A Single-Component Optogenetic System Allows Stringent Switch of

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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., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00180 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018

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A Single-Component Optogenetic System Allows Stringent Switch of Gene

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Expression in Yeast Cells

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Xiaopei Xu1,2, Zhaoxia Du1,2, Renmei Liu1,2, Ting Li1,2, Yuzheng Zhao1,2,3, Xianjun Chen1,2,3,*, Yi Yang1,2,*

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Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology,

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East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China.

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Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of

State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science, Shanghai

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and Technology, 130 Mei Long Road, Shanghai 200237, China.

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* Correspondence and requests for materials should be addressed to X.C.([email protected])

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and Y.Y. ([email protected])

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science

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ABSTRACT

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Light is a highly attractive actuator that allows spatiotemporal control of diverse cellular activities. In

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this study, we developed a single-component light-switchable gene expression system for yeast cells,

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termed yLightOn system. yLightOn system is independent of exogenous cofactors, and exhibits more

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than a 500-fold ON/OFF ratio, extremely low leakage, fast expression kinetics and high spatial

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resolution. We demonstrated the usefulness of yLightOn system in regulating cell growth and cell

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cycle by stringently controlling the expression of His3 and

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we engineered a bidirectional expression module that allows the simultaneous control of the

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expression of two genes by light. With ClpX and ClpP as the reporters, the fast, quantitative and

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spatially-specific degradation of ssrA-tagged protein was observed. We suggest that this

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single-component optogenetic system will be immensely helpful in understanding cellular gene

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regulatory networks and in the design of robust genetic circuits for synthetic biology.

ΔN

Sic1 genes, respectively. Furthermore,

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KEYWORDS: yeast; optogenetic; gene expression; protein stability.

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Optogenetics has emerged as a powerful strategy to control diverse biological systems with

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millisecond and submicron resolutions 1. Single-component optogenetics is a standard research tool

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that displays speed, simplicity and versatility and was first applied in neuroscience research to allow

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the optical control of neurons on a millisecond time scale 2. Recently, the discovery and engineering

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of new single-component optogenetic tools have shed light on diverse biological fields not limited to

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neuroscience, as with the emergence of new photoreceptors. In particular, the LOV domains of

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several photoreceptors possess several advantages, including its small size, which avoids steric

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hindrance and facilitates accurate molecular design, and its endogenous cofactor (FAD or FMN),

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which avoids addition of an exogenous cofactor or introduction of cofactor-biosynthesis genes. These

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favorable characteristics enable the LOV domains to be ideal candidates for developing novel

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optogenetic tools 3.

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Yeast serving as a model for all eukaryotes has been widely applied in the study of fundamental

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cellular processes. Precise spatiotemporal control of gene expression is an indispensable tool for

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characterization of these complex cellular processes. Several light-regulated gene expression systems

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based on multiple protein components have been reported in yeast cells

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usefulness of these systems has been minimal, probably due to technical complexities or limitations.

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We previously reported simple yet robust single-component light-switchable gene expression

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systems for mammalian cells and bacteria 12-14. Both systems consist of a single genetically encoded

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photosensitive transcription factor GAVPO or LEVI, in which the LOV domain Vivid (VVD) derived

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from the photoreceptor of Neurospora crassa rapidly forms a homodimer in response to blue light

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and promotes the binding of GAVPO or LEVI to specific DNA sequences to activate or repress gene

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transcription, respectively. In the present study, we sought to develop a compact yet robust

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light-switchable gene expression system for yeast cells based on a similar design.

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. However, the

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In this study, we developed a light-switchable gene expression system termed yLightOn for yeast

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cells. The yLightOn system consists of a single-component light-switchable transcription factor and

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shows more than 500-fold induction ratio, extremely low leakage, quantitative and spatiotemporal

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control of gene expression. We showed the usefulness of yLightOn system in controlling cell growth

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and cell cycle. Furthermore, we have also engineered a bidirectional expression module, allowing the

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simultaneous control of the expression of two genes by light. When using ClpX and ClpP as the

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reporters, we observed the fast, quantitative and spatially-specific degradation of ssrA-tagged

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proteins. The newly developed light-switchable gene expression system will be a powerful and

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convenient tool for the study of cellular gene function and gene regulatory networks and for the

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design of interesting synthetic circuits.

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RESULTS AND DISCUSSION

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Design, optimization and validation of the single-component light-switchable gene expression

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system

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We have previously created light-switchable gene expression systems based on single-component

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light sensors for mammalian cells and bacteria

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contain a DNA-binding domain (Gal4 or LexA) and a light sensing and responding domain (VVD).

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These two domains constitute a light-switchable DNA-binding fusion protein that dimerizes and

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binds to DNA sequences to directly repress gene transcription 12 or activate gene transcription by a

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fused transcriptional activation domain upon blue light exposure 13. We hypothesized that fusing the

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light-switchable DNA-binding fusion protein to a yeast transcription activation domain would create

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a light-switchable transcription factor for yeast cells, as light should induce dimerization of the fusion

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protein and binding to the upstream activation sequence (UAS) to activate transcription (Figure 1A).

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LexA protein is a repressor of the Escherichia coli SOS regulon which is orthogonal to the cellular

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components of yeast cells 15, hence reducing the possibility of cross-talk between the transactivator

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and the host chassis. We therefore fused LexA-VVD (LEVI) to the activation domain of the Gal4

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protein (Gal4AD) to obtain LEVI-Gal4AD (LVAD). We constructed a response plasmid containing the

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gene encoding mCherry fluorescent protein under the control of eight copies of the LexA-binding

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sequence and a Gal1 minimal promoter (Figure 1B, Supplementary Note). We transformed the

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. Both the light-switchable transcription factors

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response plasmid and the activator plasmid expressing LVAD driven by different promoters into

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BY4742 cells. The engineered cells exhibited distinct light-switchable mCherry expression (Figure 1C).

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Among these promoters, the truncated ADH1 promoter ADH1(410) exhibited a 23-fold ON/OFF ratio

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and ~50% activation efficiency compared to the commonly used strong constitutive promoter PMA1

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(Figure 1C).

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Owing to LVAD’s modular design, it is possible to enhance the performance of the

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light-switchable system (leakage, photosensitivity, etc.) by introducing mutations in the VVD motif.

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Previous studies showed that the variants I74V and I85V increases recovery of the photoadduct by

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~25-fold and ~23-fold relative to native VVD, respectively, whereas the double variant M135I and

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M165I decreases the recovery by ~10-fold 16. The variant Y50W forms a stable light-state cysteinyl

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adduct to promote dimerization 17,18. To this end, we carried out combinatorial mutagenesis of these

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VVD variants with varied photoadduct decays (Figure 1D). Most of the LVAD mutants exhibited

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insignificant differences in mCherry expression between light and dark conditions, with ON/OFF

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ratios lower than 5-fold (Figure S1). However, VVD mutants with the single mutation Y50W or the

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double mutations Y50W/I85V, I85V/M135I or Y50W/M135I displayed ON/OFF ratios of more than

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40-fold. In particular, VVD mutant with the triple mutations Y50W/I85V/M135I (optimized LVAD

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(LVADO)) showed a 573-fold ON/OFF ratio, which was significantly higher than the typical blue-light-

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inducible CRY2/CIB1 system (31-fold)

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CUP1(Cu2+) , 297-fold for Gal1(Galactose), 106-fold for GEV(β-estradiol))(Figure S2)20-22. LVADO also

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exhibited high activation efficiency comparable to the strong constitutive PMA1 promoter (Figure S2

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and S3A). Notably, the leakage of LVADO in dark conditions was almost the same as in the control

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cells harboring only the response plasmid (Figure S3B), demonstrating the stringent control of gene

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expression. As both the activator and reporter plasmids contain 2 μ replication origin, which may

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lead to varied number of the expression components. To better characterize the light-induced

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expression by LVADO, we transferred the PADH(410)-LVADO and 8xLexAop-PGal1mini-mCherry expressing

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cassettes to pRS315 and pRS316, respectively, which have been validated to harbor single copy in

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and the chemical-triggered systems (147-fold for

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yeast cells 23,24. Our results showed that the single copy plasmids had a 188-fold ON/OFF ratio, with

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1.3-fold lower background expression and 3.9-fold lower activation efficiency compared to the 2 μ

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plasmid (Figure S4A). Notably, we observed good homogeneity for the cells harboring single copy

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plasmids kept in dark conditions, whereas a small number of the cells harboring 2 μ plasmids

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exhibited significant leak expression(Figure S4B and 4C), probably due to much higher copy number

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of the expression components in these cells. We used LVADO in all subsequent studies, and we

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referred to the light-switchable gene expression system based on LVADO as the yeast light-on

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(yLightOn) system. Unless otherwise indicated, yLightOn system in 2 μ plasmids were used for

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subsequent studies.

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To determine the promoter configurations to fine-tune the induction characteristics of yLightOn

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system, we first replaced the Gal1 minimal promoter with other minimal promoters. A multitude of

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regulatory systems with highly diverse levels of background noise and maximal activation were

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observed (Figure 1E). We next varied the number of LexAop repeats and the spacer lengths separating

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LexAop from the Gal1 minimal promoter. Our results showed that mCherry expression levels

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decreased as the spacer length increased or the number of LexAop repeats decreased (Figure 1F and

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1G). Notably, significant activation of mCherry expression occurred even with a 500-bp spacer

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(Figure 1F), providing great potential to construct chimeric promoters that could respond to multiple

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input signals

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system can be fine-tuned by altering the promoter configurations, providing flexible options for

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specific experimental conditions.

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. Taken together, these results demonstrate that the performance of the yLightOn

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Characterization of the yLightOn system

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We first investigated the ON kinetics of yLightOn system in both 2 μ plasmids and single copy

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plasmids. Our data showed that the mCherry expression level took approximately 6 h to switch from

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OFF to fully ON state. The t1/2 (the time to reach 50% of maximal expression) was approximately 1.8 h

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for yLightOn system both in 2 μ plasmids and in single copy plasmids (Figure 2A). The ON kinetics of

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yLightOn system was faster than the LVAD (Y50W/M135I) (t1/2~2.6 h) and CRY2/CIB1 (4.7 h) (Figure

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2A), but might be slower compared to the EL222 system (t1/2