Georg Wandrey

Photocaged Arabinose: A Novel Optogenetic Switch for Rapid and Gradual Control of Microbial Gene Expression.

Controlling cellular functions by light allows simple triggering of biological processes in a non‐invasive fashion with high spatiotemporal resolution. In this context, light‐regulated gene expression has enormous potential for achieving optogenetic control over almost any cellular process. Here, we report on two novel one‐step cleavable photocaged arabinose compounds, which were applied as light‐sensitive inducers of transcription in bacteria. Exposure of caged arabinose to UV‐A light resulted in rapid activation of protein production, as demonstrated for GFP and the complete violacein biosynthetic pathway. Moreover, single‐cell analysis revealed that intrinsic heterogeneity of arabinose‐mediated induction of gene expression was overcome when using photocaged arabinose. We have thus established a novel phototrigger for synthetic bio(techno)logy applications that enables precise and homogeneous control of bacterial target gene expression.

Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform

BackgroundGenetic code expansion has developed into an elegant tool to incorporate unnatural amino acids (uAA) at predefined sites in the protein backbone in response to an amber codon. However, recombinant production and yield of uAA comprising proteins are challenged due to the additional translation machinery required for uAA incorporation.ResultsWe developed a microtiter plate-based high-throughput monitoring system (HTMS) to study and optimize uAA integration in the model protein enhanced green fluorescence protein (eGFP). Two uAA, propargyl-L-lysine (Plk) and (S)-2-amino-6-((2-azidoethoxy) carbonylamino) hexanoic acid (Alk), were incorporated at the same site into eGFP co-expressing the native PylRS/tRNAPylCUA pair originating from Methanosarcina barkeri in E. coli. The site-specific uAA functionalization was confirmed by LC-MS/MS analysis. uAA-eGFP production and biomass growth in parallelized E. coli cultivations was correlated to (i) uAA concentration and the (ii) time of uAA addition to the expression medium as well as to induction parameters including the (iii) time and (iv) amount of IPTG supplementation. The online measurements of the HTMS were consolidated by end point-detection using standard enzyme-linked immunosorbent procedures.ConclusionThe developed HTMS is powerful tool for parallelized and rapid screening. In light of uAA integration, future applications may include parallelized screening of different PylRS/tRNAPylCUA pairs as well as further optimization of culture conditions.

Newly designed and validated impedance spectroscopy setup in microtiter plates successfully monitors viable biomass online

In microtiter plates, conventional online monitoring of biomass concentration based on optical measurements is limited to transparent media: It also cannot differentiate between dead or viable biomass or suspended particles. To address this limitation, this study introduces and validates a new online monitoring setup based on impedance spectroscopy for detecting only viable biomass in 48- and 96-well microtiter plates. The setup was first validated electronically and characterized by determining the cell constants of the measuring geometry. Defined cell suspensions of Ustilago maydis, Hansenula polymorpha, Escherichia coli and Bacillus licheniformis were characterized to find, among other parameters, the most suitable frequency range and the characteristic frequency of β-dispersion for each organism. Finally, the setup was exemplarily applied to monitor the growth of Hansenula polymorpha online. As reference, three different parallel cultures were performed in established cultivation systems. This new online monitoring setup based on impedance spectroscopy is robust and enables precise measurements of microbial biomass concentration. It is promising for future high-throughput applications.

Online in vivo monitoring of cytosolic NAD redox dynamics in Ustilago maydis

Maintenance of metabolic redox homeostasis is essential to all life and is a key factor in many biotechnological processes. Changes in the redox state of NAD affect metabolic fluxes, mediate regulation and signal transduction, and thus determine growth and productivity. Here we establish an in vivo monitoring system for the dynamics of the cytosolic NADH/NAD+ ratio in the basidiomycete Ustilago maydis using the ratiometric fluorescent sensor protein Peredox-mCherry. Metabolic redox dynamics were determined in the cytosol of living cells with high time resolution under biotechnologically relevant conditions, i.e. with high cell density and high aeration. Analytical boundary conditions for reliable analysis were determined, and perturbations in C-, N- or O- availability had marked impact on the cytosolic NADH/NAD+ ratio. NAD redox dynamics could be manipulated in lines inducibly expressing a water-forming NADH oxidase as a synthetic reductant sink. The establishment of Peredox-mCherry in U. maydis and the analysis of NAD redox dynamics provides a versatile methodology for the in vivo investigation of cellular metabolism, and contributes fundamental knowledge for rational design and optimization of biocatalysts.

Light-controlled gene expression in yeast using photocaged Cu2+

The manipulation of cellular function, such as the regulation of gene expression, is of great interest to many biotechnological applications and often achieved by the addition of small effector molecules. By combining effector molecules with photolabile protecting groups that mask their biological activity until they are activated by light, precise, yet minimally invasive, photocontrol is enabled. However, applications of this trendsetting technology are limited by the small number of established caged compound-based expression systems. Supported by computational chemistry, we used the versatile photolabile chelator DMNP-EDTA, long-established in neurobiology for photolytic Ca2+ release, to control Cu2+ release upon specific UV-A irradiation. This permits light-mediated control over the widely used Cu2+-inducible pCUP1 promoter from S. cerevisiae and thus constitutes the first example of a caged metal ion to regulate recombinant gene expression. We screened our novel DMNP-EDTA-Cu system for best induction time and expression level of eYFP with a high-throughput online monitoring system equipped with an LED array for individual illumination of every single well. Thereby, we realized a minimally invasive, easy-to-control, parallel and automated optical expression regulation via caged Cu2+ allowing temporal and quantitative control as a beneficial alternative to conventional induction via pipetting CuCl2 as effector molecule.

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).

Optische Genregulation in Mikrobioreaktoren

Photolabile-caged effector molecules allow non-invasive regulation of gene expression using cell-compatible UVA-irradiation. Based on this method, independent photoregulation of gene expression was combined with optical online monitoring of important cultivation parameters in a 48-well plate format. Thereby, an innovative screening system was created which allows for cost-effective and easily automated expression profiling as well as closed-loop process control.