Redirected Stress Responses in a Genome-Minimized ‘midiBacillus’ Strain with Enhanced Capacity for Protein Secretion

Our present study showcases a genome-minimized nonpathogenic bacterium, the so-called midiBacillus, as a chassis for the development of future industrial strains that serve in the production of high-value difficult-to-produce proteins. In particular, we explain how midiBacillus, which lacks about one-third of the original genome, effectively secretes a protein of the major human pathogen Staphylococcus aureus that cannot be produced by the parental Bacillus subtilis strain. ABSTRACT Genome engineering offers the possibility to create completely novel cell factories with enhanced properties for biotechnological applications. In recent years, genome minimization was extensively explored in the Gram-positive bacterial cell factory Bacillus subtilis, where up to 42% of the genome encoding dispensable functions was removed. Such studies showed that some strains with minimized genomes gained beneficial features, especially for secretory protein production. However, strains with the most minimal genomes displayed growth defects. This focused our attention on strains with less extensive genomic deletions that display close-to-wild-type growth properties while retaining the acquired beneficial traits in secretory protein production. A strain of this category is B. subtilis IIG-Bs27-47-24, here referred to as midiBacillus, which lacks 30.95% of the parental genome. To date, it was unknown how the altered genomic configuration of midiBacillus impacts cell physiology in general, and protein secretion in particular. The present study bridges this knowledge gap through comparative quantitative proteome analyses with focus on protein secretion. Interestingly, the results show that the secretion stress responses of midiBacillus, as elicited by high-level expression of the immunodominant staphylococcal antigen A, are completely different from secretion stress responses that occur in the parental strain 168. We further show that midiBacillus has an increased capacity for translation and that a variety of critical Sec secretion machinery components is present at elevated levels. Altogether, our observations demonstrate that high-level protein secretion has different consequences for wild-type and genome-engineered Bacillus strains, dictated by the altered genomic and proteomic configurations. IMPORTANCE Our present study showcases a genome-minimized nonpathogenic bacterium, the so-called midiBacillus, as a chassis for the development of future industrial strains that serve in the production of high-value difficult-to-produce proteins. In particular, we explain how midiBacillus, which lacks about one-third of the original genome, effectively secretes a protein of the major human pathogen Staphylococcus aureus that cannot be produced by the parental Bacillus subtilis strain. This is important, because the secreted S. aureus protein is exemplary for a range of targets that can be implemented in future antistaphylococcal immunotherapies. Accordingly, we anticipate that midiBacillus chassis will contribute to the development of vaccines that protect both humans and livestock against diseases caused by S. aureus, a bacterial pathogen that is increasingly difficult to fight with antibiotics, because it has accumulated resistances to essentially all antibiotics that are currently in clinical practice.