US4828994A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Bacillus strains with reduced extracellular protease levels
Assignee: Genex Corp
•
Filing: 1985-05-13
•
Granted: 1989-05-09
•
Expires: 2005-05-13
Abstract
Abstract Bacillus strains having reduced levels of extracellular protease are produced by replacing the native chromosomal DNA sequence comprising the gene for an extracellular protease, such as subtilisin, with a partially homologous DNA sequence having an inactivating DNA segment inserted therein. The strains are useful as hosts for the expression and secretion of heterologous polypeptides or proteins.
Inventors
- 👤 Stephen R. Fahnestock
- 👤 Kathryn E. Fisher
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 14:26
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/52; C12N9/54; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N15/75; C12N15/87; C12N15/90; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/832; Y10S435/839
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
65 words
A microorganism of the genus Bacillus in which the native DNA sequence coding for the extracellular protease subtilisin has been replaced by a partially homologous DNA sequence having an alteration which renders said microorganism incapable of synthesizing subtilisin, said microorganism also containing a mutation rendering it incapable of sporulation, said mutation having the effect of decreasing the synthesis of extracellular serine proteases, other than subtilisin.
Claim 2
Dependent Claim
65 words
A microorganism of the genus Bacillus in which the native DNA sequence coding for the extracellular protease subtilisin has been replaced by a partially homologous DNA sequence having an alteration which renders said microorganism incapable of synthesizing subtilisin, said microorganism also containing a mutation rendering it incapable of sporulation, said mutation having the effect of decreasing the synthesis of extracellular serine proteases, other than subtilisin.
Claim 3
Dependent Claim
18 words
A microorganism as claimed in claim 1, wherein the microorganism is incapable of synthesizing the proteolytic enzyme subtilisin.
Claim 4
Dependent Claim
16 words
A microorganism as claimed in claim 1, which is of the genus and species Bacillus subtilis.
Claim 5
Dependent Claim
15 words
The microorganism of claim 1, wherein said mutation affecting sporulation is of the spoOA class.
Claim 6
Dependent Claim
20 words
The microorganism of claim 4, wherein said mutation affecting sporulation is due to the presence of the spoOA677 deletion allele.
Claim 7
Dependent Claim
36 words
The microorganism of claim 1, wherein said microorganism is used for the production of a protein not naturally produced by the host organism, said microorganism having been transformed with foreign DNA sequences encoding said foreign protein.
Claim 8
Dependent Claim
4 words
Bactillus subtilis strain GX4924.
Claim 9
Dependent Claim
4 words
Bactillus subtilis strain GX4924.
Claim 10
Dependent Claim
4 words
Bactillus subtilis strain BX4925.
Claim 11
Dependent Claim
4 words
Bactillus subtilis strain BX4925.
Claim 12
Dependent Claim
4 words
Bactillus subtilis strain GX4926.
Claim 13
Dependent Claim
4 words
Bactillus subtilis strain GX4926.
Claim 14
Independent Claim
131 words
A method of producing a Bacillus strain having reduced extracellular protease levels comprising: isolating a gene coding for the extracellular protease; inserting the gene for the extracellular protease onto a cloning vector; altering the gene on the cloning vector by inserting an inactivating DNA segment therein; transforming a microorganism of the Bacillus genus with the cloning vector capable of transformation and replication in one or more species of the genus Bacillus containing the insertionally inactivated gene coding for an extracellular protease of said microorganism, said strain containing a mutation rendering the organism incapable of sporulation; growing the transformants under conditions in which the replication function of the cloning vector is inactive; and selecting transformants in which the inactivated gene has replaced the native chromosomal DNA sequence coding for the extracellular protease.
Claim 15
Independent Claim
131 words
A method of producing a Bacillus strain having reduced extracellular protease levels comprising: isolating a gene coding for the extracellular protease; inserting the gene for the extracellular protease onto a cloning vector; altering the gene on the cloning vector by inserting an inactivating DNA segment therein; transforming a microorganism of the Bacillus genus with the cloning vector capable of transformation and replication in one or more species of the genus Bacillus containing the insertionally inactivated gene coding for an extracellular protease of said microorganism, said strain containing a mutation rendering the organism incapable of sporulation; growing the transformants under conditions in which the replication function of the cloning vector is inactive; and selecting transformants in which the inactivated gene has replaced the native chromosomal DNA sequence coding for the extracellular protease.
Claim 16
Dependent Claim
28 words
A method as claimed in claim 10 wherein the inactivating DNA segment comprises a functional gene coding for a protein which confers a pheotypic trait on the transformant.
Claim 17
Dependent Claim
20 words
A method as claimed in claim 11, wherein the inactivating DNA segment comprises a functional gene coding for chloramphenicol acetyltransferase.
Claim 18
Dependent Claim
23 words
A method as claimed in claim 10, wherein the extracellular protease gene which is inactivated is a gene coding for the protease subtilisin.
Claim 19
Dependent Claim
21 words
A method as claimed in claim 10, 11, 12, or 13, wherein the Bacillus strain is of the genus and species.
Claim 20
Dependent Claim
39 words
A microorganism of the genus Bacillus which exhibits no metalloprotease activity and not more than 0.07 units of serine protease activity (ΔA595 per minute per A 600 equivalent unit culture supernatant, 24 hr. culture on hide powder azure substrate).
Claim 21
Dependent Claim
39 words
A microorganism of the genus Bacillus which exhibits no metalloprotease activity and not more than 0.07 units of serine protease activity (ΔA595 per minute per A 600 equivalent unit culture supernatant, 24 hr. culture on hide powder azure substrate).
Claim 22
Dependent Claim
18 words
A microorganism as claimed in claim 15, wherein the microorganism is of the genus and species Bacillus subtilis.
Claim 23
Dependent Claim
36 words
A microorganism as claimed in claim 15 or 16 wherein the native chromosomal DNA sequence coding for the enzyme subtilisin has been replaced by a partially homologous DNA sequence having an inactivating DNA sequence inserted therein.
Claim 24
Dependent Claim
5 words
Bactillus subtilis , strain GX4931.
Claim 25
Dependent Claim
5 words
Bactillus subtilis , strain GX4931.
Claim 26
Dependent Claim
5 words
Bactillus subtilis , strain GX4937.
Claim 27
Dependent Claim
5 words
Bactillus subtilis , strain GX4937.
Claim 28
Dependent Claim
5 words
Bactillus subtilis , strain GX4939.
Claim 29
Dependent Claim
5 words
Bactillus subtilis , strain GX4939.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-05-08 | 1992-11-07 | 1993-05-09 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-05-08 | 1996-11-07 | 1997-05-09 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-05-08 | 2000-11-07 | 2001-05-09 | $7700.00 | Window Closed / Lapsed |