*4.2. Cultivation and Fermentative Limonene Production in Shake Flasks*

For the cultivation in shake flasks, precultures were grown in 5 mL of LB medium (10 g·L−<sup>1</sup> tryptone, 5 g·L−<sup>1</sup> yeast extract, and 5 g·L−<sup>1</sup> NaCl) with 100 <sup>μ</sup>g·mL−<sup>1</sup> ampicillin for 4 to 6 h at 37 ◦C and shaking at 200 rpm. LB precultures were transferred 1:500 to 250 mL baffled Erlenmeyer flasks with 50 mL M9 minimal medium (8.5 g·L−<sup>1</sup> Na2HPO4 · 2H2O, 3 g·L−<sup>1</sup> KH2PO4, 0.5 g·L−<sup>1</sup> NaCl, 1 g·L−<sup>1</sup> NH4Cl, 2 mL of 1 M MgSO4, and 1 mL of L<sup>−</sup><sup>1</sup> US\* trace element solution) with either 0.5% *w*/*v* glucose or 0.5% *w*/*v* glycerol as the sole carbon source. The cultures were incubated overnight at 30 ◦C with shaking at 200 rpm. The M9 precultures were used to inoculate 80 mL of M9 minimal medium with the 0.5% *w*/*v* carbon source in 500 mL baffled shake flasks to an optical density of 0.11 at 600 nm (OD600). The main cultivations were performed at 30 ◦C with shaking at 200 rpm. Heterologous gene expression was induced by adding 0.1 mM isopropyl <sup>β</sup>-d−1-thiogalactopyranoside (IPTG) once an OD600 of 0.4–0.6 was reached. After induction, the cultures were overlaid with 20 mL of diisononyl phthalate (DINP), and incubation was continued. For sampling, the phases were separated by centrifugation (2 min, 4 ◦C, 11,000 × *g*). Prior to gas chromatography (GC) analysis, the organic phase was diluted in diethyl ether with 0.2 mM dodecane and dried over anhydrous Na2SO4. The aqueous phase was used for HPLC analysis. The cell dry weight was determined by measurement of the optical density at a wavelength of 600 nm (Libra S11 Visible Spectrophotometer, Biochrom, Cambridge, UK). One OD600 unit corresponded to 0.312 gCDW L<sup>−</sup>1, with a linear range between 0.1 and 1.
