Fermentation at Bench-Scale and Sample Preparation

Fermentation of MG1363/pSS105 was performed as described previously (20) with minor modifications. Briefly, 5 ml pre-culture was grown at 30°C for 6h in LAB medium with 5% Glucose, 4% di-sodium glycerophosphate and 1µg/ml erythromycin by thawing one vial of frozen RCB. The fermenter (BIOFLO 310, New Brunswick Scientific) containing 1L LAB medium supplemented with 5% glucose, 5 mM cysteine, 0.5 mM cystine and 1 µg/ml erythromycin was inoculated with 0.4 ml pre-culture. After 4 h of inoculation, the fermenter was supplied with 50% glucose continuously at the rate of 8 ml/h to maintain 5% glucose in the medium until end of the fermentation. For the time-course experiment, 10 ml samples were withdrawn and used for analysis. Optical density at 600 nm (OD600) was used to assess cell density.

Cultivation was carried out at 30°C with gentle stirring (150 rpm) for 15–18 h until an OD600 of 12–15 was reached. After 18 h of growth, the bulk cell mass was removed by centrifugation (9,000×g, 4◦C, 30 min). The culture supernatant was concentrated 5-fold and buffer exchanged in buffer A (20 mM HEPES, 5%Glucose, 50 mM Sod. Borate, 10 mM L-arginine, 1 mM EDTA, pH6.5) using a QuixStand Benchtop system (Hollow fiber cartridge with cutoff at 30,000 Da, surface area 1,400 cm², GE Healthcare, Sweden) followed by filtration through a Nalgene Rapid-Flow Sterile Disposable filter units with PES membrane 0.22 μm pore size.