High-throughput GPCR-based autocrine screening for secondary metabolite
production in yeast
Abstract
Biosensors are valuable tools in accelerating the test phase of the
design-build-test-learn cycle of cell factory development, as well as in
bioprocess monitoring and control. G protein-coupled receptor
(GPCR)-based biosensors enable cells to sense a wide array of molecules
and environmental conditions in a specific manner. Due to the
extracellular nature of their sensing, GPCR-based biosensors require
compartmentalization of distinct genotypes when screening production
levels of a strain library to ensure that detected levels originate
exclusively from the strain under assessment. Here, we explore the
integration of production and sensing modalities into a single
Saccharomyces cerevisiae strain and compartmentalization using
three different methods: (1) cultivation in microtiter plates, (2)
spatial separation on agar plates, and (3) encapsulation in
water-in-oil-in-water double emulsion droplets, combined with analysis
and sorting via a fluorescence-activated cell sorting (FACS) machine.
Employing tryptamine and serotonin as proof-of-concept target molecules,
we optimize biosensing conditions and demonstrate the ability of the
autocrine screening method to enrich for high producers, showing the
enrichment of a serotonin-producing strain over a non-producing strain.
These findings illustrate a workflow that can be adapted to screening
for a wide range of complex chemistry at high throughput using
commercially available microfluidic systems.