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. An altered Bacillus strain comprising a heterologous protein of interest and a deletion of one or more indigenous chromosomal regions selected from the group consisting of a PBSX region corresponding to about 1319663 to 1348691 bp, a skin region corresponding to about 2653562 to 2699604 bp, a prophage 7 region corresponding to about 2701087 to 2749642 bp, a prophage 1 region corresponding to about 202112 to 220141 bp, a prophage 2 region corresponding to about 529067 to 569578 bp, a prophage 3 region corresponding to about 652000 to 664300 bp, a prophage 4 region corresponding to about 1262987 to 1313692 bp, a prophage 5 region corresponding to about 1879200 to 1900000 bp, a prophage 6 region corresponding to about 2046050 to 2078000 bp, a PPS region corresponding to about 1960409 to 1998026 bp, a PKS region corresponding to about 1781795 to 1857985 bp, a YVFF-YVEK region corresponding to about 3513137 to 3528896 bp, a DHB region corresponding to about 3279418 to 3292920 bp, or a fragment thereof, wherein the coordinates correspond to the B. subtilis strain 168 chromosome, and wherein said altered Bacillus strain has an enhanced level of expression of the heterologous protein of interest compared to a corresponding unaltered Bacillus strain when said altered and unaltered Bacillus strains are grown under essentially the same growth conditions.
2. The altered Bacillus strain of claim 1 , wherein said altered Bacillus strain is selected from the group consisting of B. licheniformis, B. lentus, B. subtilis, B. amyloliquefaciens B. brevis, B. stearothermophilus, B. alkalophilus, B. coagulans, B. circulars, B. pumilus, B. lautus, B. clausii, B. megaterium , and B. thuringiensis.
3. The altered Bacillus strain of claim 2 , wherein said altered Bacillus strain is a B. subtilis strain.
4. The altered Bacillus strain of claim 1 , wherein two indigenous chromosomal regions or fragments thereof have been deleted.
5. The altered Bacillus strain of claim 1 , wherein said protein of interest is a protease.
6. The altered Bacillus strain of claim 5 , wherein said protease is a subtilisin.
7. The altered Bacillus strain of claim 6 , wherein said subtilisin is selected from the group consisting of subtilisin 168, subtilisin BPN′, subtilisin Carlsberg, subtilisin DY, subtilisin 147 and subtilisin 309 and variants thereof.
8. The altered Bacillus strain of claim 1 , wherein said Bacillus host is a recombinant strain.
9. The altered Bacillus strain of claim 1 , further comprising at least one mutation in a gene selected from the group consisting of degU, degQ, degS, scoC4, spoIIE and oppA.
10. A protease producing Bacillus strain comprising a heterologous protein of interest and a deletion of an indigenous chromosomal region selected from the group consisting of a PBSX region corresponding to about 1319663 to 1348691 bp, a skin region corresponding to about 2653562 to 2699604 bp, a prophage 7 region corresponding to about 2701087 to 2749642 bp, a prophage 1 region corresponding to about 202112 to 220141 bp, a prophage 2 region corresponding to about 529067 to 569578 bp, a prophage 3 region corresponding to about 652000 to 664300 bp, a prophage 4 region corresponding to about 1262987 to 1313692 bp, a prophage 5 region corresponding to about 1879200 to 1900000 bp, a prophage 6 region corresponding to about 2046050 to 2078000 bp, a PPS region corresponding to about 1960409 to 1998026 bp, a PKS region corresponding to about 1781795 to 1857985 bp, a YVFF-YVEK region corresponding to about 3513137 to 3528896 bp, a DHB region corresponding to about 3279418 to 3292920 bp, or a fragment thereof, wherein the coordinates correspond to the B. subtilis strain 168 chromosome map, and wherein said protease producing Bacillus strain has an enhanced level of expression of the heterologous protein of interest compared to a corresponding unaltered Bacillus strain when said altered and unaltered Bacillus strains are grown under essentially the same growth conditions.
11. The protease producing Bacillus strain of claim 10 , wherein said protease is a subtilisin.
12. The protease producing Bacillus strain of claim 11 , wherein said Bacillus is a B. subtilis strain.
13. The protease producing Bacillus strain of claim 10 , wherein said protease is a heterologous protease.
14. The Altered Bacillus strain of claim 1 , wherein said indigenous chromosomal region is the PPS region.
15. The protease producing Bacillus strain of claim 10 , wherein said indigenous chromosomal region is the PPS region.