3rd-year module on Synthetic Biology

LF306

Lecture 6

Engineering Signalling Pathways

John McCarthy
Why engineer signalling pathways?

•  In order to enhance understanding of naturally evolved pathways or to build new versions

that can be applied in various ways in fundamental research, medicine, biotechnology etc.

•  Most processes that synthetic biologists want to harness occur naturally in cells.
•  Cells have evolved regulatory mechanisms to control these processes.
•  These often take the form of signalling pathways (responding to extracellular stimuli).

•  The more complex an organism, the more complex the signalling pathways.
•  More difficult to develop fully synthetic pathways in complex organisms.
•  Often easier to modify the natural signalling pathway than to create a completely new one.
Engineering the specificity of a signalling pathway
(microbial examples)

-  this helps us:

•  understand molecular mechanisms
•  build novel devices, e.g. biosensors

-  let’s look at three examples:

1. chemoreceptor specificity in E.coli
2. GPCR-based chemical biosensors in S.cerevisiae
3. yeast GPCR-based pathogen detection
1. Engineering chemoreceptor specificity in E.coli Microfluidic system*
via structure-based rational design

The objective in this study was to engineer GFP-labelled cells loaded

the Tar chemoreceptor (periplasmic domain) into central hole; potential
in order to enable it to recognize non-native attractants into peripheral
ligands. Tar naturally responds best to L-Asp. holes:

Tar exists as a homodimer – each monomer

contains a 4-helix bundle (α1-α4); α1 and α4 Asp
span the membrane. Asp

Piston model: binding of an attractant§ induces Tar

a piston-like sliding movement of α4 relative to
α1 in one monomer that can be transduced to
the cytoplasm.

Chemical compounds were screened for their

CHDCA cis-PDA
ability to act as chemoeffectors. A microfluidic
system* was used to study the responses of E.coli
cells to different molecules. Some molecules
(e.g. cis-1,2-cyclohexane-dicarboxylic acid; CHDCA)
can bind without acting as an attractant. In the case
of CHDCA, this can be converted to an attractant§
by substituting a piperidine ring for the cyclohexane
ring, which results in cis-(2R,3S)-2.3-piperidine
dicarboxylic acid (cis-PDA).
Bi et al Proc Natl Acad Sci USA 110 (2013) 16814-16819
 1. Engineering chemoreceptor specificity in E.coli
via structure-based rational design

Rational design of Tar to enable a chemotactic response to Arg

Arg is not a natural ligand of wt Tar. R73E

The binding site of Tar was engineered
via rational design. R69E and R73E
modifications enabled strong Arg
binding. Once bound, Arg could interact
with the wt residues Y149 and Q152 in
helix α4, thus triggering downstream sign-
alling. Observation of the distribution of
GFP-labelled cells across an Arg grad-
Ient reveals its acquired ability to act as R69E
a chemoattractant.

Bi et al Proc Natl Acad Sci USA 110 (2013) 16814-16819

2. Engineering novel chemical biosensor systems in yeast

In biotech applications, there is a frequent

need to optimise the production of useful
biosynthetic products by microorganisms.
HTP optimisation requires simple assays,
but how can this be achieved for chemicals
that are not calorimetrically detectable?

One useful approach is to engineer novel

biosensors – in this case, mammalian G-
protein coupled receptors (GPCRs) were
integrated into a synthetic derivative of the
yeast pheromone signalling pathway.

GPCRs are extracellular, and a number of heterologous ones can be synthesized in yeast in an active
form that communicates with the yeast pheromone sensing system. Signals from the GPCR sensing
unit can be transferred to this pathway, which processes the input to provide a signal response, i.e. in
the form of phosphorylation of a synthetic transcription factor (STF) that, in turn, activates a synthetic
promoter that drives expression of a reporter gene. In this study, different mammalian GPCRs that
bind to medium-chain (C8-C12) fatty acids were used. The resulting yeast sensor strains are then
placed in mixed cultures in HTP screens of the productivity of bacterial producer strains. Further
details are found in the publication cited below.

Mukherjee et al (2014) ACS Synthetic Biol DOI: 10.1021/sb500365m

 3. A yeast fungal pathogen biosensor
Lycopene colour assay
on a dipstick
S.cerevisiae mating receptor (Ste2)
can be replaced by equivalent trans-
membrane receptors from pathogenic
fungi, e.g. C. albicans, P. brasiliensis.

Sensitivity and specificity can be

optimised by adjusting promoter
strengths and engineering receptor
binding interfaces, respectively.

Activation via C.a. Ste2 Peptide specificity

Mating peptide recognition at the cell
surface activates biosynthesis of red
lycopene. Spots of the biosensor
strain can be deposited on a simple
filter-paper dipstick to make a low-
cost pathogen biosensor.

Ostrov et al, 2017; DOI: 10.1126/sciadv.1603221

Mammalian signalling pathways

-  more opportunities for bioengineering

-  but more complex systems
Eukaryotic (particularly higher) signalling pathways, are more complex

Prokaryotic: pyruvate-sensing network Eukaryotic: cell migration

Figure reproduced from DOI: 10.1098/rsob.180023 Figure from https://www.sinobiological.com/

pathways/actin-dynamics-signaling-pathway

The design and construction of fully synthetic eukaryotic pathways is therefore challenging.
 G protein-coupled receptor (GPCR) signalling pathways

Reviews: Liccardo et al (2022) Cells; doi: 10.3390/cells11030528

Kiel et al (2010) Cell; doi: 10.1016/j.cell.2009.12.028

•  800 GPCRs encoded in the human genome (400 dedicated to olfaction).

•  G protein Subunits: 15 a (s, i, q and 12 families), 5 b and 7 g.
•  Receptors classified by which a subunit family they activate.
Mammalian signalling cascades activated by GPCRs

Covered on previous slide

Different
Gα families

Signalling
cascades,
e.g., second
messenger

Figure modified from DOI: 10.1074/jbc.REV119.005601

Cross-talk between mammalian signalling pathways

Covered on previous slides *Hedger et al (2019)

doi: 10.1038/srep09198
*

Cross-talk:
Hur et al (2002)
Cellular Signalling 14, 397-345

Figure from https://www.creative-diagnostics.com/gpcr-pathway.htm

Physiological importance of GPCR pathways

GPCR signalling pathways are found in most eukaryotes

•  Except plants

Regulate powerful physiological responses

•  Yeast: mating pheromone response.
•  Mammals: flight or fight response.

Most important target for the pharmaceutical industry

•  30-50% of all prescription drugs target GPCRs.
•  For example, β-blockers to treat high blood pressure.

Target for many illicit drugs

•  Cannabis (cannabinoid receptor); heroin (opioid receptor); LSD (serotonin receptor).
 Engineering: orthogonal ligand / receptor pair
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)

First developed for human M3 muscarinic receptor

•  Expressed in parasympathetic nervous system.
•  Activated by the neurotransmitter acetylcholine (ACh).
ACh
Subsequently applied to several other GPCRs

Mutate receptor, so that it is: ACh CNO

•  Not activated by the native ligand (ACh). X
•  Activated by a synthetic ligand (CNO).
•  M3 DREADD: Y149C and A239G mutations.

Synthetic ligand
•  Clozapine-N-oxide (CNO).
•  Pharmacologically inert.
M3 receptor
Orthogonality
•  ACh regulates multiple receptors / processes.
•  DREADD allows stimulation of an individual GPCR pathway.
•  Study physiological role of specific pathways. CNO

Armbruster et al, 2007; DOI: 10.1073/pnas.0700293104

Engineering: orthogonal ligand / receptor pairs

DREADDs are a widely used research tool, particularly in neurobiology

In vivo (animal models)

•  Viral transduction
•  Cell-specific promoters
•  Expression of DREADDs in specific cell types

DREADDs / CNO have been used to:

•  Regulate neurones responsible for food intake
•  Regulate neurones responsible for sleep and circadian rhythms

Potential future applications in translational medicine

e.g. treat depression-related disorders in humans:
•  Express DREADD in serotonergic neurons
•  Use CNO to activate neuronal circuits implicated in depression
•  Reduced side effects compared with antidepressants
Summary of key points so far

Eukaryotic signalling pathways are complex, particularly higher eukaryotic ones

- this limits our ability to build synthetic versions

However, naturally evolved systems can be re-engineered

- allowing us to harness valuable eukaryotic cellular processes and behaviours

Different properties of a pathway can be utilised in different applications

- what property of the pathway do you want to utilise and what do you want to modify?
- DREADDs utilise a cellular response, but modify the ligand / receptor that regulates it
- engineered biosensor utilise natural diversity of receptors, but modify the cell response
Refactoring more complex systems

Refactoring means redesigning/restructuring a system in a way that maintains that

system’s functionality while simplifying it to facilitate understanding and modification

GPCR signalling pathways, especially mammalian ones, tend to be too complex

for precise modelling. Therefore, where possible, simplification is an attractive option.
 Kinetic modelling

•  Basic synthetic systems can often be Cubic ternary complex model

described by simple binding equations. Weiss et al, 1996; DOI: 10.1006/jtbi.1996.0014

•  More complex systems require more

advanced mathematical models.

•  GPCR pathways have been progressively

modelled since their discovery.

•  GPCR models have become more complex

to reflect experimental observations.

Equilibrium constants
•  However, they are still too simplistic to
Interaction states
reflect the complexity of signalling in vivo.
Model components
A = agonist
R = receptor (inactive)
R* = receptor (active)
G = G protein
Kinetic modelling

Models generally describe only signal transduction

Figure modified from DOI: 10.1074/jbc.REV119.005601

Development of a minimised GPCR pathway
- to overcome the challenges of quantitatively modelling GPCR signalling

•  Developed a minimised tuneable GPCR pathway

•  Genome engineering of the mating pheromone response pathway in yeast

•  Construct an experimental system that behaves like a mathematical model

•  Experimentally validate quantitative predictions from mathematical modelling

Shaw et al, 2019; doi: 10.1016/j.cell.2019.02.023

Development of a minimised GPCR pathway

A B
X
X
X
X
X X

X X

A. Deleted 11/15 genes from the yeast mating and glucose-sensing pathways.
•  Only core MAPK relay elements retained.

B. Minimal set of components reintroduced into a refactored pathway.

•  Refactored = Insulated pathway and tuneable components.
 Development of a minimised GPCR pathway

Expression levels of components varied in model and experimental system

•  Results from mathematical and experimental model were in close agreement

Implications
•  Computational optimisation of the system for different applications (biosensors).
•  Refactoring approach could be applied to more complex organisms.
Summary of key points regarding refactored pathways

Complex signalling pathways can be mathematically modelled

•  Improve our theoretical understanding of signal transduction
•  However, it is challenging to apply such models to native cell signalling

A minimised yeast GPCR pathway developed that is more readily modelled

•  Enables quantitative predictions from kinetic modelling to be experimentally tested
•  Modelling can then be to be used to optimise the system for different applications

Synthetic switches can be used to bypass parts of a signalling pathway

•  Simplify how pathways with multiple inputs are regulated
•  Change the way the pathway is regulated (e.g. use light instead of chemical ligands)
Signalling pathway engineering does not need to focus only
on GPCR-based systems
 Engineering other pathways: cell migration

Aim: regulate mammalian cell

migration using light.

Engineering at the receptor level

difficult due to multiple inputs.

Bypass upstream signal

transduction and relay components.

Intervene at central part of pathway

where signalling starts to converge.

Develop a light-regulated rac1

protein to control the pathway.

•  Wu et al, 2009; doi:10.1038/nature08241

 Engineering other pathways: cell migration

Aim: regulate mammalian cell

migration using light.

Engineering at the receptor level

difficult due to multiple inputs.

Bypass upstream signal

transduction and relay components.

Intervene at central part of

pathway where signalling starts to
converge.

Develop a light-regulated rac1

protein to control the pathway.

•  Wu et al, 2009; doi:10.1038/nature08241

Engineering a mammalian photo-
regulatable motility control system Irradiation makes Rac1 accessible to PAK

Rac1 is a GTPase involved in regulating actin

cytoskeletal dynamics in metazoan cells.

In the protein light switch LOV2-Jα404-547 of Avena LOV-Rac1 interface

sativa photon absorption è covalent bond between
C450 and the flavin chromophore. This causes Jα
LOV2
dissociation and helix unwinding. Covalent bonding
of LOV2-Jα404-547 to Rac1 blocks access of effector
(PAK = p21-activated kinase), relieved by light.
Rac1

Jα helix

Fusing LOV2-Jα404-547 to a mutant form of Rac1 Repeated irradiation (yellow spot)

created a photoactivatable analogue of Rac1 that
could be used to induce light-dependent localised
activation of myosin-dependent migration in time time
individual mammalian cells.

For example, PA-Rac1 activation at one spot near

the edge of a mouse embryo fibroblast (MEF) led
to local protrusion combined with retraction on the
opposite side of the cell.