Light-mediated control and analysis of recombinant protein production in microscale cultivations

The efficient production of technical or pharmaceutical proteins requires a large number of cultivation experiments during process development. These experiments are often miniaturized and parallelized and can be monitored with established measurement techniques. However, control options are still lacking in this scale. It was therefore the aim of this work to apply light, which is so far used for non-invasive optical measurements, to also reactivate photocaged inducer molecules and thereby enable optical process control. An optical online monitoring system was constructed based on the well-known BioLector setup. The capabilities of this device were demonstrated by optimizing the sequence specific incorporation of non-natural amino acids into recombinant proteins for click-chemistry applications. A sevenfold increased target protein concentration was achieved by a considerable increase of the unnatural amino acid concentration. In order to enable optical process control, the setup was expanded with a newly constructed UV-A LED module establishing an intensive yet individual illumination option for each well of a 48-well microtiter plate. The application of a 6-nitropiperonal photocaged derivate of the inducer isopropyl-D-thiogalactopyranoside (IPTG) demonstrates that a minimally invasive optical induction can be achieved and that convetional induction can be replaced this way. Light-mediated control over protein formation with caged compounds can not only replace conventional methods but also offers new control options for microscale cultivations. By releasing a second caged compound photocaged methionine (NVOC-Met) with complex illumination schemes, it is demonstrated that heterologous protein formation in a methionine repressible S. cerevisiae strain can be started, paused or stopped by optical means. A combination with the online measurements of the BioLector system establishes a closed-loop control setup for the gradual regulation of protein formation rates in each well of a microtiter plate which has not been achieved so far. Thereby, parallelized light-mediated control over recombinant protein formation is combined with light-mediated analysis, opening up a vast number of promising applications in bioprocess development. Funding, publications and contributions III Funding, publications and contributions ulatoren zur Licht031A167C) within the funding is gratefully acknowledged. Parts of this thesis have been published previously: Wandrey G, Wurzel J, Hoffmann K, Ladner T, Büchs J, Meinel L, Lühmann T. Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform. Journal of Biological Engineering, 2016, 10:11. Wandrey G, Bier C, Binder D, Hoffmann K, Jaeger K-E, Pietruszka J, Drepper T, Büchs J. Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system. Microbial Cell Factories, 2016, 15:63. Kusen P, Wandrey G, Probst C, Grünberger A, Holz M, Meyer zu Berstenhorst S, Kohlheyer D, Büchs J, Pietruszka J. Optogenetic regulation of tunable gene expression in yeast using photo-labile caged methionine. ACS Chemical Biology, 2016, 11(10), 2915 2922. Contributions to further publications and patent applications during the preparation of this thesis: Kusen P, Wandrey G, Krewald V, Holz M, Meyer zu Berstenhorst S, Büchs J, Pietruszka J. Light-controlled gene expression in yeast using photocaged Cu. Journal of Biotechnology, 2017, 258, 117-125. 1 Reprinted (adapted) with permission, Open Access CC BY 4.0, https://creativecommons.org/licenses/by/4.0/ 2 Reprinted (adapted) with permission, Open Access CC BY 4.0, https://creativecommons.org/licenses/by/4.0/ 3 Reprinted (adapted) with permission from ACS Chemical Biology. Copyright 2017 American Chemical Society. IV Funding, publications and contributions Binder D, Bier C, Grünberger A, Drobietz D, Hage-Hülsmann J, Wandrey G, Büchs J, Kohlheyer D, Loeschcke A, Wiechert W. Photocaged arabinose: A novel optogenetic switch for rapid and gradual control of microbial gene expression. ChemBioChem, 2016, 17(4), 296-299. Luchterhand B, Nolten J, Hafizovic S, Schlepütz T, Wewetzer SJ, Pach E, Meier K, Wandrey G, Büchs J. Newly designed and validated impedance spectroscopy setup in microtiter plates successfully monitors viable biomass online. Biotechnology Journal, 2015, 10(8), 1259-1268. European patent application EP16167322.3, Inventors: Büchs J, Flitsch D, Wandrey G, Giese H, Kunze M, 2016. Work in the field of biochemical engineering requires close collaboration with the cognate disciplines. While in Chapter 2 the setup of a high-throughput optical online monitoring system (BioLector) is described, Chapters 3-5 focus on the application of this system in the context of different biotechnological questions. These questions were investigated in close collaboration with external partners and some of their most relevant methods and results are included here in order to achieve a comprehensive description of the individual projects. In Chapter 3 [Wandrey et al. 2016b], cloning, protein purification, mass spectrometry, and ELISA measurements (Chapter 3.2.2-3.2.7, 3.2.11) were performed by Joel Wurzel (Institute of Pharmacy and Food Chemistry, Prof. Dr. Dr. Meinel, University of Würzburg). RSM development (Chapter 3.2.10) was headed by Tobias Ladner (AVT Biochemical Engineering, Prof. Dr.-Ing. Büchs, RWTH Aachen University). In Chapter 4 [Wandrey et al. 2016a], HPLC and NMR measurements as well as cIPTG synthesis (Chapter 4.2.2) were carried out by Claus Bier (Institute of Bioorganic Chemistry, Prof. Dr. Pietruszka, Heinrich-Heine University Düsseldorf, Forschungszentrum Jülich). Cloning and some of the offline fluorescence emission scans (Chapter 4.2.3) were performed by Dennis Binder (Institute of Molecular Enzyme Technology, Prof. Dr. Jaeger, Heinrich-Heine University Düsseldorf, Forschungszentrum Jülich). In Chapter 5 [Kusen et al. 2016], cloning, NVOC-Met synthesis, and stability testing (Chapter 5.2.1, 5.2.2) were conducted by Peter Kusen (Institute of Bioorganic Chemistry, Prof. Dr. Pietruszka, HeinrichHeine University Düsseldorf, Forschungszentrum Jülich).