Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP⁺ Reductase Systems Enables Genetically Encoded PhyB Optogenetics

Abstract

Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse, and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells

“…For example, although PCB is not naturally produced in nonphotosynthetic species, BV is widely distributed in nature including yeast and mammals. For this reason, biosynthesis of PCB has also been engineered into eukaryotic "chassis" organisms that naturally do not produce these compounds (75)(76)(77)(78). Increased expression of heme oxygenases has been used to enhance the Cyanobacterial photoactivatable adenylyl cyclases synthesis of BV in bacterial and mammalian cells (79,80).…”

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.…”

Bringing Light to Transcription: The Optogenetics Repertoire

“…These four genes were engineered to synthesize PCB at sufficient concentrations in the mitochondria to drive detectable PhyB-PIF activation ( Uda et al, 2017 ). Moreover, with a similar approach, Kyriakakis et al (2018) observed that variation in rate of expression of HO1-PcyA versus Fd-FNR in mammalian cells has a direct correlation with the production levels of PCB in mitochondria, which is further limited on the cytoplasm by heme restriction. A complementary approach proposed reducing the metabolism of biliverdin by knocking down or knocking out the biliverdin reductase A, a mammalian endogenous PCB metabolic agent, resulting, therefore, in an increase of available PCB ( Uda et al, 2017 ).…”