Soytone

Bacillus megaterium ATCC13368, the host strain of CYP106A2, as well as a respective knockout strain Bacillus megaterium ATCC13368 Δcyp106A2, were both kind gifts of Dr. R. Rauschenbach (Schering AG, Berlin, Germany). Bacillus megaterium MS941 was kindly provided by Prof. Dieter Jahn (Institute of Microbiology, TU Braunschweig, Germany). Besides, we used a xylose-inducible recombinant expression system, which has recently been established in our group, to co-express CYP106A2 and its heterologous redox-partners adrenoxodin (Adx) and adrenodoxin reductase (AdR) using the expression vector pSMF2.1_CAA in B. megaterium MS941 [18 (link)].
Unless otherwise noted, Bacillus megaterium cells were cultivated in complex medium consisting of 25 g yeast extract (Becton, Dickinson and Company, USA), 12 g soytone (Becton, Dickinson and Company, USA) and 5 ml glycerol buffered with potassium phosphate buffer (2.31 g KH₂PO₄, 12.5 g K₂HPO₄) adjusted to pH 7.4 in 1 l distilled water. Cultivations were generally conducted at 30°C and 150 rpm in baffled shake-flasks (300 ml or 2 l). For the cultivation of MS941 cells transformed with the plasmid pSMF2.1_CAA and for the cultivation of the ATCC13368 Δcyp106a2-strain the medium was supplemented with 10 mg*l^-1 tetracycline.

For precultures of the bacteria, the basic buffer recipe was 10 g/L yeast extract (Becton Dickinson, Franklin Lakes, NJ) and 10 g/L soytone (Becton Dickinson). We refer to that buffer as 1xNutrient medium. For the washing steps and the experiment itself, the medium contained 1 g/L yeast extract and 1 g/L soytone, 0.1 mM CaCl₂, 2 mM MgCl₂, 4 mg/L NiSO₄, 50 mg/L MnCl₂, and 1x Trace Metals Mixture (Teknova, Hollister, CA). We refer to that buffer as base buffer. For the different bacteria, the initial pH was either 6 or 7, and it was supplemented with phosphate buffer and/or glucose and urea as outlined in the specific experiments below. The glucose and urea were added freshly every day directly before starting the experiments, to avoid degradation of the urea. The usual concentrations were 10 g/L glucose and 8 g/L urea; deviations from that are described for the single experiments below.
All media were filter sterilized.

The all-trans MK-7 analytical standard (98.1% purity) was purchased from ChromaDex (Los Angeles, CA, USA). Glucose was obtained from Ajax Finechem Pty Ltd (Taren Point, NSW, Australia), and yeast extract, peptone, tryptone, and soytone were acquired from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Glycerol, soy peptone, K₂HPO₄, methanol, 2-propanol, and n-hexane were purchased from Merck Millipore (Burlington, MA, USA). NaCl was obtained from a domestic supplier, and CaCl₂ was acquired from Sigma-Aldrich (St. Louis, MO, USA). Nutrient agar plates were purchased from Fort Richard Laboratories (Auckland, New Zealand).

The haploid Saccharomyces cerevisiae strain BY4741 (MATahis3-1 leu2-0 met15-0 ura3-0) was maintained and grown in YPD medium (2% peptone [Oxoid], 1% yeast extract [Oxoid], 2% d-glucose). Isolates of Saitozyma podzolica, identified by internal transcribed spacer (ITS) sequencing and randomly amplified polymorphic DNA (RAPD) PCR as described by Holland et al. (39 (link)), were recovered from soil near a disused metal smelting works in the northeast of the United Kingdom (http://www.twsitelines.info/SMR/4192). S. podzolica was maintained and grown in MYP medium (7% malt extract [Sigma], 0.5% yeast extract [Oxoid], 2.5% Soytone [BD Bacto]). Where required, media were solidified with 2% (wt/vol) agar. For experiments, single colonies were used to inoculate 10 ml of medium in 50-ml Erlenmeyer flasks and incubated with orbital shaking (New Brunswick Scientific) at 120 revolutions min ⁻¹, either at 30°C for S. cerevisiae or at 24°C for S. podzolica.

Culture media (Brain heart infusion (BHI), M17, MRS, tryptic soy broth (TSB) and LB media) and inorganic nitrogen sources ((NH₄)₂SO₄, NH₆PO₄, NH₄NO₃ and NH₄Cl) were purchased from Merck (Darmstadt, Germany). Carbon sources (fructose, glucose, galactose, sucrose, lactose, maltose, sorbitol and mannitol) were obtained from Fisher Chemical (Loughborough, United Kingdom), while organic nitrogen sources (yeast extract, meat extract, peptone and soytone) were from BD (Franklin Lakes, NJ, USA).

The myxobacterial strain MSr12020 was cultivated in VY/2 medium [%, (w/v) 0.2 soytone (BD), 0.3 casitone (BD), 0.2 glucose (Sigma-Aldrich), 0.8 soluble starch (Roth), 0.15 Yeast extract (BD), 0.1 CaCl₂ x 2H₂O, 0.1 MgSO₄ x 7H₂O, 50 mM HEPES, 8 mg/L Fe-EDTA, pH adjusted to 7.2 with 10N KOH before autoclaving] containing 5% (v/v) cell inoculum and 2% (v/v) amberlite resin XAD-16 (Sigma) for 14 days at 160 rpm, 30 °C. At the end of fermentation, resin and cells were harvested together by centrifugation at 8000 rpm, 30 min, 4 °C.