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WO1993010248A1 - PROCEDE D'EXPRESSION DES GENES DANS $i(BACILLUS LICHENIFORMIS) - Google Patents

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

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Publication number
WO1993010248A1
WO1993010248A1 PCT/DK1992/000337 DK9200337W WO9310248A1 WO 1993010248 A1 WO1993010248 A1 WO 1993010248A1 DK 9200337 W DK9200337 W DK 9200337W WO 9310248 A1 WO9310248 A1 WO 9310248A1
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Prior art keywords
gene
licheniformis
process according
amylase
promoter
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PCT/DK1992/000337
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English (en)
Inventor
Steen Troels JØRGENSEN
Per Linå JØRGENSEN
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Novo Nordisk A/S
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Priority to JP5508898A priority Critical patent/JPH07503363A/ja
Priority to EP92923721A priority patent/EP0672154A1/fr
Priority to FI942227A priority patent/FI942227L/fi
Publication of WO1993010248A1 publication Critical patent/WO1993010248A1/fr

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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-(1,4- ⁇ -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.
  • CGTases Bacillus ohbensis, alkalophilic Bacillus sp., Micrococcus luteus, Micrococcus varians and Klebsiella pneumoniae. These 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. For the production of large amounts of CGTase, it is an advantage to produce it by recombinant DNA techniques.
  • 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. 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.
  • CGTase cyclodextrin glycosyl transferase
  • 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.
  • 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 B. licheniformis.
  • suitable promoters are derived from the gene coding for B. stearothermophilus maltogenic amylase (amyM), B. licheniformis ⁇ -amylase (amyL), B. amyloliquefaciens ⁇ -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 B. licheniformis ⁇ -amylase promoter variant included in the following DNA sequence GCATGCGTCC TTCTTTGTGC TTGGAAGCAG AGCCCAATAT TATCCCGAAA CGATAAAACG GATGCTGAAG GAAGGAAACG AAGTCGGCAA CCATTCCTGG GACCCATCCG TTATTGACAA GGCTGTCAAA CGAAAAAGCG TATCAGGAGA TTAACGACAC GCAAGAAATG ATCGAAAAAA TCAGCGGACA CCTGCCTGTA CACTTGCGTC CTCCATACGG CGGGATCAAT GATTCCGTCC GCTCGCTTTC CAATCTGAAG GTTTCATTGT GGGATGTTGA TCCGGAAGAT TGGAAGTACA AAAATAAGCA AAAGATTGTC AATCATGTCA TGAGCCATGC GGGACGGAAAAAAAAAAAAAAAAAAAAA TGGAAGTACA AAAATAAGCA AAAGATTGTC AATCATGT
  • 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 microorganism may 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. licheniformis host cell at the site of the B. 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. 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 ⁇ -amylase gene
  • AmyQ indicates the promoter of the B. amyloliquefaciens ⁇ -amylase 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' of the dal gene.
  • Fig. 1 is a restriction map of plasmid pNV601
  • Fig. 2 is a restriction map of plasmid pPL1878
  • Fig. 3 is a restriction map of plasmid pPL1419
  • Fig. 4 is a restriction map of plasmid pPL1489
  • Fig. 5 is a restriction map of plasmid pPL1540
  • Fig. 6 is a restriction map of plasmid pDN3000
  • Fig. 7 is a restriction map of plasmid pPL1759
  • Fig. 88 is a restriction map of plasmid pPL1892
  • Fig. 9 is a restriction map of plasmid pPL1796
  • Fig. 10 is a restriction map of plasmid pBB37
  • Fig. 11 is a restriction map of plasmid pPL1385
  • Fig. 12 is a restriction map of plasmid pPL1893
  • Fig. 13 is a restriction map of plasmid pSJ1111
  • Fig. 14 is a restriction map of plasmid pDN3060
  • Fig. 15 is a restriction map of plasmid pSJ1277
  • Fig. 16 is a restriction map of plasmid pSJ994;
  • Fig. 17 is a restriction map of plasmid pSJ1283
  • Fig. 18 is a restriction map of plasmid pSJ1342
  • Fig. 19 is a restriction map of plasmid pSJ1359
  • Fig. 20 is a restriction map of plasmid pPL1483
  • 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. 27 is a schematic presentation of the in vivo recombination between the 5' 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
  • Fig. 31 is a restriction map of plasmid pSJ1448.
  • Competent cells were prepared and transformed as described by
  • Plasmids were introduced into B. licheniformis by polyethylene glycol-mediated protoplast transformation as described by Akamatzu, 1984.
  • CGTase-producing colonies of either E. 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 30oC overnight, plates were stained by iodine vapour to show hydrolysis zones produced by the action of the CGTase on the starch.
  • BPX Potato starch 100 g/l
  • Pluronic 0.1 g/l LB agar Bacto-tryptone 10 g/l
  • 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 89/03421.
  • 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 SCS1 (frozen competent cells purchased from Stratagene, Ja Jolla, California), selecting for ampicillin resistance (200 ⁇ g/ml).
  • pPL1419 containing pPL14l9
  • Plasmid pPL1419 was partially digested with Sau3A, and fragments ligated to BglII digested pPL1489 (Fig. 4).
  • One CGTase positive, ampicillin resistant (200 ⁇ g/ml) E. coli SCS1 transformant contained pPL1540 (Fig. 5).
  • pPL1489 was derived from plasmid pKK233-2 (purchased from Pharmacia LKB Biotechnology) by insertion of a synthetic DNA linker between the PstI and HindIII sites in pKK233-2. This linker was the Pstl-HindIII fragment from pDN3000 (Fig.
  • pPL1540 was digested with HaeII and SphI, and the 2.4 kb fragment containing the cgtA gene was inserted into HaeII + SphI digested plasmid pDN1380 (Diderichsen and Christiansen, 1988).
  • a CGTase positive, chloramphenicol resistant (6 ⁇ g/ml) transformant of B. subtilis DN1885 contained pPL1878.
  • Plasmid pPL1892 (Fig. 8) was constructed by insertion of the cgtA gene excised from pPL1878 on a 2.4 kb SalI-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 kb SacI-EcoRV fragment from pBB37 (Fig. 10; J ⁇ rgensen, P. et al., 1991) into SacI + SmaI 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).
  • pSJ1283 (Fig. 17) was constructed by ligation of the 1.1 kb Sail fragment from pSJ1277 to SalI digested pSJ994, and transformation into DN1885, selecting for kanamycin (10 ⁇ g/ml) and chloramphenicol (6 ⁇ g/ml) resistance.
  • pSJ1342 (Fig. 17) was constructed by ligation of the 1.1 kb Sail fragment from pSJ1277 to SalI digested pSJ994, and transformation into DN1885, selecting for kanamycin (10 ⁇ g/ml) and chloramphenicol (6 ⁇ g/ml) resistance.
  • pSJ1359 (Fig. 19) was constructed by the actual in vivo recombination from pSJ1342. There is homology between the start of the mature part of the CGTase gene and part of the synthetic oligonucleotide extending between PstI and SalI on pSJ1342. If the plasmid undergoes a recombination event between these two homologous regions, the unique sites for Xbal, SalI and BamHI will be deleted.
  • a batch of pSJ1342 prepared from host strain DN1885 was thoroughly digested with BamHI, XbaI and SalI, 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 cells.
  • This plasmid contains the origin of replication of pUB110 (Lacey and Chopra, 1974, Gryczan et al., 1978, McKenzie et al., 1986), the pUB110 rep protein gene, the kanamycin resistance gene, and the B. licheniformis ⁇ -amylase (amyL) promoter and signal peptide 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 pUB110 kanamycin resistance gene (kan) was excised from plasmid pDN2904 (WO 91/09129), ligated to BglII digested pDN3000 (Fig. 6), transformed into E. coli SCS1 selecting ampicillin resistance (100 ⁇ g/ml), and pPL1483 (Fig. 20) was recovered from one such transformant.
  • 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 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 SalI-HindIII fragment, ligated to SalI+HindIII digested pUC19, and transformed to E. coli SJ2, selecting for ampicillin resistance (200 ⁇ g/ml).
  • One such transformant contained pSJ932 (Fig. 22).
  • Plasmid pSJ948 (Fig. 23) was obtained by insertion of a BglII linker into HindII digested pSJ932, once more selecting for ampicillin resistance (200 ⁇ g/ml) upon transformation of SJ2.
  • pSJ980 Fig.
  • pSJ1391 (Fig. 25) was constructed by ligation of the 4.0 kb BglII fragment of pSJ1359 to the 5.6 kb BglII fragment of pSJ980, selecting for kanamycin resistance (10 ⁇ g/ml) in DN1885 at 30°C.
  • This plasmid contains, on a vector temperaturesensitive 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), and then about 0.7 kb from the 3'-region of the ⁇ -amylase gene ('amyL).
  • ⁇ -amylase producing strain of B. licheniformis was transformed with pSJ1391 by the protoplast transformation procedure (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 system), 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.
  • 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 oligonucleotide corresponding to pos. 204-233 reading downstream through the amyL promoter, and another oligonucleotide corresponding 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).
  • the amplified DNA fragment from each strain was excised from an agarose gel and directly sequenced, using as sequencing primers in the dideoxy method (Sanger et al., 1977) the same oligonucleotides that were used for PCR amplification.
  • the results of the sequence analysis reveal that one or both of two point mutations in the promoter region are responsible for the large difference in CGTase production observed.
  • pDN1316 (Fig. 28) is identical to pDN1313 (B. Diderichsen, 1986) except for the orientation of the multilinker.
  • 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 Christiansen, 1988), resulting in plasmid pDN1620. The promoter region of a maltogenic amylase from B.
  • stearothermophilus (PamyM) present on pDN1620 was then transferred to SphI-BamHI digested pUC19 on an approximately 200 bp BamHI-SphI fragment, resulting in plasmid pDN2977.
  • the promoter region was excised from pDN2977 on an approximately 200 bp BglII-SacI fragment which was inserted in the polylinker region of pDN1316, thereby generating plasmid pDN3020.
  • 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 BglII 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). Strain CGTase activity, arbitrary units
  • M13 phage cloning vectors and host strains nucleotide sequences of the M13 mp18 and pUC19 vectors. Gene 33, 103-119.
  • ORGANISM Bacillus licheniformis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
  • GCATGCGTOC TTCITIGTGC TTGGAAGCAG AGOCCAATAT TATCCCGAAA OGATAAAACG 60
  • AAAATAAGCA AAAGATTGTC AATCATCTCA TGAGCCATGC GGGAGACGGA AAAATCGTCT 360
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Bacillus licheniformis
  • GCATGCGTCC TTCTTTGTGC TTGGAAGCAG AGCCCAATAT TATCCCGAAA CGATAAAACG 60

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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.
PCT/DK1992/000337 1991-11-14 1992-11-13 PROCEDE D'EXPRESSION DES GENES DANS $i(BACILLUS LICHENIFORMIS) WO1993010248A1 (fr)

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JP5508898A JPH07503363A (ja) 1991-11-14 1992-11-13 バシラス・リヘニフォルミス中で遺伝子を発現させる方法
EP92923721A EP0672154A1 (fr) 1991-11-14 1992-11-13 PROCEDE D'EXPRESSION DES GENES DANS $i(BACILLUS LICHENIFORMIS)
FI942227A FI942227L (fi) 1991-11-14 1992-11-13 Menetelmä geenien ilmentämiseksi Bacillus licheniformisissa

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994019454A3 (fr) * 1993-02-19 1994-09-29 Novo Nordisk As Enzyme amylolytique
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
US6083718A (en) * 1983-07-06 2000-07-04 Gist-Brocades, N.V. Transformed industrial bacillus strains and methods for making and using them
US6300115B1 (en) 1998-05-18 2001-10-09 Enzyme Bio-Systems Ltd. Pullulanase expression constructs containing α-amylase promoter and leader sequences
WO2001079520A1 (fr) * 2000-04-13 2001-10-25 Biotica Technology Limited Produits hybrides glycosyles, production et utilisation
WO2005098016A3 (fr) * 2004-03-31 2009-04-30 Novozymes Biopolymer As Methodes de production d'acide hyaluronique dans une cellule de bacille
CN1814755B (zh) * 2005-02-06 2010-04-28 新疆农业科学院微生物应用研究所 一种高温中性蛋白酶及其制备方法
CN113957072A (zh) * 2021-10-09 2022-01-21 湖北大学 适用于地衣芽孢杆菌的简短终止子及其在高效表达目的产物中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0284126A1 (fr) * 1987-02-27 1988-09-28 Gist-Brocades N.V. Amplification de gènes stable dans l'ADN chromosomique des prokaryotes
WO1989003421A1 (fr) * 1987-10-15 1989-04-20 Novo Industri A/S Transferase glycosyliques de cyclodextrine thermostables, sa production et son utilisation
EP0410498A2 (fr) * 1989-06-29 1991-01-30 Gist-Brocades N.V. Alpha-amylases microbiennes mutÀ©es avec une stabilité thermique, acide et/ou alcaline améliorée
WO1991002792A1 (fr) * 1989-08-25 1991-03-07 Henkel Research Corporation Enzyme proteolytique alcaline et procede de production

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0284126A1 (fr) * 1987-02-27 1988-09-28 Gist-Brocades N.V. Amplification de gènes stable dans l'ADN chromosomique des prokaryotes
WO1989003421A1 (fr) * 1987-10-15 1989-04-20 Novo Industri A/S Transferase glycosyliques de cyclodextrine thermostables, sa production et son utilisation
EP0410498A2 (fr) * 1989-06-29 1991-01-30 Gist-Brocades N.V. Alpha-amylases microbiennes mutÀ©es avec une stabilité thermique, acide et/ou alcaline améliorée
WO1991002792A1 (fr) * 1989-08-25 1991-03-07 Henkel Research Corporation Enzyme proteolytique alcaline et procede de production

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 111, No. 1, 3 July 1989, (03.07.89), (Columbus, Ohio, USA), LAOIDE, BRID M. et al., "Bacillus Licheniformis Oa -Amylase Gene, AmyL, is Subject to Promoter-Independent Catabolite Repression in Bacillus Subtilis", page 171, the Abstract No. 1695c, J. Bacteriol. 1989, 171 (5), 2435-2442. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6083718A (en) * 1983-07-06 2000-07-04 Gist-Brocades, N.V. Transformed industrial bacillus strains and methods for making and using them
WO1994019454A3 (fr) * 1993-02-19 1994-09-29 Novo Nordisk As Enzyme amylolytique
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
WO2001079520A1 (fr) * 2000-04-13 2001-10-25 Biotica Technology Limited Produits hybrides glycosyles, production et utilisation
US7482137B2 (en) 2000-04-13 2009-01-27 Biotica Technology Limited Hybrid glycosylated products and their production and use
WO2005098016A3 (fr) * 2004-03-31 2009-04-30 Novozymes Biopolymer As Methodes de production d'acide hyaluronique dans une cellule de bacille
CN1814755B (zh) * 2005-02-06 2010-04-28 新疆农业科学院微生物应用研究所 一种高温中性蛋白酶及其制备方法
CN113957072A (zh) * 2021-10-09 2022-01-21 湖北大学 适用于地衣芽孢杆菌的简短终止子及其在高效表达目的产物中的应用
CN113957072B (zh) * 2021-10-09 2023-06-27 湖北大学 适用于地衣芽孢杆菌的简短终止子及其在高效表达目的产物中的应用

Also Published As

Publication number Publication date
EP0672154A1 (fr) 1995-09-20
FI942227A7 (fi) 1994-05-13
JPH07503363A (ja) 1995-04-13
FI942227A0 (fi) 1994-05-13
FI942227L (fi) 1994-05-13

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