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 expression of a heterologous protein of interest from Bacillus comprising:
a) obtaining an altered Bacillus strain capable of producing a heterologous protein of interest, wherein said altered Bacillus strain has at least one inactivated chromosomal gene selected from the group consisting of sbo, slr, ybcO, csn, spoIISA, sigB, phrC, rapA, CssS, trpA, trpB, trpD, trpE, trpF, tdh/kbl, alsD, sigD, prpC, gapB, pckA, fbp, rocA, ycgN, ycgM, rocF, and rocD; and
b) growing said altered Bacillus strain under conditions such that said heterologous protein of interest is expressed by said altered Bacillus strain, wherein said expression of said heterologous protein of interest is enhanced compared to the expression of said heterologous protein of interest in an unaltered Bacillus host strain.
2. The method of claim 1 , wherein said protein of interest is an enzyme selected from the group consisting of proteases, cellulases, amylases, carbohydrases, lipases, isomerases, transferases, kinases, and phosphatases.
3. The method of claim 2 , wherein said protein of interest is a protease.
4. The method of claim 1 , wherein said altered Bacillus strain is obtained by deleting one or more chromosomal genes selected from the group consisting of sbo, slr, ybcO, csn, spoIISA, sigB, phrC, rapA, CssS, trpA, trpB, trpD, trpE, trpF, tdh/kbl, alsD, sigD, prpC, gapB, pckA, fbp, rocA, ycgN, ycgM, rocF, and rocD.
5. A method for enhancing expression of a protease in an altered Bacillus comprising:
a) transforming a Bacillus host cell with a DNA construct comprising an incoming sequence, wherein said incoming sequence comprises a nucleic acid encoding a heterologous protein of interest, and a selective marker flanked on each side with a homology box, wherein said homology box includes nucleic acid sequences having 80 to 100% sequence identity to the sequence immediately flanking the coding regions of at least one gene selected from the group consisting of sbo, slr, ybcO, csn, spoIISA, sigB, phrC, rapA, CssS, trpA, trpB, trpD, trpE, trpF, tdh/kbl, alsD, sigD, prpC, gapB, pckA, fbp, rocA, ycgN, ycgM, rocF, and rocD;
b) allowing homologous recombination of said DNA construct and a region of the chromosome of said Bacillus host cell, wherein at least one gene of said chromosome of said Bacillus host cell is inactivated, to produce an altered Bacillus strain; and
c) growing said altered Bacillus strain under conditions suitable for the expression of said protease, wherein the production of said protease is greater in the altered Bacillus subtilis strain compared to said Bacillus subtilis host prior to transformation in step a).
6. The method of claim 5 , wherein said protease is subtilisin.
7. The method of claim 5 , wherein said protease is a recombinant protease.
8. The method of claim 5 , wherein said inactivation is by deletion of at least one of said genes.
9. The method of claim 5 , wherein said inactivation is by insertional inactivation of said at least one of said genes.
10. The method of claim 1 , wherein said altered Bacillus strain has an inactivated sbo chromosomal gene.
11. The method of claim 1 , wherein said altered Bacillus strain has an inactivated slr chromosomal gene.
12. The method of claim 1 , wherein said altered Bacillus strain has an inactivated ybcO chromosomal gene.
13. The method of claim 1 , wherein said altered Bacillus strain has an inactivated spoIISA chromosomal gene.
14. The method of claim 1 , wherein said altered Bacillus strain has an inactivated sigB chromosomal gene.
15. The method of claim 1 , wherein said altered Bacillus strain has an inactivated phrC chromosomal gene.
16. The method of claim 1 , wherein said altered Bacillus strain has an inactivated rapA chromosomal gene.
17. The method of claim 1 , wherein said altered Bacillus strain has an inactivated pckA chromosomal gene.
18. The method of claim 1 , wherein said altered Bacillus strain has at least one inactivated chromosomal gene selected from the group consisting of csn, trpA, trpB, trpD, trpE, trpF, tdh/kbl, prpC, gapB, rocA, ycgN, ycgM, rocF, and rocD.
19. The method of claim 5 , wherein said homology box includes nucleic acid sequences having 80 to 100% sequence identity to the sequence immediately flanking the coding region of sbo.
20. The method of claim 5 , wherein said homology box includes nucleic acid sequences having 80 to 100% sequence identity to the sequence immediately flanking the coding region of slr.
21. The method of claim 5 , wherein said homology box includes nucleic acid sequences having 80 to 100% sequence identity to the sequence immediately flanking the coding region of ybcO.
22. The method of claim 5 , wherein said homology box includes nucleic acid sequences having 80 to 100% sequence identity to the sequence immediately flanking the coding region of spoIISA.
23. The method of claim 5 , wherein said homology box includes nucleic acid sequences having 80 to 100% sequence identity to the sequence immediately flanking the coding region of sigB.
24. The method of claim 5 , wherein said homology box includes nucleic acid sequences having 80 to 100% sequence identity to the sequence immediately flanking the coding region of phrC.
25. The method of claim 5 , wherein said homology box includes nucleic acid sequences having 80 to 100% sequence identity to the sequence immediately flanking the coding region of rapA.
26. The method of claim 5 , wherein said homology box includes nucleic acid sequences having 80 to 100% sequence identity to the sequence immediately flanking the coding region of pckA.
27. The method of claim 5 , wherein said homology box includes nucleic acid sequences having 80 to 100% sequence identity to the sequence immediately flanking the coding regions of at least one gene selected from the group consisting of csn, trpA, trpB, trpD, trpE, trpF, tdh/kbl, prpC, gapB, rocA, ycgN, ycgM, rocF, and rocD.