Hyperproduction of an intracellular heterologous protein in a sacUh mutant of Bacillus subtilis.

The sacR regulatory region which controls inducible expression of sacB, the gene for extracellular levansucrase, was isolated on a 500-bp fragment of Bacillus subtilis chromosomal DNA. The region was separated into two components: a 325-bp fragment which carries a constitutive promoter and a 175-bp fragment which carries a stem-loop structure presumably involved in the induction process. The constitutive sacR promoter was used to drive expression of the plasmid-borne xylE gene, coding for intracellular catechol 2,3-dioxygenase (C23O), with its Pseudomonas ribosome-binding site, in otherwise isogenic sacU-, sacU+ and sacUh mutant strains of B. subtilis. Mutations at the sacU locus have been previously shown to affect multiple cell functions, particularly production of extracellular enzymes. The presence of the sacUh mutation allowed for hyperproduction of C23O to levels exceeding 25% of total cellular protein. This represents a 50- to 100-fold enhancement over levels observed in sacU- and sacU+ host cells. A 5- to 10-fold improvement of C23O production in sacUh cells was observed when the subtilisin (aprA) promoter was used in place of sacR. In contrast, a bacteriophage T5 synthetic promoter was found to be independent of host strain for high-level synthesis of C23O. Hyperproduction of an intracellular protein in sacUh cells suggests that enhancement of exoenzyme production, previously observed in these mutants, occurs prior to the secretion event. Therefore, hyperproduction is most likely due to elevated transcription or translation of specific nucleotide sequences.