2.1 Media and culture conditions
Plasmids and strains used in this study can be found in Table S1 in the supplementary information. For cloning purposes, Escherichia coliand P. taiwanensis cells were cultivated at 37 or 30 °C, respectively, either in liquid LB medium containing 5 g L−1 sodium chloride or on solid LB agar plates (with 1.5 % (w/v) agar). After conjugational mating procedures, Pseudomonads were isolated on cetrimide agar (Sigma Aldrich) plates supplemented with 10 mL L−1 glycerol. Kanamycin (50 mg L−1) or gentamicin (20 mg L−1) was added to cultures or plates when necessary. Growth and production experiments were performed in mineral salts medium (MSM) [9] with 20 mM glucose or 40 mM glycerol as sole carbon source.
In production experiments, liquid cultures of P. taiwanensis were performed in MSM with 20 mM glucose or 40 mM glycerol without the addition of antibiotics. Main cultures were inoculated at an OD600 of ~0.2, from seed cultures grown in MSM containing glucose. Batch production experiments were performed in 500 mL Erlenmeyer flasks with a culture volume of 50 mL, cultivated in a rotary shaker with a frequency of 200 rpm and a throw of 50 mm. Fed batch fermentations were performed in DASbox® mini-bioreactors (Eppendorf) according to the setup and procedure described in Otto et al. [8].