Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His(6) tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L(−1) could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process

Application of bipolar electrodialysis to E.coli fermentation for simultaneous acetate removal and pH control

International audienceThe application of bipolar electrodialysis (BPED) for the simultaneous removal of inhibitory acetate and pH control during fermentation was investigated. A two cell pair electrodialysis module, consisting of cation exchange, anion exchange and bipolar membranes with working area of 100 cm each, was integrated with a standard 7 l stirred tank bioreactor. Results showed that BPED was beneficial in terms of in situ removal of inhibitory acetate and a reduction in the amount NHOH used for pH control. In batch and fed-batch BPED fermentations, base additions were decreased by up to 50% in both cases compared to electrodialysis (ED) fermentations with pH controlled at 6.7 ± 0.1. Consequently, the final biomass (34.2 g DCW l) and recombinant protein (5.5 g l) concentrations obtained were increased by up to 37 and 20%, respectively

Screening for conditions of enhanced production of a recombinant beta-glucanase secreted into the medium by Escherichia coli

Spexard M, Beshay U, Risse JM, Miksch G, Flaschel E. Screening for conditions of enhanced production of a recombinant beta-glucanase secreted into the medium by Escherichia coli. BIOTECHNOLOGY LETTERS. 2010;32(2):243-248.The extracellular production of a hybrid bacterial beta-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a beta-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength. By far the highest extracellular glucanase activity (> 200 U ml(-1)) was achieved when a strain harbouring the kil gene under control of a strong synthetic stationary-phase promoter and the glucanase gene under control of a strong synthetic constitutive promoter was cultivated on a complex medium mainly composed of casein peptone, yeast extract, and glycerol