(340a) Adaptive Laboratory Evolution and Rational Engineering Enables Osmolysis-Based Recovery of Biomacromolecules from Cupriavidus Necator

Intracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products in bacterial hosts. A key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material demand, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparations such as the purification of polyhydroxbutyrate (PHB) from extreme halophiles and protein products from mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis. To overcome this limitation, we sought to increase the susceptibility of non-halotolerant hosts to osmolysis using the lithoautotroph Cupriavidus necator as a prototypical strain. C. necator was evolved to increase its tolerance to NaCl through adaptive laboratory evolution. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% w/v NaCl. The cells were then made more susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (MscL) gene in C. necator. Greater than 99% osmolytic efficiency was observed upon osmotic downshock in this strain, indicating that this method serves as a simple and highly effective way to lyse cells for the purification of intracellular biomacromolecules.