Methods for producing a polypeptide in a bacillus cell

A Bacillus cell comprising a nucleic acid construct which comprises (a) a tandem promoter in which each promoter sequence of the tandem promoter is operably linked to a single copy of a nucleic acid sequence encoding a polypeptide, and optionally (b) an mRNA processing/stabilizing sequence located downstream of the tandem promoter and upstream of the nucleic acid sequence encoding the polypeptide.

A method for obtaining a Bacillus host cell, comprising introducing into a Bacillus cell a nucleic acid construct comprising (i) a tandem promoter in which each promoter sequence of the tandem promoter is operably linked to a single copy of a nucleic acid sequence encoding a polypeptide and alternatively also (ii) an mRNA processing/stabilizing sequence located downstream of the tandem promoter and upstream of the nucleic acid sequence encoding the polypeptide.

A method for producing a selectable marker-free mutant of a Bacillus cell, comprising deleting a selectable marker gene of the Bacillus cell, wherein the Bacillus cell comprises a nucleic acid construct comprising (i) a tandem promoter in which each promoter sequence of the tandem promoter is operably linked to a single copy of a nucleic acid sequence encoding a polypeptide and alternatively also (ii) an mRNA processing/stabilizing sequence located downstream of the tandem promoter and upstream of the nucleic acid sequence encoding the polypeptide.

A method for producing a polypeptide, comprising:

• (a) cultivating a Bacillus cell in a medium conducive for the production of the polypeptide, wherein the Bacillus cell comprises a nucleic acid construct comprising (i) a “consensus” promoter having the sequence TTGACA for the “−35” region and TATAAT for the “−10” region operably linked to a single copy of a nucleic acid sequence encoding the polypeptide and (ii) an mRNA processing/stabilizing sequence located downstream of the “consensus” promoter and upstream of the nucleic acid sequence encoding the polypeptide;
• and
• (b) isolating the polypeptide from the cultivation medium.

A Bacillus cell comprising a nucleic acid construct which comprises (a) a “consensus” promoter having the sequence TTGACA for the “−35” region and TATAAT for the “−10” region operably linked to a single copy of a nucleic acid sequence encoding the polypeptide and (b) an mRNA processing/stabilizing sequence located downstream of the “consensus” promoter and upstream of the nucleic acid sequence encoding the polypeptide.

A method for obtaining a Bacillus host cell, comprising introducing into a Bacillus cell a nucleic acid construct comprising (i) a “consensus” promoter having the sequence TTGACA for the “−35” region and TATAAT for the “−10” region operably linked to a single copy of a nucleic acid sequence encoding the polypeptide and (ii) an mRNA processing/stabilizing sequence located downstream of the “consensus” promoter and upstream of the nucleic acid sequence encoding the polypeptide.

A method for producing a selectable marker-free mutant of a Bacillus cell, comprising deleting a selectable marker gene of the Bacillus cell, wherein the Bacillus cell comprises a nucleic acid construct comprising (i) a “consensus” promoter having the sequence TTGACA for the “−35” region and TATAAT for the “−10” region operably linked to a single copy of a as nucleic acid sequence encoding the polypeptide and (ii) an mRNA processing/stabilizing sequence located downstream of the “consensus” promoter and upstream of the nucleic acid sequence encoding the polypeptide.

An isolated “consensus” amyQ promoter sequence having the nucleic acid sequence contained in SEQ ID NO. 26 or SEQ ID NO. 27.