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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
  • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination

Definitions

• the present invention relates to a method for the targeted genetic modification of Ashbya gossypii by homologous recombination, and to the Ashbyagossypii strains modified by this method.
• the filamentous Hemiascomycet Ashbya gossypii is of great interest, for example, as a ribo-flavin-producing microorganism.
• homologous recombination has been observed in filamentous ascomycetes such as Aspergillus or Neurospora; however, the proportion of homologous versus non-homologous recombination is often low. In addition, no reliable parameter is specified which • leads preferentially or almost exclusively to homologous recombination (Microbiological Reviews, 53, 148-170, 1989).
• the object was therefore to provide a method which allows the targeted genetic modification of Ashbya gossypii by homologous recombination.
• the gene of the translation elongation factor EF-l ⁇ (TEF) from Ashbya gossypii is contained in a 4.6 kb EcoRI-EcoRI fragment and a 6.3 kb BamHI-BamHI fragment, which are derived from Ashbya DNA libraries by hybridization with a TEF sample Have Saccnaromyces cerevisiae (EMBO J.3, 3311-3315, 1984) isolated (Fig. 12).
• the coding part of the TEF gene lies within the overlapping 2.1 kb EcoRI-BamHI fragment, the sequence of which is shown in the DNA sequence listing SEQ ID NO: 1 together with the adjacent sequences.
• Ashoya gossypii strains modified by this process represent microorganisms with valuable properties.
• the transformation of Ashbya gossypii means the transfer of recombined DNA into Ashbya gossypii nuclei or other DNA-bearing organelles using different methods. Protoplast transformation and electroporation are particularly suitable methods of transmission.
• the protoplast transformation is expediently carried out in such a way that protoplasts are produced from the mycelium with the aid of enzymes such as zymolyase.
• the protoplasts are suspended in an aqueous buffer containing calcium ions in a concentration of 1 to 10 ⁇ 10 8 / ml, preferably 3 to 5 ⁇ 10 ° / ml, and incubated with the purified DNA.
• the purified DNA is added in a ratio of 1 to 50 ⁇ g, preferably 10 to 25 ⁇ g, per 100 ml protoplast suspension.
• the transformed Ashbya gossypn cells are incubated in nutrient medium and spread on agar plates. If a selection marker, for example an antibiotic resistance gene, is used, the successfully transformed cells can be recognized particularly easily by growth on antibiotic-containing soil. Clonal purification via spore isolation is then advisable.
• Recombinant DNA constructions which contain the DNA to be recombined flanked by one or more gene regions of Ashbya gossypii are used as vectors for the transformation.
• a flanking gene region that is particularly well suited for homologous recombination is the TEF gene region.
• the DNA to be recombined is flanked, for example, as shown in the plasmids pAG-102 and pAG-145 (FIGS. 4 and 11).
• the DNA to be recombined can also come from Ashbya gossypii; however, DNA from other organisms of both prokaryotic and eukaryotic origin or synthetic DNA can also be used.
• the DNA to be recombined can be coding or non-coding DNA; preference is given to using coding DNA.
• the vectors used in the method according to the invention can also contain further DNA sequences such as selection markers.
• Shuttle vectors which replicate in bacteria and are used for the transformation of Ashbya gossypii are preferably used.
• these vectors usually have a bacterial origin of DNA replication and one or more antibiotic resistance genes.
• the plasmid pAG-102 and the plasmid pAG-145 or derivatives derived therefrom are particularly preferred as the vector (FIGS. 4 and 11). Derivatives are to be understood as those plasmids which, in addition to or instead of the G418 resistance gene, contain further DNA sequences flanked by A. gossypii DNA.
• circular DNA is used as a vector, it is linearized before the transformation of Ashbya gossypii in such a way that fragments with a terminal A. gossypii sequence are formed which initiate homologous recombination.
• the Ashbya gossypii strains modified in a targeted manner by this method can be identified by the fact that vector DNA has been integrated at the predetermined locations in the homology region (eg TEF gene locus). This can be demonstrated, for example, by Southern blotting with a vector sample. The isolation of modified Ashbya gos- sypii strains if a selection marker is contained in the vector DNA. Then only the changed strains can be isolated under selective conditions, for example by antibiotic selection.
• strains genetically modified with the plasmid pAG-102 by homologous recombination have the designations LU8334 to LU8341. These strains were deposited with the German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, under the following numbers:
• LU 8334 DSM 6661 LU 8335: DSM 6662 LU 8336: DSM 6663 LU 8337: DSM 6664 LU 8338: DSM 6665 LU 8339: DSM 6666 LU 8340: DSM 6667 LU 8341: DSM 6668
• the method according to the invention makes it possible to construct genetic changes, such as insertions, deletions and substitutions which are accurate to the base pair, in a simple and genetically stable or unstable manner as desired.
• the Hemiascomycet Ashbya gossypii is just as easily accessible as the single-cell yeast S. cerevisiae for genetic engineering processes which aim, for example, at genetically stable overexpression by homologous promoters.
• TEF gene sample comprises nucleotides 363 to 1235 of the 1377 bp long open reading frame of the S. cerevisiae TEF2 gene (Schirmaier and Philippsen, EMBO J. 3 (1984), 3311-3315).
• a 4.6 kb EcoRI fragment and a 6.3 kb BamHI fragment hybridized to the heterologous TEF gene sample.
• SEQ ID NO: 1 contains the open reading grid of 1377 bp (SEQ ID NO: 2), 436 bp of the 5'-non-coding region and 686 bp of the 3'-non-coding region.
• the promoter region was then isolated as a 403 bp HindIII / Hincll fragment which, in addition to the 379 bp before the start codon, also carries the first 24 bp of the open reading frame of the TEF gene, and was used for the constructions of pAG-100.
• pAG-1 (Fig. 1) (deposited DSM 6010), a derivative of the vector pEX4 was according to Ernst and Chan, J.Bacteriol. 163 (1985), 8-14.
• pAG-1 contains a 1.7 kb SalI fragment with the G418 resistance gene (G418r) of the transposon Tn903 coding for the aminoglycoside phosphotransferase (APH (3 ') I).
• G418r G418 resistance gene
• APH (3 ') I aminoglycoside phosphotransferase
• pAG-1 contains the Saccharomyces cerevisiae ARS elements ARS1 and 2 ⁇ ARS and replicates autonomously in Ashbya gossypii.
• pAG-2 (Fig. 2).
• the 1.7 kb Sall fragment with the G418 resistance gene was excised from pAG-1 and inserted into the Sall site of S. cerevisiae-E. coli shuttle vectors YEp24 (Botstein et al., Gene 8 (1979), 17-24; New England Biolabs Inc., Beverly, MA, USA, 1988-1989 Catalog, 112-113).
• the structure of the newly formed plasmid - pAG-2 - was checked by restriction endonuclease mapping, the Xhol interface located in the 1.7 kb SalI fragment being used to check the insert orientation.
• pAG-2 contains the Saccharomyces cerevisiae ARS element 2 ⁇ ARS and replicates autonomously in Ashbya gossypii. (Wright and Philippsen, Gene 109, 99-105 (1991) c) pAG-100 (Fig. 3).
• pAG-2 contains the Saccharomyces cerevisiae ARS element 2 ⁇ ARS and replicates autonomously in Ashbya gossypii. (Wright and Philippsen, Gene 109, 99-105 (1991) c) pAG-100 (Fig. 3).
• a 403 bp Hindlll / Hincll fragment which the promoter region and contains the first 24 bp of the open reading frame of the gene for the translation elongation factor EF-l ⁇ (TEF gene) from A. gossypii.
• pAG-100 contains the Saccharomyces cerevisiae ARS element 2 ⁇ ARS and replicates autonomously in Ashbya gossypii.
• pAG-102 (Fig. 4).
• the starting vector is pUC19, in which the BamHI site was inactivated after cutting, filling and religating.
• a 940 bp fragment was inserted into the HindIII site of pAG-121, which carries the additional 5 'end of the G418 resistance gene, fused to the TEF promoter.
• This fragment was obtained by digesting pAG-100 with HindIII. By inserting this fragment, a complete G418 resistance gene is created again.
• pAG-103 was partially digested with HindIII and the linearized plasmid isolated from an agarose gel.
• the protruding ends were blunt-ended by filling in using the Klenow fragment of DNA polymerase I and a 1.64 kb BamHI / Sall fragment from pAG-122 with the kanamycin resistance gene under control of the TEF promoter after filling of the protruding ends inserted.
• the fusion of the filled Sall and Hindlll interfaces again results in Hindlll interfaces.
• Transformation of A. gossypii with TEF gene region vectors The transformations were carried out according to the following scheme:
• MA2 Peptone (Gibco casein hydrolyzate, No. 140): 10 g / 1 yeast extract (Gibco): 1 g / 1
• SMA2 agar Sorbitol peptone yeast extract glucose myo-inositol agar (Gibco)
• SMA2 top layer Like SMA2 agar, instead of agar 0.8% agarose
• EDTA ethylenediaminetetraacetic acid
• Tris Tris (hydroxymethyl) aminomethane
• PTC40 40% (w / v) polyethylene glycol 4000 (Merck);
• the plasmid also carries a G418 resistance gene under the control of the TEF promoter, which gives Ashbya gossypii transformants resistance to the aminoglycoside G418.
• the plasmid has no signals for autonomous replication Ashbya gossypii. pAG-102 DNA was found within the
• TEF homology region (3 'end of the TEF gene) cut at the BamHI site and used to transform Ashbya gossypii protoplasts according to Example 3. This allows homologous recombination 3 'to be induced by the TEF gene.
• the eight independently obtained G418-resistant transformants (LU8334-LU8341, deposited with DSM, Braunschweig) retained their G418 resistance after clonal cleaning and without selection pressure.
• the pAG-102 DNA had been stably integrated into the Ashbya gossypii genome.
• FIG. 5a shows a chromosome separation of the eight transformants and the wild-type strain (middle lane).
• the TEF gene is located on the largest of the five visible chromosomes, in which the plasmid was also integrated in all eight cases (FIG. 5b).
• DNA fragments containing the novel uoints between chromosomal and plasmid DNA were cloned from three of the eight transformants as a control.
• a sequence analysis in the area of the BamHI sites showed no change compared to the wild type DNA.
• A. gossypii protoplasts were transformed with BamHI cut plasmid pAG-145. As a result, homologous recombination 5 'and 3' of the TEF gene can be induced simultaneously.
• Five transformants were clonally purified and examined for the integration of the kanamycin resistance gene using Southern analyzes. The analyzes of EcoRI-cleaved DNA were carried out with two clonally purified strains of each transformand, the analyzes of BglII-cleaved DNA with one clonally purified strain of each transformande. The results of these analyzes are shown in FIGS. 13 and 14; the data are interpreted using the model in FIG. 15.
• a stabilization test was carried out with the clonally purified transformants 1 to 5. Mycelium was incubated for six days on non-selective medium and then mycelium was transferred from the edge of each colony onto a new plate of non-selective medium and onto a plate with selective medium. After an incubation of a further three days on non-selective medium, mycelium was again transferred to new plates. After six days of non-selective growth, all of the transformants were still G418-resistant. After nine days, transformants 1, 2 and 3 had lost their resistance to G418. The data from the heart indicate that the loss is due to a homologous recombination between the TEF promoter fragments. The relatively high deletion frequency of the heterologous marker (G418 resistance gene) observed with this construction may be advantageous in the case of cotransformations.
• Cultivation in a liquid culture or on a plate is achieved by inoculating with mycelium or spores.
• Medium 1 liter
• MA-2 1 g peptone 1 g yeast extract
• MA-2-G418 MA-2 + 200 mg G418 / ml
• the gels thus obtained were transferred to Hybond-N membranes by Southern transfer and baked at 80 ° C. for 2 hours. Prehybridization, hybridization and the subsequent washing were carried out non-radioactively under stringent conditions ('Non-radioactive Labeling and Detection' Applications Manual, Boehringer Mannheim, order number: 1093 657).
• Running conditions 20 h running time, 48 s pulse duration
• Fig. 5 a Chromosome separation with OFAGE.
• Chromosomal DNA is characterized by thick lines, the plasmid DNA by thin or double lines. Arrows show the location of the TEF reading frame.
• B BamHI
• FIG. 12 Comparison of the homology regions used for the integration of pAG-102 and pAG-145, a: Homology region of pAG-102: 4.6 kb EcoRI fragment b: Homology region of pAG-145: 6.3 kb BamHI fragment Der Arrow indicates at which point in the genome the foreign DNA is integrated after homologous recombination.
• Figure 13 Southern with BglII-digested DNA from five pAG-145 transformants
• Genomic DNA from five clonally purified transformants of the plasmid pAG-145 and the untransformed parent strain was cleaved with BglII, separated on a 0.4% agarose gel and hybridized against radioactively labeled pAG-145 DNA in a Southern experiment. All transformants (lanes 1 to 5) show a signal with a length of about 17 kb, which also occurs in the wild-type DNA lane 6). In addition, all transformants have a fragment with a length of approximately 18.6 kb, which indicates integration of the G418 resistance gene into the 17 kb BglII fragment.
• FIG. 14 Southern with EcoRI digested DNA from five pAG-145 transformants
• Genomic DNA from two clonally purified strains of five transformants of the plasmid pAG-145 and the untransformed starting strain was split with EcoRI, separated on a 0.8% agarose gel and in a Southern experiment against radioactively labeled pAG-103 DNA hybridizes.
• the transformants 1 (lanes 1, 2), 3 (lanes 5, 6), 4 (lanes 7, 8) and 5 (lanes 9, 10) no longer have the fragment which occurs in the wild-type DNA (lane 11) of 1.55 kb, but instead a 1.32 kb fragment and additionally a 1.6 kb fragment that corresponds to the G418 resistance gene.
• MOLECULE TYPE DNA (genomic)
• HYPOTHETICAL NO
• ANTI-SENSE NO
• ORIGINAL.SOURCE

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• 1992
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