Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1994-05-10
1997-12-16
Vogel, Nancy T.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
43525231, 4353201, 536 241, C12P 2106, C12N 121, C12N 1575, C12N 1511
Patent
active
056984154
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national stage application of PCT/DK92/00338 filed Nov. 13, 1992, the contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to a Bacillus licheniformis promoter, a DNA construct comprising said promoter, A host cell transformed with said DNA construct and a method of producing a protein in Bacillus by means of the promoter.
BACKGROUND OF THE INVENTION
Various promoter sequences of the Bacillus licheniformis .alpha.-amylase gene have been described previously. Thus, M. Sibakov and I. Palva, Eur. J. Biochem. 145, 1984, pp. 567-572, describe the isolation and determination of the 5' end of the Bacillus licheniformis .alpha.-amylase gene, including the promoter sequence; T. Yuuki et al., J. Biochem. 98, 1985, pp. 1147-1156, show the complete nucleotide sequence of the Bacillus licheniformis .alpha.-amylase gene, including the promoter sequence; and B. M. Laoide et al., J. Bacteriol. 171(5), 1989, pp. 2435-2442, discuss catabolite repression of the Bacillus licheniformis .alpha.-amylase gene from a region around the 5' end of the gene and show the sequence of this region.
SUMMARY OF THE INVENTION
The present inventors have surprisingly found that a novel promoter homologous to the previously published promoter sequences gives rise to a dramatically increased yield of a protein when the gene coding for the protein is transcribed from the promoter.
Accordingly, the present invention relates to a Bacillus promoter included in the following DNA sequence ##STR1## wherein each of N.sup.1 -N.sup.9 is A, T, C or G with the exception that N.sup.2 -N.sup.9 do not together form the sequence ATGTTTCA or GTGTTTCA, or a functional homologue of said sequence.
In the the previously published sequences, N.sup.1 is either T (cf. T. Yuuki et al., supra) or C (B. M. Laoide et al., supra), while N.sup.2 -N.sup.9 is either ATGTTTCA (T. Yuuki et al., supra, and B. M. Laoide et al., supra) or GTGTTTCA (cf. M. Sibakov, supra). Several papers discuss catabolite repression of Bacillus genes, including the B. licheniformis .alpha.-amylase gene. Thus, B. M. Laoide et al, supra, and B. M. Laoide and D. J. McConnell, J. Bacteriol. 171, 1989, pp. 2443-2450, map the cis sequences essential for mediation of catabolite repression of amyL in B. subtilis to a 108 bp region downstream from the promoter and upstream from the signal sequence cleavage site. They identify an inverted repeat sequence, TGTTTCAC-20 bp-ATGAAACA, in this region but note that deletion into the left-hand part of this sequence either abolished or altered expression without affecting catabolite repression. They identify sequences homologous to the left-hand part of the amyL inverted repeat (5'-A/T T G T N A/T-3') around the transcription initiation sites in a number of B. subtilis catabolite-repressible genes.
Y. Miwa and Y. Fujita, Nucl. Acids Res. 18, pp. 7049-7053, limit the cis sequences involved in catabolite repression of the B. subtilis gnt operon to a 11 bp region. Within this 11 bp region is a 8 bp sequence, ATTGAAAG, which the authors claim could be a consensus sequence involved in catabolite repression in the genus Bacillus, as it was found in other catabolite repressible Bacillus genes. Interestingly, in the B. licheniformis .alpha.-amylase gene, the consensus sequence shown above immediately follows the left-hand part of the inverted repeat sequence identified by Laoide et al.
M. J. Weickert and G. H. Chambliss, Proc. Natl. Acad. Sci. USA 87, pp. 6238-6242, describe site-directed mutagenesis of a catabolite repression operator sequence in B. subtilis from the amyE gene. They observe that hyperproduction and catabolite repression of amylase were both affected by mutations in the same region, and sometimes by the same mutation. They found that the B. subtilis .alpha.-amylase catabolite repression operator shares significant homology with sequences in other Bacillus amylase gene regulatory regions and with other catabolite repres
REFERENCES:
patent: 4769327 (1988-09-01), Stephens et al.
Laoide et al., J. Bacteriol., vol. 171, pp. 2435-2442 (1989).
Sibakov et al., Eur. J. Biochem., vol. 145, pp. 567-572 (1984).
Yuuki et al., J. Biochem. vol. 98, No. 5, pp. 1147-1156 (1985).
Diderichsen B.o slashed.rge Krag
J.o slashed.rgensen Steen Troels
Gregg, Esq. Valeta
Novo Nordisk Als
Vogel Nancy T.
Zelson Esq. Steve T.
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