An improved high cell density cultivation-iHCDC-strategy for leucine auxotrophic K12 ER2507.

Fed-batch processes are commonly used tools in high cell density cultivation of . However, the setup of a fed-batch process can be challenging, especially when working with genetically modified strains. This work deals with the design of a specific strategy for the leucine auxotrophic strain K12 ER2507 that is of interest for arabinose-inducible gene expression systems due to its mutation. To set up this process the optimum yield coefficient of biomass from leucine was determined by use of a design of experiments tool. Unfortunately, the yield coefficient is negatively influenced by both leucine and glucose concentrations. Furthermore, an inhibitory effect of leucine reduced the specific growth rate even at low concentration. Under consideration of these problems, an improved high cell density cultivation (iHCDC) for K12 ER2507 was established. The start medium was set up without any C-source to omit a batch phase with high glucose concentration and substrate feeding was initiated directly upon inoculation. Due to the poor solubility of leucine in feed medium, the amino acid was dissolved in the base. With the improved high cell density cultivation process a final cell density of more than 90 g/L was obtained reproducibly.