Genome engineering offers the possibility to create completely novel cell factories with enhanced properties for biotechnological application. In recent years, the possibilities for genome engineering have been extensively explored in the Gram-positive bacterial cell factory Bacillus subtilis, where up to 42% of the genome, encoding dispensable functions has been removed. Such studies have shown that some strains with minimized genomes gained beneficial features, for instance in protein production. However, strains with the most minimal genomes also showed particular growth defects. This has focused our attention on strains with less extensive genome deletions that show close-to-wild-type growth properties, while retaining the acquired beneficial traits in secretory protein production of strains lacking larger genomic segments. A strain of the latter category is B. subtilis IIG-Bs27-47-24, here referred to as midiBacillus II, which lacks 30.95% of the parental genome. To date, it was unknown how the altered genomic configuration of midiBacillus II impacts on cell physiology at large, and protein secretion in particular. Therefore, the present study was aimed at bridging this knowledge gap through an in-depth proteomics analysis with special focus on protein secretion stress responses. Interestingly, the results show that the secretion stress response of midiBacillus II as elicited by high-level expression of a staphylococcal antigen is completely different from the secretion stress responses that occur in the parental strain 168. This implies that high-level protein secretion has different implications for wild-type and genome-engineered Bacillus strains, dictated by the altered genomic and proteomic configurations.