posted on 2022-01-03, 23:03authored byJustin
I. Yoo, Tejas A. Navaratna, Patrick Kolence, Michelle A. O’Malley
The directed evolution
of proteins comprises a search of sequence
space for variants that improve a target phenotype, yet identification
of desirable variants is inherently limited by library size and screening
ability. Selections that couple protein phenotype to cell viability
accelerate identification of promising variants by depleting libraries
of undesirable variants en masse. Here, we introduce
GPCR-FEX, a stringent selection platform that couples G-protein coupled
receptor (GPCR) signaling to expression of a fluoride ion exporter
(FEX)-GFP fusion gene and concomitant cellular fluoride
tolerance in yeast. The GPCR-FEX platform works to deplete inactive
GPCR variants from the library prior to high-throughput fluorescence-based
cell sorting for rapid, inexpensive screening of receptor libraries
that sample an expanded sequence space. Using this system, FEX1 was placed under the control of either PFUS1 or PFIG1, promoters activated upon agonist binding by
the native yeast GPCRs, Ste2p or Ste3p. Addition of a C-terminal degron
to FEX1p enhanced the dynamic range of cell growth between agonist-treated
and untreated cells. Using deep sequencing to enumerate population
members, we show rapid selection of a previously engineered Ste2p
receptor mutant strain over wild-type Ste2p in a model library enrichment
experiment. Overall, the GPCR-FEX platform provides a mechanism to
rapidly engineer GPCRs, which are important cellular sensors for synthetic
biology.