Improvement of Phycocyanobilin Synthesis for Genetically Encoded Phytochrome-Based Optogenetics

Abstract

Optogenetics is a powerful technique using photoresponsive proteins, and light-inducible dimerization (LID) systems, an optogenetic tool, allow to manipulate intracellular signaling pathways. One of the red/far-red responsive LID system, phytochrome B (PhyB)-phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phytochromobilin or phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (synPCB), and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely, PcyA, HO1, Fd, and Fnr, were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated PhyB-PIF and synPCB into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.

Optogenetic Tools for Manipulating Protein Subcellular Localization and Intracellular Signaling at Organelle Contact Sites

“…However, some dimerizers require the addition of cofactors that are not synthesized by mammalian cells, such as the cofactors required for phytochromes. In this case, cell media should be supplemented with phycocyanobilin (PCB) to ensure the functionality of the photosensitive protein (Toettcher, Gong, Lim, & Weiner, 2011a), or the user should use cell lines engineered for the synthesis of PCB (Uda et al., 2020).…”

Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin

“…The cDNAs of PcyA, HO1, Fd , and Fnr were originally derived from Thermosynechococcus elongatus BP-1 as previously described (Uda et al, 2020). The mitochondrial targeting sequence (MTS; MSVLTPLLLRGLTGSARRLP) was derived from human cytochrome C oxidase subunit VIII.…”

PhotoGal4: A Versatile Light-Dependent Switch for Spatiotemporal Control of Gene Expression in Drosophila Explants

“…Another potential issue is that the supplemented PCB may not reach proper concentrations in certain tissues in vivo, which may affect the potency of PhotoGal4. However, it has been shown that mammalian cultured cells expressing cyanobacterial enzymes PcyA and HO1 can successfully transform endogenous heme groups into PCB (Kyriakakis et al, 2018;Muller et al, 2013b;Uda et al, 2017;Youichi Uda et al, 2020). We strongly believe that genetic synthesis of PCB will pave the way for more practical applications of PhotoGal4.…”