Exploring GPCR biosensing in yeast

Bettina Lengger

Research output: Book/ReportPh.D. thesis

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Abstract

Many key pharmaceuticals on the market are extracted from plant material, including anticancer drugs. Low yields and plant pests, as well as ethical and environmental concerns contribute to an unstable supply of critical drugs. Chemical synthesis is often not possible, or only possible in low yields too, as many of the drugs found in plants exhibit complex chemical structures.

Microbial production of pharmaceuticals has been on the rise since it offers a stable and scalable on-demand production alternative. Development of microbial production strains is however costly and time-consuming, partly due to the larger number of strains that have to be engineered and evaluated for their production potential.

Biosensors have taken a promising role as a tool in shortening the cost and time needed, as they can serve as an alternative fast and low-cost screening method.

G protein-coupled receptors (GPCRs) are eukaryotic membrane proteins, that allow cells to sense their environments, and have been used to create biosensors in yeast. In this thesis, the exploration of GPCR biosensors with the aim of developing them as screening tools for faster metabolic engineering endeavours is presented.

Chapter 2 summarises the current state of the GPCR sensing in yeast and highlights applications in the medical and biotechnological space. Chapter 3 focuses on serotonin sensing, also in the context of examining a library of sensing strains and explores important parameters influencing the sensor’s behaviour. Owing to the Jensen group’s special interest in alkaloid production, and to develop a better mechanistic understanding of GPCR functional modes, a GPCR is evolved using an in vivo mutagenesis system, with the goal of creating a biosensor capable of sensing alkaloids in an agonistic way (Chapter 4). Finally, turning to droplet microfluidics, chapter 5 summarises the work on encapsulating biosensing yeast cells in double-emulsion droplets, with the aim of establishing a high-throughput single cell workflow for the efficient screening of large numbers of production strains.

This thesis lays the groundwork for successfully using GPCR biosensing in metabolic engineering applications and hopes to contribute to the faster development of production strains for critical drugs.
Original languageEnglish
PublisherTechnical University of Demark
Number of pages146
Publication statusPublished - 2022

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