| ubtilisin synthesis by the spore-forming bacterium B. subtilis was studied as a model non-growth-associated (stationary-phase) protein production system. Since subtilisin gene expression is regulated similarly to sporulation initiation, this inquiry was conducted in the context of B. subtilis development. Host strain genotype, media development, and reactor design were considered, with an ongoing reflection on the interactions among these elements. Combining this multifaceted approach within a statistically-designed experimental framework not only facilitated the development of high-level subtilisin production systems but also prompted a reexamination of some of the basic tenets surrounding B. subtilis growth and development.;The formulation of a suite of simple, chemically-defined, minimal media fostering balanced growth, stationary-phase biomass stability, subtilisin production, and sporulation was a critical enabling technology, allowing relationships between nutrient availability and cellular responses to be probed. For example, nitrogen starvation triggered subtilisin production (and developmental initiation), and a combination of excess glucose and an appropriate metal ion milieu stabilized the nongrowing cell populations. This, and other, experimentation also suggested that the repressing effects of glucose had been overemphasized by others. In addition, metal ions (including magnesium, calcium, and manganese) profoundly influenced subtilisin gene expression, production, and culture medium stability.;Another central strategy was to temporally extend subtilisin production by incorporating defined mutations to arrest the spore formation process. The benefits of stage II-blocked hosts were demonstrated, though multiple asymmetric septation and loss of productivity issues remained. Mutational dissection of elements of the subtilisin regulatory circuits in nonseptating stage 0-blocked hosts, through inactivation of AbrB and incorporation of known hyperproducing mutations (either the scoC4 allele of hpr or the ;This research also generated significant contributions to the Bacillus developmental biology field. For example, both AbrB and Hpr were implicated in determining whether a nutrient-sufficient B. subtilis cell will continue to grow or initiate sporulation. In addition, immunotransmission electron microscopy was employed to demonstrate that the... |
