Methods for producing biological substances in pigment-deficient mutants of
View Patent ↗The present invention relates to methods of producing a heterologous biological substance, comprising: (a) cultivating a mutant of a parent Bacillus cell under conditions conducive for the production of the heterologous biological substance, wherein (i) the mutant cell comprises a first nucleic acid sequence directing synthesis of the heterologous biological substance and a second nucleic acid sequence comprising a modification of at least one of the genes cypX and yvmC, which are involved in the production of a red pigment, and (ii) the mutant cell is deficient in the production of the red pigment compared to the parent Bacillus cell when cultivated under the same conditions; and (b) recovering the heterologous biological substance from the cultivation medium. The present invention also relates to mutants of Bacillus cells and methods for producing the mutants.
1. A method of producing a heterologous protein, comprising:
(a) cultivating a mutant Bacillus subtilis cell transformed with a nucleic acid construct comprising a nucleic acid directing synthesis of the heterologous protein in a medium suitable for the production of the heterologous protein, wherein the mutant cell comprises a deletion mutation in a cypX gene comprising SEQ ID NO: 1, a yvmC gene comprising SEQ ID NO: 7, or both genes, in which the deletion mutation(s) renders the cell deficient in red pigment compared to a wild-type Bacillus subtilis cell comprising the cypX gene comprising SEQ ID NO: 1, the yvmC gene comprising SEQ ID NO: 7, or both genes; and
(b) recovering the heterologous protein from the cultivation medium.
2. The method of claim 1 , wherein the heterologous protein is involved in the biosynthesis of a biopolymer.
3. The method of claim 1 , wherein the heterologous protein is involved in the biosynthesis of a metabolite.
4. The method of claim 1 , wherein the mutant cell produces no detectable red pigment compared to the parent Bacillus subtilis cell when cultured under identical conditions.
5. A isolated mutant Bacillus subtilis cell, comprising a deletion mutation in a cypX gene comprising SEQ ID NO: 1, a yvmC gene comprising SEQ ID NO: 7, or both genes, in which the deletion mutation(s) renders the cell deficient in red pigment compared to a wild-type Bacillus subtilis cell comprising the cypX gene comprising SEQ ID NO: 1, the yvmC gene comprising SEQ ID NO: 7, or both genes, transformed with a nucleic acid construct comprising a nucleic acid directing synthesis of the heterologous protein.
6. The mutant cell of claim 5 , wherein the heterologous protein is involved in the biosynthesis of a biopolymer.
7. The mutant cell of claim 5 , wherein the heterologous protein is involved in the biosynthesis of a metabolite.
8. The mutant cell of claim 5 , which produces no detectable red pigment compared to the parent Bacillus subtilis cell when cultured under identical conditions.
9. A method of producing a an isolated mutant Bacillus subtilis cell, comprising: making a deletion mutation in a cypX gene comprising SEQ ID NO: 1, a yvmC gene comprising SEQ ID NO: 7, or both genes, of a Bacillus subtilis cell, in which the deletion mutation(s) renders the cell deficient in red pigment compared to a wild-type Bacillus subtilis cell comprising the cypX gene comprising SEQ ID NO: 1, the yvmC gene comprising SEQ ID NO: 7, or both genes, and transforming the cell with a nucleic acid construct comprising a nucleic acid directing synthesis of a heterologous protein.
10. The method of claim 9 , wherein the heterologous protein is involved in the biosynthesis of a biopolymer.
11. The method of claim 9 , wherein the heterologous protein is involved in the biosynthesis of a metabolite.
12. The method of claim 1 , wherein the mutant cell is further deficient in the production of protease.
13. The method of claim 1 , wherein the mutant cell is further deficient in the production of surfactin.
14. The mutant cell of claim 5 , which is further deficient in the production of protease.
15. The mutant cell of claim 5 , which is further is deficient in the production of surfactin.
16. The method of claim 9 , wherein the mutant cell produces no detectable red pigment when compared to the parent Bacillus subtilis cell when cultured under identical conditions.
17. The method of claim 9 , wherein the mutant Bacillus subtilis cell is further deficient in the production of protease.
18. The method of claim 9 , wherein the mutant Bacillus subtilis cell is further-deficient in the production of surfactin.
19. The method of claim 1 , wherein the mutant Bacillus subtilis cell does not produce spores.
20. The mutant cell of claim 5 , which does not produce spores.
21. The method of claim 9 , wherein the mutant Bacillus subtilis cell does not produce spores.
22. The method of claim 1 , wherein the mutant Bacillus subtilis cell is further deficient in the production of amylase.
23. The mutant cell of claim 5 , which is further deficient in the production of amylase.
24. The method of claim 9 , wherein the mutant Bacillus subtilis cell is further deficient in the production of amylase.