MiniBacillus - the Construction of a Minimal Organism

The MiniBacillus project is a minimal genome project. It attempts to reduce the genome of the model organism Bacillus subtilis in a top-down approach by deleting not necessary parts of the genome step by step. In this work, a MiniBacillus strain was created with a genome reduction of 40.51%, which is the most extensive genome reduction, achieved in a top-down approach. The data obtained from previous multi-omics experiments were used to adapt the deletion process and maintain a stable strain. Furthermore, new proteome data were analysed. A glycolytic cassette was inserted as a first attempts of a defragmentation approach to counteract the deceleration of the deletion process. Another important goal of the MiniBacillus project is to gain more knowledge about the mechanisms in the cell. The final minimal cell will be able to utilize glucose as the single carbon source. Therefore, glycolysis and the pentose-phosphate pathway will remain in the cell and the TCA cycle will be deleted. To analyse the effect of a deletion of this central pathway, a TCA cycle mutant in the wild type strain was created. This strain is viable, but shows severe problems in sporulation, competence and cell morphology. Especially the reduced competence is a disadvantage for the MiniBacillus project. The final minimal cell will import all amino acids from the complex medium and all biosynthesis pathways will be deleted. However, not for every amino acid the particular importers are characterized. In this work, the three new serine/ threonine transporters YbeC, BcaP and YbxG could be identified. The YbeC transporter seems to be the low-affintiy serine transporter, which transports the major part of serine into the cell at high serine concentrations. BcaP and YbxG have just a minor function. Furthermore, the major import of threonine is mediated by BcaP, which also transports isoleucine and valine into the cell. In contrast, YbeC and YbxG have a minor threonine import function. This information can be used for the MiniBacillus project and the biosynthesis pathways of serine and threonine can be deleted in the final strain. Furthermore, BcaP will remain in the MiniBacillus to ensure the necessary import of serine and threonine.To analyse the serine import, the toxicity of high serine concentrations on wild type cells was utilized. However, the mechanism and physiological role of serine inhibition was unknown. The results of this work indicate a function in the inhibition of the threonine pathway. Serine might bind to the homoserine dehydrogenase protein and inhibit its activity. This causes a reduced level of threonine in the cell. The information of this work was incorporated into the Minibacillus project and a blueprint 2.0 was created.