Understanding protein secretion stress in Bacillus subtilis for biotechnological applications

The bacterium Bacillus subtilis is a prolific producer of high-value proteins, especially enzymes that are applied in processes ranging from food processing to the cleaning of dirty laundry. A major advantage of B. subtilis is that it secretes such proteins in massive amounts into its growth medium, which facilitates their downstream processing and reduces production costs. Unfortunately, not all proteins are secreted equally well, as high-level enzyme secretion can be stressful for bacteria. Therefore, the aim of the research described in this PhD thesis was to (i) pinpoint specific advantages of B. subtilis over other microorganisms and mammalian cells that are also used for protein production, and (ii) to uncover the stress responses and metabolic changes that B. subtilis exhibits during recombinant protein production. Altogether, the results show that the application potential of B. subtilis is huge, but that particular hurdles in the protein secretion process need to be overcome. This can be achieved by engineering the bacterial machinery for protein quality control, such that misfolded proteins are not degraded but repaired. Importantly, this results in a concomitant increase in bacterial fitness. Alternatively, improved protein production may be achieved by deleting genes for proteolytic enzymes that regulate the bacterial quality control machinery. Here, a novel finding was that removal of a proteolytic enzyme that is active inside the bacteria enhances stability of secreted proteins outside the bacteria. Lastly, the results focus attention on the enhancement of protein production by genome reduction, which simultaneously eliminates multiple counterproductive processes from B. subtilis.