Phosphate limited inducible promoter and a
An evolvable production strain of B. subtilis exhibiting continuous or high level expression during protein evolution is described. An evolved Bacillus subtilis pstS promoter facilitates screening and production of secreted proteins.
1. An isolated nucleic acid comprising a Bacillus subtilis PstS promoter variant, said PstS promoter variant comprising a sequence corresponding to nucleotides −100 to +106 of PstS promoter from B. subtilis strain W168, wherein said sequence comprises at least one mutation chosen from A17T, A26G, A32G and T86C to provide enhanced production of a polypeptide of interest.
2. A Bacillus subtilis host cell comprising the nucleic acid of claim 1 .
3. The host cells of claim 2 , wherein said nucleic acid is integrated into the chromosome of said host cell.
4. The host cells of claim 2 , further comprising a nucleic acid encoding a polypeptide of interest under the transcriptional control of said PstS promoter variant.
5. An expression construct comprising an isolated nucleic acid encoding a B. subtilis PstS promoter variant and a nucleic acid molecule encoding a polypeptide of interest, wherein said promoter variant comprises a sequence corresponding to nucleotides −100 to +106 of the PstS promoter from B. subtilis strain W168, wherein said sequence comprises at least one mutation chosen from A17T, A26G, A32G and T86C to provide enhanced production of a polypeptide of interest.
6. A Bacillus host cell comprising the nucleic acid of claim 1 , wherein said Bacillus host cell expresses a heterologous sequence under nutrient limited conditions.
7. The Bacillus host cell of claim 6 , wherein said expression is prolonged.