Cultivation and maintenance of bacterial strains

For strain maintenance and cloning experiments, the strains P. putida KT2440 (DSM6125, ATCC47054), E. coli DH5α (New England Biolabs, Ipswich, MA, USA), and E. coli PIR2 (ThermoFisher Scientific, Waltham, MA, USA) were routinely cultivated in LB medium containing 10 g L⁻¹ peptone, 5 g L⁻¹ yeast extract, and 10 g L⁻¹ NaCl. P. putida was cultivated at 30°C and E. coli at 37°C. If required, 50 μg mL⁻¹ kanamycin or 30 μg mL⁻¹ gentamycin were added to the medium to avoid loss of plasmid. After mating procedures, P. putida strains were selected on cetrimide agar (Sigma-Aldrich, St. Louis, MO, USA). Growth and production experiments were performed using M9 minimal medium with a final composition (per L) of 8.5 g Na₂HPO₄x2H₂O, 3 g KH₂PO₄, 0.5 g NaCl, 1 g NH₄Cl, 2 mM MgSO₄, 4.87 mg FeSO₄x7H₂O, 4.12 mg CaCl₂x2H₂O, 1.5 mg MnCl₂x4H₂O, 1.87 mg ZnSO₄x7H₂O, 0.3 mg H₃BO₃, 0.25 mg Na₂MoO₄x2H₂O, 0.15 mg CuCl₂x2H₂O, 0.84 mg Na₂EDTAx2H₂O (Sambrook and Russell, 2001 ), and 10 g glucose for pre-cultures or 10 g xylose for main cultures. Growth experiments were performed in 500 mL shake flasks with 10% filling volume at 200 rpm and in 24-deep well-plates (SystemDuetz; Enzyscreen B.V., Heemstede, The Netherlands) with 1 mL filling volume at 300 rpm.