Introduction 1 2 3 4 ® Thermobifida fusca Thermomonospora fusca 5 6 7 bta1 bta2 6 T. fusca co 8 8 9 T. fusca E. coli bta1 6 omp Materials and methods 6 Escherichia coli Thermobifida fusca supE hsdΔ5 thiΔ(lac-proAB) [traD36 proAB +  lacI q  lacZΔM15] 10 Omp -bta 6 Nde Eco 6 bta bta rbta 6 rbta 6 Nco Bam Omp bta1 6 T. fusca Omp bta1- 6 E. coli R L Media -1 −1 −1 −1 E. coli 11 −1 12 E. coli 4 2 4 2 4 4 2 2 2 2 2 2 2 3 3 2 4 2 3 2 2 4 −1 −1 4 2 2 2 2 2 2 2 3 3 2 4 2 3 2 2 Cultivation −1 3 3 4 p 2 2 E. coli Omp bta 6 In batch cultivation, induction of rTfH production was started at an OD of 0.5–0.6 by raising the cultivation temperature from 30 to 39°C or 42°C, respectively, within about 15 min. p 2 set −1 F glc −1 1 13 1 \documentclass[12pt]{minimal}
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$$ F_{\rm glc} = \frac{{\mu_{\rm set}} {\text{(}}XV{\text{)}}_0 {\text{e}}^{{\mu_{\rm set} t_{\rm f} }} } {{Y_{{\rm X/S}} S}}$$\end{document} XV 0  t f Y X/S −1 S −1 1 set −1 −1 1 set −1 Analytical methods g g 2 4 −1 Electrophoresis ® 11 14 ® Hydrolase activity p p 15 p p p −1 −1 −1 p p 2 −1 p 8 8 p Localization of rTfH g 16 g 4 11 g g g Purification of rTfH 11 g Localization of rTfH −1 −1 Protein determination ® Results  Batch cultivations E. coli Omp bta 6 1 Fig. 1 E. coli a 2 b open square open circle ) continuous line c  1 2 1 2 2 −1 1 R L bta 2 −1 2 −1 −1 2 −1 Fig. 2 E. coli a 2 b open square open circle continuous line c  E. coli. 17 2 2 18 1 Table 1 Summary of results on rTfH production in batch cultivations  Complex media Defined media Induction temperature(°C) 39 42 39 42 DCW −1 2.2 2.1 9.8 9.6 Induction time (h) 9.9 6 23 23.7 3 −1 8.08 4.43 7.5 2.84 3 DCW −1 3.67 2.11 0.77 0.30 3 −1 0.82 0.74 0.33 0.12 3 DCW −1 0.373 0.352 0.034 0.013 3 19 2 3 20 E. coli Fig. 3 a b dashed line  3 4 21 21 X Fig. 4 underlined arrows  Fed-batch cultivation Q p −1 2 \documentclass[12pt]{minimal}
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$$ Q_{\rm p} = q_{\rm p} X $$\end{document} q p DCW  −1 20 E. coli set max 1 Cultivation 5 Fig. 5 E. coli dashed line a −1 glc −1 b set −1  −1 DCW −1 5 max  −1 6 −1 1 Y X/S −1 5 DCW −1 glc −1 −1 2 F glc 1 set −1 Y X/S −1 −1 set −1 −1 2 DCW −1 Fig. 6 5  6 7 −1 −1 7 1 2 Fig. 7 DCW −1  8 Fig. 8 SDS-PAGE: EFs of different cultivations are shown after increasing the temperature to 39°C to induce rTfH production, HCDC (Run A)  p 5 2 1 Table 2 − 1 T T ind Cultivation  Run A Run B −1 38.5 42 Feed rate glc −1 −1 −1 0.03 0.07 Total induction time (h) 22.4 11.4 −1 53.2 67.8 Supernatant 3 −1 12.04 5.16 3 DCW −1 0.227 0.076 Periplasm 3 −1 74.22 51.69 3 DCW −1 1.395 0.762 Cytoplasm (soluble) 3 −1 141.24 156.74 3 DCW −1 2.655 2.312 Total 3 −1 227.5 213.59 3 DCW −1 4.28 3.15 3 −1 a 0.19 0.28 a Purification 6 bta Localization of rTfH DCW 2+ 9 −1 6 Fig. 9 E. coli a 2+ b line  9 2+ 10 3 3 Fig. 10 1 2 2+ 3  Table 3 Summary of two routes used to recover and purify rTfH from HCDC; the highly purified product was obtained from the periplasmic fraction (Run A), the low grade product was recovered from the cytoplasm and periplasm by ultrasonification (Run B) Purification step 3 −1 −1 −1 Purification-factor Yield (%) High grade rTfH  Supernatant with periplasmic protein 8.57 587 15 1 100 2+ 6.53 15 436 29 71  Gel filtration 4.89 11 445 30 57 Low grade rTfH  Crude extract 26.52 2,570 10.32 1 100  Heat treatment 22.51 1,470 15.3 1.5 85  Ultrasonification + heat treatment 28.89 1,410 20.5 2 a a T. fusca 7 7 6 E. coli 2+ −1 DCW −1 Localization of rTfH DCW −1 −1 Protein −1 7 3 Discussion E. coli Omp bta 6 T. fusca T. fusca E. coli T. fusca −1 E. coli E. coli 6 7 6 −1 −1 E. coli 22 23 2 1 2 6 24 2 Localization of rTfH E. coli 25 20 25 26 21 27 2+ 2+ 28 E. coli 26 20 E. coli In all cultivations performed in this study, rTfH could be detected in the medium without applying any additional measures to permeabilize the outer cell membrane. Cell lysis as a possible mechanism of rTfH release can be excluded as the cleavage of the OmpA sequence in the extracellularly found hydrolase indicates transport across the cytoplasmic membrane by the Sec-translocation mechanism. Any significant cell lysis would also release cytoplasmic rTfH still containing the complete signal sequence. 1 2 26 29 30 31 3 20 1 2 3 20 3 8 20 8 7 2 These mechanisms, which govern the local distribution of rTfH are not yet understood. Though cell-specific productivities are in a comparable range, product distributions differ largely. Our results suggest that the outer membrane permeability is related with the growth rate and hence all rTfH can migrate into the extracellular medium at high growth rates (batch runs). Therefore, final product release across the cell envelope could play an important role in the overall translocation process as product accumulation in the periplasm seemingly reduces the translocation rate from the cytoplasm by the Sec pathway. This hypothesis requires further investigations, which are presently in preparation. Therefrom, new strategies for cultivation and downstream processing with improved product qualities are expected.