Genetically Intact Bioengineered Spores of Bacillussubtilis

Developments in genome editing offer potential solutions to challenges in agriculture, industry, medicine, and the environment. However, many technologies remain unexploited due to limitations in the use of genetically altered organisms. In this study, we use B. subtilis spores to explore the possibility of bioengineering organisms while leaving their genome intact. Taking advantage of the differential expression between the mother cell and the fore-spore compartments during sporulation, we created plasmids programmed to modify the spore phenotype from the mother cell compartment, but to "self-digest" in the fore-spore. At the end of sporulation, the mother cell undergoes lysis and releases the phenotypically engineered, genetically unaltered spores. Using this approach, we demonstrated the potential to express foreign proteins in B. subtilis spores without genome alterations by producing spores expressing GFP in their protective coats, where approximately 90% of the spore population had no detectable plasmid or chromosome alterations. In a separate demonstration, we programmed KinA overexpression during vegetative growth to artificially induce sporulation, and also obtained spores with nearly 90% of them free of detectable plasmid. Artificial induction of sporulation could potentially simplify the bioprocess for industrial spore production, as it reduces the number of steps involved. Overall, these findings demonstrate the potential to create genetically intact bioengineered organisms.