Enhanced protein expression in Bacillus
OwnerEugenio Ferrari, Carole Harbison +2inventors
InventorsEugenio Ferrari, Carole Harbison, M. Harunur Rashid, Walter Weyler
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Abstract
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.
Claims27
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.
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).
Dependencies are inferred from the claim text.