Enhanced protein expression in
The present invention provides cells that have been genetically manipulated to have an altered capacity to produce expressed proteins. In particular, the present invention relates to Gram-positive microorganisms, such as Bacillus species having enhanced expression of a protein of interest, wherein one or more chromosomal genes have been inactivated, and preferably wherein one or more chromosomal genes have been deleted from the Bacillus chromosome. In some further embodiments, one or more indigenous chromosomal regions have been deleted from a corresponding wild-type Bacillus host chromosome.
1. A method for enhancing the expression of a protein of interest in Bacillus comprising:
a) introducing a DNA construct including a selective marker and an inactivating chromosomal segment into a Bacillus host strain, wherein said DNA construct is integrated into the chromosome of said Bacillus host strain, resulting in the deletion of an indigenous chromosomal region or fragment thereof from said Bacillus host cell to produce an altered Bacillus strain, wherein said indigenous chromosomal region is the prophage 2 region; and
b) growing said altered Bacillus strain under suitable conditions, wherein expression of a protein of interest is greater in the altered Bacillus strain compared to the expression of the protein of interest in a Bacillus host cell that has not been altered.
2. The method of claim 1 , further comprising recovering said protein of interest.
3. The method of claim 1 , further comprising the step of excising said selective marker from the altered Bacillus strain.
4. The method of claim 1 , wherein said altered Bacillus strain comprises deletion of at least a second indigenous chromosomal region or fragment thereof.
5. The method of claim 1 , wherein said protein of interest is an enzyme.
6. The method of claim 1 , wherein said Bacillus host strain is selected from the group consisting of B. licheniformis, B. lentus, B. subtilis, B. amyloliquefaciens B. brevis, B. stearothermophilus, B. clausii, B. alkalophilus, B. coagulans, B. circulans, B. pumilus and B. thuringiensis.
7. A method for obtaining a protein of interest from a Bacillus strain comprising:
a) transforming a Bacillus host cell with a DNA construct comprising a selective marker and an inactivating chromosomal segment, wherein said DNA construct is integrated into the chromosome of the Bacillus strain resulting in deletion of an indigenous chromosomal region or fragment thereof, to produce an altered Bacillus strain, wherein said indigenous chromosomal region is the prophage 2 region,
b) culturing said altered Bacillus strain under suitable growth conditions to allow the expression of a protein of interest, and
c) recovering said protein of interest.
8. The method of claim 7 , wherein said protein of interest is an enzyme.
9. The method of claim 7 , wherein said Bacillus host comprises a heterologous gene encoding a protein of interest.
10. The method of claim 7 , wherein said Bacillus host cell is selected from the group consisting of B. licheniformis, B. lentus, B. subtilis, B. amyloliquefaciens B. brevis, B. stearothermophilus, B. clausii, B. alkalophilus, B. coagulans, B. circulans, B. pumilus and B. thuringiensis.
11. The method of claim 7 , wherein said altered Bacillus strain further comprises at least one mutation in a gene selected from the group consisting of degU, degQ, degS, sco4, spollE and oppA.
12. The method of claim 7 , wherein said protein of interest is an enzyme selected from the group consisting of proteases, cellulases, amylases, carbohydrases, lipases, isomerases, transferases, kinases, and phosphatases.
13. The method of claim 12 , wherein said enzyme is a protease.
14. The method of claim 4 , wherein said at least second indigenous chromosomal region or fragment thereof is selected from the group consisting of: PBSX, SKIN, prophage 7, SPR, prophage 1, prophage 3, prophage 4, prophage 5, prophage 6, PPS, PKS, YVFF-YVEK, DHB, and combinations thereof.
15. The method of claim 7 , wherein said altered Bacillus strain comprises deletion of at least a second indigenous chromosomal region or fragment thereof.
16. The method of claim 15 , wherein said at least second indigenous chromosomal region or fragment thereof is selected from the group consisting of: PBSX, SKIN, prophage 7, SPβ, prophage 1, prophage 3, prophage 4, prophage 5, prophage 6, PPS, PKS, YVFF-YVEK, DHB, and combinations thereof.