PT  - JOURNAL ARTICLE
AU  - Youichi Uda
AU  - Haruko Miura
AU  - Yuhei Goto
AU  - Kazuhiro Aoki
TI  - Improvement of phycocyanobilin synthesis for genetically encoded phytochrome-based optogenetics
AID  - 10.1101/2020.01.15.908343
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.01.15.908343
4099  - http://biorxiv.org/content/early/2020/01/16/2020.01.15.908343.short
4100  - http://biorxiv.org/content/early/2020/01/16/2020.01.15.908343.full
AB  - 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.PhyBphytochrome BPIFphytochrome-interacting factorPCBphycocyanobilinPcyAphycocyanobilin:ferredoxin oxidoreductaseHO1heme oxygenaseFdferredoxinFNRferredoxin-NADP+-reductaseBVbiliverdin IX-alphaBVRAbiliverdin reductase A