Mutant aprE promotor

Details Mutant aprE promotor Mutant aprE promotor

2000-01-07

2003-01-21

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S252300, C435S471000, C435S320100, C435S069100, C536S023100, C536S024100

Reexamination Certificate

active

06509185

ABSTRACT:

FIELD OF THE INVENTION
The present invention is in the field of molecular biology and relates to the production of proteins in Bacillus species. In particular the present invention relates to a mutant aprE promotor and its use in methods for the production of proteins.
BACKGROUND OF THE INVENTION
Genetic engineering has allowed the improvement of microorganisms used as industrial bioreactors, cell factories and in food fermentations. The Bacillus genera produce and secrete a large number of useful proteins and metabolites (Zukowski 1992 Zukowski M M (1992) Production of commercially valuable products. In: Doi R H, McGlouglin M (eds) Biology of bacilli: applications to industry. Butterworth-Heinemann, Stoneham. Mass pp 311-337). The most common bacilli used in industry are
B. licheniformis, B. amyloliquefaciens
and
B. subtilis.
Furthermore, because of its GRAS (generally recognized as safe) status,
B. subtilis
is a natural candidate for the production of proteins utilized in the food and phamaceutical industries.
The aprE gene of
B. subtilis
codes for the extracellular protease subtilisn, a valuable enzyme manufactured by the biotechnology industry (Debadov V G (1982) The Industrial Use of Bacilli. In: Dubnau D A (ed) The Molecular Biology of the Bacilli. Academic Press: New York/London, vol 1, pp 331-370). The development of recombinant protein production systems using
B. subtilis
as a host organism, especially those driven by the subtilisin promoter, provides an important tool for research and commercial production in this area (Oyama et al. (1989) Secretion of
Escherichia coli
Aminopeptidase P in
Bacillus subtilis
using the Prepro-Structure Coding Region of Subtilisin Amylosacchariticus. J. Ferment. Bioeng. 68: 289-292). Although subtilisin synthesis is not required for sporulation (Stahl and Ferrari (1984)Replacement of the
Bacillus subtilis
Subtilisin Structural Gene With an In Vitro-Derived Deletion Mutation. J Bacteriol. 158: 411-418), its production is triggered by mechanisms common to those events responsible for the sporulation initiation, and hence, it has served as a model for developmentally-associated gene expression (Sonenshein A L (1989) Metabolic Regulation of Sporulation and Other Stationary-Phase Phenomenon. In: Smith I, Slepecky R A, Setlow P (eds) Regulation of Procaryotic Development. American Society for Microbiology, Washington, D.C. pp 109-130). The aprE gene is transcribed by sigma A (&sgr;
A
) and its expression is highly controlled by several regulators, such as: DegU/DegS, AbrB, Hpr and SinR (Valle and Ferrari (1989) In: Smith I, Slepecky R A, Setlow P (eds) Regulation of Procaryotic Development. American Society for Microbiology. Washington, D.C. pp 131-146). A consensus sigma A promoter has been identified (Helman et al., 1995, Nucleic Acid Research, Vol. 23, pp. 2351-2360). In spite of advances in the understanding of production of proteins in host cells, there remains a need for methods for increasing expression of proteins in host cells, such as Bacillus host cells.
SUMMARY OF THE INVENTION
The present invention relates to the use of a mutant aprE promoter in the production of proteins. The present invention is based upon the unexpected finding that a hundred fold increase in the production of a desired protein occurred in a host cell which contained the mutant aprE promoter. The present invention is also based upon the unexpected finding that the mutant aprE promoter having the nucleotide sequence as shown in SEQ ID NO:1 was able to enhance transcription of both heterologous and homologous proteins and remained regulatable during production of the proteins. Accordingly, the present invention provides an isolated mutant aprE promoter and in another embodiment, provides an isolated mutant aprE promoter having the nucleotide sequence as given in SEQ ID NO: 1. The present invention also provides for host cells comprising an isolated mutant aprE promoter and methods for using such host cells to produce desired proteins. In one embodiment, the host cell is a Bacillus species and in another embodiment, the Bacillus species includes
B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillus thuringiensis.
In another embodiment, the desired protein is subtilisn.
In yet another embodiment, the host cell comprising an isolated mutant aprE promoter, and in particular the isolated mutant aprE promoter of SEQ ID NO: 1, further comprises nucleic acid encoding a desired protein which may be homologous or heterologous to the host cell. The nucleic acid may encode therapeutically significant proteins or peptides, such as growth factors, cytokines, ligands, receptors and inhibitors, as well as vaccines and antibodies. The nucleic acid may encode commercially important industrial proteins or peptides, such as proteases, including subtilisn, carbohydrases such as amylases and glucoamylases, cellulases, oxidases and lipases. The nucleic acid may be a naturally occurring gene, a mutated gene or a synthetic gene. Examples of industrial proteins include enzymes such as hydrolases including proteases, cellulases, amylases, carbohydrases, and lipases; isomerases such as racemases, epimerases, tautomerases, or mutases; transferases, kinases and phophatases. In one embodiment, the protein is heterologous to the cell and in another it is homologous to the cell. In one illustrative embodiment disclosed herein, the protein is &bgr;-galactosidase and in another illustrative embodiment disclosed herein, the protein is subtilisn.
The present invention provides methods for producing a desired protein in a Bacillus species comprising, culturing a Bacillus comprising an isolated aprE promoter said Bacillus further comprising nucleic acid encoding the desired protein and optionally recovering said desired protein. In one embodiment, the isolated mutant aprE promoter has the sequence as shown in SEQ ID NO:1. In one embodiment of the method, said nucleic acid encoding the desired protein is integrated into the Bacillus genome and in another embodiment, nucleic acid encoding the desired protein is present on a replicating plasmid. The present invention also provides for methods for producing host cells comprising an isolated mutant aprE promoter.


REFERENCES:
Jan, et al. FEMS Microbiology Letters, 183: 9-14, Feb. 2000.*
Park,et al. Journal of Bacteriology, vol. 171, No. 5, pp. 2657-2665, May 1989.*
Ferrari, et al. Journal of Bacteriology, vol. 170, No. 1, pp. 289-295, Jan. 1988.*
Bakhiet, Nouna and Donald P. Stahly, “Studies on Transfection and Transformation of Protoplasts ofBacillus larvae, Bacillus subtilis, andBacillus popilliae,” Applied and Environmental Microbiology, pp. 577-581, Mar. 1985.
Bron, Sierd,Molecular Biological Methods for Bacillus, ed. Harwood and Cutting, John Wiley & Sons, pp. 75-174, 1990.
Chang, Shing et al., “High Frequency Transformation ofBacillus subtilisProtoplasts,” Molec. Gen. Genet., vol. 168, pp. 111-115,1979.
Contente, Sara et al., “Marker Rescue Transformation by Linear Plasmid DNA inBacillus subtilis,” Plasmid, vol. 2, pp. 555-571, 1979.
Debabov, V.G., “The Industrial Use of Bacilli,”The Molecular Biology of the Bacilli, Dubnau, D.A., ed., Academic Press, vol. 1, pp. 331-370, 1982.
Fischer, Hans-Martin, et al., “Introduction of plasmid pC194 intoBacillus thuringiensisby protoplast transformation and plasmid transfer,” Arch. Microbiol., vol. 139, pp. 213-217, 1984.
Genex Corp., Sequence Accession No. N60475, Database EMBL Online, “Sequence of the apr 'BSU! gene encoding subtilisin” XP-002166627, Aug. 24, 1991.
Haima, Peter, et al., “Novel plasmid marker rescue transformation system for molecular cloning inBacillus subtilisenableing direct selection of recombinants,” Mol. Gen. Genet, vol. 223, pp. 185-181, 1990.
Henner, Dennis et al., “Localization ofBacillus subilissacU(Hy) Mutations to Two Linked Genes with Similarities to the Conserved Procaryotic Family of Two-Component Signalling System,” Journal of Bacteriology, vol. 170, N

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