Methods and compositions for enhanced protein production in bacillus cells

A nucleic acid encoding a signal sequence comprising SEQ ID NO:

• 6.

A polynucleotide comprising an upstream (5′) nucleic acid encoding a signal sequence comprising SEQ ID NO:

• 2 operably linked to a downstream (3′) nucleic acid encoding a protein of interest (POI).

A polynucleotide comprising an upstream (5′) nucleic acid encoding a signal sequence comprising SEQ ID NO:

• 6 operably linked to a downstream (3′) nucleic acid encoding a protein of interest (POI).

A polynucleotide comprising an upstream (5′) promoter operably linked to a downstream nucleic acid encoding a signal sequence comprising SEQ ID NO:

• 2 operably linked to a downstream (3′) nucleic acid encoding a protein of interest (POI).

A polynucleotide comprising an upstream (5′) promoter sequence operably linked to a downstream nucleic acid encoding a signal sequence comprising SEQ ID NO:

• 6 operably linked to a downstream (3′) nucleic acid encoding a protein of interest (POI).

(canceled)

(canceled)

11-12. (canceled)

A recombinant Bacillus cell expressing at least two introduced polynucleotides encoding a heterologous protein of interest (POI), wherein the introduced polynucleotides comprises an upstream (5′) nucleic acid encoding a signal sequence comprising SEQ ID NO:

• 2 operably linked to a downstream (3′) nucleic acid encoding the POI.

14-15. (canceled)

A recombinant Bacillus cell expressing at least two introduced polynucleotides encoding a heterologous protein of interest (POI), wherein the first and second introduced polynucleotides comprise an upstream (5′) nucleic acid encoding a signal sequence comprising SEQ ID NO:

• 2 operably linked to a downstream (3′) nucleic acid encoding the POI, and an upstream (5′) nucleic acid encoding a signal sequence comprising SEQ ID NO:
• 6 operably linked to a downstream (3′) nucleic acid encoding the POI. respectively.

A method for expressing a heterologous protein of interest (POI) in a Bacillus cell comprising:

• (a) obtaining or constructing a Bacillus cell comprising an introduced polynucleotide comprising an upstream (5′) promoter operably linked to a downstream nucleic acid encoding a modified SacB signal sequence (modSacBss) comprising SEQ ID NO:
• 2 operably linked to a downstream (3′) nucleic acid encoding the POI, and (b) fermenting the Bacillus cell under suitable conditions for the expression of the POI.

18-20. (canceled)

A method for expressing a heterologous protein of interest (POI) in a Bacillus cell comprising:

• (a) obtaining or constructing a Bacillus cell comprising an introduced polynucleotide comprising an upstream (5′) promoter operably linked to a downstream nucleic acid encoding a modified Bli03445 signal sequence (modBli03445) comprising SEQ ID NO:
• 6 operably linked to a downstream (3′) nucleic acid encoding the POI, and (b) fermenting the Bacillus cell under suitable conditions for the expression of the POI.

22-24. (canceled)