Modified messenger RNA stabilizing sequences for expressing genes in bacterial cells
The present invention relates to methods of producing a polypeptide having biological activity in a bacterial cell, comprising: (a) cultivating a bacterial host cell in a medium conducive for production of the polypeptide, wherein the bacterial host cell comprises a nucleic acid construct comprising a promoter region operably linked to a polynucleotide sequence encoding the polypeptide and a modified mRNA processing/stabilizing sequence located downstream of the promoter region and upstream of the ribosome binding site of the polynucleotide sequence encoding the polypeptide, wherein the modified mRNA processing/stabilizing sequence promotes higher expression of the polynucleotide sequence compared to an unmodified mRNA processing/stabilizing sequence; and (b) isolating the polypeptide having biological activity from the cultivation medium. The present invention also relates to such modified mRNA processing/stabilizing sequences, nucleic acid constructs, and bacterial host cells and to methods of obtaining such bacterial host cells.
1. A method for producing a polypeptide having biological activity in a bacterial host cell, comprising:
(a) cultivating the bacterial host cell in a medium conducive for production of the polypeptide having biological activity, wherein the bacterial host cell comprises one or more copies of a nucleic acid construct comprising a promoter region operably linked to a polynucleotide sequence encoding the polypeptide having biological activity and a modified mRNA processing/stabilizing sequence located downstream of the promoter region and upstream of the ribosome binding site of the polynucleotide sequence encoding the polypeptide having biological activity, wherein the modified mRNA processing/stabilizing sequence is a cryIIIA mRNA processinq/stabilizing sequence comprising at least one additional copy of a Shine-Dalgamo sequence or a SP82 mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgamo sequence; and wherein the modified mRNA processing/stabilizing sequence promotes higher expression of the polynucleotide sequence compared to an unmodified mRNA processing/stabilizing sequence; and
(b) isolating the polypeptide having biological activity from the cultivation medium.
2. The method of claim 1 , wherein the bacterial host cell is a Bacillus cell.
3. A bacterial cell comprising a nucleic acid construct that comprises one or more copies of a nucleic acid construct comprising a promoter region operably linked to a polynucleotide sequence encoding the polypeptide having biological activity and a modified mRNA processing/stabilizing sequence located downstream of the promoter region and upstream of the ribosome binding site of the polynucleotide sequence encoding the polypeptide having biological activity, wherein the modified mRNA processing/stabilizing sequence is a cryIIIA mRNA processinq/stabilizinq sequence comprising at least one additional copy of a Shine-Dalqarno sequence or a SP82 mRNA processinq/stabilizinq sequence comprising at least one additional copy of a Shine-Dalqarno sequence; and wherein the modified mRNA processinq/stabilizinq sequence promotes higher expression of the polynucleotide sequence compared to an unmodified mRNA processing/stabilizing sequence.
4. The bacterial cell of claim 3 , which is a Bacillus cell.
5. A method for producing a selectable marker-free mutant of a bacterial cell, comprising deleting a selectable marker gene of the bacterial cell, wherein the bacterial cell comprises one or more copies of a nucleic acid construct comprising a promoter region operably linked to a polynucleotide sequence encoding the polypeptide having biological activity and a modified mRNA processing/stabilizing sequence located downstream of the promoter region and upstream of the ribosome binding site of the polynucleotide sequence encoding the polypeptide having biological activity, wherein the modified mRNA processing/stabilizing sequence is a cryIIIA mRNA processinq/stabilizinq sequence comprising at least one additional copy of a Shine-Dalqarno sequence or a SP82 mRNA processinq/stabilizinq sequence comprising at least one additional copy of a Shine-Dalqarno sequence; and wherein the modified mRNA processinq/stabilizinq sequence promotes higher expression of the polynucleotide sequence compared to an unmodified mRNA processing/stabilizing sequence.
6. The method of claim 5 , wherein the bacterial host cell is a Bacillus cell.
7. A method for obtaining a bacterial host cell, comprising introducing into a bacterial host cell one or more copies of a nucleic acid construct comprising a promoter region operably linked to a polynucleotide sequence encoding the polypeptide having biological activity and a modified mRNA processing/stabilizing sequence located downstream of the promoter region and upstream of the ribosome binding site of the polynucleotide sequence encoding the polypeptide having biological activity, wherein the modified mRNA processing/stabilizing sequence is a cryIIIA mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalqarno sequence or a SP82 mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence; and wherein the modified mRNA processing/stabilizing sequence promotes higher expression of the polynucleotide sequence compared to an unmodified mRNA processing/stabilizing sequence.
8. The method of claim 7 , wherein the bacterial host cell is a Bacillus cell.
9. The method of claim 1 , wherein the modified mRNA processing/stabilizing sequence is a cryIIIA mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence.
10. The method of claim 1 , wherein the modified mRNA processing/stabilizing sequence is a SP82 mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence.
11. The method of claim 1 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAG.
12. The method of claim 1 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAGG.
13. The method of claim 1 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises SEQ ID NO: 1.
14. The bacterial cell of claim 3 , wherein the modified mRNA processing/stabilizing sequence is a cryIIIA mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence.
15. The bacterial cell of claim 3 , wherein the modified mRNA processing/stabilizing sequence is a SP82 mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence.
16. The bacterial cell of claim 3 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAG.
17. The bacterial cell of claim 3 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAGG.
18. The bacterial cell of claim 3 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises SEQ ID NO: 1.
19. The method of claim 5 , wherein the modified mRNA processing/stabilizing sequence is a cryIIIA mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence.
20. The method of claim 5 , wherein the modified mRNA processing/stabilizing sequence is a SP82 mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence.
21. The method of claim 5 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAG.
22. The method of claim 5 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAGG.
23. The method of claim 5 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises SEQ ID NO: 1.
24. The method of claim 7 , wherein the modified mRNA processing/stabilizing sequence is a cryIIIA mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence.
25. The method of claim 7 , wherein the modified mRNA processing/stabilizing sequence is a SP82 mRNA processing/stabilizing sequence comprising at least one additional copy of a Shine-Dalgarno sequence.
26. The method of claim 7 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAG.
27. The method of claim 7 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises the sequence GGAGG.
28. The method of claim 7 , wherein the at least one additional copy of a Shine-Dalgarno sequence comprises SEQ ID NO: 1.