EP0672154A1 - PROCEDE D'EXPRESSION DES GENES DANS $i(BACILLUS LICHENIFORMIS) - Google Patents

PROCEDE D'EXPRESSION DES GENES DANS $i(BACILLUS LICHENIFORMIS)

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Publication number
EP0672154A1
EP0672154A1 EP92923721A EP92923721A EP0672154A1 EP 0672154 A1 EP0672154 A1 EP 0672154A1 EP 92923721 A EP92923721 A EP 92923721A EP 92923721 A EP92923721 A EP 92923721A EP 0672154 A1 EP0672154 A1 EP 0672154A1
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Prior art keywords
gene
licheniformis
process according
amylase
promoter
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EP92923721A
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German (de)
English (en)
Inventor
Steen Troels Jorgensen
Per Lina Jorgensen
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Novo Nordisk AS
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Novo Nordisk AS
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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to a process for expressing genes derived from anaerobic and/or thermophilic microorganisms in Bacillus licheniformis, as well as to a process for producing cyclodextrin glycosyl transferase in Bacillus licheniformis.
  • Cyclodextrin glycosyl transferases (1,4- ⁇ -D-glucan 4- ⁇ :-D-(l,4- ⁇ -D-glucano)transferase, EC 2.4.1.19), hereinafter termed CGTases, have previously been employed in the liquefaction of starch or starch hydrolysate, and for the formation of cyclodextrins by cyclisation.
  • the CGTases so far used for this purpose are produced by such microorganisms as Bacillus macerans, Bacillus circulans. Bacillus stearothermophilus, Bacillus megaterium. Bacillus ohbensis, alkalophilic Bacillus sp.
  • CGTases suffer from the disadvantage that they are not sufficiently stable at temperatures above 60°C to be useful in the production of cyclodextrins at sufficiently elevated temperatures to avoid microbial contamination. More recently, CGTases derived from a strain of Thermoanaerobacter or Thermoanaerobium have been isolated, as described in WO 89/03421. These CGTases have a temperature optimum at pH 5.0 of about 95°C.
  • the present invention relates to a process for expressing genes derived from anaerobic and/or thermophilic microorganisms in Bacillus licheniformis. in which process a suitable strain of B_i_ licheniformis transformed with a DNA sequence which includes a gene derived from an anaerobic and/or thermophilic microorganism, which DNA sequence is preceded by a promoter sequence capable of effecting transcription of said gene, is cultured under suitable conditions to obtain gene expression.
  • the present invention relates to a process for producing a cyclodextrin glycosyl transferase (CGTase) in B. licheniformis, in which process a suitable strain of B. licheniformis transformed with a DNA sequence which includes a gene coding for a CGTase, which DNA sequence is preceded by a promoter sequence capable of effecting transcription of said gene, is cultured under suitable conditions for the production of the CGTase, and the CGTase is recovered from the culture.
  • CGTase cyclodextrin glycosyl transferase
  • B ⁇ . licheniformis is an advantageous microorganism to use for the production of recombinant enzymes as at least some strains of B. licheniformis produce large amounts of enzyme protein. It is therefore possible to obtain a higher yield of CGTase and other enzymes derived from anaerobic organisms in B____ licheniformis than in for instance B____ subtilis.
  • the DNA sequence including the anaerobic and/or thermophilic gene should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in B. licheniformis and may be derived from a gene encoding a protein homologous or heterologous to J . licheniformis.
  • suitable promoters are derived from the gene coding for B. stearothermophilus maltogenic amylase (amyM) , B ⁇ licheniformis ⁇ -amylase (amyL) , B_ s _ amyloli ⁇ uefaciens ⁇ -amylase (amyQ) , B.
  • subtilis alcaline protease or the B ⁇ pumilus xylosidase promoter or the hybrid SPOl/lac promoter (D.G. Yansura and D.J. Henner, Proc. Natl. Acad. Sci. USA 81, 1984, pp. 439-443).
  • a particularly preferred promoter for use in the present process is a JL. licheniformis ⁇ -amylase promoter variant included in the following DNA sequence
  • thermophilic donor microorganism may be a strain of Archaebacterium and, more specifically, the gene derived from the thermophilic microorganism may therefore suitably be one encoding a Pyrococcus sp. pullulanase or ⁇ -amylase.
  • the Pyrococcus sp. pullulanase and ⁇ -amylase may, for instance, be the one described in PCT/DK91/00219 and WO 90/11352, respectively.
  • the anaerobic donor microorganism may be one which is also thermophilic, and the gene derived from the thermophilic and anaerobic microorganismmay therefore suitably be one encoding Thermoanaerobacter sp. or Thermoanaerobium sp. cyclodextrin glycosyl transferase, Thermotoga sp. glucose isomerase.
  • the DNA sequence including the gene derived from an anaerobic and/or thermophilic microorganism is present on an autonomously replicated expression vector.
  • the vector further comprises a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19 (C. Yanisch-Perron et al., Gene 33, 1985, pp. 103-119), pACYC177 (A.C.Y. Chang and
  • the vector may also comprise a selectable marker, e.g. a gene whose product confers antibiotic resistance such as ampcillin, chloramphenicol, kanamycin or tetracyclin resistance, or the dal genes from B. subtilis or B ⁇ licheniformis (B. Diderichsen, 1986) .
  • a selectable marker e.g. a gene whose product confers antibiotic resistance such as ampcillin, chloramphenicol, kanamycin or tetracyclin resistance, or the dal genes from B. subtilis or B ⁇ licheniformis (B. Diderichsen, 1986) .
  • the procedures used to ligate the DNA sequence coding for the gene from the anaerobic and/or thermophilic microorganism, promoter and origin of replication are well known to persons skilled in the art (cf. , for instance, Sambrook et al.. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, 1989) .
  • the DNA sequence including the gene derived from an anaerobic and/or thermophilic microorganism may be present on the chromosome of the B ⁇ licheniformis host cell. This is often an advantage as the DNA sequence is more likely to be stably maintained in the host cell. Integration of the DNA sequence into the host chromosome may be performed according to conventional methods, e.g. by homologous recombination. In one embodiment, said DNA sequence may be present in two or more copies on the chromosome of the B ⁇ _ licheniformis host cell.
  • said DNA sequence is present on the chromosome of the B__j_ licheniformis host cell at the site of the B_j_ licheniformis ⁇ - amylase gene, and is expressed by means of the expression signals of the B_____ licheniformis ⁇ -amylase, including the amyL promoter, in particular the amyL promoter variant described above, and the amylase signal peptide.
  • the B_j_ licheniformis host cell is one which is protease and/or amylase deficient as, generally speaking, it is an advantage that as few proteins as possible are present in the culture medium, thus facilitating the purification of the protein of interest.
  • An expressed protease might also degrade at least part of the gene product of interest, and an expressed amylase (insofar as the gene product of interest is a starch-degrading enzyme such as CGTase) might not be tolerated in the final product and might make the subsequent purification of the product particularly difficult, either case resulting in a decreased yield of the product of interest.
  • Protease and/or amylase deficiency may for instance be obtained by deletions or insertions in the genes encoding the protease or amylase, e.g. by introducing the DNA sequence encoding a CGTase into the host chromosome at the site of the ⁇ -amylase gene, as indicated above.
  • the product of the expressed gene is preferably recovered from the culture.
  • Recovery of the product may be done by conventional procedures including separating the cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt., e.g. ammonium sulphate, followed, if necessary, by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.
  • pBR322 indicates pBR322-derived DNA
  • PhamyM indicates the promoter of the B ⁇ . sterothermophilus maltogenic amylase gene (Diderichsen and Christiansen, 1988) ;
  • PTK233-2 indicates pKK233-2 derived DNA
  • PamyL indicates the promoter of the B_-_ licheniformis a- a ylase gene
  • amyL-cgtA indicates the fusion gene comprising the signal peptide coding part of the B ⁇ licheniformis ⁇ -amylase gene and the part of the Thermoanaerobacter CGTase gene coding for the mature enzyme;
  • ori pE194" indicates the plus origin of replication and rep gene containing region of pE194;
  • dfs indicates a sequence immediately 3 1 of the dal gene.
  • Fig. 21 is a restriction map of plasmid pPL1487;
  • Fig. 22 is a restriction map of plasmid pSJ932;
  • Fig. 23 is a restriction map of plasmid pSJ948;
  • Fig. 24 is a restriction map of plasmid pSJ980;
  • Fig. 25 is a restriction map of plasmid pSJ1391;
  • Fig. 26 is a schematic presentation of the exchange, by homologous recombination, between the chromosomal ⁇ -amylase gene and the amyL-cgtA fusion gene carried on plasmid pSJ1391;
  • Fig. 26 is a schematic presentation of the exchange, by homologous recombination, between the chromosomal ⁇ -amylase gene and the amyL-cgtA fusion gene carried on plasmid pSJ1391; Fig.
  • FIG. 27 is a schematic presentation of the .in vivo recombination between the 5 1 ends of the mature parts of cgtA;
  • Fig. 28 is a restriction map of plasmid pDN1316;
  • Fig. 29 is a restriction map of plasmid pDN3020;
  • Fig. 30 is a restriction map of plasmid pSJ1446; and
  • Fig. 31 is a restriction map of plasmid pSJ1448.
  • Competent cells were prepared and transformed as described by
  • Transformation of B. licheniformis Plasmids were introduced into B. licheniformis by polyethylene glycol-mediated protoplast transformation as described by Akamatzu, 1984. CGTase-producing colonies of either L . coli, B_-_ subtilis or B____ licheniformis were identified by plating transformants on LB agar plates supplemented with 1% soluble starch. After incubation at either 37°C or 30"C overnight, plates were 5 stained by iodine vapour to show hydrolysis zones produced by the action of the CGTase on the starch.
  • E. coli plasmid pNV601 (Fig. 1) , carrying the Thermoanaerobacter sp. ATCC 53627 CGTase gene referred to in the following as cgtA, is disclosed in WO 2589/03421.
  • the B. subtilis plasmid pPL1878 (Fig. 2), containing the cgtA gene, is disclosed in WO 91/09129. It was constructed as follows:
  • pNV601 was digested partially with Sau3A, then religated and transformed into E. coli SCSI (frozen competent cells purchased
  • pPL1489 was derived from plasmid pKK233-2 (purchased from Pharmacia LKB Biotechnology) by insertion of a synthetic DNA linker between the PstI and Hindlll sites in pKK233-2. This linker was the Pstl-Hindlll fragment from
  • pPL1540 was digested with Haell and SphI, and the 2.4 kb fragment containing the cgtA gene was inserted into Haell + SphI digested plasmid pDN1380 (Diderichsen and Christiansen, 1988) .
  • Plasmid pPL1892 (Fig. 8) was constructed by insertion of the cgtA gene excised from pPL1878 on a 2.4 kb Sall-NotI fragment into Sail + NotI digested pPL1759, and transformation of DN1885 to kanamycin resistance (10 ⁇ g/ml) .
  • Plasmid pPL1796 (Fig. 9) was constructed by insertion of a 0.5 0 kb SacI-EcoRV fragment from pBB37 (Fig. 10; J ⁇ rgensen, P. et al., 1991) into SacI + Smal digested pPL1385 (Fig. 11; Diderichsen et al., 1990), and transformation of DN1885 to chloramphenicol resistance (6 ⁇ g/ml) .
  • Plasmid pPL1893 (Fig. 12) was constructed by insertion of the CGTase gene excised from pPL1878 on a 2.4 kb BamHI-NotI fragment into BamHI + NotI digested pPL1796, and transformation of DN1885 to chloramphenicol resistance (6 ⁇ g/ml) .
  • oligonucleotide linker was syn ⁇ thesized and ligated into Sail digested pUC19 (Yanish-Perron et al., 1985), giving pSJllll (Fig. 13) upon transformation of E. coli SJ2 (Diderichsen et al., 1990) and selection for ampicillin resistance (200 ⁇ g/ml) :
  • pSJ994 (Fig. 16) was constructed by ligation of the 0.6 kb Notl-Ncol fragment from pPL1893 to the 5.4 kb Notl-Ncol fragment from pPL1892, and transformation into B. subtilis DN1885, selecting for kanamycin resistance (10 ⁇ g/ml) .
  • pSJ1283 (Fig. 17) was constructed by ligation of the 1.1 kb Sail fragment from pSJ1277 to Sail digested pSJ994, and transformation into DN1885, selecting for kanamycin (10 ⁇ g/ml) and chloramphenicol (6 ⁇ g/ml) resistance.
  • pSJ1342 (Fig. 18) was constructed by deletion of the 1.1 kb PstI fragment from pSJ1283, and transformation into DN1885, selecting for kanamycin resistance (10 ⁇ g/ml) .
  • pSJ1359 (Fig. 19) was constructed by the actual in vivo recom- bination from pSJ13 2. There is homology between the start of the mature part of the CGTase gene and part of the synthetic oligonucleotide extending between PstI and Sail on pSJ1342. If the plasmid undergoes a recombination event between these two homologous regions, the unique sites for Xbal, Sail and BamHI will be deleted.
  • a batch of pSJ1342 prepared from host strain DN1885 was thoroughly digested with BamHI, Xbal and Sail, and the digested plasmid was directly (i.e. without ligation) transformed into competent cells of DN1885, selecting for kanamycin resistance (10 ⁇ g/ml) .
  • This procedure strongly enriches for recombined plasmids, as linearized plasmid monomers are unable to transform B. subtilis competent cells (Mottes et al., 1979). Recombined plasmids would not be cleaved by the restriction enzymes, and thus exist as a mixture of monomeric and oligomeric forms well able to transform competent B. subtilis 5 cells.
  • This plasmid contains the origin of replication of pUBllO (Lacey and Chopra, 1974, Gryczan et al., 1978, McKenzie et al., 1986), the pUBllO rep protein gene, the kanamycin resistance gene, and the B. licheniformis ⁇ -amylase (amyL) promoter and signal peptide 10 coding region perfectly fused to the DNA encoding the mature part of the CGTase from Thermoanaerobacter sp. ATCC 53627.
  • a 1.4 kb BamHI fragment containing the pUBllO kanamycin resistance gene (kan) was excised from plasmid pDN2904 (WO 1591/09129) , ligated to Bglll digested pDN3000 (Fig. 6) , transformed into E. coli SCSI selecting ampicillin resistance (100 ⁇ g/ml) , and pPL1483 (Fig. 20) was recovered from one such transformant.
  • This plasmid was then combined with a Bacillus vector
  • AccI, pE194 digested with Clal the two linearized plasmids mixed, ligated, and transformed into B. subtilis DN1885 selecting kanamycin resistance (10 ⁇ g/ml) at 30 °C.
  • One such 5 transformant contained pPL1487 (Fig. 21) .
  • a 3*-terminal fragment of the amyL gene was excised from plasmid pDN1528 (J ⁇ rgensen, S. et al., 1991) as a 0.7 kb Sall- Hindlll fragment, ligated to Sall+Hindlll digested pUC19, and transformed to E. coli SJ2, selecting for ampicillin resistance 0 (200 ⁇ g/ml).
  • One such transformant contained pSJ932 (Fig. 22).
  • Plasmid pSJ948 (Fig. 23) was obtained by insertion of a Bglll linker into Malawi digested pSJ932, once more selecting for ampicillin resistance (200 ⁇ g/ml) upon transformation of SJ2.
  • pSJ980 (Fig. 24) was constructed by ligation of the 5.1 kb Hindlll fragment of pPL1487 to Hindlll digested pSJ948, selecting for kanamycin resistance (10 ⁇ g/ml) in B. subtilis DN1885 at 30 °C.
  • pSJ1391 (Fig. 25) was constructed by ligation of the 4.0 kb Bglll fragment of pSJ1359 to the 5.6 kb Bglll fragment of pSJ980, selecting for kanamycin resistance (10 ⁇ g/ml) in DN1885 at 30 °C.
  • This plasmid contains, on a vector temperature- sensitive for replication and conferring resistance to kanamycin and erythromycin, the promoter and upstream region (about 0.4 kb) from the B.
  • licheniformis ⁇ -amylase gene (amyL)
  • the ⁇ -amylase/CGTase fusion gene (amyL-cgtA)
  • 'amyL the 3'-region of the ⁇ -amylase gene
  • ⁇ -amylase producing strain of B. licheniformis was transformed with pSJ1391 by the protoplast transformation pro ⁇ cedure (Akamatzu, 1984) .
  • One regenerating, kanamycin resistant colony was isolated, and was found to produce both ⁇ -amylase and CGTase.
  • Production of the two enzymes can be easily distinguished by separating proteins in the culture supernatant from shake flask cultures in BPX medium (WO 91/09129) on isoelectric focusing gels (e.g. using the Pharmacia Phast sys ⁇ tem) , followed by overlayering with an agarose gel containing 1 % soluble starch and subsequent staining by iodine vapour.
  • the CGTase activity was detected at pi 4.5, the ⁇ -amylase activity at pi 8.
  • this transformant was analyzed for its plasmid content, it turned out that a recombination event between the incoming plasmid and the chromosome had taken place: A double recombination had exchanged the chromosomal ⁇ -amylase (amyL) gene and the plasmid borne amyL-cgtA fusion gene, so that the plasmid isolated carried the amyL gene (B. subtilis DN1885 transformed with this plasmid produced ⁇ -amylase) whereas the amyL-cgtA fusion gene now resided on the chromosome (Fig. 26) .
  • the original B. licheniformis transformant was also subjected to experimental conditions to ensure chromosomal integration and subsequent excision of the plasmid, in order to promote recombination events.
  • the transformant was plated on LB agar (WO 91/09129) with 10 ⁇ g/ml kanamycin at 50 °C, individual colonies restreaked a few times at 50 °C, and each then grown in successive overnight TY cultures at 30 °C without kanamycin to permit plasmid excision and loss.
  • Kana s isolates from each original 50 °C colony were incubated in BPX shake flasks and production of either ⁇ -amylase or CGTase determined by analysis on isoelectric focusing gels as above.
  • the plasmid free strains analyzed all produced either CGTase or ⁇ -amylase.
  • CGTase producing isolates are e.g. SJ1561-62, 1580-83, 1586-91 and
  • SJ1608 appeared to produce CGTase in larger amounts than the others.
  • amyL promoter sequence of the B. licheniformis host strain is shown in SEQ ID#2.
  • the promoter region from a number of the CGTase producing B. licheniformis strains was amplified from chromosomal DNA by the PCR technique (Saiki et al., 1988) , using as primers one oligo- 0 nucleotide corresponding to pos. 204-233 reading downstream through the amyL promoter, and another oligonucleotide corre ⁇ sponding in sequence to the 5'-end of the DNA encoding the mature CGTase and reading upstream.
  • the sequence of this second oligonucleotide was 5'-CCTGTTGGATTATTACTGGG-3' (SEQ ID#4) .
  • pDN3020 (Fig. 29) is a derivative of pDN1316 constructed by inserting a synthetic SphI site containing oligonucleotide linker into the EcoRI site of plasmid pDN1380 (Diderichsen and
  • plasmid pDN1620 25 Christiansen, 1988, resulting in plasmid pDN1620.
  • the promoter region of a maltogenic amylase from B_-_ stearothermophilus (Pa yM) present on pDN1620 was then transferred to Sphl-BamHI digested pUC19 on an approximately 200 bp BamHI-SphI fragment, resulting in plasmid pDN2977.
  • the promoter region was excised
  • Strain DN1686 is a Spo " derivative of DN1280 which contains a chromosomal deletion in the dal gene (Diderichsen, 1986) . DN1686 was derived from DN1280 by traditional mutagenesis procedures and was used as the host in the following experiment.
  • amyL-cgtA fusion gene was excised from pSJ1360 (identical to pSJ1359 shown in Fig. 19) as a 4 kb Bglll fragment and ligated to BamHI digested pDN3020, resulting in pSJ1446 (Fig. 30) and pSJ1448 (Fig. 31) on transformation of DN1686 to chloramphenicol resistance (6 ⁇ g/ml) .
  • Integrant strains SJ1454 and SJ1455 were subsequently isolated by transformation of DN1686 with pSJ1446 and pSJ1448, respectively, and isolation of transformants that were CGTase- producing, but chloramphenicol sensitive. These strains were incubated in BPX shake flasks at 37°C for 6 days, and the CGTase activity was measured (in arbitrary unit, as in example 4).
  • Ml3 phage cloning vectors and host strains nucleotide sequences of the M13 mpl ⁇ and pUC19 vectors. Gene 33, 103-119.
  • ORGANISM Bacillus licheniformis
  • TCAGOGGACA (XTGCCTGTA CACITGCGTC CTCCATAOGG OGGGATCAAT GATTCCGICC 240 GCTOGCITTC C&ATCTGAAG GTTTCATIGT GGGATGTTGA TOOGGAAGAT TGGAAGTACA 300
  • AAAATAAGCA AAAGAI CTC AATCATCTCA.
  • CTAGAG 66 (2) INFOEMAnCN FOR SEQ ID NO: 4:

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Abstract

Procédé d'expression des gènes dérivés de micro-organismes anaérobies et/ou thermophiles dans Bacillus licheniformis, dans lequel une souche adéquate de Bacillus licheniformis transformée avec une séquence d'ADN qui comprend un gène dérivé d'un micro-organisme anaérobie et/ou thermophile, laquelle séquence d'ADN est précédée par une séquence activante capable d'effectuer la transcription de ce gène, est cultivée dans des conditions adéquates pour obtenir l'expression du gène.
EP92923721A 1991-11-14 1992-11-13 PROCEDE D'EXPRESSION DES GENES DANS $i(BACILLUS LICHENIFORMIS) Withdrawn EP0672154A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
WOPCT/DK91/00344 1991-11-14
DK9100344 1991-11-14
PCT/DK1992/000337 WO1993010248A1 (fr) 1991-11-14 1992-11-13 PROCEDE D'EXPRESSION DES GENES DANS $i(BACILLUS LICHENIFORMIS)

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EP0672154A1 true EP0672154A1 (fr) 1995-09-20

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EP (1) EP0672154A1 (fr)
JP (1) JPH07503363A (fr)
FI (1) FI942227A0 (fr)
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US5624829A (en) * 1984-07-03 1997-04-29 Gist-Brocades, B.V. Transformed industrial bacillus strains and methods for making and using them
AU6107094A (en) * 1993-02-19 1994-09-14 Novo Nordisk A/S An amylolytic enzyme
WO1996023887A1 (fr) * 1995-01-30 1996-08-08 E.I. Du Pont De Nemours And Company Procede pour produire les enzymes thermostables xylanase et beta-glucosidase a partir de bacteries
US6300115B1 (en) 1998-05-18 2001-10-09 Enzyme Bio-Systems Ltd. Pullulanase expression constructs containing α-amylase promoter and leader sequences
JP5037771B2 (ja) 2000-04-13 2012-10-03 バイオチカ テクノロジー リミテッド グリコシル化されたハイブリッド産物およびその産生方法と利用
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JPH07503363A (ja) 1995-04-13

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