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Aug 29, 2018 - KEYWORDS: yeast, optogenetic, gene expression, protein stability ... submicrometer resolutions.1 Single-component optogenetics is...
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Letter Cite This: ACS Synth. Biol. 2018, 7, 2045−2053

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A Single-Component Optogenetic System Allows Stringent Switch of Gene Expression in Yeast Cells Xiaopei Xu,†,‡ Zhaoxia Du,†,‡ Renmei Liu,†,‡ Ting Li,†,‡ Yuzheng Zhao,†,‡,§ Xianjun Chen,*,†,‡,§ and Yi Yang*,†,‡

ACS Synth. Biol. 2018.7:2045-2053. Downloaded from pubs.acs.org by UNIV OF TEXAS AT EL PASO on 11/06/18. For personal use only.

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Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, and §Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China ‡ CAS Center for Excellence in Brain Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China S Supporting Information *

ABSTRACT: Light is a highly attractive actuator that allows spatiotemporal control of diverse cellular activities. In this study, we developed a single-component light-switchable gene expression system for yeast cells, termed yLightOn system. The yLightOn system is independent of exogenous cofactors, and exhibits more than a 500-fold ON/OFF ratio, extremely low leakage, fast expression kinetics, and high spatial resolution. We demonstrated the usefulness of the yLightOn system in regulating cell growth and cell cycle by stringently controlling the expression of His3 and ΔNSic1 genes, respectively. Furthermore, we engineered a bidirectional expression module that allows the simultaneous control of the expression of two genes by light. With ClpX and ClpP as the reporters, the fast, quantitative, and spatially specific degradation of ssrA-tagged protein was observed. We suggest that this single-component optogenetic system will be immensely helpful in understanding cellular gene regulatory networks and in the design of robust genetic circuits for synthetic biology. KEYWORDS: yeast, optogenetic, gene expression, protein stability

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minimal, probably due to technical complexities or limitations. We previously reported simple yet robust single-component light-switchable gene expression systems for mammalian cells and bacteria.12−14 Both systems consist of a single genetically encoded photosensitive transcription factor GAVPO or LEVI, in which the LOV domain Vivid (VVD) derived from the photoreceptor of Neurospora crassa rapidly forms a homodimer in response to blue light and promotes the binding of GAVPO or LEVI to specific DNA sequences to activate or repress gene transcription, respectively. In the present study, we sought to develop a compact yet robust light-switchable gene expression system for yeast cells based on a similar design. In this study, we developed a light-switchable gene expression system termed yLightOn for yeast cells. The yLightOn system consists of a single-component light-switchable transcription factor and shows more than a 500-fold induction ratio, extremely low leakage, and quantitative and spatiotemporal control of gene expression. We showed the usefulness of the yLightOn system in controlling cell growth and cell cycle. Furthermore, we have also engineered a

ptogenetics has emerged as a powerful strategy to control diverse biological systems with millisecond and submicrometer resolutions.1 Single-component optogenetics is a standard research tool that displays speed, simplicity, and versatility and was first applied in neuroscience research to allow the optical control of neurons on a millisecond time scale.2 Recently, the discovery and engineering of new singlecomponent optogenetic tools have shed light on diverse biological fields not limited to neuroscience, as with the emergence of new photoreceptors. In particular, the LOV domains of several photoreceptors possess several advantages, including its small size, which avoids steric hindrance and facilitates accurate molecular design, and its endogenous cofactor (FAD or FMN), which avoids addition of an exogenous cofactor or introduction of cofactor-biosynthesis genes. These favorable characteristics enable the LOV domains to be ideal candidates for developing novel optogenetic tools.3 Yeast serving as a model for all eukaryotes has been widely applied in the study of fundamental cellular processes. Precise spatiotemporal control of gene expression is an indispensable tool for characterization of these complex cellular processes. Several light-regulated gene expression systems based on multiple protein components have been reported in yeast cells.4−11 However, the usefulness of these systems has been © 2018 American Chemical Society

Received: May 1, 2018 Published: August 29, 2018 2045

DOI: 10.1021/acssynbio.8b00180 ACS Synth. Biol. 2018, 7, 2045−2053

Letter

ACS Synthetic Biology

Figure 1. Design, optimization and validation of the single-component optogenetic system. (A) Schematic representation of the single-component optogenetic system. (B) Configuration of the single-component light-switchable gene expression system. (C) Light-inducible mCherry expression by LVAD driven by different promoters. The data were normalized to the mCherry expression from the strong constitutive promoter PMA1 in darkness. (D) Crystal structure of the VVD homodimer (PDB 3RH8). The mutation sites for VVD variants are indicated. (E−G) Fine tuning yLightOn system by altering the promoter configurations, including different minimal promoters (E), spacer lengths between the operator and the minimal promoter (F) and LexAop copy number (G). The data in panels C and E−G are the means of three independent experiments, and the error bars indicate the standard deviation.

systems based on single-component light sensors for mammalian cells and bacteria.12,13 Both the light-switchable transcription factors contain a DNA-binding domain (Gal4 or LexA) and a light sensing and responding domain (VVD). These two domains constitute a light-switchable DNA-binding fusion protein that dimerizes and binds to DNA sequences to directly repress gene transcription12 or activate gene transcription by a fused transcriptional activation domain upon blue light exposure.13 We hypothesized that fusing the lightswitchable DNA-binding fusion protein to a yeast transcription activation domain would create a light-switchable transcription factor for yeast cells, as light should induce dimerization of the fusion protein and binding to the upstream activation sequence (UAS) to activate transcription (Figure 1A). LexA protein is a

bidirectional expression module, allowing the simultaneous control of the expression of two genes by light. When using ClpX and ClpP as the reporters, we observed the fast, quantitative, and spatially specific degradation of ssrA-tagged proteins. The newly developed light-switchable gene expression system will be a powerful and convenient tool for the study of cellular gene function and gene regulatory networks and for the design of interesting synthetic circuits.

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RESULTS AND DISCUSSION Design, Optimization, and Validation of the SingleComponent Light-Switchable Gene Expression System. We have previously created light-switchable gene expression 2046

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Figure 2. Characterization of the yLightOn system. (A) ON kinetics of mCherry expression by the light-inducible systems. The engineered cells transformed with the activator plasmid expressing LVADO, LVAD (Y50W M135I) or CRY2/CIB1 and the corresponding response plasmid were first cultured in dark conditions and then transferred to blue light illumination. The mCherry fluorescence at the indicated times was quantified. (B) OFF kinetics of mCherry expression by the light-inducible systems. The same engineered cells in panel A were first cultured in light illumination and then were transferred to dark conditions. The mCherry fluorescence at the indicated times was quantified. Inset, OFF kinetics of the 0−200 min. (C) Light intensity dependent activation by the light-inducible systems. (D) Light-inducible gene activation by a single blue-light pulse of varying durations. The engineered cells transformed with yLightOn system plasmids using mCherry as the reporter were illuminated with pulses of blue light of varying durations and then were transferred to dark conditions. The mCherry fluorescence was determined at time point 4 h. (E) Spatial control of gene by yLightOn system. The same engineered cells in panel D were grown on solid medium and irradiated by blue light using a mask with a specific image (left panel). mCherry fluorescence was imaged and quantified (middle and right panel) after 36 h. The orange circle indicated the glass bottom of the dish, where the cells were attached. Scale bar, 1 cm. Data in (A−D) are means of three independent experiments, and error bars indicate the standard deviation.

variant M135I and M165I decreases the recovery by ∼10fold.16 The variant Y50W forms a stable light-state cysteinyl adduct to promote dimerization.17,18 To this end, we carried out combinatorial mutagenesis of these VVD variants with varied photoadduct decays (Figure 1D). Most of the LVAD mutants exhibited insignificant differences in mCherry expression between light and dark conditions, with ON/OFF ratios lower than 5-fold (Figure S1). However, VVD mutants with the single mutation Y50W or the double mutations Y50W/I85V, I85V/M135I or Y50W/M135I displayed ON/ OFF ratios of more than 40-fold. In particular, VVD mutant with the triple mutations Y50W/I85V/M135I (optimized LVAD (LVADO)) showed a 573-fold ON/OFF ratio, which was significantly higher than the typical blue-light-inducible CRY2/CIB1 system (31-fold)4,19 and the chemical-triggered systems (147-fold for CUP1(Cu2+), 297-fold for Gal1(Galactose), 106-fold for GEV(β-estradiol))(Figure S2).20−22 LVADO also exhibited high activation efficiency comparable to the strong constitutive PMA1 promoter (Figure S2 and S3A). Notably, the leakage of LVADO in dark conditions was almost the same as in the control cells harboring only the response plasmid (Figure S3B), demonstrating the stringent control of gene expression. As both the activator and reporter plasmids

repressor of the Escherichia coli SOS regulon which is orthogonal to the cellular components of yeast cells,15 hence reducing the possibility of cross-talk between the transactivator and the host chassis. We therefore fused LexA-VVD (LEVI) to the activation domain of the Gal4 protein (Gal4AD) to obtain LEVI-Gal4AD (LVAD). We constructed a response plasmid containing the gene encoding mCherry fluorescent protein under the control of eight copies of the LexA-binding sequence and a Gal1 minimal promoter (Figure 1B, Supplementary Note). We transformed the response plasmid and the activator plasmid expressing LVAD driven by different promoters into BY4742 cells. The engineered cells exhibited distinct lightswitchable mCherry expression (Figure 1C). Among these promoters, the truncated ADH1 promoter ADH1(410) exhibited a 23-fold ON/OFF ratio and ∼50% activation efficiency compared to the commonly used strong constitutive promoter PMA1 (Figure 1C). Owing to LVAD’s modular design, it is possible to enhance the performance of the light-switchable system (leakage, photosensitivity, etc.) by introducing mutations in the VVD motif. Previous studies showed that the variants I74V and I85V increases recovery of the photoadduct by ∼25-fold and ∼23fold relative to native VVD, respectively, whereas the double 2047

DOI: 10.1021/acssynbio.8b00180 ACS Synth. Biol. 2018, 7, 2045−2053

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Figure 3. Optical control of cell growth and cell cycle. (A) Histidine auxotrophy assays by the yLightOn system. The engineered BY4742 cells transformed with yLightOn system plasmids using His3 as the reporter were serially diluted (1:10; first spot approximately 104 cells) and grown on solid medium in the presence or absence of histidine under light or dark conditions. The BY4742 cells transformed with empty vectors or the plasmid constitutively expressing HIS3 were used as the controls. (B−C) Cell cycle progression controlled by the yLightOn system. (B) The engineered TUB1/YML085C (Tub-GFP) cells transformed with the yLightOn system plasmids using ΔNSic1 as the reporter were serially diluted (1:10; first spot approximately 105 cells) and grown in solid medium under light or dark conditions. The BY4742 cells transformed with empty vectors were used as the controls. (C) The same yeast cells as in (B) were cultured in light or dark conditions for 10 h before imaging. Circular graphs (inner ring, dark conditions; outer ring, light conditions) show the mean distribution of cell cycle stages obtained from six biological replicates counting at least 100 cells for each replicate. Scale bars, 5 μm.

contain 2 μ replication origin, which may lead to varied number of the expression components. To better characterize the light-induced expression by LVADO, we transferred the PADH(410)-LVADO and 8xLexAop-PGal1 mini-mCherry expressing cassettes to pRS315 and pRS316, respectively, which have been validated to harbor single copy in yeast cells.23,24 Our results showed that the single copy plasmids had a 188-fold ON/OFF ratio, with 1.3-fold lower background expression and 3.9-fold lower activation efficiency compared to the 2 μ plasmid (Figure S4A). Notably, we observed good homogeneity for the cells harboring single copy plasmids kept in dark conditions, whereas a small number of the cells harboring 2 μ plasmids exhibited significant leak expression (Figure S4B and 4C), probably due to much higher copy number of the expression components in these cells. We used LVADO in all subsequent studies, and we referred to the light-switchable gene expression system based on LVADO as the yeast light-on (yLightOn) system. Unless otherwise indicated, the yLightOn system in 2 μ plasmids was used for subsequent studies. To determine the promoter configurations to fine-tune the induction characteristics of the yLightOn system, we first replaced the Gal1 minimal promoter with other minimal promoters. A multitude of regulatory systems with highly diverse levels of background noise and maximal activation were observed (Figure 1E). We next varied the number of LexAop repeats and the spacer lengths separating LexAop from the Gal1 minimal promoter. Our results showed that mCherry expression levels decreased as the spacer length increased or the number of LexAop repeats decreased (Figure 1F,G). Notably, significant activation of mCherry expression occurred even with a 500-bp spacer (Figure 1F), providing great potential to construct chimeric promoters that could respond to multiple input signals.14 Taken together, these results demonstrate that the performance of the yLightOn system can be fine-tuned by altering the promoter configurations, providing flexible options for specific experimental conditions. Characterization of the yLightOn System. We first investigated the ON kinetics of yLightOn system in both 2 μ

plasmids and single copy plasmids. Our data showed that the mCherry expression level took approximately 6 h to switch from OFF to the fully ON state. The t1/2 (the time to reach 50% of maximal expression) was approximately 1.8 h for the yLightOn system both in 2 μ plasmids and in single copy plasmids (Figure 2A). The ON kinetics of the yLightOn system was faster than the LVAD (Y50W/M135I) (t1/2 ∼ 2.6 h) and CRY2/CIB1 (4.7 h) (Figure 2A), but might be slower compared to the EL222 system (t1/2 < 10 min for mRNA detection) and the chemical inducible GEV system (t1/2 < 1 h using GFP as the reporter).21,25 We next tested the OFF kinetics by transferring the cells from light illumination to dark conditions. We found that mCherry expression continued to increase within 10 min for LVAD (Y50W M135I) and yLightOn systems (Figure 2B), indicating that these systems remain active for some time after switching off the light, as it takes time for the decay of LVADO photoadduct and degradation of the accumulated mRNA.16 In comparison, yLightOn system had faster decay kinetics than LVAD (Y50W M135I), consistent with the fact that the I85V variant can significantly accelerate recovery of the photoadduct.16 CRY2/ CIB1 system exhibited no increase of mCherry expression and had faster decay kinetics compared to the yLightOn system (Figure 2B). These results were further validated by a pulse experiment (Figure S5). We next tested the ability of the yLightOn system to induce graded protein expression by controlling the light intensities (Figure 2C). The gene activation by the yLightOn system is well fitted by a Hill function (Hill coefficient n = 2.60 ± 0.75) with light sensitivity (half-maximal response, k) of 0.65 W·m−2 (Table S1), showing much less sensitivity to blue light than the LVAD (Y50W M135I) (k = 0.06 W·m−2) and CRY2/CIB1 system (k = 0.27 W·m−2). In addition, a strong dose dependence on the duration of a single pulse was observed for the yLightOn system (Figure 2D), even a 10 min pulse of blue light could lead to marked activation of a reporter expression. Thus, activation levels controlled by the yLightOn system can be continuously adjusted by either light intensity 2048

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Figure 4. Spatiotemporal control of protein stability. (A) Schematic representation and design of the bidirectional expression module containing two facing transcription units. (B) Light-induced mCherry and sfGFP expression from the bidirectional expression module or from the yLightOn system. (C) Schematic representation and design of the spatiotemporal control of protein stability. ClpX and ClpP are assembled to form a protease complex that can recognize ssrA-tagged protein substrates, recruit the degradable substrates and unfold their tertiary structure. (D) Lightinduced degradation of mCherry-ssrA. Non-ssrA-tagged mCherry was used as the control. Scale bars, 5 μm. (E) Western blot validation of the light-induced degradation of mCherry-ssrA. (F) Temporal kinetics of the light-induced degradation of mCherry-ssrA or mCherry-psd. (G) Light intensity-dependent degradation of mCherry-ssrA or mCherry-psd. (H) Spatial control of mCherry-ssrA degradation. The engineered BY4742 cells were grown on solid medium and irradiated by blue light using a mask with a specific image (left panel). Imaging of mCherry fluorescence was taken (right panel). The orange circle indicates the glass bottom of the dish, where the cells were attached. Scale bar, 1 cm. Data in panels B, F, and G are means of three independent experiments, and error bars indicate the standard deviation.

(Figure 2C) or duration of illumination pulse (Figure 2D). To spatially control gene expression, the engineered cells transformed with yLightOn system plasmids using mCherry as the reporter were grown on solid medium and irradiated by blue light using a mask with a specific image. The mCherry fluorescence image of the cells had the pattern of the original image used as the mask (Figure 2E). These data demonstrate that the yLightOn system is a robust tool for the rapid, reversible, quantitative, and spatiotemporal control of gene expression in yeast cells. Optical Control of Cell Growth and Cell Cycle. Because of the extremely low leakage in noninducing conditions, the

yLightOn system is well suited to stringent control of the expression of arbitrary target genes. In a preliminary proof-ofprinciple study, we utilized the yLightOn system to control cell growth. The engineered BY4742 cells transformed with yLightOn system plasmids using His3 gene as the reporter were cultured in the absence or presence of histidine. Our data exhibited the light-dependent rescue of His3 expression and Histidine auxotrophy (Figure 3A), which was not observed for the control cells (Figure 3A). To implement cell cycle control by light, we utilized the yLightOn system to stringently control the expression of the ΔN Sic1 gene, a shortened version of Sic1 lacking the SCFCdc42049

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protein degradation system provides a useful tool for the quantitative and spatiotemporal control of protein degradation.

dependent degradation sequence located within the N-terminal 105 amino acids that imposes a lengthened G1 phase.26−28 In darkness, the engineered yeast cells grew similarly to the control cells (Figure 3B). Upon light illumination, lightinduced high-level ΔNSic1 expression led to the accumulation of ΔNSic1 and significantly prolonged cell cycle (Figure 3B). Analysis of the cells revealed that the behaviors of the engineered yeast cells in dark conditions had a small difference compared to the control cells (Figure 3C), indicating that a small number of cells had significant ΔNSic1 leak expression in dark conditions, which was consistent with previous results and the growth rate measurement (Figures S4 and S6). In comparison, the engineered cells harboring yLightOn system in single copy plasmids expressing ΔNSic1 in darkness exhibited almost the same behavior with the control cells (Figures S6 and S7). Upon blue light illumination, the majority of both the cells displayed large buds with short spindles localized at the bud neck (Figure 3C, Figure S7B), which is the typical phenotype of G1/S transition. Spatiotemporal Control of Protein Stability. The regulation of protein stability is required to ensure the proper signaling and growth of living cells. Previously, a genetic photosensitive degron (psd) module has been developed to allow the spatiotemporal control of protein stability in yeast cells.29 However, the relatively large size of the degron (20 kDa) may lead to potential interference with the target protein. The E. coli ssrA, an11-amino-acid degradation tag (AANDENYALAA) mediated by the protease complex ClpX/ClpP,30 has been repurposed in yeast cells in a galactose-inducible manner.31 To construct a light-regulated protein degradation system based on ssrA for yeast cells, we first designed a lightswitchable bidirectional expression module (Figure 4A). Robust light-induced mCherry and sfGFP expression was observed (Figure 4B). The activation efficiencies of both reporters were similar to those from the single-direction module in the yLightOn system (Figure 4B), indicating that the activation of one transcription unit did not significantly affect the other. We next replaced the mCherry and sfGFP genes with ClpP and ClpX genes, respectively. The reporter plasmid was obtained by fusing the ssrA tag to mCherry protein driven by the ADH1 constitutive promoter (Figure 4C). We transformed the reporter plasmid and the bidirectional expression module into BY4742 cells. Significantly decreased mCherry fluorescence was observed for the cells illuminated with blue light, compared to the cells kept in darkness (Figure 4D,E, Figure S8). The dark/light ratio for mCherry-ssrA could reach 28-fold, which was significantly higher than the 9-fold of the psd system (Figure S9). We next investigated the kinetics of mCherry-ssrA depletion after exposure to blue light. Our data showed that mCherry-ssrA had a slower depletion half-life (62 min) than mCherry-psd (33 min) (Figure 4F), probably because it took additional time for the accumulation of ClpX and ClpP proteins. Graded protein depletion was observed upon exposing the cells to different blue light intensities (Figure 4G). The depletion curve is well fitted by a Hill function (Hill coefficient n = 2.05 ± 0.14) with a light sensitivity (half-maximal response, k) of 0.07 W·m−2 (Table S2), reflecting higher sensitivity than the psd system (halfmaximal response of 0.29 W·m−2). The ability of the lightinduced protein degradation system to spatially control protein stability was examined by using a plate-based assay (Figure 4H). Thus, the bidirectional expression module based tunable

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CONCLUSION During recent decades, several inducible systems with chemicals as the triggers have become available for controlling gene expression in yeast cells. However, as small chemicals diffuse freely and are difficult to remove, it is impossible to precisely control gene expression at an exact location and time. Compared to chemicals, light is an ideal inducer of gene expression as it is easy to obtain, highly tunable, nontoxic, and most importantly, has high spatiotemporal resolution. Several light-regulated gene expression systems based on multiple protein components have been developed in yeast cells and applied in a few synthetic biology studies.4−11 However, these systems suffer from a few drawbacks, including a low ON/OFF ratio,4,6,8,9,11 limited portability due to multiple protein components,4−11 and/or dependence on extraneous cofactors or appropriate cofactor synthesis genes.4−8 During our preparation of the manuscript, a light-sensitive transcription factor based on EL222 from Erythrobacter litoralis was developed to engineer metabolic pathways in yeast cells.32 Nevertheless, this system has a low ON/OFF ratio (43-fold) and activation efficiency (similar to ADH1 promoter), which may limit its usage in some specific applications, for example, large-scale fermentation to produce recombinant proteins. The yLightOn system developed in this study possesses the following advantages. First, the yLightOn system is very simple and compact, as it consists of only one protein component. This design will prove useful for synthetic biologists in the construction of complex yet interesting genetic circuits. Second, the light-switchable transactivator LVADO in the yLightOn system utilizes the endogenous FAD as its cofactor, not requiring the addition of exogenous cofactors or the introduction of cofactor-biosynthesis genes. Third, the yLightOn system exhibits extremely low leakage and high activation efficiency (similar to PMA1 strong promoter). Fourth, the induction characteristics of the yLightOn system can be finetuned by altering the promoter configurations. Fifth, both the LexA and VVD modules in LVADO are heterogeneous, which reduces the possibility of interaction with the host chassis. Gal4AD domain in LVADO is derived from yeast itself and may be affected by Gal80 activity.33 To test this possibility, we measured the yLightOn system in AH109 cells (whose Gal80 gene has been knocked out). Our results showed that the yLightOn system had a ON/OFF ratio of 251-fold in AH109 cells (Figure S10), slightly lower than 573-fold in BY4742 cells (containing Gal80 gene), indicating that the yLightOn system has good compatibility to different yeast strains without knockout of specific genes. Modularity is a concept that is widely used in biological science, especially in synthetic biology studies. In this study, the light-switchable transactivator LVADO was designed on the basis of a modular principle to contain a DNA-binding domain (LexA), a light-sensing and dimerization domain (VVD), and a transactivation domain (Gal4AD). Theoretically, changing any module in LVADO may lead to different induction characteristics. Our data showed that LVAD (Y50W M135I) exhibited extreme sensitivity to blue light, and even weak 33 mW·m−2 blue light could result in ∼100-fold activation of gene expression (Figure 2C), demonstrating that the LVADO can be further designed to obtain novel transcription factors with diverse induction characteristics. 2050

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cells were illuminated with the indicated blue-light pulse of varying durations before moving to dark conditions. mCherry fluorescence and OD600 were measured by a Synergy 2 multimode microplate reader (BioTek) (ex = 590/20 nm, em = 645/40 nm). The fluorescence was normalized to OD600 of each sample. To spatially control gene expression by the yLightOn system, the BY4742 cells transformed with pADH1(410)LVADO and pL8Gal1-mCherry were grown in darkness overnight. The cells were then mixed with SD medium containing 1.5% agar (ensuring that the temperature of the SD medium was lower than 50 °C and that the final OD600 concentration was approximately 0.2), immediately poured into a Petri dish with a 90 mm internal diameter, and allowed to harden at room temperature for 30 min. A photomask made of 0.2 mm thick transparency film was placed on the dish. The dish was incubated at 30 °C and illuminated from below with a blue LED array lamp. The blue light irradiation applied to the culture was approximately 2.5 W·m−2. After 36 h, the mCherry fluorescence image was acquired by a Kodak In-Vivo Multispectral System FX (Carestream Health) with a 600/20 nm excitation and a 670/50 nm emission filters. The mCherry expression of the cells on the plate was quantified by the ImageJ image processing program. Light-controlled protein stability was analyzed in BY4742 cells transformed with pADH1(410)-LVADO-ADH1mCherry-ssrA and pBi-ClpX-ClpP or pADH1-mCherry-psd. The quantitative and spatial control of the degradation of ssrAtagged proteins were analyzed by using similar procedures to those described above. Measurement of the degradation kinetics was performed in exponential growing culture. The overnight cultures were diluted to a density of OD600 ∼ 0.0001 and cultured under dark conditions for 4 h before transferring to light illumination. mCherry fluorescence at the indicated time points were determined by a CytoFLEX-S flow cytometer (Beckman Coulter). Optical Control of Cell Growth and Cell Cycle. BY4742 cells were transformed with pADH1(410)-LVADO and pL8Gal1-His3. TUB1/YML085C (Tub-GFP) cells were transformed with pADH1(410)-LVADO and pL8Gal1-ΔNSic1. The engineered cells were cultured in SD minimal medium at 30 °C overnight in dark conditions. Serial dilutions of overnight cells (1:10; first spot about 104 cells for HIS3 and 105 for ΔNSic1) were spotted on SD minimal medium plates and incubated for 2 days at 30 °C in the absence or presence of blue light (460 nm, 4 W·m−2). Images of clones on the plate were acquired using a Kodak In-Vivo Multispectral System FX (Carestream Health). Images of cell cycle stages were acquired using an S Plan Fluor ELWD 40×, 0.95 numerical aperture (NA) objective and a digital sight camera on an Eclipse Ti inverted microscope system (Nikon). Western Blot. Equal amounts of the total lysate protein (50 μg) were electrophoresed on 12% SDS-PAGE gel, and then transferred onto polyvinylidene fluoride (PVDF) membranes (PALL) using an electroblotter. After blocking with 0.5% casein, the membranes were probed with primary antibodies (Anti-HA-tag rabbit mAb (1:1000; Cell Signaling Technology), Anti-mCherry tag mouse mAb (1:1000; Abbkine), or Antiyeast beta Actin mouse mAb (1:1000; CMCTAG)). Subsequently, the cells were treated with the secondary antibodies (Antimouse lgG, HRP linked Antibody for Anti-mCherry and Antibeta Actin (1:2000; Cell Signaling Technology)). Immunoreactivity was detected using a BM

Overall, we have provided an example for the construction and systematic optimization of a novel optogenetic tool based on the homodimerization of the LOV domain in yeast cells. We have shown that the yLightOn system, as a new addition to the synthesis biology toolbox, is a robust and versatile tool for the rapid, reversible, quantitative, and spatiotemporal control of gene expression in yeast cells and will be a powerful and convenient tool for the study of cellular gene function and gene regulatory networks.

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METHODS AND MATERIALS Molecular Biology. Construction of the vectors is given in detail in Table S3. Cell Growth and Blue Light Irradiation. Escherichia coli TOP10 (Invitrogen) strain was used for cloning and was grown in LB medium. Plasmids were transformed into yeast cells using the lithium acetate method.34 All the experiments were carried out using yeast strain BY4742 (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0) or AH109 (MATα, ura3−52, his3− 200, ade2−101, trp1−901, leu2−3,112, gal4Δ, met‑, gal80Δ, URA3:: GAL1UAS-GAL1TATA-lacZ), TUB1/YML085C (TubGFP) (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0), or Y187 (MATα, ura3--52, his3--200, trp1--901, leu2--3,112, gal4Δ, met-, gal80Δ, LYS2::GAL1UAS--GAL1TATA--HIS3, GAL2UAS-GAL2TATA--ADE2, URA3::MEL1UAS--MEL1TATA--lacZ). Yeast cells were cultured in yeast extract Peptone Dextrose Adenine (YPDA)-rich medium or in appropriate synthetic dextrose (SD) media at 30 °C. For detection of lightregulated gene expression, cells were cultured in a 48-well plate with 1 mL of medium in a shaking incubator at 300 rpm. The cells were illuminated by 4 W·m−2 blue light emitting from an LED lamp (460 nm peak) or remained in the dark for 24 h before characterization. Neutral density filters were used to adjust the light intensity. Light intensities were measured with a Luminometer (Sanwa, LX-2). A red (620−630 nm) LED lamp was used for dark manipulations. To test the ON kinetics of the light-inducible systems in batch culture, the BY4742 cells were cotransformed with pADH1(410)-LVADO and pL8Gal1-mCherry, or with pL8Gal1-mCherry and pADH1(410)-LVAD (Y50W M135I). The AH109 cells were cotransformed with pU5Gal1-mCherry, pGal4BD-CRY2 and pGal4AD-CIB1. The above engineered cells were first grown in darkness overnight. The overnight cultures were then diluted to a density of OD600 ∼ 0.01 and transferred to blue light illumination. The aliquots were taken at the indicated time points and protein synthesis was stopped by the addition of 1 mM cycloheximide (chx). To detect the OFF kinetics of the light-inducible systems in exponential growing culture, the above engineered cells were first cultured in dark conditions overnight. The overnight cultures were diluted to a density of OD600 ∼ 0.0001 and cultured under light illumination for 4 h before transferring to dark conditions. The aliquots were taken at the indicated time points and protein synthesis was stopped by the addition of chx as described above. To detect the OFF kinetics using a pulse experiment, the engineered cells were treated with the indicated durations of dark conditions, and the mCherry fluorescence was measured at 6 h. To allow mCherry maturation, the aliquots were incubated in dark conditions for 2 h prior to fluorescence measurements. The mCherry fluorescence was analyzed by a CytoFLEX-S flow cytometer (Beckman Coulter). To detect light-induced gene expression by a single blue-light pulse of varying durations, the engineered 2051

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Chemiluminescence blotting kit (Roche Diagnostics) according to the manufacturer’s protocol on a Kodak In-Vivo Multispectral System X (Carestream Health). Imaging and Flow Cytometry. For fluorescence imaging of mCherry, images were acquired using an S Plan Fluor ELWD 40 × , 0.95 numerical aperture (NA) objective and a digital sight camera on an Eclipse Ti inverted microscope system (Nikon), using a Texas Red filter. For fluorescence imaging of Tub-GFP, images were acquired using a Leica SP8 confocal laser scanning microscope equipped with HCXPL APO 63.0 × 1.47 OIL objective and a HyD detector, using an excitation of 488 nm and an emission of 495−595 nm. For analyzing the fluorescence of mCherry by flow cytometry, FACS was performed with a CytoFLEX-S flow cytometer (Beckman Coulter), using an excitation of 561/10 nm and an emission of 610/20 nm for mCherry. Processing and analysis of the data were performed using the Cytexpert program (Beckman Coulter).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.8b00180. Figures S1 to S10, Tables S1 to S3, and Supplementary

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Letter

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Xianjun Chen: 0000-0002-8475-332X Author Contributions

X.J.C. and Y.Y. designed the experiment. X.P.X., Z.X.D., R.M.L., and T.L. performed the experiments. X.J.C., Y.Y., X.P.X., and Y.Z.Z. analyzed the data. X.P.X., X.J.C., and Y.Y. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Miaowei Mao, Zhengda Chen, Xie Li, Zengmin Du, Fangting Zuo and Ni Su for technical assistance. We thank Prof. Christof Taxis at Philipps-Universität Marburg for providing sequence information on psd construct. We thank Prof. David Botstein at Princeton University for providing sequence information on GEV constructs. We thank Prof. Junbiao Dai at the Center for Synthetic Biology Engineering Research for providing TUB1/YML085C yeast strain. This research was supported by the National Key Research and Development Program of China (2017YFA050400 to Y.Y.), NSFC (31225008 and 31470833 to Y.Y., 31600688 to X.C.), the Shanghai Science and Technology Commission (18JC1411900, 14XD1401400, and 16430723100 to Y.Y.), China Postdoctoral Science Foundation (2016M59027 and 2017T00277 to X.C.), the State Key Laboratory of Bioreactor Engineering to Y.Y., the Fundamental Research Funds for the Central Universities to Y.Y. and X.C. 2052

DOI: 10.1021/acssynbio.8b00180 ACS Synth. Biol. 2018, 7, 2045−2053

Letter

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DOI: 10.1021/acssynbio.8b00180 ACS Synth. Biol. 2018, 7, 2045−2053