of the sequence mentioned in SEQ ID NO: 4 are understood to mean, for example, variants which have at least 95% homology at the derived amino acid level, preferably at least 96% homology, particularly preferably at least 97 or 98% homology, very particularly preferably at least 99 or Have 99.9% homology.
• the homology was calculated over the entire amino acid range.
• the PileUp program was used (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153). Homology is to be understood as identity for the present invention. Both terms are synonymous.
• An operative link is understood to mean the sequential arrangement of, for example, the promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence.
• These regulatory nucleotide sequences can be of natural origin or can be obtained by chemical synthesis.
• any promoter that can control gene expression in the corresponding host organism is suitable as a promoter.
• chemically inducible promoters are preferred, by means of which the expression of the genes underlying them in the host cell can be controlled at a specific point in time.
• the ⁇ -galacosidase, arabinose or xylose-inducible system may be mentioned here as an example.
• the xylose-inducible system and here the xylA promoter from pWH1520 is preferred.
• the invention therefore also includes an integrative vector of the type described above, in which the gene expression is under the control of the xylA promoter.
• sequences for selection, replication and / or integration into the chromosome of the host cell have been described in the literature.
• Various selection markers are known, e.g. Genes that confer resistance to ampicillin, tetracycline, kanamycin or erythromycin.
• this list is not exhaustive or limiting for the present invention.
• Sequences which are advantageous according to the invention for selection are the ampicillin resistance gene for selection of the vector in E. coli or the erythromycin resistance gene for selection in B. megaterium.
• an origin of replication in E. coli are pBR322 (Sutcliffe, JG, 1979, Cold Spring Habor Symp. Quant. Biol., 43, Pt 1: 77-90 or in B. megaterium pE194ts or repF.
• the temperature sensitive Origin pE194ts for ß. megaterium only allows replication below 40 ° C, which means that a selection pressure for integration into the chromosome can be built up above this "permissive" temperature (Rygus et. al, 1992).
• the repF gene product is described as an in-trans acting element required for replication of the plasmid in Gram-positive bacteria (Villafane et al., 1987).
• an integrative vector according to the invention is characterized in that it has at least one temperature-sensitive origin of replication.
• An integrative vector which has the temperature-sensitive origin of replication pE194ts is preferred.
• Another variant of the present invention comprises an integrative vector which is characterized in that it contains a genetically modified nucleotide sequence of the hemA gene (hemA [KK]) which codes for a feedback-resistant glutamyl-tRNA synthase, the amino acid sequence of which is an insertion of at least two positively charged amino acids.
• the genetically modified nucleotide sequence contained in the integrative vector preferably codes for a feedback-resistant glutamyl tRNA which has 2 to 6, preferably 2 to 4 and particularly preferably two additional amino acids. These additional amino acids can be obtained at the level of the nucleotide sequence encoding them by inserting two or, accordingly, up to 6 triplets according to procedures known to the person skilled in the art, e.g. via PCR, into the coding nucleotide sequence.
• a preferred variant of the integrative vector contains a genetically modified nucleotide sequence of the hemA gene (hemA [KK]) which codes for a feedback-resistant glutamyl-tRNA synthase, the amino acid sequence of which at positions 3 and 4 of the N-terminus has an insertion of two has positively charged amino acids.
• the positively charged amino acids are preferably lysine residues.
• a feedback-resistant form of an enzyme is to be understood as a protein whose activity is no longer inhibited by the end product of the metabolic pathway (or a metabolic branch). According to the invention, the enzyme of a feedback-resistant glutamyl tRNA reductase with the amino acid sequence according to SEQ ID No. 5 encoded by the hemA [KK] gene from B. megaterium.
• the genetically modified nucleotide sequence of the hemA gene includes naturally occurring variants of the hemA sequence described here as well as an artificial, e.g. B. obtained by chemical synthesis, and optionally adapted to the codon use of the host organism nucleotide sequence. Genetic changes include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
• sense mutations which can lead to the exchange of conserved amino acids at the protein level, but which do not lead to a fundamental change in the activity of the protein and are therefore function-neutral.
• certain amino acids can be replaced by those with similar physicochemical properties (space filling, basicity, hydrophobicity, etc.).
• arginine residues are exchanged for lysine residues, valine residues for isoleucine residues or aspartic acid residues for glutamic acid residues.
• 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 affecting the catalytic function of the protein, but probably the regulation of the activity.
• the present invention comprises a vector containing part of the nucleotide sequence according to SEQ ID No. 1 (hemZ) as antisense RNA
• Chromosome of the host cell Chromosome of the host cell.
• a preferred embodiment of the vector according to the invention contains an antisense RNA (ashemZ) according to SEQ ID No. 3 and also operatively linked sequences for induced gene expression, selection, replication and / or integration into the chromosome of the host cell.
• antisense RNA ashemZ
• antisense RNA preferably an antisense RNA (ashemZ) according to SEQ ID No. Third
• the number of copies of the corresponding genes can be increased 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 can act in the same way. With inducible promoters it is also possible to increase expression in the course of vitamin B12 production.
• genes or gene constructs can either be present in plasmids with different copy numbers or can be integrated and amplified in the chromosome.
• the activity of the enzyme itself can also be increased, for example by an increased catalytic activity or a deregulated or feedback-desensitive (feedback-resistant) activity Inhibitors or enhanced by preventing the breakdown of the enzyme protein.
• overexpression of the genes in question can be achieved by changing the media composition and culture management.
• the (expressed) antisense RNA formed is attached to the corresponding (complementary) Range of the mRNA coding for the coproporphyrinogen III oxidase. It thereby preferably blocks the ribosome binding site of the hemZ gene and in this way inhibits the translation and expression of the key enzyme involved in heme biosynthesis. This in turn results in reduced heme biosynthesis, with the advantage of an increased flow of metabolic metabolites in the direction of vitamin B12 production.
• the present invention furthermore relates to a vector comprising part of the nucleotide sequence according to SEQ ID No. 1 (hemZ) as antisense-RNA (ashemZ), preferably an antisense-RNA (ashemZ) according to SEQ ID No. 3, in which the gene expression is under the control of the xylA promoter.
• this vector can in principle also integrate into the chromosome of the host cell, for example if it is equipped with a temperature-sensitive origin of replication.
• a variant of this vector has at least one temperature-sensitive origin of replication.
• Such a vector variant preferably has the temperature-sensitive origin of replication pE194ts.
• the vectors according to the invention are produced by fusion of the aforementioned components, such as promoter, coding gene segments, origin of replication, genes for selection, etc., according to 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.
• adapters or linkers can be attached to the fragments.
• the present invention furthermore relates to the use of the integrative vector containing the hemA [KK] gene of the type described above for the production of a Bacillus megaterium strain genetically modified according to the invention.
• the present invention also includes the use of a nucleotide sequence according to SEQ ID No. 1 for the production of an antisense RNA (ashemZ) according to SEQ ID No. 3.
• an antisense RNA (ashemZ) according to SEQ ID No. 3 for the production of a vector of the aforementioned type containing part of the hemZ gene according to SEQ ID No. 1 as antisense RNA (ashemZ) according to SEQ ID No. 3 in the present invention.
• the present invention furthermore relates to the use of a vector containing part of the hemZ gene according to SEQ ID No. 1 as antisense RNA (ashemZ) according to SEQ ID No.
• the integrative vector containing the hemA [KK] gene and the vector containing an antisense RNA (ashemZ) can also be transferred into a suitable Bacillus megaterium strain and the resulting genetically modified strain can be used to produce vitamin B12.
• the present invention thus also relates to the use of a genetically modified Bacillus megaterium strain of the type described for the production of vitamin B12.
• the present invention furthermore relates to a process for the production of vitamin B12 by means of a culture comprising a genetically modified Bacillus megaterium strain of the type described, the fermentation being carried out under aerobic conditions.
• a transition from aerobic to anaerobic fermentation conditions takes place in the exponential growth phase of the aerobically fermented cells. With this so-called shift or a two-stage fermentation process, vitamin B12 production can be increased even further.
• a method is advantageous 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.
• the optical density is usually determined at 570-600 nm.
• anaerobic conditions are understood to mean 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. This means that after the transfer 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 prevail 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.
• the oxygen can also be actively expelled via gassing with inert gas, such as nitrogen.
• inert gas such as nitrogen.
• strictly anaerobic conditions can also be created, for example, by adding reducing agents to the culture medium.
• 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.
• the inoculum is taken directly from the stock and used to produce vitamin B12 under anaerobic conditions.
• the fermentation of the genetically modified Bacillus megaterium strain according to the invention can be carried out in a batch approach. Variants in which the fermentation takes place in a fed-batch approach or in continuous culture are also included according to the invention.
• a method is advantageous in which the expression of the hemA [KK] XCDBL operon and / or the expression of the nucleotide sequence (ashemZ) coding for an antisense RNA of the hemZ gene is induced by adding xylose to the fermentation medium.
• the present invention also relates to a variant of the method mentioned for the production of vitamin B12, in which the expression of the hemA [KK] XCDBL operon according to SEQ ID No. 4 and / or the expression of the nucleotide sequence (ashemZ) coding for an antisense RNA of the hemZ gene according to SEQ ID No. 3 is induced by the addition of xylose to the fermentation medium.
• concentrations of xylose of approximately 0.1 to 1% have proven to be advantageous.
• An addition of about 0.2 to 0.5% xylose to the culture medium is preferred. It is particularly preferred to add about 0.20-0.25%, in particular 0.23%, xylose under aerobic and 0.4-0.5%, in particular 0.5% under anaerobic fermentation conditions.
• megaterium strain which has the ⁇ em-4 [KK] XCDßL operon integrated in the chromosome under the induced control of the xylA promoter (“integrated strain” ) leads to an increase in the vitamin B-
• integral strain XCDßL operon integrated in the chromosome under the induced control of the xylA promoter
• a comparison strain is to be understood as a B. megaterium strain which also carries a vector, but without an ashemZ insert.
• the culture medium at least cobalt and / or 5-aminolevulinic acid is added to the culture medium.
• the fermentation is advantageously carried out under aerobic conditions with the addition of about 250 ⁇ M cobalt; Under anaerobic conditions, the addition of up to 500 ⁇ M cobalt is advantageous.
• 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 can be raised.
• the vitamin BT2 formed can be processed from the fermentation medium. Measures to do this are part of common laboratory practice and will not be discussed further here.
• Titration reagent was KOH solution.
• the titration reagent was NaOH solution.
• MgCI 2 20.0 mM titration reagent was NaOH solution.
• the titration reagent was NaOH solution.
• Luria-Bertani Broth (LB) was used with full medium as in Sambrook et. al (1989). For solid media, an additional 15 g agar per liter was added.
• Additives such as carbon sources, amino acids, antibiotics or salts were either added to the media and autoclaved together with them 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 usually used were as follows, but this does not exclude a variation:
• Aerobic bacterial cultures were incubated in baffled flasks at 37 ° C and a speed of 180 rpm. The incubation times were varied according to the desired optical densities of the bacterial cultures. Growth conditions for Bacillus megaterium
• Aerobic cultures were incubated in baffled flasks at 250 rpm and 37 ° C for the best possible aeration.
• Anaerobic cultures were cultivated in a volume of 150 ml in 150 ml anaerobic bottles at 37 ° C. and 100 rpm. In both cases, attention was paid to inoculation in a ratio of 1: 100 from overnight cultures, and the use of constant conditions for the overnight cultures.
• ß Megaterium cultures pre-incubated aerobically and switched to anaerobic growth conditions at a desired density, ß. Megaterium was first incubated in Shikan flasks at 250 rpm and 37 ° C. In the middle of the exponential growth or at the beginning of the stationary phase, the entire culture was transferred to a 150 ml anaerobic bottle and cultivated further at 37 ° C. and 100 rpm.
• Bacteria were removed from a glycerol culture using a sterile inoculation loop and fractionally streaked on an LB agar plate to which an appropriate antibiotic had been added, so that individual colonies can be seen on the plate after incubation at 37 ° C. overnight were. If bacteria from a liquid culture were used, they were spread on the LB agar plate with a Drygalski spatula and then incubated at 37 ° C. overnight.
• the cell density of a bacterial culture was determined by measuring the optical density (OD) at 578 nm, it being assumed that an OD 57 8 of one corresponds to a cell count of 1x10 9 cells.
• Glycerol cultures produced. 850 ⁇ l of a bacterial Overnight culture mixed thoroughly with 150 ⁇ l sterile 85% glycerol and then stored at -80 ° C.
• the transformation was carried out by electroporation using a gene pulser with connected pulse controller (BioRad). In addition, 40 ⁇ l competent £ were added. co // - cells and 1 ⁇ g plasmid DNA transferred into a transformation cuvette and exposed in the Gene Pulser to a field strength of 12 kV / cm at 25 ⁇ F and a parallel resistance of 200 ⁇ . In the event that more than 2 ⁇ l of the plasmid DNA had to be added, dialysis was carried out.
• the transformed cells were incubated in a 1 ml LB medium at 37 ° C. on the thermal shaker for half an hour immediately after the transformation. From the approaches were then spread different volumes on LB plates with the appropriate antibiotic additive and incubate overnight at 37 ° 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 carefully and the suspension was centrifuged (3000 x g; 5 min; RT). Immediately afterwards, the supernatant was removed and the barely visible sediment was resuspended in 500 ⁇ l SMMP buffer. The suspension was incubated at 37 ° C. for 90 min with gentle shaking. Then 50-200 ⁇ l of the transformed cells were 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 one day incubation at 37 ° C.
• PCR primer 1 5'-TTTATATTCATATTCCATTTTG-3 'PCR primer 2: 5'-GGTAATCCAAAAATAAAATC-3'
• a 480 bp PCR fragment was amplified, which has 65.1% identity with the hemZ gene from B. subtilis and is a partial sequence of the hemZ gene from B. megaterium.
• a unidirectional PCR i.e. a so-called vectorette PCR, carried out with the vector system from Sigma-Genosys.
• Vectorette PCR enables the amplification of unknown DNA areas that border on known sequence sections.
• a first primer is designed based on the known DNA sequence.
• the genome is cut with a restriction endonuclease and all the resulting ends are connected with a known short DNA sequence. After synthesis of the primary strand, this short sequence (vectorette) serves as the target sequence of the second primer.
• the entire restriction digest fused with the vectorette units can be regarded as a kind of gene bank, the vectorette bank, with the aid of which any sequence can be amplified.
• the PCR primer complementary to it for the second round of amplification can only hybridize when the primer specific for the known sequence region has been extended and the complementary sequence has been formed. This guarantees that the desired DNA is only amplified.
• a prerequisite for a successful vectorette PCR is an amplifiable fragment size of the genomic DNA sought. The fragment size should not exceed 6-7 kb so that a special DNA polymerase (Taq) can synthesize the fragment without termination until the end.
• Taq DNA polymerase
• An adequate restriction enzyme for digesting the genomic DNA is determined by Southern blot pre-analysis. This was the Restriction enzyme Clal selected.
• the fragment size determined by the Southern blot analysis allows the size of the expected PCR fragment to be calculated and thus facilitates its identification.
• a strand of the complete hemZ gene from Bacillus megaterium was isolated by the vectorette PCR.
• the entire hemZ gene could be completely amplified and sequenced from this sequence by inverse PCR.
• the sequence is according to SEQ ID No. 1 shown.
• the starting plasmids were pWH1967E (Schmiedel, D. et al., 1997, Appl. Micorbiol. Biotechnol., 47 (5): 543-546) and pMM1520 (Malten, Marco, 2002, production and secretion of a dextran sucrase in Bacillus megaterium, doctoral thesis , Institute of Microbiology (Prof. Dr. D. Jahn), Technical University Braunschweig).
• the two plasmids were first cut with Pstl and Hindill. The complete batch was then applied to an agarose gel and the fragments of interest eluted.
• the eluted fragment of pWH1967E (4198 bp) contained erythromycin resistance, the repF gene, the temperature sensitive origin pE194ts and half an ampicillin resistance.
• the fragment of pMM1520 (1485 bp) contained the xylA 'promoter from ⁇ . megaterium, directly upstream of the promoter, a multiple cloning site, the origin from pBR322 and the second part of the ampicillin resistance gene, which complements the ampicillin resistance.
• the cohesive ends of the two fragments have now been ligated.
• the resulting plasmid was named pHBintE.
• the cloning strategy is shown schematically in FIG. 1.
• the cloned plasmid pHBintE (Fig. 1) thus has the following properties. It has ampicillin resistance for selection in E. coli and erythromycin resistance for selection of ß. megater / um transformants.
• the temperature sensitive Origin pE194 ts for ß. megaterium permits replication only below 40 ° C., which means that a selection pressure for integration into the chromosome can be built up above this “permissive” temperature (Rygus et al, 1992).
• the repF gene product is described as a trans-acting element, required for replication of the plasmid in gram positive bacteria (Villafane et al., 1987).
• the plasmid also contains the xylA 'promoter with a multiple cloning site directly upstream. This promoter enables the induction of inserted into the multiple cloning site Genes with xylose.
• the first 27 amino acids of the alignment report for HemA are of ⁇ . Megaterium with "KK-deregulated HemA", ß. megaterium INildtyp and from S. typhimurium. There is again clarified at which 15 place the insertion should take place.
• the HemA [KK] mutant was cloned by means of PCR. Chromosomal DNA from ß served as template. megaterium. Since the sequence of the hemA gene from ß. megaterium was known, primers could be derived. The sequences of the primers are shown below:
• the derived primers had no complete homology to ß. megate / m sequence.
• 6 bases were exchanged for a Spel interface (italics).
• the codon usage indicates the probability with which a particular base triplet codes for an amino acid in the genome of the organism.
• ß. Megaterium is the most common triplet for lysine "AAA" with a percentage usage of 76%.
• a Kpnl interface was inserted into the reverse primer. The primers were synthesized by MWG, Ebersbach.
• the PCR-amplified hemA [KK] mutant was purified using a Quiagen PCR purification kit, cut with Spei and Kpnl and purified again.
• the plasmid pHBintE was also cut with Spei and Kpnl and purified with a PCR purification kit from Quiagen. After determining the concentration, these two fragments were ligated with a vector / insert ratio of 1: 4 to the integration vector called pHBiHemAKK.
• the cloning strategy for the plasmid pHBiHemAKK is shown schematically in FIG. 3.
• pHBiHemAKK differs from pHBintE essentially only by the insertion of the hemA [KK] mutant, it retains its properties. There is also ampicillin and erythromycin resistance for selection in E. coli and in ß. megaterium available. The origin pBR322 and in ß are used for replication in E. coli. megaterium of the temperature-sensitive Origin pE194ts and repF. Xyl A 'and the hemA [KK] mutant were ligated to a translation fusion and are under the control of the xyl promoter. Due to the hemA [KK] insertion, pHBiHemAKK has a homologous section to the ß. megaterium chromosome and thus additionally the possibility to integrate into the chromosome via "single crossing over recombination".
• the temperature-sensitive Origin pE194ts is important for the selection of this integration event. Since the plasmid is replicated at 30 ° C, ß. megater / tym transformants on erythromycin can be selected at this temperature. When the temperature is raised to 42 ° C, the plasmid can no longer be replicated. This means that only the Transformants can grow that have integrated the plasmid and thus the erythromycin resistance into their chromosome. The integration of pHBiHemA [KK] in the ß. megater / wm chromosome thus enables xylose-inducible overexpression of the entire ⁇ emAXODß operon.
• the B. megaterium strain DSMZ509 with integrated plasmid pHBiHemA [KK] is referred to below as HBBml.
• Primer forward contains BamHi interface 5'-GCGGGATCCCTTGAACTGAGCACCTTGACCGG-3 'primer reverse (ashemZ): contains Spel interface 5'-TCGACTAGTCGGACGTAAAAAACGTTCATCTTCTATACC-3'
• the amplified BamHI / Spel antisense RNA fragment was then purified and cloned into the vector pWH1520 (Rygus, T. et al., 1991, Appl. Microbiol. Biotechnol., 35: 594-599), which had previously been identified with the Restriction endonucleases Spei and BamHI was linearized.
• the resulting plasmid pHBasHemZ which contains an antisense hemZ RNA under the control of the xylA promoter, is shown in FIG. 4.
• the inserted antisense hemZ RNA according to SEQ ID No. 3 has a length of 129 bp, which begins 82 nucleotides before the start codon of the actual hemZ gene.
• An overview of the location of the antisense hemZ RNA is given below:
• the transcript of the antisense RNA consequently forms a double-stranded RNA with the ⁇ emZ mRNA and thus blocks the ribosome binding site of the hemZ gene for the ribosomes.
• ß. Megaterium DSMZ509 first transformed with pHBiHemAKK using protoplast transformation.
• the transformed strain was cultured on agar plates with the addition of erythromycin (1 ⁇ g / ml and 75 ⁇ g / ml). 30 ° C was chosen as the cultivation temperature because the plasmid can be replicated at this temperature. After 24 hours of growth, colonies were identified at all concentrations of erythromycin indicated. Inoculation of some colonies on agar plates with an erythromycin additive (75 ⁇ g / ml) also led to growth under the conditions mentioned after 24 hours. This was the transformation of pHBiHemAKK into ß. megaterium DSMZ509 successful.
• a 110 ml LB culture with 5 ⁇ g / ml erythromycin and 0.23% xylose was inoculated with these transformants and incubated at 30 ° C. under aerobic conditions (250 rpm shaking). After approx. 12 h, the temperature was raised to 42 ° C., so that no further replication of the plasmid could take place and a pressure for integration was built up. After a further 12 h, inoculation was carried out for a total of 3 days in new LB medium and incubation was continued at 42 ° C. After this time, there was good growth of the LB medium, which indicated the integration of the plasmid into the chromosome, since transformants with freely replicable plasmids under these conditions would not have been able to pass on the plasmid by replication.
• FIG. 5 shows that when 298 ⁇ M ALA and 250 ⁇ M CoCl 2 are added, the growth of ⁇ . megaterium DSMZ509-pHBasHemZ (-! -) is significantly worse over the entire course than with the comparative transformer DSMZ509-pWH1520 (- + -). This means that the heme formation has been reduced by the antisense RNA.
• ALA the precursor of all tetrapyrroles
• the growth disadvantage of the transformants in comparison to the non-transformed strain is due to the additional replication of the plasmids and the addition of antibiotics in the medium.
• Figure 6 confirms that the antisense hemZ RNA formed inhibits growth.
• the growth of DSMZ509-pHBasHemZ (-! -) is always worse than that of the comparative transformant (- + -).
• the antisense-ftemZ-RNA transformant thus reaches a maximum OD 578 of 8.3, while the maximum OD 578 of the comparative transformant is 10.0. This means that the coproporphyrinogen III oxidase has been inhibited.
• S. typhimurium metE cysG double mutants were incubated overnight on minimal medium containing methionine and cysteine at 37 ° C., scraped off the plate and washed with 40 ml of isotonic NaCl solution. After centrifugation, the cell sediment was resuspended in isotonic saline. The washed bacterial culture was thoroughly mixed with 400 ml of 47-48 ° C minimal medium agar containing cysteine.
• Vitamin B12 determination with the ELISA test 10 ⁇ l of the ß resuspended in deionized, sterile water and boiled in a water bath for 15 min. Megater / um samples were placed on the cooled plates and incubated at 37 ° C for 18 h. The diameters of the grown Salmonella colonies are then proportional to the content of vitamin B in the applied B. megaterium samples. By comparison with a calibration curve, made from the addition of 0.01, 0.1, 1, 10 and 40 pmol vitamin B12, the content of vitamin B12 in the examined samples was inferred. This standard method allows the detection of small amounts of vitamin B12 in biological materials quickly and very reproducibly. Vitamin B12 determination with the ELISA test
• the basis is an antigen-antibody reaction, in which wells
• Microtiter plate are coated with specific antibodies against vitamin B12. After the addition of enzyme-labeled vitamin B12 (enzyme conjugate) and sample solutions or vitamin B12 standard solutions, free and enzyme-labeled vitamin B12 compete for the vitamin B12 antibody binding sites (competitive enzyme immunoassay). Unbound enzyme-labeled vitamin B12 is then removed in a washing step. The detection is done by adding substrate / chromogen solution (tetramethylbenzidine / urea peroxide). Bound enzyme conjugate converts the chromogen into a blue end product. The addition of the stop reagent leads to a color change from blue to yellow. The measurement is carried out photometrically at 450 nm. Thus, the absorbance of the solution is inversely proportional to the vitamin B12 concentration in the sample.
• enzyme-labeled vitamin B12 enzyme conjugate
• sample solutions or vitamin B12 standard solutions free and enzyme-labeled vitamin B12 compete for the vitamin B12 antibody binding sites (competitive enzyme immunoassay). Unbound enzyme-
• the mixture was mixed (shaking function in the fusion device) and incubated (15 min; RT). Following the incubation, the wells were emptied by tapping the microtiter plate and washed with 250 ⁇ l wash buffer per well. The cavities were emptied again by tapping and the washing step was repeated twice. Equitemporally, two drops of stop reagent were added per cavity, mixed and incubated for 10 min at room temperature in the dark. After the equitemporal addition of two drops of the stop reagent per cavity, the absorbance at 450 nm was measured in the Packard fusion device. The percentage absorbance was calculated as follows for evaluation:
• a calibration line could now be determined by plotting the absorbance in% over log (ppb). Using the straight line equation, the dilution factor and the known cell density (OD578), the vitamin B12 content of the samples could then be given in ⁇ g / (l x OD).
• FIG. 10 show the results of the vitamin B 2 determination when growing with glucose (1, 2, 5, 6) and when growing with glucose with the addition of 298 ⁇ M ALA and 250 ⁇ M CoCI 2 (3, 4, 7 , 8).
• Figure 9 shows the vitamin B- ⁇ 2 concentrations based on the cell density of the respective culture. It can be seen there that in three out of four cases (No. 2, 6 and 8) DSMZ509-pHBasHemZ produced more vitamin B 12 than the comparative transformant (No. 1, 5 and 7).
• DSMZ509-pHBasHemZ forms six hours after induction (No. 8) 10% more vitamin B 12 than DSMZ509-pWH1520.
• the vitamin B 12 content is shown in pmol / OD 578 and in ⁇ g / l (FIGS. 11-12).
• Vitamin B ⁇ 2 synthesis is coming.
• FIG. 12 The presentation of the results in ⁇ g per liter of bacterial culture (FIG. 12) shows that the antisense- ⁇ emZ-RNA transcribing transformant (No. 3, 6 and 9) also produced the most vitamin B- ⁇ 2 overall, although this
• the samples were resuspended in 1 ml of sterile, deionized water and the optical densities were then adjusted by dilution with water. 1 ml of these adjusted samples were now mixed with 50 ⁇ l lysozyme (1 mg / ml) and incubated at 37 ° C. for 30 min in a shaker at 300 rpm. The samples were then placed in an ultrasonic bath for 10 minutes and then centrifuged at 4000 ⁇ g for 3 minutes. The fluorescence measurement was now carried out with the supernatant. The following settings were made:
• Em Slit 12 nm
• Scan speed 200 nm / min
• the growth curves with the addition of CoCI 2 and ALA gave the first indications of an inhibition of heme synthesis by antisense- ⁇ emZ-RNA.
• the antisense ⁇ emZ-RNA inducible by xylose inhibits the ribosome binding site by occupying the hemZ-mR A and thus prevents translation to HemZ. This leads to a reduced formation of the HemZ protein, which catalyzes the reaction of coproporphyrinogen III to protoporhyrinogen IX. Since the actual metabolite flow is interrupted at this point, coproporphyrinogen III accumulates.
• coproporphyrinogen III The direct detection of coproporphyrinogen III in samples proves to be difficult since coproporphyrinogen III is oxidized to coproporphyrin III in air. Preliminary tests showed that the fluorescence spectrum of coproporphyrin III has emission peaks at approximately 579 nm and approximately 620 nm. Relative amounts of coproporphyrinogen III should therefore be able to be detected indirectly using fluorescence spectra, the oxidized form (coproporphyrin III) being measured.
• Figure 1 shows the schematic representation of the cloning of the integrative plasmid pHBintE for ß. megaterium.
• the starting plasmids pWH1967E and pMM1520 were cut with the endonucleases Pstl and Hindi II.
• the 4198 bp fragment (between Pstl-2786 and Hindlll-6984) from pWH1967E and the 1485 bp fragment (between Hindlll-7212 and Pstl-1307) from pMM1520 were eluted and ligated.
• FIG. 2 shows a representation of the first 27 amino acids of the alignment report for 1.) S. typhimurium HemA, 2.) ß. megaterium HemA and 3.) ß. megaterium HemAKK.
• FIG. 3 shows a schematic representation of the cloning strategy of the plasmid pHBiHemAKK.
• the hemA [KK] mutant amplified by PCR and the vector pHBintE were cut with Spei and Kpnl, respectively, and the resulting cohesive ends were ligated to the integration vector pHBiHemAKK.
• Figure 4 shows a schematic representation of the plasmid pHBasHemZ.
• the interfaces Spei and BamHI indicated in the illustration were used to insert the antisense RNA.
• Figure 5 shows the growth behavior of the ß. megater / um strain DSMZ509 and transformants of this strain at 37 ° C in Mopso minimal medium with glucose as carbon source and the addition of 298 ⁇ M ALA and 250 ⁇ M CoCI 2 . Transfer (shift) from aerobic to anaerobic growth took place at the end of the exponential phase (after 11 h). Growth of DSMZ509 untransformed (-A-), DSMZ509 pWH1520 (- + -) and DSMZ509 pHBasHemZ (-! -). The gene expression of the xylA promoter on pHBasHemZ and pWH1520 was induced by adding 0.5% (w / v) xylose after 10 h of growth. Samples were taken at the times indicated and the optical density at 578 nm was determined.
• Figure 6 shows the growth behavior of the ß. megater / tm strain DSMZ509-pWH1520 (- + -) and DSMZ509-pHBasHemZ (-! -) in Mopso minimal medium with glucose as a carbon source and the addition of 298 ⁇ M ALA and 250 ⁇ M CoCI 2 under aerobic growth conditions. Induction was carried out by adding 0.5% (w / v) xylose at an OD 5 8 of 2. Samples were taken at the times indicated and the optical density at 578 nm was determined.
• Figure 7 shows the content of vitamin B ⁇ 2 in ⁇ g / POD under aerobic growth conditions of ß.
• Megaterium DSMZ509 (1), DSMZ509-pWH1520-cobA (2) and DSMZ509 with integrated pHBiHemAKK (3) in LB medium, measured with an ELISA test. Induction was carried out with 0.5% (w / v) xylose after 5 h of growth; the cell harvest after 10 h of growth. 1 DSMZ509
• FIG. 8 shows the content of vitamin B 12 in ⁇ g / l under aerobic growth conditions of ⁇ .
• FIG. 3 DSMZ509 with integrated pHBiHemAKK
• Figure 9 shows the vitamin B 12 production in the transfer experiment of ß. megaterium DSMZ509-pWH1520 and DSMZ509-pHBasHemZ in Mopso minimal medium with glucose as carbon source measured with an ELISA test. Induction was carried out with 0.5% (w / v) xylose after 9 h (1, 2, 5, 6,) or 10 h (3, 4, 7, 8) growth. The transfer from aerobic to anaerobic ⁇ took place one hour after induction. The content of vitamin B 12 is given in ⁇ g per liter of bacterial culture and OD 578 .
• Figure 10 shows the vitamin B- ⁇ 2 production in the transfer experiment of ß. megaterium DSMZ509-pWH1520 and DSMZ509-pHBasHemZ in Mopso minimal medium with glucose as carbon source measured with an ELISA test. Induction was carried out with 0.5% (w / v) xylose after 9 h (1, 2, 5, 6,) or 10 h (3, 4, 7, 8) growth. The transfer from aerobic to anaerobic took place one hour after induction. The content of vitamin B 12 is given in ⁇ g per liter of bacterial culture.
• DSMZ509-pWH1520 with the addition of 250 ⁇ M CoCI 2 and 298 ⁇ M ALA, 3 h after induction.
• DSMZ509-pHBasHemZ with the addition of 250 ⁇ M CoCI 2 and 298 ⁇ M ALA, 3 h after induction.
• 6 DSMZ509-pHBasHemZ without additives, 6 h after induction.
• 7 DSMZ509-pWH1520 with the addition of 250 ⁇ M CoCI 2 and 298 ⁇ M ALA, 6 h after induction.
• Figure 11 shows the vitamin B- 2 production in the transfer experiment of ß. megaterium DSMZ509, DSMZ509-pWH1520 and DSMZ509-pHBasHemZ in Mopso minimal medium with glucose as carbon source.
• the transfer from aerobic to anaerobic took place one hour after induction.
• Induction was carried out with 0.5% (w / v) xylose after 9 h growth.
• the vitamin B 2 content per cell mass is given in pmol / OD 578 .
• Figure 12 shows the vitamin B-
• Figure 13 shows the vitamin B 2 production in the transfer experiment of ß. Megaterium DSMZ509, DSMZ509-pWH1520 and DSMZ509-pHBasHemZ in Mopso minimal medium with glucose as a carbon source with the addition of 298 ⁇ M ALA and 250 ⁇ M CoCI 2 .
• the transfer from aerobic to anaerobic took place one hour after induction.
• Induction was carried out with 0.5% (w / v) xylose after 10 h growth.
• the content of vitamin B 12 per cell mass is given in pmol / OD 578 .
• Figure 14 shows the vitamin B 12 production in the transfer experiment of ß. Megaterium DSMZ509, DSMZ509-pWH1520 and DSMZ509-pHBasHemZ in Mopso minimal medium with glucose as a carbon source with the addition of 298 ⁇ M ALA and 250 ⁇ M CoCI 2 .
• the transfer from aerobic to anaerobic took place one hour after induction.
• Induction was carried out with 0.5% (w / v) xylose after 10 h growth. It is the content of vitamin B- t2 in ⁇ g per liter Bacteria culture indicated.
• FIG. 15 shows the difference fluorescence spectrum of B.megaterium DSMZ509-pHBasHemZ minus the fluorescence spectrum of DSMZ509-pWH1520 with excitation at 409 nm.
• the emission peaks at 579 nm and 618 nm indicate coproporphyrin III and thus an accumulation of the metabolite pH in DSMasH50Z compared to DSMZ509-pWH1520.

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