Proteases from gram-positive organisms

Details Proteases from gram-positive organisms Proteases from gram-positive organisms

2000-02-22

2003-02-18

Achutamurthy, Ponnathapu (Department: 1651)

Chemistry: molecular biology and microbiology

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

Bacteria or actinomycetales; media therefor

C435S069100, C435S252310, C435S320100, C435S472000, C536S023200, C536S023700

Reexamination Certificate

active

06521440

ABSTRACT:

This application is filed pursuant to 35 USC §371 based upon PCT/US98/1867, filed Sep. 8, 1998, which claims priority to GB 9719637.2 filed Sep. 15, 1997.
FIELD OF THE INVENTION
The present invention relates to metallo-proteases derived from gram-positive microorganisms. The present invention provides nucleic acid and amino acid sequences of metallo-proteases identified in Bacillus. The present invention also provides methods for the production of the metallo-protease in host cells as well as the production of heterologous proteins in a host cell having a mutation or deletion of part or all of metallo-proteases of the present invention.
BACKGROUND OF THE INVENTION
Gram-positive microorganisms, such as members of the group Bacillus, have been used for large-scale industrial fermentation due, in part, to their ability to secrete their fermentation products into the culture media. In gram-positive bacteria, secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external media usually maintaining their native conformation.
Various gram-positive microorganisms are known to secrete extracellular and/or intracellular protease at some stage in their life cycles. Many proteases are produced in large quantities for industrial purposes. A negative aspect of the presence of proteases in gram-positive organisms is their contribution to the overall degradation of secreted heterologous or foreign proteins.
The classification of proteases found in microorganisms is based on their catalytic mechanism which results in four groups: the serine proteases; metallo-proteases; cysteine proteases; and aspartic proteases. These categories can be distinguished by their sensitivity to various inhibitors. For example, the serine proteases are inhibited by phenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate (DIFP); the metallo-proteases by chelating agents; the cysteine enzymes by iodoacetamide and heavy metals and the aspartic proteases by pepstatin. The serine proteases have alkaline pH optima, the metalloproteases are optimally active around neutrality, and the cysteine and aspartic enzymes have acidic pH optima (
Biotechnology Handbooks, Bacillus.
vol. 2, edited by Harwood, 1989 Plenum Press, New York).
Metallo-proteases form the most diverse of the catalytic types of proteases. About half of the families comprise enzymes containing the His-Glu-Xaa-Xaa-His (or HEXXH) motif which has been shown by X-ray crystallography to form part of the site for binding of the metal (normally zinc) atom. In one family of metalloproteases, a glutamic acid residue completes the metal-binding site, HEXXH+E. This family contains the most well characterized of the metallo-proteases, thermolysin. The three dimensional structure of thermolysin shows that, in the HEXXH motif, the His residues are zinc ligands and the Glu residue has a catalytic function. (Methods in Enzymology, vol. 248, Academic Press, Inc. 1994).
Fujimura-Kamada et al. (1997, J. Cell Biol. 136: 271-285) disclose a new subfamily of proteins that appear to function as intracellular, membrane-associated zinc metalloproteases. They disclose the
Saccharomyces cerevisiae
STE24 gene product which contains a zinc metalloprotease motif (HEXXH), as well as multiple predicted membrane spans. They further disclose that STE24 is required for the first NH2-terminal proteolytic cleavage event during biogenesis of the a-factor precursor.
SUMMARY OF THE INVENTION
The present invention relates to the discovery of a heretofore unknown metallo-protease (MP) found in gram positive microorganisms, uses of the MP in industrial applications, and advantageous strain improvements based on genetically engineering such microorganisms to delete, underexpress or overexpress that MP. The present invention is based in part upon the discovery that MP has overall amino acid relatedness to
S. cerevisiae
STE24 (Fujimura-Kamada et al., supra) and in part upon the unexpected discovery that nucleic acid encoding gram positive microorganism MP is found immediately downstream of nucleic acid encoding the major alkaline protease putative transcriptional terminator in gram-positive microorganisms.
The present invention is also based, in part, upon Applicant's discovery that the characteristic metallo-protease amino acid motif HEXXH+E and putative transmembrane domains exist in
Bacillus subtilis
MP. The present invention is also based in part upon Applicant's discovery that
Bacillus subtilis
MP homologs are found in
Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis
and
Bacillus amyloliquifaciens.
Applicant's discovery, in addition to providing a new and useful group of proteases and methods of detecting DNA encoding such proteases in a gram positive microorganism, provides several advantages which may facilitate optimization and/or modification of strains of gram positive microorganisms, such as Bacillus, for expression of desired, e.g. heterologous, proteins. Such optimizations, as described below in detail, allow the construction of strains having decreased proteolytic degradation of desired expression products.
Due to the relatedness of MP to STE24, a zinc metallo-protease which has been shown to be involved in processing events, and the unexpected conserved structural arrangement and proximity of gram positive MPs to the major alkaline protease of multiple Bacillus species, it appears that MP may play a role in regulating and/or processing the major alkaline protease in Bacillus. Furthermore, MP can serve as a marker for identification of the major alkaline protease in Bacillus species.
In one embodiment, the metallo-protease is obtainable from a gram-positive microorganism which is a Bacillus. In another embodiment, the metallo-protease is obtainable from a Bacillus which is preferably selected from the group consisting of
Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis
and
Bacilliis amyloliquifaciens.
The present invention encompasses the naturally occurring MP encoded by nucleic acid found immediately downstream from the transcriptional terminator of the major alkaline protease of a Bacillus species as well as the nucleic acid and amino acid molecules having the sequences disclosed in the Figures.
In a preferred embodiment, the present invention encompasses the naturally occurring MP nucleic acid molecule having the sequence found in
Bacillus subtilis
I-168 strain (Bacillus Genetic Stock Center, accession number 1A1, Columbus, Ohio) in the region of about 1102 kb from the point of origin and immediately downstream of the putative transcriptional terminator of the aprE gene. In another preferred embodiment, the
Bacillus subtilis
MP nucleic acid and amino acid molecules have the sequences as shown in
FIGS. 1A-1E
.
The present invention is also based in part upon the unexpected discovery of nucleic acid encoding portions of
Bacillus subtilis
MP homologs found in at least 3 non
B. subtilis
Bacillus species, including
Bacillus stearothermophilus, Bacillus licheniformis
and
Bacillus amyloliquifaciens.
The
Bacillus stearothermophilus, Bacillus licheniformis
and
Bacillus amyloliquifaciens
MP is found downstream of the major alkaline protease of each Bacillus.
The present invention encompasses the naturally occurring
Bacillus stearothermophilus, Bacillus licheniformis
and
Bacillus amyloliquifaciens
MP. In a preferred embodiment, the MP is encoded by the nucleic acid molecules having the nucleic acid sequence that is immediately downstream of the putative transcriptional terminator of the major alkaline protease or subtilisn in the genome of
Bacillus stearothermophilus, Bacillus licheniformis
or
Bacillus amyloliquifaciens.
In one preferred embodiment, the
Bacillus stearothermophilus
MP comprises the amino acid sequence as shown in FIG.
3
. In another preferred embodiment, the
Bacillus licheniformis
MP comprises the amino acid sequence as shown in FIG.
4
. In another preferred embodiment, the
Bacillus amyloliquifa

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