Development of a high cell-density protocol for the production of pregallidermin, a non-toxic precursor of the lantibiotic gallidermin.

Gallidermin, produced by Staphylococcus gallinarum Tü 3928, is a type-A lantibiotic with potential for the treatment of multidrug-resistant infections from Gram-positive pathogens such as methicillin-resistant S. aureus. In order to eliminate product inhibition as a reason for so far very modest product titers in S. gallinarum cultivations, we recently developed a novel two-stage production strategy based on the production of a non-toxic gallidermin precursor - pregallidermin - by an engineered strain and the subsequent conversion of the precursor to gallidermin in a separate step. This directed our efforts to the identification and alleviation of cultivation constraints for the engineered strain in fed-batch cultivations based on complex media supplemented with carbon sources, reasoning that extending the biomass production phase would lead to an extended pregallidermin production and higher titers. Substantial accumulation of acetate occurred in fed-batch cultivations with either maltose or glycerol - but not succinate - as an additional carbon source. Reductions in feeding rate to limit acetate accumulation led in turn to increased product degradation. Based on these observations, we developed an optimized exponential feeding strategy that allowed the process to reach a biomass concentration of 120gL(-1) and a product concentration of 1.23gL(-1) pregallidermin, corresponding to 0.780gL(-1) mature gallidermin, a 2.5-fold increase over previous processes.