Optimization of recombinant in protein production in an E.coli-based process with limited oxygen transfer
Publikation/Tidskrift/Serie: Biotechnology 2000 : the World Congress on Biotechnology, 11th International Biotechnology Symposium and Exhibition, 3 - 8 September 2000, ICC Berlin, book of abstracts
E. coli continues to be a popular host organism for production of recombinant proteins. To obtain a high volumetric productivity at the cultivation stage, it is important to achieve a high cell density and a high specific productivity per cell. In many fed-batch processes, the substrate feed rate eventually becomes limited by the oxygen transfer capacity of the bioreactor. While biomass can continue to increase, parameters like specific substrate uptake and specific growth rate will decrease with time. As the specific productivity of a particular protein may depend critically on the growth rate and the substrate supply, the time for induction of the recombinant production becomes a key parameter for process optimization.A probing technique enabled the development of a substrate feeding strategy that automatically avoids overflow metabolism as well as oxygen limitation due to reactor constraints. It is based on a standard dissolved oxygen sensor and does not require any strain-, product-, or media-specific knowledge. This gives a powerful tool for process development as it allows the user to experiment with different constructs and media compositions in a flexible way.The probing feeding strategy was employed when investigating the influence of induction time and media composition in a process for production of a recombinant xylanase in E. coli BL21(DE3) where oxygen transfer quickly became a limiting factor. It is shown how the specific productivity increases with increasing induction OD, that is, decreasing substrate uptake. For higher induction OD, the enzyme activity tend to decrease after 1.5-2 h, which could be correlated to a significantly higher protease activity for low specific glucose uptakes. As has been suggested previously, addition of complex media components (TSB) to the feed medium reduced the protease activity and improved the xylanase production further to approximately 10 g xylanase per liter broth. The results suggest that a "late" induction is preferred for the xylanase process (increased specific productivity and increased cell mass) and that a production phase of 2 h should be enough. If a complex medium can be used, the productivity can be improved further.In processes with limited oxygen transfer, the described approach, with the probing feeding startegy and changing the induction time, gives a simple but powerful approach for process optimization. A limited number of experiments give information that can improve the process productivity significantly, in the described example more than three-fold. The method will be further evaluated, and is currently applied to a process for production of a 50 kDa fusion protein.References1) Hellmuth, K. et al., "Effect of growth rate on stability and gene expression of recombinant plasmids during continuous and high cell density cultivation of Escherichia coli TG1", J. Biotechnol. 32:289-298,1994.2) Wong, H. et al., "Effect of post-induction feeding strategies on the production of bioadhesive protein in Escherichia coli", Biotechnol. Bioeng. 60:271-276, 1998.3) Åkesson, M. et al., "On-line detection of acetate formation in Escherichia coli cultures using dissolved oxygen responses to feed transients", Biotechnol. Bioeng. 64:590-598, 1999.4) Åkesson, M., "Probing control of glucose feeding in Escherichia coli cultures", Ph.D. thesis, ISRN LUTFD2/TFRT-1057-SE, Lund Institute of Technology, Lund, Sweden. 1999.5) Tsai, L. et al., "The effect of organic nitrogen and glucose on the production of recombinant human insulin-like growth-factor in high-cell-density Escherichia coli fermentations", J. Ind. Microbiol. 2:181-187, 1987.
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