GPCR-based biosensor for synthetic biology applications

Project Information

GPCR-Sensor

Grant agreement ID: 845835

DOI

10.3030/845835

Project closed

EC signature date 23 April 2019

Start date 1 June 2020

End date 31 May 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 207 312,00

EU contribution

€ 207 312,00

Coordinated by

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 1 - GPCR-Sensor (GPCR-based biosensor for synthetic biology applications)

Reporting period: 2020-06-01 to 2022-05-31

Summary of the context and overall objectives of the project

Human senses such as sense of smell, taste and sight rely on G-protein-coupled receptors (GPCRs). In addition, GPCRs play key roles in endocrine signalling and the regulation of the immune system. Also eukaryotic microbes such as baker’s yeast (Saccharomyces cervisiae) have similar receptors that they use to sense mating pheromones. Furthermore, it has been previously shown, that these pheromone receptors can be replaced by human GPCRs giving the yeast cells the ability to sense the corresponding external signals. Such cell lines could be developed into sensitive and economical biosensors with uses in biomedicine, industrial biotechnology and even consumer applications. The objectives of this work are to develop a biosensor platform capable of utilizing different GPCR receptors, optimize the different components of the biosensor platform to reach high sensitivity and dynamic range, and finally, to demonstrate proof-of -concept for different real-life applications.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In the course the MSCA project “GPCR-sensor” we established a biosensor platform and demonstrated its use by constructing a sensitive cannabinoid biosensor. We started by engineering a chassis host yeast strain capable of housing the different interchangeable biosensor components. The first biosensors were established by adding either the A2A human adenosine receptor or the CB2 human cannabinoid receptor together with a GFP reporter construct. The latter can react to the activation of the yeast pheromone pathway triggered by activation of the inserted receptor and results in measurable green fluorescence. The CB2 cannabinoid biosensor was further developed by engineering the receptor itself for increased sensitivity and by fitting the biosensor with colorimetric or luminometric reporters. Finally, we performed three proof of concept experiments demonstrating the use the biosensor in real-life applications: 1) robotic high-throughput screening of a synthetic chemical library of 1600 compounds, 2) bioactivity-guided screening of medicinal plants for discovery of novel natural cannabinoids and 3) development of a portable biosensor device for cannabinoid detection outside of laboratory settings.

As an outcome of this project, we developed a modular GPCR biosensor platform in yeast that can be armed with different GPCR receptors for sensing a variety of different signals. The CB2 based cannabinoid biosensor was engineered to reach sensitivity on par with that of mammalian cell lines expressing the same receptor. Proof-of -concept experiments resulted in demonstrating the functionality of the biosensor in real-life applications. By screening a library of 1600 random synthetic compounds, we were able to find 4 novel potent synthetic cannabinoids. Screening of 54 medicinal plans resulted in the discovery of one new natural cannabinoid, dugesialactone. Finally, we demonstrated the portable detection of cannabinoids from reconstituted urine, saliva and serum samples using the biosensor with a dedicated prototype device and a cellular phone.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

GPCRs from humans and other organisms have previously been successfully connected to the yeast pheromone pathway. However, in order to such transgenic strains to be used as biosensors the balance between different components has to be optimized. In addition, human GPCRs do not always perform optimally in a yeast system as they are. In this work we show that fusing the yeast signal peptide (excretion signal) from the alpha mating factor to the CB2 receptor improves the correct localization of the receptor in yeast cell resulting in improved biosensor performance. In addition, we report the development of a novel colorimetric reporter system based on biosynthesis of a betalain compound (the color from beetroot) for use in yeast. Finally, we describe novel synthetic cannabinoids discovered using the biosensor.

In this work we describe the development of a modular biosensor platform where different GPCRs can be inserted to create different biosensors capable of sensing different signals. We demonstrate the capabilities of this platform by implementing a cannabinoid biosensor. This work serves as a proof-of-concept for the use of yeast based GPCR biosensors in real-life applications and provides a platform for non-specialist labs to harness the sensing potential of GPCRs. The CB2 cannabinoid biosensor developed in this work can be used as it is for example for the discovery of novel CB2 agonists and antagonists (e.g. anti-inflammatory or immunomodulatory drugs), monitoring of medicinal cannabis treatments or for intercepting novel cannabinoid designer drugs for which no detection method has been yet developed.

This work was published in the top tier generalist journal Nature Communications (2022) https://doi.org/10.1038/s41467-022-31357-6. In addition, this work has been disseminated at international conferences including the 4th International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology (2020, online) and at Synthetic Biology: Engineering, Evolution & Design (SEED) 2021 (online).

Overview of the GPCR sensor design