Subscriber access provided by UNIV OF DURHAM
Letter
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 26 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
ACS Synthetic Biology
1
A Single-Component Optogenetic System Allows Stringent Switch of Gene
2
Expression in Yeast Cells
3
Xiaopei Xu1,2, Zhaoxia Du1,2, Renmei Liu1,2, Ting Li1,2, Yuzheng Zhao1,2,3, Xianjun Chen1,2,3,*, Yi Yang1,2,*
4 5
1
6
Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology,
7
East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China.
8
2
9
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
10
3
11
and Technology, 130 Mei Long Road, Shanghai 200237, China.
12
* Correspondence and requests for materials should be addressed to X.C.(
[email protected])
13
and Y.Y. (
[email protected])
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science
14 15 16 17 18 19 20 21 22 23 24 25 26 27
ACS Paragon Plus Environment
ACS Synthetic Biology 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
Page 2 of 26
1
ABSTRACT
2
Light is a highly attractive actuator that allows spatiotemporal control of diverse cellular activities. In
3
this study, we developed a single-component light-switchable gene expression system for yeast cells,
4
termed yLightOn system. yLightOn system is independent of exogenous cofactors, and exhibits more
5
than a 500-fold ON/OFF ratio, extremely low leakage, fast expression kinetics and high spatial
6
resolution. We demonstrated the usefulness of yLightOn system in regulating cell growth and cell
7
cycle by stringently controlling the expression of His3 and
8
we engineered a bidirectional expression module that allows the simultaneous control of the
9
expression of two genes by light. With ClpX and ClpP as the reporters, the fast, quantitative and
10
spatially-specific degradation of ssrA-tagged protein was observed. We suggest that this
11
single-component optogenetic system will be immensely helpful in understanding cellular gene
12
regulatory networks and in the design of robust genetic circuits for synthetic biology.
ΔN
Sic1 genes, respectively. Furthermore,
13 14
KEYWORDS: yeast; optogenetic; gene expression; protein stability.
15 16 17 18 19 20 21 22 23 24 25 26
ACS Paragon Plus Environment
Page 3 of 26 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
ACS Synthetic Biology
1
Optogenetics has emerged as a powerful strategy to control diverse biological systems with
2
millisecond and submicron resolutions 1. Single-component optogenetics is a standard research tool
3
that displays speed, simplicity and versatility and was first applied in neuroscience research to allow
4
the optical control of neurons on a millisecond time scale 2. Recently, the discovery and engineering
5
of new single-component optogenetic tools have shed light on diverse biological fields not limited to
6
neuroscience, as with the emergence of new photoreceptors. In particular, the LOV domains of
7
several photoreceptors possess several advantages, including its small size, which avoids steric
8
hindrance and facilitates accurate molecular design, and its endogenous cofactor (FAD or FMN),
9
which avoids addition of an exogenous cofactor or introduction of cofactor-biosynthesis genes. These
10
favorable characteristics enable the LOV domains to be ideal candidates for developing novel
11
optogenetic tools 3.
12
Yeast serving as a model for all eukaryotes has been widely applied in the study of fundamental
13
cellular processes. Precise spatiotemporal control of gene expression is an indispensable tool for
14
characterization of these complex cellular processes. Several light-regulated gene expression systems
15
based on multiple protein components have been reported in yeast cells
16
usefulness of these systems has been minimal, probably due to technical complexities or limitations.
17
We previously reported simple yet robust single-component light-switchable gene expression
18
systems for mammalian cells and bacteria 12-14. Both systems consist of a single genetically encoded
19
photosensitive transcription factor GAVPO or LEVI, in which the LOV domain Vivid (VVD) derived
20
from the photoreceptor of Neurospora crassa rapidly forms a homodimer in response to blue light
21
and promotes the binding of GAVPO or LEVI to specific DNA sequences to activate or repress gene
22
transcription, respectively. In the present study, we sought to develop a compact yet robust
23
light-switchable gene expression system for yeast cells based on a similar design.
4-11
. However, the
24
In this study, we developed a light-switchable gene expression system termed yLightOn for yeast
25
cells. The yLightOn system consists of a single-component light-switchable transcription factor and
26
shows more than 500-fold induction ratio, extremely low leakage, quantitative and spatiotemporal
ACS Paragon Plus Environment
ACS Synthetic Biology 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
1
control of gene expression. We showed the usefulness of yLightOn system in controlling cell growth
2
and cell cycle. Furthermore, we have also engineered a bidirectional expression module, allowing the
3
simultaneous control of the expression of two genes by light. When using ClpX and ClpP as the
4
reporters, we observed the fast, quantitative and spatially-specific degradation of ssrA-tagged
5
proteins. The newly developed light-switchable gene expression system will be a powerful and
6
convenient tool for the study of cellular gene function and gene regulatory networks and for the
7
design of interesting synthetic circuits.
8 9
RESULTS AND DISCUSSION
10
Design, optimization and validation of the single-component light-switchable gene expression
11
system
12
We have previously created light-switchable gene expression systems based on single-component
13
light sensors for mammalian cells and bacteria
14
contain a DNA-binding domain (Gal4 or LexA) and a light sensing and responding domain (VVD).
15
These two domains constitute a light-switchable DNA-binding fusion protein that dimerizes and
16
binds to DNA sequences to directly repress gene transcription 12 or activate gene transcription by a
17
fused transcriptional activation domain upon blue light exposure 13. We hypothesized that fusing the
18
light-switchable DNA-binding fusion protein to a yeast transcription activation domain would create
19
a light-switchable transcription factor for yeast cells, as light should induce dimerization of the fusion
20
protein and binding to the upstream activation sequence (UAS) to activate transcription (Figure 1A).
21
LexA protein is a repressor of the Escherichia coli SOS regulon which is orthogonal to the cellular
22
components of yeast cells 15, hence reducing the possibility of cross-talk between the transactivator
23
and the host chassis. We therefore fused LexA-VVD (LEVI) to the activation domain of the Gal4
24
protein (Gal4AD) to obtain LEVI-Gal4AD (LVAD). We constructed a response plasmid containing the
25
gene encoding mCherry fluorescent protein under the control of eight copies of the LexA-binding
26
sequence and a Gal1 minimal promoter (Figure 1B, Supplementary Note). We transformed the
12,13
. Both the light-switchable transcription factors
ACS Paragon Plus Environment
Page 4 of 26
Page 5 of 26 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
ACS Synthetic Biology
1
response plasmid and the activator plasmid expressing LVAD driven by different promoters into
2
BY4742 cells. The engineered cells exhibited distinct light-switchable mCherry expression (Figure 1C).
3
Among these promoters, the truncated ADH1 promoter ADH1(410) exhibited a 23-fold ON/OFF ratio
4
and ~50% activation efficiency compared to the commonly used strong constitutive promoter PMA1
5
(Figure 1C).
6
Owing to LVAD’s modular design, it is possible to enhance the performance of the
7
light-switchable system (leakage, photosensitivity, etc.) by introducing mutations in the VVD motif.
8
Previous studies showed that the variants I74V and I85V increases recovery of the photoadduct by
9
~25-fold and ~23-fold relative to native VVD, respectively, whereas the double variant M135I and
10
M165I decreases the recovery by ~10-fold 16. The variant Y50W forms a stable light-state cysteinyl
11
adduct to promote dimerization 17,18. To this end, we carried out combinatorial mutagenesis of these
12
VVD variants with varied photoadduct decays (Figure 1D). Most of the LVAD mutants exhibited
13
insignificant differences in mCherry expression between light and dark conditions, with ON/OFF
14
ratios lower than 5-fold (Figure S1). However, VVD mutants with the single mutation Y50W or the
15
double mutations Y50W/I85V, I85V/M135I or Y50W/M135I displayed ON/OFF ratios of more than
16
40-fold. In particular, VVD mutant with the triple mutations Y50W/I85V/M135I (optimized LVAD
17
(LVADO)) showed a 573-fold ON/OFF ratio, which was significantly higher than the typical blue-light-
18
inducible CRY2/CIB1 system (31-fold)
19
CUP1(Cu2+) , 297-fold for Gal1(Galactose), 106-fold for GEV(β-estradiol))(Figure S2)20-22. LVADO also
20
exhibited high activation efficiency comparable to the strong constitutive PMA1 promoter (Figure S2
21
and S3A). Notably, the leakage of LVADO in dark conditions was almost the same as in the control
22
cells harboring only the response plasmid (Figure S3B), demonstrating the stringent control of gene
23
expression. As both the activator and reporter plasmids contain 2 μ replication origin, which may
24
lead to varied number of the expression components. To better characterize the light-induced
25
expression by LVADO, we transferred the PADH(410)-LVADO and 8xLexAop-PGal1mini-mCherry expressing
26
cassettes to pRS315 and pRS316, respectively, which have been validated to harbor single copy in
4,19
and the chemical-triggered systems (147-fold for
ACS Paragon Plus Environment
ACS Synthetic Biology 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
1
yeast cells 23,24. Our results showed that the single copy plasmids had a 188-fold ON/OFF ratio, with
2
1.3-fold lower background expression and 3.9-fold lower activation efficiency compared to the 2 μ
3
plasmid (Figure S4A). Notably, we observed good homogeneity for the cells harboring single copy
4
plasmids kept in dark conditions, whereas a small number of the cells harboring 2 μ plasmids
5
exhibited significant leak expression(Figure S4B and 4C), probably due to much higher copy number
6
of the expression components in these cells. We used LVADO in all subsequent studies, and we
7
referred to the light-switchable gene expression system based on LVADO as the yeast light-on
8
(yLightOn) system. Unless otherwise indicated, yLightOn system in 2 μ plasmids were used for
9
subsequent studies.
10
To determine the promoter configurations to fine-tune the induction characteristics of yLightOn
11
system, we first replaced the Gal1 minimal promoter with other minimal promoters. A multitude of
12
regulatory systems with highly diverse levels of background noise and maximal activation were
13
observed (Figure 1E). We next varied the number of LexAop repeats and the spacer lengths separating
14
LexAop from the Gal1 minimal promoter. Our results showed that mCherry expression levels
15
decreased as the spacer length increased or the number of LexAop repeats decreased (Figure 1F and
16
1G). Notably, significant activation of mCherry expression occurred even with a 500-bp spacer
17
(Figure 1F), providing great potential to construct chimeric promoters that could respond to multiple
18
input signals
19
system can be fine-tuned by altering the promoter configurations, providing flexible options for
20
specific experimental conditions.
14
. Taken together, these results demonstrate that the performance of the yLightOn
21 22
Characterization of the yLightOn system
23
We first investigated the ON kinetics of yLightOn system in both 2 μ plasmids and single copy
24
plasmids. Our data showed that the mCherry expression level took approximately 6 h to switch from
25
OFF to fully ON state. The t1/2 (the time to reach 50% of maximal expression) was approximately 1.8 h
26
for yLightOn system both in 2 μ plasmids and in single copy plasmids (Figure 2A). The ON kinetics of
ACS Paragon Plus Environment
Page 6 of 26
Page 7 of 26 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
ACS Synthetic Biology
1
yLightOn system was faster than the LVAD (Y50W/M135I) (t1/2~2.6 h) and CRY2/CIB1 (4.7 h) (Figure
2
2A), but might be slower compared to the EL222 system (t1/2
