WO2005095446A1 - Der faktor reca aus bacillus licheniformis und reca-inaktivierte sicherheitsstämme für die biotechnologische produktion - Google Patents
Der faktor reca aus bacillus licheniformis und reca-inaktivierte sicherheitsstämme für die biotechnologische produktion Download PDFInfo
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
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- the present invention relates to the RecA factor from Bacillus Hcheniformis and to microorganisms as security strains for biotechnological production, which are characterized in that they have functional deletions in the associated recA gene. Furthermore, RecA is available for further molecular biological approaches.
- the present invention is in the field of biotechnology, in particular the production of valuable substances by fermentation of microorganisms which are capable of forming the valuable substances of interest.
- microorganisms which are capable of forming the valuable substances of interest.
- These include, for example, the production of low molecular weight compounds, such as food supplements or pharmaceutically relevant compounds, or proteins, for which there is in turn a wide range of technical applications due to their diversity.
- the metabolic properties of the microorganisms in question are used and / or changed to produce the valuable substances;
- cells are used which express the genes of the proteins of interest. In both cases, these are mostly genetically modified organisms (GMOs).
- the application relates to EP 369817 A1 ⁇ / us strains, in particular ⁇ . subtilis, for the production and secretion of proteins in which the genes for extra- and intracellular proteases, namely epr, rp-l, rp-ll, isp-1, apr and / or npr, have been functionally inactivated by point mutations or insertions of inactive gene copies ,
- the purpose of these genetic modifications is to minimize the protease activities which are harmful to the proteins of interest produced with these strains.
- the strains in question may additionally have mutations which prevent sporulation and thus the formation of equally harmful sporulation proteases.
- the application WO 92/16642 A1 pursues the same approach: it discloses that by inactivating the protease genes apr, npr, isp-1, epr, bpr, rsp and mpr from Bacillus, a large part of the extracellular protease activity is switched off, and teaches that this can be further improved by inactivating the newly described gene vpr for the residual protease III.
- the possibility of inactivating spoOA is pointed out in order to prevent the formation of intracellular proteases.
- Sporulation of gram-positive bacteria is a development process for the formation of permanent forms, the so-called spores, for surviving adverse environmental influences. It is controlled via a complex regulatory cascade with probably more than 100 genes and with the participation of special sigma factors. The connection of this process with the cell cycle of ß.
- subtilis describes the publication "Cell cycle and sporulation in Bacillus subtilis” (1998) by PALevin and A.D. Grossmann in Curr. Opin. Microbiol, Volume 1, pages 630 to 635.
- the transcription factor SpoOA is presented as a control element for initiating sporulation.
- EP 492274 A2 discloses that the prior art has already succeeded in inactivating sporulation genes via unspecific mutagenesis, as a result of which asporogenic mutants (spo-minus phenotype) have been obtained.
- EP 492274 A2 itself describes a ⁇ treated by targeted mutagenesis in the early sporulation gene spollD. su // s strain, which with a reversion rate of less than 10 "8 is practically no longer able to form spores.
- This application teaches that this strain is only leu after inactivation of the other genes (for the leucine synthesis), pyrD1 (for uracil synthesis), apr and npr for the production of valuable materials for biotechnological production, because this is associated with advantages in production as well as safety aspects.
- the application WO 97/03185 A1 also deals with the inactivation of the sporulation ability of ßac /// us species with the exception of ß. subtilis and use of these strains for the biotechnological production of valuable materials.
- the early gene spollAC coding for the sigma factor F is to be functionally inactivated, advantageously in combination with deletions in genes of the sporulation gene groups spo2, spo3, which were also activated early.
- spo2, spo3 which were also activated early.
- an irreversible inactivation of the relevant chromosomal sections for spollAC is described.
- the application WO 02/097064 A1 (EP 1391502 A1) relates to microorganisms in which genes from stages II, III, IV or V of sporulation have been deleted or inactivated. These are the genes sigE, sigF, spollE, spollSB and sigG of ß. subtilis that within the locus from spolVCB to spolllC from ß. lie subtilis.
- SubtiList database accessible via http://genolist.pasteur.fr/SubtiList/genome.cgi
- this can range from approx. 2,642,000 kb to approx. 2,700,000 kb of the now known total genome of B to be narrowed down.
- the gene recA coding for the factor RecA described in prokaryotes is very well known in molecular biology, but so far in particular from a context other than that of the production of safety strains.
- This factor binds specifically and cooperatively to single-stranded DNA and, under ATP hydrolysis, ensures partial unwinding of double-stranded DNA.
- This process enables the genetic process of recombination, that is, the strand exchange between similar DNA molecules.
- It is a common procedure in molecular biology to use the recA gene by inactivating a defective copy of recA via a corresponding genetic construct and thus creating a rec / ⁇ - minus phenotype which is no longer used Recombination is capable.
- deletion mutants generated by crossing-over are genetically stabilized by subsequent inactivation of recA.
- references to recA appear in a wide variety of molecular biological contexts.
- DE 10011358 A1 which deals with L-shaped bacterial strains, also mentions that, in addition to numerous other possible modifications, it is also possible, among other things, to mutate recA in order to achieve improved transformation and plasmid stability.
- a biochemical description of the RecA from Escherichia coli is provided, for example, by the publication "C-terminal deletions of the Escherichia coli RecA" by SLLusetti et al. (2003; J. Biol. Chem., Volume 278, number 18, pages 16372-16380) It follows that, in particular, the C-terminus of this molecule mediates single-strand binding and that corresponding deletion mutants in this area, in addition to other biochemical properties, have an increased sensitivity to mitomycin, which is known to impair DNA synthesis and thereby have a bactericidal effect.
- the N-terminal region is more involved in the binding of DNA double strands.
- RecA The state of the art for RecA can be summarized to the extent that this protein has hitherto been known predominantly from genetic contexts. The use of this factor or the inactivation of the gene in question to generate safety strains of GMOs has so far been rejected due to its physiological importance. Successful described examples of this are only recA-minus mutants of the gram-negative species Sinorhizobium meliloti and the gram-positive Bacillus megaterium, the latter only in combination with three other safety-relevant mutations.
- the task was therefore to develop a further suitable security system for genetically modified gram-positive bacteria, for which a suitable factor and / or a suitable gene had to be identified as the basis.
- a partial aspect of this task was the isolation of a genetic element that can be used for this purpose, possibly a gene, and the amino acid sequence of one of these, if applicable encoded factor in order to make this system accessible to corresponding molecular biological constructions for use in production strains, in particular in combination with one or more further safety mechanisms serving for safety purposes.
- a subtask was to define another such security system to be combined with it, preferably one that would not require any further mutations in addition to these two systems.
- a maximum of these two mutations should be sufficient to create a gram-positive safety strain that meets extensive requirements for reducing environmental viability, that is, should lead to a minimal reversion rate. Because a lower number than four side-by-side systems means an increasingly lower amount of work to produce these strains.
- a side aspect of this task was to find such a security system that is not so special that it could not be used in other molecular biological approaches.
- This task is carried out by the factor RecA with an amino acid sequence that corresponds to that in SEQ ID NO. 2 specified amino acid sequence is at least 96% identical, or by the nucleic acid coding for a factor RecA, the nucleotide sequence of which in SEQ ID NO. 1 specified nucleotide sequence is at least 85% identical.
- the in SEQ ID NO. Amino acid and nucleotide sequences given in 2 and 1 are those for RecA. All positions from 1 to 1047 code for the protein; the last three represent the stop codon. They are referred to as the gene and protein recA and RecA, respectively. They come from the Bacillus Hcheniformis strain deposited with the number DSM 13 at the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b, 38124 Braunschweig (http://www.dsmz.de). Solutions to the task according to the invention represent all factors or nucleic acids which have sufficient homology for this, as defined by the respective percentages.
- the corresponding factor can be from ß. amyloliquefaciens can be viewed.
- the corresponding complete DNA and amino acid sequences are published in the NCBI database of the National Institutes of Health of the USA (http://www.ncbi.nlm.nih.gov) under the accession number AJ515542, with a partial sequence located near the C-terminus additionally emerges from the entry AY147924.
- RecA from ß. Hcheniformis DSM 13 has a homology of 94.0% identity at the amino acid level and an agreement of 81.2% identity at the nucleic acid level. Both comparisons emerge from the alignments of FIGS. 1 and 2, where the sequences of ⁇ . amyloliquefaciens are shown in the second line.
- the next similar enzymes were RecA from ß. subtilis and RecE from ß. subtilis, each with 93.4% identity. They have homology values of 81.0% and 81.2% identity at the DNA level. They are also published in the NCBI database under registration numbers Z99112 (region 161035 to 162078) and X52132. The amino acid and DNA comparisons with these factors are also shown in FIGS. 1 and 2 (lines 3 and 4, respectively).
- Example 1 of the present application illustrates how other factors RecA can suitably be obtained which lie within the homology range designated here.
- a molecular-biological procedure is shown there, according to which genes or gene sections relating to specific PCR primers, in particular the specifically disclosed (SEQ ID NO. 25 to 30) oligonucleotides, can be obtained from chromosomal DNA preparations of the species concerned. If necessary, if these primers cannot be used successfully, similar primers can be used in which - controlled by the reaction conditions in the primer synthesis - individual positions are varied.
- These PCR products can - if only partial sequences have been obtained when using appropriate primers (cf. FIG. 3B) - can be combined to form coherent DNA sequences by customary methods (using overlaps).
- the amino acid sequence of that of the obtained RecA-encoded factor RecA gene can be used as probes in order to isolate relevant genes from gene banks by methods known per se.
- RecA This opens up a wide technological and commercially relevant area for RecA and especially for the associated recA gene, namely the production of valuable substances by fermentation of genetically modified gram-positive bacteria. They can be improved not only in terms of their genetic stability but also in terms of their safety through mutations in recA. As explained further below, this applies in particular to strains which are actually used in biotechnological production, such as, for example, Bacillus hcheniformis.
- this is preferably done in connection with one and particularly preferably no further security-relevant deletions.
- This is also preferably done in strains that are as closely related as possible.
- B. megaterium should not be used, on the one hand because, as described in the introduction, the species-specific recA genes or the mutants deleted therein are already available, and on the other hand because these species, which are characterized in particular by their large cells and thus associated microbiological peculiarities, is generally not used for large-scale fermentation.
- Objects of the present invention thus lie in the factor RecA (SEQ ID NO. 2) and the associated gene recA (SEQ ID NO. 1) from ⁇ . Hcheniformis DSM 13 or close relatives.
- recAGens and / or one that is related to SEQ ID NO. 31 is sufficiently related to the functional inactivation of recA in a gram-positive bacterium, preferably in combination with the functional inactivation of a gene active in phase IV of sporulation of gram-positive microorganisms, preferably spolV, yqfD or homologues thereof.
- a gene active in phase IV of sporulation of gram-positive microorganisms preferably spolV, yqfD or homologues thereof.
- the gram-positive microorganisms obtained in this way represent a corresponding subject of the present invention; likewise the fermentations carried out with these organisms, in particular for the production of valuable materials.
- the present application provides a RecA protein which can be used in molecular-biological approaches or for modulating the molecular-biological activities of cells, in particular in connection with DNA polymerization or recombination processes.
- the first subject matter of the invention includes each factor RecA defined above with an amino acid sequence that corresponds to that in SEQ ID NO. 2 specified amino acid sequence is increasingly preferably at least 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% and very particularly preferably 100% identical.
- This is preferably a factor RecA, which is encoded by a nucleic acid whose nucleotide sequence corresponds to that in SEQ ID NO. 1 specified nucleotide sequence is at least 85% identical.
- the relevant factors or genes are available via the nucleic acids for the transformation into other, preferably related species or for modifications. As explained in more detail below, this includes in particular mutations of the genes in question. With an increasing degree of identity to the given sequence, the success with such species should be greater, that too ß. Hcheniformis are increasingly related, especially in the species ß, which is particularly important for biotechnological production. Hcheniformis itself.
- nucleic acids coding for a factor RecA the nucleotide sequence of which corresponds to that in SEQ ID NO. 1 specified nucleotide sequence is at least 85% identical.
- nucleic acids whose nucleotide sequence corresponds to that in SEQ ID NO. 1 specified nucleotide sequence is increasingly preferably at least 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99% and very particularly preferably 100% identical. This is because these can be used via appropriate constructions for transformation and / or mutagenesis, an increasing similarity making the desired success all the more likely.
- nucleic acid of this type which codes for a previously described factor RecA.
- a mutation in a single position is sufficient to switch off the gene or the factor in its natural function via a nonsense mutation.
- a separate subject of the invention is the use of a nucleic acid coding for a factor RecA for the functional inactivation of the gene recA in a gram-positive bacterium which is not Bacillus megaterium.
- a gram-positive bacterium which is not Bacillus megaterium.
- B. megaterium there are already the studies mentioned in the introduction, in which it is proposed to delete recA at the same time as several other mutations in order to arrive at safety strains.
- other gram-positive bacteria such as those of the genera Bacillus, Staphylococcus, Corynebacterium and Clostridium, are more important host organisms in the prior art for the biotechnological production of valuable substances (see below).
- functional inactivation means any type of modification or mutation, after which the function of a RecA as a single-strand DNA-binding factor is prevented.
- the "use" of this factor or gene according to this embodiment which was discussed at the outset, consists in the fact that it does not function naturally in the cell in question. According to this object of the invention, this is achieved on a genetic level in that the The gene in question is switched off, and example 2 of the present application describes one way in which this can be achieved technically.
- nucleic acid coding for a non-active protein with a point mutation is used.
- nucleic acids can be generated by methods known per se for point mutagenesis. Such are described, for example, in relevant manuals such as that of Fritsch, Sambrook and Maniatis, "Molecular cloning: a laboratory manual”, Cold Spring Harbor Laboratory Press, New York, 1989.
- oligonucleotides are synthesized with individual exchanges (mismatch primers) and hybridized with the single-stranded gene, followed by DNA polymerization to give corresponding point mutants Due to the high homologies it is possible and particularly advantageous according to the invention to use the reaction with SEQ ID NO provided sequence or possibly the other sequences of related species shown in FIG. 2. These sequences can also be used to design corresponding mismatch primers for related species, in particular on the basis of the conserved regions which can be identified in the alignment of FIG.
- a nucleic acid with a deletion or insertion mutation is used, preferably comprising the edge sequences of the region coding for the protein each comprising at least 70 to 150 nucleic acid positions.
- Example 2 Such an approach was chosen in Example 2: As explained there, SEQ ID NO. 31 two flanking regions of approx. 340 bp each were used to ß the part of the recA gene in between. delete licheniformis stem (see FIG. 3B). In example 3 below, the success of this deletion is checked at the genetic level. Figure 4 (A and B) thus shows that the relevant DNA fragment has been shortened accordingly by the deletion. The phenotypic description of the mutants obtained in this way is given in the examples below. Accordingly, recA inactivated strains are significantly more UV-sensitive than those with an intact recAGen (example 6).
- nucleic acid positions are required in each of the two edge sequences for the mismatched part, the part in between not being important. Accordingly, those embodiments are preferred which only comprise two flanking regions with at least these sizes.
- nucleic acids with a total of two nucleic acid segments are used, each comprising at least 70 to 150 nucleic acid positions and thus flanking at least partially, preferably completely, the region coding for the protein.
- the marginal areas of the genes in question are also suitable for this, which naturally perform a different function (promoter, terminator, enhancer etc.) or merely represent non-functional intergenic sections.
- the functional inactivation can also consist in the deletion of the promoter, for which it is necessary to use flanking, non-coding sections in the case of a deletion mutation of this embodiment. Depending on the individual case, it may also be sensible to select those sections for the flanking regions which partly extend into the protein-coding region and partly lie outside.
- Such areas can be used for ⁇ .
- Hcheniformis for example SEQ ID NO. 31 are removed.
- subtilis can be found in the database entries given above.
- it is possible to open up the relevant non-coding areas using PCR-based methods from a preparation of the genomic DNA; as illustrated in Example 1. These methods (for example anchored PCR with primers pointing outwards into an unknown region) are established in the prior art.
- the known gene segments which serve to open up the still unknown regions, serve as starting points for this.
- the primers required for this can be identified using SEQ ID NO. 1 and 31 also for other species of gram-positive bacteria and in particular for those of the genus Bacillus can be designed, possibly with the introduction of variable positions, as has already been explained above.
- a corresponding use is accordingly one of the previously described nucleic acids according to the invention and / or a nucleic acid whose nucleotide sequence corresponds to that in SEQ ID NO. 31 indicated nucleotide sequence in positions 369 to 1415 corresponds to at least 1045, preferably at least 1046, very particularly preferably 1047 of these 1047 positions, or around the at least partially non-coding flanking regions for these nucleic acids.
- SEQ ID NO. 1 is based on the commercially available strain DSM 13; SEQ ID NO. 31 was by reworking the invention based on an arbitrary ß. Hcheniformis strain obtained (Example 1). Since they match in 1044 positions, the embodiment described as successful in the examples can be given an increasing preference for 1045, 1046 and very particularly 1047 corresponding positions to SEQ ID NO. 31 describe.
- the gram-positive bacterium is preferably one of the genera Clostridium or Bacillus and one which is naturally capable of sporulation, in which a gene from phase IV of the sporulation is functionally inactivated simultaneously with recA.
- Example 1 shows the procedure for functional inactivation and example 3 shows its success (FIG. 4). The hoped for phenotypic sporulation defects are demonstrated by Example 4 and Figure 5.
- the inactivated gene from phase IV of sporulation is in the nomenclature of ⁇ . subtilis is one of the genes spolVA, spolVB, spolVCA, spolVCB, spoIVFA, spoIVFB or yqfD or a gene homologous thereto, preferably in the case of ⁇ . subtilis for the yqfD gene, in the case of Bacillus Hcheniformis for the spolV gene and in any other case for a homologous gene.
- the ß. subtilis-Ger spolVA codes for phase IV sporulation protein A, which is available in the databases Swiss-Prot (Geneva Bioinformatics (GeneBio) SA, Geneva, Switzerland; http://www.genebio.com/sprot.html) and NCBI ( see above) under number P35149. It plays a role in the formation and assembly of an intact spore shell.
- the amino acid sequence of the associated SpolVA factor is shown in SEQ ID NO. 8 of the present application, as the translation of the previous DNA sequence generated by the Patentin program.
- nucleotide sequence can be found in the Subtilist database of the Pasteur Institute, Paris, France (http://genolist.pasteur.fr/SubtiList genome.cgi) under number BG10275 and is in the sequence listing in SEQ ID NO. 7, together with the 200 nucleotides located before the 5 'end and the 197 nucleotides behind the 3' end. Notwithstanding the fact that these marginal sequences may well contain useful genetic information, in particular regulatory elements or sections of other genes, the complete under SEQ ID NO. 7 indicated nucleotide sequence from 1 to 1876 referred to according to the invention as gene spolVA.
- the coding area extends from positions 201 to 1679; the first codon, ie positions 201 to 203, are translated in vivo not as leucine but as methionine.
- the ß. subtilis gene spolVB codes for the phase IV sporulation protein B, which is stored in the Swiss-Prot and NCBI databases under the number P17896. It is important for the sigma factor K-dependent transition point during sporulation or its activation in the mother cell. It plays a role in inter-compartmental signal transmission, probably via the hydrophobic one N-terminus.
- the amino acid sequence of the factor SpolVB is shown in SEQ ID NO. 10 of the present application, as the translation of the previous DNA sequence generated by the Patentin program.
- the associated nucleotide sequence can be found in the database Subtilist under number BG10311 and is in the sequence listing in SEQ ID NO.
- the complete under SEQ ID NO. 9 specified nucleotide sequence from 1 to 1675 according to the invention referred to as gene spolVB.
- the coding area extends from positions 201 to 1478.
- the ß. subtilis gene spolVCA codes for a putative site-specific DNA recombinase, which is stored in the Swiss-Prot and NCBI databases under the number P17867. It probably plays a role in recombining the spolllC and spolVCB genes, from which the sigma factor K emerges.
- the amino acid sequence of this recombinase is shown in SEQ ID NO. 12 of the previous application, as the translation of the previous DNA sequence generated by the Patentin program.
- the associated nucleotide sequence can be found in the Subtilist database under number BG10458 and is in the sequence listing in SEQ ID NO.
- the complete under SEQ ID NO. 11 indicated nucleotide sequence from 1 to 1900 referred to according to the invention as gene spolVCA.
- the coding area extends from positions 201 to 1703; the first codon, ie positions 201 to 203, are translated in vivo not as valine but as methionine.
- the ß. subtilis gene spolVCB codes for the RNA polymerase sigma factor K precursor, which is stored in the Swiss-Prot and NCBI databases under number P12254.
- the rest of this factor is encoded by the gene spolllC, which is approximately 10 kb away on the chromosome, the region in between being referred to as SKIN.
- the active sigma factor K is obtained, which in turn is called Transcription factor works.
- the amino acid sequence of the sub-factor SpolVCB is shown in SEQ ID NO. 14 of the present application, as the translation of the previous DNA sequence generated by the Patentin program.
- nucleotide sequence can be found in the database Subtilist under the number BG 10459 and is in the sequence listing in SEQ ID NO. 13, together with the 200 nucleotides before the 5 'end and the 197 nucleotides behind the 3' end. Notwithstanding the fact that these marginal sequences may well contain useful genetic information, in particular regulatory elements or sections of other genes, the complete under SEQ ID NO. 13 indicated nucleotide sequence from 1 to 868 referred to according to the invention as gene spolVCB. The coding area extends from positions 201 to 671.
- the ß. subtilis gene spoIVFA codes for the phase IV sporulation protein FA. This factor, which is presumably able to form a heterodimer with SpoIVFB (see below), probably fulfills the task of stabilizing this factor but also inhibiting it at the same time. That is why SpoIVFA is formed earlier, probably in phase II.
- the amino acid sequence of SpoIVFA is stored in the Swiss-Prot and NCBI databases under number P26936. It is listed in SEQ ID NO. 16 of the present application, as the translation of the previous DNA sequence generated by the Patentin program.
- the associated nucleotide sequence can be found in the Subtilist database under number BG10331 and is in the sequence listing in SEQ ID NO.
- SEQ ID NO. 15 indicated nucleotide sequence from 1 to 1192 referred to as gene spoIVFA according to the invention.
- the coding area extends from positions 201 to 995.
- the ß. subtilis gene spoIVFB codes for the phase IV sporulation protein FB. It is a membrane-associated metalloprotease that is probably responsible for the processing from Pro-Sigma K to Sigma K; it is also formed in phase II of the sporulation.
- the amino acid sequence of SpoIVFB is in the Swiss-Prot and NCBI databases stored under number P26937. It is listed in SEQ ID NO. 18 of the present application, as the translation of the previous DNA sequence generated by the Patentin program.
- the associated nucleotide sequence can be found in the database Subtilist under the number BG 10332 and is in the sequence listing in SEQ ID NO. 17 together with the 200 nucleotides before the 5 'end and the 197 nucleotides behind the 3' end.
- SEQ ID NO. 17 indicated nucleotide sequence from 1 to 1264 according to the invention referred to as spoIVFB gene.
- the coding area extends from positions 201 to 1067; the first codon, ie positions 201 to 203, are translated in vivo not as leucine but as methionine.
- the region of nucleotides 1 to 1792 is referred to as gene spo / V
- the The actual SpolV coding section comprises positions 140 to 1336.
- the edge sequences may in turn contain other genetic elements such as regulatory elements or parts of other genes, below which the first codon GTG of positions 140 to 142 is translated in vivo not as valine but as methionine.
- subtilis which, with a homology of 68% identity at the amino acid level, is regarded as the closest similar protein known to date.
- This factor is specified in the Swiss-Prot database under number P54469; Both the amino acid sequence and the DNA sequence with both approximately 200 bp flanking regions can be found in the database Subtilist under the number BG11654. The entry there notes that it is an unknown protein, but due to the existing sequence homologies it can be regarded as similar to the phase IV sporulation protein.
- the associated sequences can be SEQ ID NO. 5 and 6 of the present application.
- the range of nucleotides 1 to 1594 is referred to as gene yqfD according to the invention, the actual protein-coding section comprising positions 201 to 1397.
- the first codon GTG of positions 201 to 203 is made as in spolV from ß. Hcheniformis translated in vivo not as valine but as methionine.
- one of these genes is inactivated simultaneously with recA in the production strain in order to obtain appropriate safety strains therefrom.
- deletion mutagenesis those in SEQ ID NO. 3, 5, 7, 9, 11, 13, 15 or 17 specified nucleic acids themselves used for inactivation. It is not even necessary to identify the relevant homologous genes from the species used for production. It can be expected that these deletions will be more successful the narrower the species in question with ß. subtilis or ß. Hcheniformis are related. Because this should be associated with an increasing homology of the genes in question. For this reason, the approx.
- 200 bp marginal sequences are also given in the sequence listing, because in accordance with the statements made for recA, constructs can be formed that completely cover the areas of at least 70 to 150 positions required for a crossing-over Contain areas and can be used with a certain probability of success for the deletion of the relevant sections in microorganisms not characterized in this regard.
- active systems that prevent viability and are to be regulated accordingly stringently, these also include those that were referred to in the introductory state of the art as "passive" systems for generating GMOs, in particular include inactivating mutations in one or more of the following Gene: epr, rp-l, rp-ll, isp-1, apr, npr, spoOA, bpr, rsp, mpr, vpr, spoOA, spollD, spollAC, spo2, spo3, sigE, sigF, spollE, spollSB, sigG, spolVCB, spolllC, nprM and the gene for isopropylmalate dehydrogenase (leuB).
- sporulation defects in the genes spolVA, spolVB, spolVCA, spolVCB, spoIVFA, spoIVFB or yqfD are identified in the nomenclature of ⁇ . subtilis or in the case of Bacillus Hcheniformis in the gene spolV or the genes that are homologous to it depending on the host cell.
- this homology can be inferred from a sequence comparison.
- the gene in question can be inactivated in the present microorganism strain intended for biotechnological production and the functional agreement of the genes in question can be checked by restoring the phenotype (rescue). If the parallel provision of a spolVA, spolVB, spolVCA, spolVCB, spoIVFA, spoIVFB, yqfD or spo / V copy relevant to the invention converts the knock-out mutant in question into a sporulation-positive phenotype, this would prove that there is a functional interchangeability of the genes under consideration.
- sporulation genes are therefore understood in particular to be those which are accessible to such a "rescue”. If this is possible, it is a preferably used sporulation gene.
- This control is possible in particular with a reasonable amount of work because, on the one hand, such a functionally inactive one Mutant is to be generated and, on the other hand, the sequences in question from ß. Subtilis and the particularly preferred thereof from ß. Hcheniformis are made available via the sequence listing for the present application, via which such a rescue can be carried out.
- the use according to the invention described so far for the functional inactivation of the genes spolVA, spolVB, spolVCA, spolVCB, spoIVFA, spoIVFB, yqfD or spolV or the genes which are homologous thereto is carried out using the sequences SEQ ID NO. 3, 5, 7, 9, 11, 13, 15, or 17 or parts thereof, preferably using parts that comprise at least 70 to 150 contiguous nucleic acid positions, particularly preferably using two such parts that enclose an intermediate part of the gene.
- microorganisms obtained with the methods described are a separate subject of the present invention. In its most general formulation, it is therefore a gram-positive bacterium which is not Bacillus megaterium, in which the recA gene is functionally inactivated.
- Gram-positive bacteria in which the gene recA has been functionally inactivated by genetic engineering, i.e. artificial work steps, are primarily intended here.
- gram-positive bacteria are the most important microorganisms for biotechnology, for example due to their ability to secrete valuable substances and / or their comparatively easy fermentability.
- different species are preferred for the different fields of application, so low-molecular compounds such as amino acids are used to a particularly large extent produced by Corynebacteria; Bacillus and especially ß.
- Hcheniformis is particularly valued for the production of extracellular proteins. According to the invention, they are all accessible, at least in principle, to functional inactivation of RecA.
- bacteria according to the invention are distinguished by the recombination defects described and therefore, under natural conditions, in particular in competition with other microorganisms, have disadvantages with regard to their survivability and are therefore suitable as security strains for biotechnological production. For the reasons stated above, this is not Bacillus megaterium.
- Gram-positive bacteria are also preferred, in which the functional inactivation was carried out via a nucleic acid according to the invention coding for RecA and / or via a nucleic acid whose nucleotide sequence corresponds to that in SEQ ID NO. 31 indicated nucleotide sequence in positions 369 to 1415 corresponds to at least 1045, preferably at least 1046, very particularly preferably 1047 of these 1047 positions, or via the at least partially non-coding flanking regions to these nucleic acids.
- nucleic acids with the homology values described above are around SEQ ID NO. 1 or, as already explained, to SEQ ID NO. 31 preferred accordingly.
- Microorganisms in which the functional inactivation using the in SEQ ID NO. 1 or 31 specified nucleic acid or sections thereof are the most preferred microorganisms in this regard.
- edge sequences of at least 70 to 150 bp each were preferably used, which can be checked by sequencing the relevant chromosomal sections.
- this includes in particular those genetic defects which have been carried out via biotechnological work steps.
- Gram-positive bacteria are preferred, among which the inactivated gene from phase IV of sporulation is in the nomenclature of ⁇ . subtilis is one of the genes spolVA, spolVB, spolVCA, spolVCB, spoIVFA, spoIVFB or yqfD or a gene homologous thereto, preferably in the case of ⁇ . subtilis for the yqfD gene, in the case of Bacillus Hcheniformis for the spolV gene and in any other case for a homologous gene.
- this is preferably a gram-positive bacterium in which - in addition to the RecA inactivation - exactly one gene from phase IV of the sporulation is functionally inactivated.
- a gram-positive bacterium is furthermore preferred in which the functional inactivation of the genes spolVA, spolVB, spolVCA, spolVCB, spoIVFA, spoIVFB, yqfD or spolV or the genes which are homologous to each other with the aid of one of the sequences SEQ ID NO. 3, 5, 7, 9, 11, 13, 15, or 17 or parts is effected thereof, preferably by means of parts which comprise at least 70 to 150 contiguous nucleic acid positions, particularly preferably by means of two such 'parts of an intermediate part Enclose the gens.
- the respective flanking sequences can be used as primers for this, the size of the PCR product providing information about the presence and possibly the size of inserts. This procedure is exemplified for spolV in the examples for the present application.
- those which are representatives of the genera Clostridium or Bacillus are particularly preferred, in particular those of the species Bacillus subtilis, B. Hcheniformis, B. amyloliquefaciens, B. stearothermophilus, B. globigii , B. clausii or ß. lentus, and especially around strains of ß. Hcheniformis.
- RecA preferably in combination with the preferred embodiments shown, is not active and the strain in question presents a significantly minimized safety risk in the event of an accidental release into the environment of the plant.
- Appropriate safety requirements are made for fermentation processes so that they can only be carried out if they meet these requirements.
- Example 5 shows that such a strain is not fundamentally disadvantageous under the optimal conditions during fermentation; this also applies to the double mutant described there.
- the inactivation of recA leads however, to a significantly reduced viability under the influence of UV. This is a common environmental factor that bacteria are confronted with when they leave the production plant.
- UV radiation is a common sterilization method for laboratories and biotechnological production facilities.
- the inactivation of spolV leads to a drastically reduced sporulation rate.
- both approaches can be combined with each other in the same bacterial strain.
- both "passive" systems for the generation of technically usable security strains complement each other due to their different active principles.
- the low-molecular compound is a natural product, a food supplement or a pharmaceutically relevant compound.
- compositions include, for example, amino acids or vitamins that are used particularly as food supplements.
- Pharmaceutically relevant compounds can be preliminary or intermediate stages to medication or even these themselves. In all these cases one speaks of biotransformation, according to which the metabolic properties of the microorganisms are used to completely or at least in individual steps to replace the otherwise complex chemical synthesis.
- the protein is an enzyme, in particular one from the group of ⁇ -amylases, proteases, cellulases, lipases, oxidoreductases, peroxidases, laccases, oxidases and hemicellulases.
- Industrial enzymes that are produced using such processes are used, for example, in the food industry.
- ⁇ -amylases are used to prevent bread from becoming stale or to clarify fruit juices.
- Proteases are used to break down proteins. All of these enzymes have been described for use in detergents and cleaning agents, with the subtilisin proteases already naturally produced by gram-positive bacteria occupying a prominent place. In particular in the textile and leather industry, they are used to process natural raw materials. Furthermore, all of these enzymes can in turn be used as catalysts for chemical reactions in the sense of biotransformation.
- the use is preferred for stabilizing single-stranded DNA, in particular in the case of DNA polymerization, in recombination processes taking place in vitro, or for converting double-stranded DNA into single-stranded DNA or vice versa.
- RecA is a DNA single-strand binding protein which, as explained, also has a certain affinity for double-stranded DNA. This function comes into play in the natural process of crossing over in the course of homologous recombination.
- RecA can be added to a PCR or a preparation of phage DNA in order to stabilize the single strands. If in vitro RecA processes can be understood, for example when introducing mutations (this also applies to the mutations according to the invention described above), this can be supported by RecA.
- the conversion of double-stranded DNA into single-stranded DNA or vice versa means a function that supports gyrase or gyrase. This can be used to influence the DNA topology, for example when working with plasmid DNA.
- Vectors which contain a previously described nucleic acid according to the invention, represent a separate subject of the invention.
- the present invention is also implemented in this form.
- This DNA can be processed or stored in the form of molecular biology in the form of cloning vectors.
- Such a vector is preferably an expression vector. This is because this can be used to produce a RecA according to the invention and to supply it to the mentioned applications of the factor.
- SEQ ID NO. 1 preferably a corresponding expression vector and more preferably by fermentation of a host containing this nucleic acid or this expression vector.
- the present invention is achieved in that a cell receives and translates such a gene in the form of a chromosomal copy.
- a cell receives and translates such a gene in the form of a chromosomal copy.
- the availability of this gene in the form of a plasmid appears to be easier to control, which may provide for the formation of this factor in several copies.
- a separate subject of the invention is the use of the nucleic acid according to the invention coding for a factor RecA for the expression of this factor. This is because, in accordance with what has been said above, the present invention is achieved in at least one aspect. This preferably serves to produce this factor yourself, in particular in a method described above. Alternatively, the intracellular expression can also be used to modulate the molecular biological activities of the cells in question, in particular in the case of recombination processes taking place in vivo.
- the inactivation by an antisense or RNA interference approach is intended, according to which the mRNA coding for RecA is specifically switched off or only partially translatable.
- the expression of this factor can be modulated in a very targeted manner. This applies both to biotechnological production strains and to laboratory approaches for studying molecular biological aspects.
- the present invention is also used in the use of the nucleic acid coding for a factor RecA described above according to the invention and / or a nucleic acid coding for a factor RecA, the nucleotide sequence of which corresponds to that in SEQ ID NO. 31 indicated nucleotide sequence in positions 369 to 1415 corresponds to at least 1045, preferably at least 1046, particularly preferably 1047 of these 1047 positions, for inactivating this factor or the recA gene in an in vitro approach, in particular via interaction with an associated nucleic acid.
- One embodiment of the present invention is thus a nucleic acid coding for a partial sequence of recA or for a partial sequence adjacent to recA in vivo, preferably less than 1,000 bp, particularly preferably less than 500 bp, according to one of SEQ ID NO. 25 to 30. Because these are partial sequences of recA or those that may lie just a few hundred intermediate base pairs away from recA. Increasingly preferred are 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100 bp up to an immediate neighborhood, that is to say a location at the beginning or end of recA, preferably in the areas not yet coding for protein. They can be obtained in accordance with Example 1 for obtaining a recA from a strain not further characterized in this regard, the probability of success increasing with increasing relatives. Hcheniformis increases.
- a preferred possible use here is to first prepare the most externally binding primers (for example recA6 in combination with recA5 according to FIG. 3B or, if these should not work, recA1 and / or recA4) in order to obtain the area in between. Then, to produce the specific deletion construct, oligonucleotides which bind further to the inside can be used as primers, for example recA2 (for example in combination with recA1) and recA3 (for example in combination with recA4), the nucleotide sequences of the inner primers being optionally based on the sequences obtained by the preceding PCR can be corrected. If this procedure fails, it is also possible, as is known per se and has already been said above, to use PCR primers with sequence variations. The success of this approach is confirmed by the sequencing of the fragment obtained, which corresponds to that in SEQ ID NO. 1 and / or 31 or sequences shown in Figure 2 should have clear homologies if it is a recAGen as desired.
- This is preferably a use for the amplification of a recA gene, since the aspect of inactivating this gene according to the present invention can thus be realized.
- these are preferably such uses for producing a gram-positive bacterium which is not Bacillus megaterium, in which the recA gene is functionally inactivated, including the embodiments of this aspect of the invention described above.
- a nucleic acid coding for a partial sequence of spolV or for a partial sequence adjacent to spolV in vivo preferably less than 1,000 bp, particularly preferably less than 500 bp away according to one of SEQ ID NO. 19 to 24;
- sequences for rec, 4 and spolV from ⁇ can in principle be given with the sequence listing.
- Hcheniformis DSM 13 (SEQ ID NO. 1 or 3) can be started. This was started earlier and initially a PCR-based method for isolating these genes from a Bacillus strain was used, as described in the publication “A general method for cloning recA genes of Gram-positive bacteria by polymerase chain reaction” (1992) Duwat et al. In J. Bacteriol., Volume 174 (No. 15), pp. 5171-5175.
- the coding region comprises positions 140 to 1336 (including the stop codon) shown there, the first three coding for the start codon GTG, which is translated in vivo as methionine.
- the entire section of 1,792 bp shown here is referred to as the gene spolV because it contains not only the protein-coding part but also regulatory elements which can be assigned to this gene. This gene designation is also made regardless of the fact that sections which are primarily attributable to other genes may extend into it. This seems justified because gene areas are sometimes overlapping. In the case of the recARegion, a DNA with a length of 1,557 bp was obtained, which in the sequence with SEQ ID NO.
- the two loci spolV and recA obtained in this way were stored in the database GenBank (National Center for Biotechnology Information NCBI, National Institutes of Health, Bethesda, MD, USA) under the accession numbers AJ616332 (for spolV) and AJ511368 (for recA).
- Part A shows the introduction of the deletion in spolV and thus the derivation of the strain ß. Hcheniformis A.1 ( ⁇ .spolV) from ß. Hcheniformis A.
- Part B shows the further development of ß. Hcheniformis A.1 ( ⁇ spolV) to ß. Hcheniformis A.2 ( ⁇ .spolV, ärecA).
- the gene location in question, including the genes immediately flanking it, is also designated, as are important restriction sites and the binding regions for the primers listed in Example 1.
- flank regions from the chromosomal DNA were amplified with the oligonucleotides shown in FIG. 3 and used to construct corresponding deletion cartridges. These were first created in the E. co // vector pUCBM21. This is below http://seq.yeastgenome.org/vectordb/vector_descrip/PUCBM21.html (viewed on January 14, 2005) and commercially available from the company, Röche Diagnostics GmbH, Röche Applied Science, Sandhofer Str. 116, 68305 Mannheim (formerly Boehringer) available. They were later cloned into the Bacillus ector pE194.
- a gene disruption with the help of such integrative vectors is generated by recombination events over the corresponding homologous flank areas.
- the original plasmid-localized, in vitro mutated copy of the gene to be disrupted is replaced by the native, intact copy in the bacterial chromosome by means of two successive single crossover events. Since the Bacillus effector has a temperature-sensitive origin of replication, the plasmid parts can be removed from the cells later after the disruption under non-permissive conditions (42 ° C.), which enables the establishment of a stable mutant line.
- the oligonucleotides spo3 and spo4 as well as spo7 and spo6 were used to construct the spo / V deletion cartridge; both sides are approximately 450 bp in size and frame an area of 740 bp (size of the later deletion).
- the oligonucleotides recA " ⁇ and rec.42 as well as recA3 and recA ⁇ were used to construct the recADeletion cartridge; both flanks are approx. 340 bp in size and frame an area of 852 bp (size of the later deletion).
- the transformant was subcultured at 42 ° C over several cultivation passages, with a corresponding ⁇ recA mutant ( ⁇ spo / V / ⁇ recA double mutant, referred to as B. Hcheniformis A.2) with the aid of a screening for mitocyc C sensitivity (0, 03 ⁇ g / ⁇ l) could be identified.
- this phenotypic finding was verified by a PCR using the oligonucleoCde recAS and recA5.
- the three strains were subjected to a Souf ⁇ ern analysis.
- 2 ⁇ g each of chromosomal DNA was cut with the restriction endonuclease C / al and after gel electrophoretic separation according to standard methods with a DIG-labeled PCR product (created with the primers spo3 and spo4 based on the starting DNA) hybridized.
- the larger of the two detected C / al fragments appeared in both strain A.1 and strain A.2 at a height which was lower than the original strain A by an amount corresponding to the deletion (FIG. 4B, left part).
- the DNA was digested with the restriction endonuclease Sspl and hybridized in an analogous manner with a DIG-labeled PCR product (prepared in an analogous manner with the primers recA and rec> 42).
- the corresponding Sspl fragment was located in strain A.2 due to the Deletion also at a correspondingly lower height than in parental strain A.1 or wild-type strain A (FIG. 4B right part).
- the sporulation tests were grown in 200 ml Schaeffer's sporulation medium (16.0 g LB medium, 2.0 g KCI, 0.5 g MgSO 4 x 7 H 2 O, ad 993.0 ml distilled water; pH 7.0; the solution is autoclaved and then supplemented with the following components: 1 ml Ca (NO 3 ) 2 (0.1 M), 1 ml MnCl 2 (0.1 M), 1 ml FeSO 4 (1 mM) , 4 ml glucose (20% (w / v)) in 500 ml two-chicane flasks, for each of the three strains to be tested, three flasks were inoculated 0.25% from an LB preculture and at 30 ° C and about 120 rpm (Innova 4230, New Brunswick Scientific, Edison, NY, USA).
- the cells were diluted to the one shown in Figure 6 the time of the logarithmic growth phase for a quantitative comparison of their UV-sensitivity each with 15 mM NaCl solution to a level of 10 "4 and 100 ul aliquots of the dilutions on
- the remaining LB plates were subsequently irradiated with UV light of wavelength 254 nm for different lengths: the plates were placed under a UV lamp, whose power was 100 ⁇ W / cm 2 , placed, covered after various irradiation times and wrapped in aluminum foil to protect against further exposure to light.
- Figure 1 Amino acid sequence alignment of SEQ ID NO. 2 with those of the closest similar rec factors described in the prior art.
- Mean RecA factor from ß. Hcheniformis DSM 13 (SEQ ID NO. 2) factor RecA from ß. amyloliquefaciens (AJ515542 in NCBI) factor RecA from ß. subtilis (Z99112 in NCBI; region 161035 to 162078) factor RecE from ß. subtilis (X52132 in NCBI)
- Figure 2 Nucleic acid sequence alignment of SEQ ID NO. 1 with those of the closest similar rec genes described in the prior art. Here mean: gene recA from ß. Hcheniformis DSM 13 (SEQ ID NO. 1) gene recA from ß. amyloliquefaciens (AJ515542 in NCBI) gene recA from ß. subtilis (Z99112 in NCBI; region 161035 to 162078) gene recE from ß. subtilis (X52132 in NCBI)
- Figure 3 Schematic representation of the genetic organizations of the wild-type and mutant loci of spolV (A) and recA (B), including the binding sites for the SEQ ID NO. 19 to 30 specified primers.
- Figure 4 Genotypic investigation of the mutant strains A.1 and A.2 in comparison with the parent strain ß. Hcheniformis A using PCR (A) and Southern analysis (B) (see Example 3).
- Figure 5 Graphical plot of the living cell numbers and the spore titer of the ß. licheniformis cultivation. Each culture was examined in three parallel experiments. Each experiment was statistically verified by four-fold determination (see example 4). Mean:
- Figure 6 Growth curve of a cultivation of the three ß. licheniformis strains in minimal medium (see example 5).
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JP2007503212A JP2007533306A (ja) | 2004-03-19 | 2005-02-16 | 生物工学的産生のために使用される、バシラス・リケニホルミス由来のRecA因子およびRecA不活性化安全な系統 |
US10/593,425 US20070212693A1 (en) | 2004-03-19 | 2005-02-16 | Factor reca from bacillus licheniformis and reca-inactivated safety stems used for biotechnological production |
EP05715348A EP1725582A1 (de) | 2004-03-19 | 2005-02-16 | Der faktor reca aus bacillus licheniformis und reca-inaktivierte sicherheitsstämme für die biotechnologische produktion |
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Title |
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KUNST F ET AL: "THE COMPLETE GENOME SEQUENCE OF THE GRAM-POSITIVE BACTERIUM BACILLUS SUBTILIS", NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 390, 1997, pages 249 - 256, XP000919353, ISSN: 0028-0836 * |
VEITH B ET AL.: "The complete genome sequence od Bacillus Lichenifirmis DSM13, an organism with great industrial potential", JOURNAL OF MOLECULAR MICROBIOLOGY AND BIOTECHNOLOGY, vol. 7, no. 4, 31 March 2004 (2004-03-31), pages 204 - 211, XP009047713 * |
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