Cultivation of recombinant E. coli strains for the production of ectoines.

For the heterologous production of ectoine and hydroxyectoine in E. coli, various strains were transformed with different plasmids carrying either the wild-type ectABCD-ask_ect gene cluster or mutant derivatives of it that either carried alterations in the ect promoter sequence or lacked the ectD gene (Table 6). Twenty-milliliter cultures (in 100-ml baffled Erlenmeyer flasks) of these plasmid-carrying E. coli strains were cultivated in MMA with the addition of different concentrations of NaCl and sodium aspartate and with chloramphenicol (30 μg ml⁻¹) to select for the recombinant plasmids. The cultures were inoculated with a preculture grown in MMA overnight at 37°C to an OD₅₈₇ of 0.15 and incubated for 24 or 48 h (as indicated in the individual experiments) in a shaking water bath (set to 220 rpm and 37°C). Two-milliliter samples were collected from these cultures, and the cells were harvested by centrifugation (16,000 × g for 10 min) in an Eppendorf tabletop centrifuge. The supernatant was separated from the pellet, and both samples were stored at −20°C until further analysis.