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

Abstract

Our present study showcases a genome-minimized nonpathogenic bacterium, the so-called midi Bacillus , 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 midi Bacillus , 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.

Let There Be Light: Genome Reduction Enables Bacillus subtilis to Produce Disulfide-Bonded Gaussia Luciferase

“…Indeed, there is evidence for both options as a previous proteomics analysis revealed changes in amino acid and nitrogen metabolism in the IIG-Bs27-47-24 strain, as well as an altered oxidative stress response. 59 ■ CONCLUSIONS Our present study shows that genome-reduced "midiBacillus" strains offer excellent opportunities for the expression of difficult-to-produce POIs, even those with multiple disulfide bonds. This is underscored by the more than 3000-fold increase that we observed for the production of active GLuc in the IIG-Bs27-47-24 strain.…”

“…After the already steep 336-fold activity increase between the TS10 and IIG-Bs27-31 strains, the additional 9.4-fold increase between the IIG-Bs27-31 and IIG-Bs27-47-24 strains was particularly surprising. Judged by the results from our previous study on the secretion of a difficult-to-produce staphylococcal protein by the IIG-Bs27-47-24 strain, the massive increase of more than 3000-fold overall in secretory GLuc production may also originate from other beneficial traits of this strain. These include an improved capacity for translation, increased levels of Sec secretion machinery components and chaperones, increased levels of the quality control proteases HtrA and HtrB that degrade misfolded proteins, and decreased competition for the Sec pathway by other Sec-dependently secreted proteins of which the genes had been removed.…”

“…Moreover, a possible limitation in the activity of disulfide reductases like ResA or StoA in genome-reduced strains could potentially be caused by reduced expression of the respective genes or a lowered availability of reducing equivalents due to metabolic rearrangements. Indeed, there is evidence for both options as a previous proteomics analysis revealed changes in amino acid and nitrogen metabolism in the IIG-Bs27-47-24 strain, as well as an altered oxidative stress response …”

“…Previous studies have shown that large-scale genome reduction confers novel traits to the B. subtilis cell factory that can be beneficial for recombinant protein production. ,, In the present study, we wanted to explore whether this also applies to a protein with multiple disulfide bonds, since such proteins are notoriously difficult to produce in bacteria . The POI that we selected for this purpose was the luciferase of Gaussia princeps (GLuc), which has five disulfide bonds.…”

Heterologous expression of pediocin/papA in Bacillus subtilis

“…In addition, B. subtilis strains are generally regarded as safe (GRAS) because toxic byproducts such as lipopolysaccharides are not formed in their cells. The biochemistry, physiology, and genetics of B. subtilis are well known [ 32 ], which makes it a promising microbial cell factory for use with the developing genetic manipulation tools. WB800N is a B. subtilis strain with eight extracellular proteases ( nprE , nprB , aprE , epr , mpr , bpr , vpr , WprA ) knockout [ 33 ], which can secrete protein directly into the culture medium, no need to worry about degradation.…”