Prozessnahe Hochdurchsatzoptimierung der heterologen Proteinproduktion in alternativen Wirtsorganismen

  • High-throughput optimization of heterologous protein production in alternative hosts

Rohe, Peter; Wiechert, Wolfgang (Thesis advisor)

Jülich : Forschungszentrum Jülich, Zentralbibliothek (2012, 2013)
Dissertation / PhD Thesis

In: Schriften des Forschungszentrums Jülich : Reihe Gesundheit 58
Page(s)/Article-Nr.: 165 S. : Ill., graph. Darst.

Zugl.: Aachen, Techn. Hochsch., Diss., 2012


This work includes the development of automated high-throughput methods for optimizing heterologous protein production. In the first part of the work, the automated cultivation platform named “Jülich Bioprocess Optimization System” (JuBOS) is designed and validated. The two main parts are a microtiter plate (MTP)-based cultivation system and a pipetting robot platform. The robot enables fluid transfer in and out of the MTP wells during cultivation. Further instrument integrations enable automated microbial work on the JuBOS, like sample handling and enzyme activity assays. MTP cultivations of various protein producer strains are successfully scaled up to laboratory bioreactors with volumes of 1 L and 20 L showing the validity of the results. In the second part, four methods for enhancing bioprocess optimization have been developed and applied. Mostly cutinase from Fusarium solani pisi secreted with Corynebacterium glutamicum was used as recombinant model protein. In the first method an automated strain screening results in an appropriate selection of signal peptide/cutinase combinations with higher precision than hitherto screening methods. As first biochemical engineering parameter, optimal combinations of IPTG concentration and induction time are evaluated with the Induction Profiling. Besides an impact of the two parameters on the product yield, also the target protein itself influences the optimum. The third method of this work is the automated media optimization shown for CG XII minimal media optimized for protein production. A screening of 19 media components and a subsequent algorithm-based optimization leads to a new minimal medium, which increases the yield by factor 1.5. Furthermore, the possibility of miniaturization of fed-batch processes using an enzymatic glucose release medium is shown. In the simplest case, these optimization methods are carried out subsequently. In such a sequence, the throughput of a bioprocess optimization is enhanced by factor 17 compared to parallel bioreactor cultivations.