Tryptone

The pre-cultures for the inoculation of the bioreactors were cultivated in 500 mL shake flasks containing 100 mL xylose mineral medium (composition as indicated above) until exponential phase. The cells were harvested by centrifugation (4,500×g, Sorvall STR40) and used to inoculate 0.5 l bioreactors (MiniBio, Applikon) that contained 250 mL medium at an OD₆₀₀ of ~1.2. The composition of the fermentation medium was similar to the mineral medium used in the shake flask experiments, except that it contained 55 g/L (d)-xylose, 2 g/L Na₂HPO₄ · 12H₂O, 0.8 g/L KH₂PO₄, 6 g/L (NH₄)₂HPO₄, 0.4 g/L (NH₄)₂SO₄, 1 g/L tryptone (Biokar), 0.5 g/L yeast extract (Biokar), 0.4 mL/L polypropylene glycol as antifoaming agent, 1 mM IPTG, and no MOPS. The pH of the cultures was kept at 7.0 by the addition of 10 M KOH, and reactors were aerated with air at 1 vvm. Dissolved oxygen tension was regulated by adjusting the appropriate agitation speed (300–1,200 rpm, Rushton rotor, 28 mm diameter), and was either kept at 40 % to impose fully aerobic conditions, or at 2 % to impose micro-aerobic conditions.

The bacterial strain Escherichia coli K12-MG1655 (ATCC 700926; genotype, F-lambda-ilvG-rfb-50 rph-1) was used as a model organism. Bacteria were cultivated according to Bittel et al. [29 (link)]. Luria-Bertani (LB) medium was used, which was prepared as follows: 1 L of distilled water was supplemented with 10 g tryptone (Biokar Diagnostics, Allonne, France, ref A1401HA), 5 g yeast extract (Biokar Diagnostics, ref A1202HA) and 5 g NaCl (Carlo Erba Reagents, Milan, Italy, ref 479687). Sterilization was performed by autoclaving at 120 ${}^{\circ }$ C for 20 min. Starting from 10 mL overnight precultures, 50 mL cultures were generated with an optical density ( $O{D}_{620nm}$ ) of = 0.1, with both cultures shaken at 250 rpm at 30 ${}^{\circ }$ C (Eppendorf Innova^® 42 Benchtop incubator shaker, Eppendorf, France). Growth was monitored over time on bacterial culture diluted 1/10 in LB media measured by a spectrophotometer (SAFAS UVmc2) at 620 nm in disposable cuvettes (Brand GmBH, ref 759015) until it reached $O{D}_{620nm}$ = 0.4, which corresponds to the middle of the exponential growth phase.

E. coli LMG2092 and Nissle 1917 strains were selected as negative and positive control for pks+ bacteria, respectively. Both strains were grown in Luria-Bertani (LB) broth containing 10 g/L tryptone (Biokar Diagnostics, France), 5 g/L yeast extract (Biokar Diagnostics, France), 10 g/L of NaCl (Merck, KGaA, Darmstadt, Germany), and 1 L of deionized H₂O. All cultures were grown on the surface of agar plates at 37°C in an MG500 anaerobic chamber (Don Whitley Scientific, West Yorkshire 100, UK) with an atmosphere of 10% (v/v) H₂, 10% CO₂, and 80% N₂ for 48 h. After that, 1 single colony was inoculated in 4 mL of fresh liquid media and all cultures were incubated at 37°C for 24 h in anaerobic, aerobic, and aerobic with shaking conditions. The next day, 100 µL of the bacteria suspension was inoculated into 4 mL of fresh medium and incubated at 37°C until an optical density (OD₆₀₀) of about 0.6 after approximately 3 h. After that, cultures were harvested by centrifugation, washed once with bacterial FC buffer (Miltenyi, Bergisch Gladbach, Germany) and resuspended in the same buffer to an OD₆₀₀ = 0.2, which represents around 1 × 10⁸ CFUs/mL.
The bacteria were also grown in fresh liquid media of Nutrient broth (Oxoid, Ltd., Basingstoke, Hampshire, UK) and Nutrient broth supplemented with 2% of glucose (Sigma-Aldrich, St. Louis, MO, USA).

Terrific Broth (TB) powder and tryptone were from Biokar Diagnostics (Allone, France). Yeast extract was from Biolife (Milano, Italy), glycerol was from Labchem (Zelienople, PA, USA), and L-arabinose was from Alfa Aesar (Tewksbury, MA, USA). The Luria Broth (LB) powder and kanamycin sulfate were from Fisher BioReagents (Pittsburgh, PA, USA).