Figure 2

Methanol pathway promoter-controlled GFP production in recombinant H. polymorpha strains. A. Screening of CBS4732-derived transformants producing GFP under control of the FMD promoter. Screening of 44 different H. polymorpha pC10-FMD (P_FMD-GFP) clones cultured under different cultivation modes; screening experiments were performed in deep-well-plates for batch and in FeedBead96-plates for fed-batch cultivation. Gas permeable sealings were used as sterile closures. To reduce the evaporation during the cultivation, water-saturated air was used for gassing. To compare the results from the different cultivation modes an identical carbon source concentration of 16.63 g/L were applied to each fermentation (batch with glycerol, batch with glucose, fed-batch with glucose). The batch cultivations were stopped when the stationary growth phase (glycerol 21 h, glucose 16 h) was reached and the fed-batch cultivations were stopped when 16.63 g/L glucose were released from the FeadBeat96-plate (16 h). Dry cell weight (DCW) was calculated from optical density measurements in a PowerWave ×340 micro titer plate reader. Green Fluorescent Protein (GFP) was measured at 485 nm excitation and 520 nm emission wavelengths. Cultivation conditions: shaking frequency 400 rpm, shaking diameter 50 mm, temperature 37°C, filling volumes 300 μL per well; media: SYN6-MES with 16.63 g/L of different carbon sources. Carbon sources: batch mode with glycerol (black line), batch mode with glucose (grey bars), fed-batch mode with glucose (black bars); inocula ratio 1:30. B. GFP production in a DL-1-derived transformant under control of the MOX promoter. H. polymorpha transformants producing GFP under control of MOX promoter were cultured for 12 h on YP medium containing 10 g/L methanol (YP+1%M) or 0.5 g/L glucose (YP+0.05%D) and then analyzed by confocal microscopy.