Optogenetic Regulation of Tunable Gene Expression in Yeast Using

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Optogenetic Regulation of Tunable Gene Expression in Yeast using Photo-Labile Caged Methionine Peter M. Kusen, Georg Wandrey, Christopher Probst, Alexander Grünberger, Martina Holz, Sonja Meyer zu Berstenhorst, Dietrich Kohlheyer, Jochen Büchs, and Jörg Pietruszka ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00462 • Publication Date (Web): 29 Aug 2016 Downloaded from http://pubs.acs.org on September 7, 2016

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ACS Chemical Biology

Optogenetic Regulation of Tunable Gene Expression in Yeast using Photo-Labile Caged Methionine Peter M. Kusen◊, Georg Wandrey†, Christopher Probst‡, Alexander Grünberger‡, Martina Holz◊, Sonja Meyer zu Berstenhorst◊, Dietrich Kohlheyer‡, Jochen Büchs†, Jörg Pietruszka*,◊,‡ ◊

Institute for Bioorganic Chemistry, Heinrich Heine University Düsseldorf at the Forschungszentrum Jülich, 52426 Jülich, Germany † AVT – Biochemical Engineering, RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany ‡ Institute of Bio- and Geosciences (IBG-1: Biotechnology), Forschungszentrum Jülich GmbH, 52426 Jülich, Germany KEYWORDS: caged methionine, caged compounds, photo-labile protecting group, gene expression, optogenetics.

ABSTRACT: Light-mediated gene expression enables the non-invasive regulation of cellular functions. Apart from their classical application of regulating single cells with high spatiotemporal resolution, we highlight the potential of light-mediated gene expression for biotechnological issues. Here, we demonstrate the first light-mediated gene regulation in Saccharomyces cerevisiae using the repressible pMET17 promoter and the photo-labile NVOC methionine that releases methionine upon irradiation with UVA light. In this system, the expression can be repressed upon irradiation and is reactivated due to consumption of methionine. The photolytic release allows precise control over the methionine concentration and therefore over the repression duration. Using this light regulation mechanism we were able to apply an in-house constructed 48-well cultivation system which allows parallelized and automated irradiation programs as well as online detection of fluorescence and growth. This system enables screening of multiple combinations of several repression/derepression intervals to realize complex expression programs, (e.g., a stepwise increase of temporally constant expression levels, linear expression rates with variable slopes, and accurate control over the expression induction, although we used a repressible promoter.) Thus, we were able to control all general parameters of a gene expression experiment precisely, namely start, pause and stop at desired time points, as well as the ongoing expression rate. Furthermore, we gained detailed insights into single-cell expression dynamics with spatiotemporal resolution by applying microfluidics cultivation technology combined with fluorescence time-lapse microscopy.

Photo-regulated gene expression in pro- and eukaryotic microorganisms as well as in mammalian cell cultures is a fast developing field of research of high topicality.1–7 Classical applications are based on the high spatiotemporal resolution and focus on single-cell regulation, cell-cell communication, or spatial patterning of multicellular organisms.8–12 Moreover, further outstanding characteristics like the non-invasive stimulation, fast modes of action and a simplified parallel control that can be easily automated, make this technology very attractive for biotechnological issues, which is in line with our results presented here.6,13–15 In general, several strategies have been applied to realize light-mediated gene expression in different organisms reaching from prokaryotes (e.g., E. coli) to eukaryotes (e.g., S. cerevisiae, zebra fish and mouse embryos).12,13,16–18 Already well described in this context are yeast two-hybrid systems using the Gal4p-transcription activator which is genetically split into two domains and reconstituted in vivo via lightdependent interaction of two fusion proteins.16,19–23 Other approaches based on recombinant photoreceptors demonstrated that light-activated sensor kinases can also be used to activate diverse cellular functions. Activation of gene expression, for instance, was enabled by light-controlled phosphorylation of a response regulator followed by activation of an intracellu-

lar signaling cascade leading, inter alia, to induction of gene expression.24,25 An alternative method for photo-regulated expression is based on chemical modifications of biological effector molecules which are then called caged compounds.26 The biological activity masked by the chemical modifications can be regained by irradiation. The transferability of the light-dependent regulation on widely used and well characterized expression systems is a main advantage of caged compounds in comparison to genetically encoded systems and demonstrates their broad flexibility. For instance, a photo-labile IPTG derivative tightly regulates the classical lac-promoter controlled gene expression in E. coli. Only after irradiation with UVA light, the modified IPTG led to a strong and tunable induction of gene expression.15,27–29 Notwithstanding photo-regulation techniques, most gene expression systems currently applied in biotechnology appear to be rather basic. Gene expression can be irreversibly switched on or off with these tools, but more precise regulation is usually not possible. However, recently more advanced regulations of protein production were described and the need of more complexity has been recognized. Likewise automated control of expression levels was shown by Fracassi et al. using a tetracycline dependent promoter to gain oscillating gene expression around a preset expression level in mammalian

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cells.14 Also a light-activated transcription factor was demonstrated by Milias-Argeitis et al. using an in silico feedback control to adjust the expression level in S. cerevisiae.30 Nevertheless, the establishment of complex gene regulation for biotechnologically relevant microorganisms is still at its beginning and requires the design of new (optical) regulation systems that provide great flexibility. In this, caged compounds are very intriguing. Taking account of the wide range of available photo-labile caging groups and their photo-sensitivity to different wavelengths, it becomes evident that caged compounds might offer a huge flexibility regarding their wavelength used for uncaging and for potential multichromatic combinations.3,17,31–33 On the other hand, photo-regulated expression systems comprise the opportunity to regulate several gene activities in parallel, while tuning the expression strength of each gene individually, reversibly and in real-time. This would represent a substantive component on the way towards mimicking complex regulation networks from nature making it possible to use the current cell factories much more efficiently. Interestingly, all photoregulated gene expression strategies applied in yeast up to now are based on genetically encoded systems, but caged compounds were to the best of our knowledge never combined with optogenetic expression in these model organisms. Hence, we focused on transferring the strategy of caged compounds to the repressible pMET17-promoter expression system in S. cerevisiae, which was already described for heterologous protein production in yeast.34 Prior to the application of optical regulation, we characterized our pMET17 expression system by using microfluidics cultivation technology combined with time-lapse imaging in methionine-free and methionine-containing medium. The highly resolved expression dynamics at single-cell resolution visualized a homogenous responsiveness of the expression system towards the inducer. Furthermore, the application of the consumable repressor methionine led to perfect reversibility of the gene repression. In expression cultures an extensive screening of irradiation conditions and expression dynamics was facilitated by an innovative in-house constructed 48-well cultivation system equipped with an UVA array.15 Moreover, a precise control over gene repression was possible using UVA light and a caged methionine called (S)-N-(4,5-dimethoxy-2nitrobenzyloxycarbonyl)-methionine (NVOC-Met).35

cleaved only under defined irradiation conditions and not by cell culture components (e.g., enzymes), the stability of NVOC-Met was tested in cultivation experiments. Therefore, cell cultures of S. cerevisiae supplemented with non-irradiated NVOC-Met were cultivated over five days in the dark. The NVOC-Met concentration, determined via RPHPLC, was unaffected by the cells and remained nearly constant over time (figure 2A). Apart from staying within standard deviation, a slight increase was observed, probably caused by evaporation during prolonged shaking. Hence, it can be assumed that NVOC-Met is stable in darkened cell culture and undergoes no unwanted decomposition or cellular degradation which could lead to uncontrolled release of methionine followed by repression of the pMet17-promoter system.

RESULTS AND DISCUSSION

Only a controlled release of methionine under irradiation conditions guarantees a precise light-driven gene expression. Therefore, the kinetic of methionine release was characterized by studying the photocleavage of NVOC-Met under UVA light illumination at 365 nm (3 – 6 mW/cm2). The chosen wavelength addresses 90% of the maximum absorbance (figure 2B) and enables good access to commercially available light sources. Determination of the NVOC-Met concentration during irradiation using RP-HPLC showed a roughly exponential decay (figure 2C). Based on these data an efficient and complete photocleavage of NVOC-Met can be presumed which makes it a suitable reagent to control the methionine concentration in the culture medium via irradiation. Next, realization of the light-mediated regulation of gene expression as well as quantification of the protein production required the design of suitable reporter strains. Hence, S. cerevisiae cells were transformed with an expression vector containing a yellow fluorescent protein (YFP) gene controlled by the pMET17 promoter from S. cerevisiae. We obtained a high-

NVOC-Met Characterization The caged methionine used for gene regulation in this study is (S)-N-(4,5-dimethoxy-2-nitrobenzyloxycarbonyl)methionine which was originally developed for peptide synthesis.35 It consists of the amino acid methionine coupled via a carbamate to the photo-labile 4,5-dimethoxy-2-nitrobenzyl protecting group (NVOC). It was synthesized in 65% yield according to a one-step procedure published by Chen et al. in 2012 for coupling of L-threonine using the commercially available 4,5-dimethoxy-2-nitrobenzyl-chloroformate (NVOCCl) in a 1:1-mixture of dioxane/H2O with NaHCO3 (figure 1A and Supporting Information).36 The low synthetic effort and costs of caged methionine guarantees good access to this essential regulator. In addition, the well-studied NVOC protecting group was reported to undergo a one-step photocleavage mechanism (figure 1B).37–39 In order to ensure that the photo-labile protecting group is

Figure 1: (A) Synthesis of (S)-N-(4,5-dimethoxy-2-nitro-benzyloxycarbonyl)-methionine (NVOC-Met) using NVOC-chloroformate. a) NaHCO3 (2 eq.), 1,4-dioxane/CH2Cl2 (1:1), room temperature, 24 h. (B) Generally proposed one-step photocleavage mechanism of NVOC-compounds transferred to methionine.37–39


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ly regulated recombinant expression system, allowing a noninvasive fluorescence-based quantification of protein production, which is activated (“induced”) under methionine starvation conditions and tightly deactivated (“repressed”) under methionine overflow.

Figure 2: (A) Stability test of NVOC-Met in cell culture and sterile SC medium. (B) Absorption spectrum of NVOC-Met (0.1 mg/mL) in buffered H2O (50 mM Tris-HCl, pH 7.4). (C) Photocleavage of NVOC-Met (1.75 mM) under UVA light (365 nm, 3 - 6 mW/cm2) irradiation in buffered H2O (Tris-HCl 50 mM, pH 7.4). Concentrations of NVOC-Met were determined in triplicates using RP-HPLC.

Characterization of pMET17-Expression System For further characterization of the methionine repression system in S. cerevisiae microfluidic single-cell cultivations combined with time-lapse imaging (MSCA)40 were performed to study the expression homogeneity and dynamics at the single-cell level. Instead of NVOC-Met and irradiation, a microfluidic cultivation device (details in Supporting Information) with integrated microfluidic valves enabled defined medium switches during continuous flow. This allowed quick changes between no methionine and methionine-containing medium during single-cell cultivations. 10 µM was identified as lower experimental limit for a sufficient repression of gene expression during microfluidic cultivations (see supplementary figure S3) and was thus used for the following experiments. Figure 3 depicts the mean fluorescence intensities of three parallel growth chambers each (colony 1 = red, colony 2 = blue, colony 3 = green) and the corresponding time-lapse series of an exemplary microcolony. Averaging the mean fluorescence of all cells in a growth chamber at each frame provided the mean fluorescence values. Cells grew with no addition of methionine for 2 h accompanied with a minor YFP fluorescence increase. The following period under a methionine concentration of 10 µM (after shift S1) led to a complete

repression and a YFP production plateau until the chambers were long filled with cells. After that, the changes to methionine-free medium after 21 h (S2) and 26 h (S4) resulted in an immediate increase in YFP production and fluorescence intensities. In contrast, a change back (S3) to methionine-containing medium caused a direct decrease of fluorescence explainable by intracellular depletion, cell division and the fact that higher fluorescent cells were overgrown by lower fluorescent cells with higher growth rate (see supplementary movie). For the reference cultivation without any methionine (see supplementary figure S3) just a direct increase in YFP production was observed, reaching a saturation level after the growth chambers were filled with cells (approx. 10 h). As visualized in the photos of figure 3, the fluorescence of the microcolony (i.e., all cells in the chamber) seemed relatively homogenous at the beginning of cultivation (3 h), but population heterogeneity increased at a later stage (28 h). Hence, we investigated the expression dynamics of individual cells inside one growth chamber, to show that despite the YFP production heterogeneity, the response to a methionine concentration change affects each cell. In figure 4 two cells, a high fluorescent and a low fluorescent cell were selected and dynamics in the mean single-cell fluorescence were observed. During various medium changes over 10 h both cells followed comparable dynamics, but with different magnitudes (S2-S3 and S4-S5: w/o methionine, S3-S4: with methionine). Hence, we conclude that with a suitable methionine concentration a tight regulator response occurred in all individually analyzed cells independent from the absolute expression level, which probably arises from natural population heterogeneity. 41–43 Summing up the microfluidic experiments, the pMET17promoter system enabled a reversible expression regulation of a desired gene in which methionine-induced expression could either be completely stopped or paused for a desired time interval. As a proof of principle, the constant activation of the YFP production observed in methionine-free medium almost directly stagnated upon changing to medium containing 10 µM of methionine followed by a sustaining decrease of the YFP fluorescence signal approx. 30 min after medium change. Moreover, the expression system showed perfect reversibility after changing back to methionine-free medium, indicated by a fast and strong derepression (i.e., reactivation) of gene expression within approx. half an hour. Finally, the whole cell culture was simultaneously affected by repression and derepression events demonstrated by a uniform response pattern of all cells. Characterization of Light-Mediated-Regulation After the detailed characterization of the pMET17 expression system on microscopic scale, YFP formation was monitored on macroscopic scale with an innovative in-house constructed 48-well cultivation system, which allowed diverse irradiation programs as well as online detection of fluorescence and growth. While the culture plate was attached to the shaking table, online monitoring of YFP formation was performed through the transparent bottom and optical regulation was realized by an LED array on top of the multiwell plate including 48 single and individually controlled LEDs (for details see Methods and supplementary figure S1).



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Figure 3: Microfluidic single-cell cultivations and analysis of the S. cerevisiae methionine repression system. Detailed study of colony responsiveness and homogeneity of YFP expression for 10 µM methionine and n = 3 growth chambers (red, blue, green) including a time lapse image series. The protocol for the performed shifts in methionine concentration is visualized in the lower panel: phases S0-S1, S2-S3 and S4-S5: w/o methionine, S1-S2 and S3-S4: with 10 µM methionine. Scale bar: 10 µm.

Figure 4: Microfluidic analysis of single-cell dynamics with different starting fluorescence (low, green line and high, blue line). Varying methionine concentrations as in figure 3. Scale bar: 5 µm.

Figure 5A graphs the YFP fluorescence intensity during cultivation with different concentrations of uncaged inducer. In the early growth phase (before methionine addition) fluorescence of all cell cultures increased exponentially since YFP expression was not repressed. After 5 h, different concentrations of non-modified methionine were added and the YFP fluorescence reached an almost stagnant level indicating successful repression of YFP formation. This repression phase began approximately 30 min after addition of the repressor methionine. Such a response time is to be expected since already formed mRNA was still translated and the oxygendependent maturation of YFP is slow.44 The duration of the repression phase could be controlled by the methionine concentration: The more was added, the longer it took the culture to metabolize the methionine surplus until it fell below the

physiological threshold value, which led to de novo activation of the pMET17 promoter. This reversible repression of gene expression was not only possible with manual addition of methionine solution but also with photo uncaging of NVOC-Met (figure 5B). At the start of the cultivation, NVOC-Met was added as a dormant reservoir together with the other medium components. After 5 h, cultures were automatically irradiated for varying durations with an UVA LED array (see Methods). Methionine was released from its photo-cage and repressed YFP formation as visible from the stagnant fluorescence intensity. When cultures were irradiated for longer durations, more methionine was released and the duration of the repression phase increased accordingly. This demonstrates that reversible optical gene repression can be gradually controlled with the here presented system. The delay until reactivation of gene expression after the manual addition of methionine solutions was similar to the delay after optical repressions showing a roughly linear correlation with 1 s of UVA irradiation being equivalent to the addition of 1 µM methionine under the given conditions (figure 5C). However, the expression delay did not double when methionine concentrations or UVA irradiation duration were doubled because the cultures continued to grow during the phase of methionine surplus and more cells consume the remaining methionine faster. For the same reason higher methionine concentrations or longer UVA irradiations were required when the repression was performed later in the cultivation when more biomass was already present (figure 6A and additional data not shown).


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Figure 5: Online-monitoring of YFP formation. Repression of YFP formation after (A) addition of 0 – 500 µM methionine (Met) or (B) 0 – 600 s UVA irradiation of 500 µM NVOC-Met, and (C) correlation of the expression delay. YFP formation is repressed after 5 h of cultivation by either manual addition of Met solutions or by photo-uncaging of 500 µM NVOC-Met with high intensity UVA irradiation (I = 52.7 mW/cm2, λmax = 368 nm). The methionine concentration (cMet) required to delay YFP formation for a specific amount of time is correlated to the duration of UVA exposure (dUVA) to achieve the same result (C). Cultivation conditions: 800 µL SC-medium per well in 48-FlowerPlates, 30 °C, shaking frequency = 1000 rpm.

Treating microorganisms with UVA light, photo-release byproducts (e.g., nitroso-compounds) or high methionine concentrations like in figure 5 could include the risk of influencing cellular fitness. Therefore, growth behaviors of cell cultures were monitored after both irradiation in NVOC-Met containing medium and methionine addition (see supplementary figure S4), but no negative effect on cell growth could be observed. Summarizing figure 5, the combination of an LED array for individual and automated illumination with a setup for online monitoring of YFP fluorescence in parallelized experiments allowed the in-depth characterization of the reversible optical repression with NVOC-Met. NVOC-Met irradiation and the resulting methionine release can perfectly mimic the manual addition of methionine regarding the repression behavior of the pMET17-based expression system. Complex Light-Mediated Regulation After the basic characterization of the pMET17-promoter system, the applicability of NVOC-Met in combination with UVA irradiation was realized for complex expression schemes. The red line in figure 6A, for example, shows that YFP production was repressed stepwise for about 1 h each, with an adjusted irradiation pattern (after 4 h cultivation: 20 s, 8 h: 40 s, 12 h: 60 s). The expression was automatically derepressed (i.e., reactivated) after each irradiation as soon as the released methionine was consumed. Thus, it was demonstrated that multiple repression and derepression steps could be combined sequentially using consecutive UVA irradiations. This extraordinary flexibility offers a great advantage of our NVOC-Met based expression system in contrast to most published caged compound-dependent regulation systems that can only activate gene expression irreversibly.8,12,18,28,29,45 Nevertheless, the presented pMET17 expression system originally enabled only control over expression deactivation. However, this putative drawback was overcome and temporal control over the expression activation was gained by converting the light-controlled OFF-switch into an ON-switch. Figure 6B shows that precise activation was realized with short (3 – 8 s) half-hourly irradiation pulses at the beginning of the cultivation. The irradiation time was adjusted to the growth phase and cell density, namely UVA exposure every 30 min for 3 s (0 – 2.5 h of cultivation), 4 s (3 – 5.5 h of cultivation) or 8 s (6 – 8.5 h of cultivation). Thereby, only small amounts of methionine were released, which caused a repression of the expression just until the next pulse. After stopping the irradiation pulses, YFP expression started after some minutes caused by methionine depletion. Thus, the successive photolytic release of small methionine amounts from NVOC-Met allowed a flexible "induction" (activation) of the expression at any time. Applying the constructed irradiation setup, the repression system was successfully transformed into an “induction” (activating) system using the reversibility and excellent accuracy of optical repression with methionine released from NVOCMet using an UVA LED array. This deliberate control of expression activation mimics inducible expression systems with very low basal expression in the early cultivation phase and specific induction in a later growth phase. In contrast, a single “batch-like” addition of uncaged methionine at the beginning of the cultivation would just define a rigid repression time without any flexibility.



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repress gene expression if the YFP production rate exceeded a threshold value. Curves of individual cultures: response to the closed-loop control, dashed lines: preset slopes for the YFP production during the controlled period (3 – 13 h). Cultivation conditions: 800 µL SC-medium, 500 µM NVOC-Met per well in 48-FlowerPlates, T = 30 °C, shaking frequency = 1000 rpm.

Figure 6: Optical regulation of YFP formation. (A) Multi-step repression–derepression of YFP formation, (B) temporal control over initiation of YFP formation with short light pulses and (C) automated gradient control. (A) Repeated repression of YFP formation by photo-uncaging of NVOC-Met after 4, 8 and 12 h (black arrows at x, y, z). (B) Repression of YFP formation via half-hourly light pulses uncaging just enough NVOC-Met to repress YFP formation until the next light pulse and derepression (“induction”) if irradion process is stopped. Horizontal arrows indicate the duration of repression/pulsing (from 1 h (blue) to 8 h (red)). Duration of half-hourly UVA exposure was gradually increased from 4 s at 0 h to 15 s at 8 h. In (A) and (B), mean YFP fluorescence intensities and error bars for standard deviations of triplicates are given. (C) Automated control of YFP formation rate in a closed-loop setup. Light pulses were triggered to temporarily

After demonstrating the light-mediated regulation of expression start, pause and stop at desired time points, subsequent experiments aimed at analyzing the expression rate as the hitherto missing parameter. Usually, controlled maximization of a hetero- or homologous overexpression is the best choice to gain as much product as possible. Several issues that lead to cellular stress responses, misfolding, or accumulation of toxic intermediates can occur and require individualized downregulation of expression rates to be overcome. To optimize the light-mediated expression rates, a closed-loop control using the monitored fluorescence intensities as input and the photolytic release of methionine from NVOC-Met as output was established (figure 6C). In a stand-alone operation, YFP formation was analyzed and slightly repressed by short light pulses (from 4 s after 0 h of cultivation up to 32 s after 13 h of cultivation), if the observed production rate exceeded a preset value (see Methods for details). Thus, applying NVOC-Met assisted photo-regulation, a fully automated process was created for defined down-regulation of gene expression. As depicted in figure 6C almost constant production rates were realized which roughly correlated with the predetermined slopes. Conclusion Concluding all abovementioned results, the pMET17 expression system was characterized on microscopic scale and could be highly regulated by low methionine concentrations (10 µM). The system also showed perfect reversibility and a uniform response of all cells independent of the absolute expression level, enabling multiple activation and deactivation intervals. Based on that, a light-mediated expression system was designed using the S. cerevisiae pMET17 promoter combined with a photo-labile caged methionine (NVOC-Met). This pMET17-NVOC-Met expression system was applied with an in-house constructed 48-well cultivation system allowing parallelized and automated irradiation as well as online fluorescence and growth detection. Thereby, precise time-limited gene repression followed by derepression was achieved by photolytic release and subsequent cellular consumption of methionine. The combination of several repression and derepression steps demonstrated outstanding flexibility of the expression system. The problem of controlling the “induction” time with high flexibility, while using a repressible expression system, was solved by an accurate sequential repression control at the beginning of the cultivation. Thus, not only a pause or stop but also the start of a heterologous protein expression could be controlled flexibly. Finally, predefined linear expression rates were realized in a closed-loop approach using a fully automated online fluorescence detection and irradiation device on a multi-well scale. The here described light-mediated regulation of gene expression based on the pMET17 promotor system and caged methionine thus represents a novel and powerful tool which in combination with our optical multi-well cultivation system enables a new level of regulation control for heterologous protein expression in yeast. The results of this work show


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clear benefits of our advanced expression technique over standard expression methods: First, without irradiation NVOC-Met remains stable during cultivation and thus can be supplied as a deposit without influencing the cells natural metabolism or being metabolized like added methionine. Second, the methionine release by irradiation is a noninvasive process reducing, i.a., the risks of culture contamination and evaporation, and maintaining even special culture atmospheres (e.g., anaerobic) without additional effort using NVOC-Met as a stable deposit right from the beginning. As extraordinary features of our light-mediated regulation system, the release process can be performed stepwise and repeated multiple times without the mechanical inference needed for recurring effector addition. Third, the release from the NVOC-Met deposit by irradiation enables perfect synchronization of the effector supplementation in separated parallel cultures (e.g., in multi-well plates) without time lags caused by alternate pipetting procedures and washing steps of the tips. In our cultivation system, all wells can be irradiated simultaneously. Forth, apart from not yet being available commercially, the UVA array remarkably reduces the technical effort and is extremely cheaper in acquisition and maintenance than a pipetting robot unit is. Moreover, the use of separated and independently controllable LEDs clearly simplifies the parallel and/or diverse regulation of multiple cultivations: triggering of a single well without affecting neighbors is no problem in contrast to automated (multichannel) pipetting. Fifth, by using our UVA array-equipped cultivation system and a defined irradiation setup, the parallelization of complex regulations was successfully managed on a multi-well scale using a set of 48 individually controlled LEDs. Thereby, 48 parallel cultivations were independently regulated at very different time points. Sixth, the expression system can be converted from a lightcontrolled OFF-switch into an ON-switch using light-mediated methionine release for a rigid repression of pMET17 at the beginning of the cultivation. Compared with a feasible initial addition of methionine, the defined photo-regulated effector release provides a higher flexibility of gene expression activation since the irradiation can be individually fine-tuned for each culture. Seventh, the great flexibility of controlling the gene expression start, pause and stop offers a surpassing advantage of our NVOC-Met based expression system in contrast to most published caged compound-dependent regulation systems that can only activate gene expression irreversibly. Finally, even linear protein production rates with predefined slopes were achieved in a multi-well scale in a closed-loop approach using a fully automated online fluorescence detection and irradiation device for the exceptional fine-tuned modulation of the effector release from NVOC-Met that exceeds the capabilities of recurring methionine addition. Taking all these advantages together, the constructed screening system in combination with the LED array and NVOC-Met represents a relatively easy adjustable optical device that allows precise monitoring and highly flexible control of protein production. Therefore, our highly advanced novel light-dependent expression system is an attractive tool for numerous applications, especially in the field of parallel

screening for optimal induction/repression conditions. Using the highly fine-tunable and non-invasive photo-cleavage of NVOC-Met instead of (repeated) methionine addition facilitates even sophisticated tasks were the culture medium and atmosphere should remain untouched, e.g., synchronization of enzyme-based reaction cascades in vivo with all their intermediate metabolites, studying and regulation of cellular functions, or optimization of the production of challenging (e.g., toxic) proteins. Moreover, upscaling of the technique is already envisaged.

METHODS See Supporting Information for additional methods. For the NVOC-Met stabilization test and precultivation of microfluidic experiments, yeast cells were cultivated in synthetic complete (SC) medium containing 6.70 g/L Yeast Nitrogen Base with (NH4)2SO4 (Carl Roth), 76 mg/L Histidine, 360 mg/L Leucine, 76 mg/L Lysine and 2% (w/v) Glucose with starting pH of 5.6 in test tubes at 30 °C and 600 rpm. The absorption spectrum of NVOC-Met (figure 2) was measured with a Shimadzu Photometer UV1800 at a concentration of 0.1 mg/mL in buffered H2O (50 mM Tris-HCl, pH 7.4). Photocleavage characterization of NVOC-Met (1.75 mM) in buffered H2O (Tris-HCl 50 mM) was performed using a VL-315.BL lamp (UVA light source, Vilber Lourmat Deutschland GmBH) equipped with three 15 W 365 nm blacklight tubes placed 3 cm above a FlowerPlate (m2p-labs GmbH) leading to a light intensity of 3 – 6 mW/cm2. The FlowerPlate was mounted on a microplate mixer (MixMate, Eppendorf AG) for vigorous shaking (1000 rpm). HPLC analytics were carried out with a Hyperclone 5µ ODS C18, 125 x 4 mm (Phenomnex Inc.) with 30% (v/v) acetonitrile and 0.1% (v/v) acetic acid in H2O, 0.5 mL/min flow rate, 25 °C. The absorption spectrum was measured with a M1000 Pro fluorescence microtiter plate reader (Tecan). Stability of NVOC-Met (figure 2) was tested by supplementing medium and cell cultures of S. cerevisiae with nonirradiated NVOC-Met, followed by cultivation in the dark over five days. Samples were taken after 0, 2 and 5 days and NVOC-Met concentration was determined via RP-HPLC. For studies of the pMET17-promoter dependent recombinant protein production yeast cells were cultivated in synthetic complete (SC) medium as described above. Precultures with 10 mL SC medium in 250 mL shake flasks were inoculated from cryo-stock cultures to an initial OD600 of 0.1 at 600 nm. Precultivation was performed for 16 h at 30 °C with a shaking frequency of 300 rpm and a shaking diameter of 50 mm. The main cultivation was then started in 48-well FlowerPlates (MTP-48-B, lot 1509, m2p-labs). Each well was filled with 800 µL SC medium from a master mix inoculated to an initial optical density (600 nm) of 0.1. Details on the microfluidic cultivations and setup are given in the Supporting Information. For optical repression experiments (figure 5), 500 µM NVOCMet was added from a 50 mM NVOC-Met stock solution in DMSO. Plates were sealed with a gas-permeable, transparent polyolefin sealing foil (900371, HJ-Bioanalytik) as sterile barrier that also reduced evaporation. Cultivation was performed at 30 °C with a shaking frequency of 1000 rpm and a shaking diameter of 3 mm. Online-monitoring of YFP formation was performed with an in-house constructed screening system based on the established BioLector setup (m2p-labs) and optical regulation was



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achieved with an in-house constructed LED array attached to the top of the FlowerPlates (see Supporting Information for detailed descriptions). For closed-loop control (figure 6C), the current YFP formation rate was calculated and irradiation was activated if the rate exceeded the preset threshold (see Supporting Information for details).

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Construction of reporter strain, Optical multi-well cultivation system, Microfluidic cultivation and full-length video corresponding to photos of figure 3, Analysis on cellular fitness, Synthesis and NMR-spectra of NVOC-Met.

AUTHOR INFORMATION Corresponding Author * email: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources This work was supported by grants from the German Federal Ministry of Education and Research (OptoSys: FKZ 031A16, FKZ 031A167A, FKZ 031A167B).

ACKNOWLEDGMENT The authors thank K.-E. Jaeger and T. Drepper for providing the pRhotHi-2-YFP plasmid containing a S. cerevisiae-optimized gene of YFP. We also thank J. Ernst and I. Eichhof for providing the pIE3 vector. In addition, a very special thanks to Susanne for her great support.

ABBREVIATIONS NVOC-Met, 6-nitroveratryloxycarbonyl methionine; pMET17, MET17 promoter.

REFERENCES (1) Drepper, T., Krauss, U., Meyer zu Berstenhorst, S., Pietruszka, J., and Jaeger, K.-E. (2011) Lights on and action! Controlling microbial gene expression by light. Appl. Microbiol. Biotechnol. 90, 23–40. (2) Motta-Mena, L. B., Reade, A., Mallory, M. J., Glantz, S., Weiner, O. D., Lynch, K. W., and Gardner, K. H. (2014) An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat. Chem. Biol. 10, 196–202. (3) Fournier, L., Gauron, C., Xu, L., Aujard, I., Le Saux, T., Gagey-Eilstein, N., Maurin, S., Dubruille, S., Baudin, J.-B., Bensimon, D., Volovitch, M., Vriz, S., and Jullien, L. (2013) A Blue-Absorbing Photolabile Protecting Group for in Vivo Chromatically Orthogonal Photoactivation. ACS Chem. Biol. 8, 1528–1536. (4) Mayer, G., and Heckel, A. (2006) Biologically Active Molecules with a “Light Switch.” Angew. Chem. Int. Ed. 45, 4900–4921. (5) Brieke, C., Rohrbach, F., Gottschalk, A., Mayer, G., and Heckel, A. (2012) Light-Controlled Tools. Angew. Chem. Int. Ed. 51, 8446–8476. (6) Young, D. D., and Deiters, A. (2007) Photochemical control of biological processes. Org. Biomol. Chem. 5, 999–1005.

Page 8 of 10

(7) Ellis-Davies, G. C. R. (2007) Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat. Methods 4, 619–628. (8) Cambridge, S. B., Geissler, D., Keller, S., and Cürten, B. (2006) A Caged Doxycycline Analogue for Photoactivated Gene Expression. Angew. Chem. Int. Ed. 45, 2229–2231. (9) Hayashi, K., Hashimoto, K., Kusaka, N., Yamazoe, A., Fukaki, H., Tasaka, M., and Nozaki, H. (2006) Caged gene-inducer spatially and temporally controls gene expression and plant development in transgenic Arabidopsis plant. Bioorg. Med. Chem. Lett. 16, 2470– 2474. (10) Tsien, R. Y., and Zucker, R. S. (1986) Control of cytoplasmic calcium with photolabile tetracarboxylate 2-nitrobenzhydrol chelators. Biophys. J. 50, 843–853. (11) Adams, S. R., and Tsien, R. Y. (1993) Controlling Cell Chemistry with Caged Compounds. Annu. Rev. Physiol. 55, 755–784. (12) Lin, W., Albanese, C., Pestell, R. G., and Lawrence, D. S. (2002) Spatially Discrete, Light-Driven Protein Expression. Chem. Biol. 9, 1347–1353. (13) Cruz, F. G., Koh, J. T., and Link, K. H. (2000) LightActivated Gene Expression. J. Am. Chem. Soc. 122, 8777–8778. (14) Fracassi, C., Postiglione, L., Fiore, G., and di Bernardo, D. (2015) Automatic control of gene expression in mammalian cells. ACS Synth. Biol. (15) Wandrey, G., Bier, C., Binder, D., Hoffmann, K., Jaeger, K.E., Pietruszka, J., Drepper, T., and Büchs, J. (2016) Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system. Microb. Cell Factories 15, 63. (16) Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002) A light-switchable gene promoter system. Nat. Biotechnol. 20, 1041–1044. (17) Lee, H., Larson, D. R., and Lawrence, D. S. (2009) Illuminating the Chemistry of Life: Design, Synthesis, and Applications of “Caged” and Related Photoresponsive Compounds. ACS Chem. Biol. 4, 409–427. (18) Shi, Y., and Koh, J. T. (2004) Light-Activated Transcription and Repression by Using Photocaged SERMs. ChemBioChem 5, 788– 796. (19) Sorokina, O., Kapus, A., Terecskei, K., Dixon, L. E., KozmaBognar, L., Nagy, F., and Millar, A. J. (2009) A switchable lightinput, light-output system modelled and constructed in yeast. J. Biol. Eng. 3, 15. (20) Yazawa, M., Sadaghiani, A. M., Hsueh, B., and Dolmetsch, R. E. (2009) Induction of protein-protein interactions in live cells using light. Nat. Biotechnol. 27, 941–945. (21) Kennedy, M. J., Hughes, R. M., Peteya, L. A., Schwartz, J. W., Ehlers, M. D., and Tucker, C. L. (2010) Rapid blue-lightmediated induction of protein interactions in living cells. Nat. Methods 7, 973–975. (22) Melendez, J., Patel, M., Oakes, B. L., Xu, P., Morton, P., and McClean, M. N. (2014) Real-time optogenetic control of intracellular protein concentration in microbial cell cultures. Integr. Biol. 6, 366– 372. (23) Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009) Spatiotemporal control of cell signalling using a lightswitchable protein interaction. Nature 461, 997–1001. (24) Tabor, J. J., Levskaya, A., and Voigt, C. A. (2011) Multichromatic Control of Gene Expression in Escherichia coli. J. Mol. Biol. 405, 315–324. (25) Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K., and Möglich, A. (2012) From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol. 416, 534–542. (26) Jack H. Kaplan, Forbush, B., and Hoffman, J. F. (1978) Rapid photolytic release of adenosine 5’-triphosphate from a protected analog: utilization by the sodium:potassium pump of human red blood cell ghosts. Biochemistry (Mosc.) 17, 1929–1935. (27) Binder, D., Bier, C., Grünberger, A., Drobietz, D., HageHülsmann, J., Wandrey, G., Büchs, J., Kohlheyer, D., Loeschcke, A., Wiechert, W., Jaeger, K.-E., Pietruszka, J., and Drepper, T. (2016) Photocaged Arabinose: A Novel Optogenetic Switch for Rapid and


Page 9 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Gradual Control of Microbial Gene Expression. ChemBioChem 17, 296-299. (28) Binder, D., Grünberger, A., Loeschcke, A., Probst, C., Bier, C., Pietruszka, J., Wiechert, W., Kohlheyer, D., Jaeger, K.-E., and Drepper, T. (2014) Light-responsive control of bacterial gene expression: precise triggering of the lac promoter activity using photocaged IPTG. Integr. Biol. 6, 755–765. (29) Young, D. D., and Deiters, A. (2007) Photochemical Activation of Protein Expression in Bacterial Cells. Angew. Chem. Int. Ed. 46, 4290–4292. (30) Milias-Argeitis, A., Summers, S., Stewart-Ornstein, J., Zuleta, I., Pincus, D., El-Samad, H., Khammash, M., and Lygeros, J. (2011) In silico feedback for in vivo regulation of a gene expression circuit. Nat. Biotechnol. 29, 1114–1116. (31) San Miguel, V., Bochet, C. G., and del Campo, A. (2011) Wavelength-Selective Caged Surfaces: How Many Functional Levels Are Possible? J. Am. Chem. Soc. 133, 5380–5388. (32) Priestman, M. A., Sun, L., and Lawrence, D. S. (2011) Dual Wavelength Photoactivation of cAMP- and cGMP-Dependent Protein Kinase Signaling Pathways. ACS Chem. Biol. 6, 377–384. (33) Klán, P., Šolomek, T., Bochet, C. G., Blanc, A., Givens, R., Rubina, M., Popik, V., Kostikov, A., and Wirz, J. (2013) Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy. Chem. Rev. 113, 119–191. (34) Solow, S. P., Sengbusch, J., and Laird, M. W. (2005) Heterologous protein production from the inducible MET25 promoter in Saccharomyces cerevisiae. Biotechnol. Prog. 21, 617–620. (35) D.-S. Shin, and Y.-S. Lee. (2009) Synthesis of Pentafluorophenyl Esters of Nitroveratryloxycarbonyl-Protected Amino Acids. Synlett 3307–3310. (36) Chen, S., Fahmi, N. E., Nangreave, R. C., Mehellou, Y., and Hecht, S. M. (2012) Synthesis of pdCpAs and transfer RNAs activat-

ed with thiothreonine and derivatives. Bioorg. Med. Chem. 20, 2679– 2689. (37) Amit, B., Zehavi, U., and Patchornik, A. (1974) Photosensitive Protecting Groups — A Review. Isr. J. Chem. 12, 103–113. (38) H. Schupp, W. K. Wong, and W. Schnabel. (1987) Mechanistic studies of the photorearrangement of o-nitrobenzyl esters. J. Photochem. 36, 85–97. (39) Il’ichev, Y. V., Schwörer, M. A., and Wirz, J. (2004) Photochemical Reaction Mechanisms of 2-Nitrobenzyl Compounds: Methyl Ethers and Caged ATP. J. Am. Chem. Soc. 126, 4581–4595. (40) Grünberger, A., Probst, C., Helfrich, S., Nanda, A., Stute, B., Wiechert, W., von Lieres, E., Nöh, K., Frunzke, J., and Kohlheyer, D. (2015) Spatiotemporal microbial single-cell analysis using a highthroughput microfluidics cultivation platform. Cytometry A 87, 1101– 1115. (41) Raser, J. M., and O’Shea, E. K. (2004) Control of Stochasticity in Eukaryotic Gene Expression. Science 304, 1811–1814. (42) Avery, S. V. (2006) Microbial cell individuality and the underlying sources of heterogeneity. Nat. Rev. Microbiol. 4, 577–587. (43) Levy, S. F., Ziv, N., and Siegal, M. L. (2012) Bet Hedging in Yeast by Heterogeneous, Age-Correlated Expression of a Stress Protectant. PLoS Biol 10, e1001325. (44) Gordon, A., Colman-Lerner, A., Chin, T. E., Benjamin, K. R., Yu, R. C., and Brent, R. (2007) Single-cell quantification of molecules and rates using open-source microscope-based cytometry. Nat. Methods 4, 175–181. (45) Feil, R., Brocard, J., Mascrez, B., LeMeur, M., Metzger, D., and Chambon, P. (1996) Ligand-activated site-specific recombination in mice. Proc. Natl. Acad. Sci. 93, 10887–10890.



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Optogenetic Regulation of Tunable Gene Expression in Yeast Using

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