Multitron shaker

Plasmids pPA2, pANA, pBUG, pFLU, pAMB, pDAD, pPLY, and pHAL were separately transformed in E. coli. For HAA production, a 100-ml shake flask with 10 ml of LB containing 50 μg/ml kanamycin was inoculated with a freshly transformed E. coli strain. The cells were grown overnight at 37°C in a Multitron shaker (Infors HT, Bottmingen, Switzerland) at 200 rpm with a throw of 25 mm and humidity of 80%. HAA synthesis was conducted in 50 ml of the same medium in a 500-ml shake flask without baffles. The culture was inoculated to an optical density at 600 nm (OD₆₀₀) of 0.1, and cells were grown at 37°C until the OD₆₀₀ was between 0.5 and 0.9. Expression of rhlA was induced by the addition of 0.5 mM IPTG, at which point the temperature was lowered to 30°C and the shaking speed raised to 300 rpm to ensure that sufficient oxygen was supplied. Next, 2 and 20 h after the cultures were induced, 200 μl of 50 g/liter glucose was added, and the supernatant for HPLC-MS/MS was harvested 28 h after induction. In the experiment shown in Fig. 5, the cultures received 0.2% (wt/vol) glucose 2, 20, 22, 24, and 26 h after induction.

Tris was purchased from Carl Roth (Karlsruhe, Germany), isopropyl β-d-1-thiogalactopyranoside (IPTG) from Serva (Heidelberg, Germany), methacrylonitrile from Fluka (Buchs, Switzerland) and CoCl₂ and FeSO₄*7H₂O from Merck. HPLCMS grade acetonitrile was purchased from J.T.Baker/Avantor Performance Materials (Deventer, The Netherlands). All other chemicals were obtained from Sigma–Aldrich (St. Luis, MO, USA) and used without further purification.
E. coli cells were cultivated in an RS 306 shaker (Infors, Bottmingen, Switzerland), a Multitron shaker (Infors AG Bottmingen, Switzerland) and a Certomat BS-1, and the cells were harvested with an Avanti J-20 XP centrifuge (Beckman Coulter, Brea, CA, USA). Cell pellets were disrupted by a 102C converter with a Sonifier 250 (Branson, Danbury, CT, USA), and the cell-free extract was obtained by centrifugation in an Avanti J-20 XP centrifuge (Beckman Coulter). Reactions were performed on a Thermomixer comfort (Eppendorf, Hamburg, Germany). HPLC/MS analysis was carried out on an Agilent Technologies (Santa Clara, CA, USA) 1200 Series equipped with G1379B degasser, G1312B binary pump SL, G1367C HiP-ALS SL autosampler, a G1314C VWD SL UV detector, G1316B TCC SL column oven and a G1956B MSD. A positive electrospray ionization mode was used as ionization method.