Real-Time Tracking of Intracellular Prenyl Phosphate Pools in the Marine Diatom Phaeodactylum tricornutum with a...

Metabolite-responsive, protein-based biosensors are powerful tools for monitoring cellular metabolite dynamics in vivo and accelerating strain engineering workflows in microorganisms. In this study, we introduced a previously developed protein-based biosensor, computationally designed to detect farnesyl diphosphate (FPP), in the marine diatom Phaeodactylum tricornutum. We expressed two versions of the biosensor constitutively, under a strong promoter-terminator pair using extrachromosomal episomes, and we parametrized the capacity of both designs in detecting intracellular metabolite levels. Initial assays revealed that the two versions of the biosensor we investigated, S3-2D and S3-3A, had specificity not only for FPP but also for other exogenously supplied prenyl phosphates such as geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP) in a dose-dependent manner, showcasing broader specificity for multiple prenyl phosphates. We further demonstrated the capacity of S3-3A to track perturbations in the endogenous prenyl phosphate pools by testing it in the presence of pharmacological inhibition of the mevalonate pathway. Moreover, S3-3A generated dot-like, fluorescent signal "hotspots" in the cytosol of diatoms, suggesting a complex subcellular organization of the isoprenoid biosynthesis in P. tricornutum. These findings lay the groundwork for developing metabolite-responsive biosensors as useful tools for monitoring and investigating prenyl phosphate dynamics in diatoms, providing a foundation for advanced metabolic engineering of microalgae.