Expression of recombinant full‐length plant phytochromes assembled with phytochromobilin in Pichia pastoris

Abstract

a b s t r a c tWe have successfully developed a system to produce full-length plant phytochrome assembled with phytochromobilin in Pichia pastoris by co-expressing apophytochromes and chromophore biosynthetic genes, heme oxygenase (HY1) and phytochromobilin synthase (HY2) from Arabidopsis. Affinity-purified phytochrome proteins from Pichia cells displayed zinc fluorescence indicating chromophore attachment. Spectroscopic analyses showed absorbance maximum peaks identical to in vitro reconstituted phytochromobilin-assembled phytochromes, suggesting that the co-expression system is effective to generate holo-phytochromes. Moreover, mitochondria localization of the phytochromobilin biosynthetic genes increased the efficiency of holophytochrome biosynthesis. Therefore, this system provides an excellent source of holophytochromes, including oat phytochrome A and Arabidopsis phytochrome B.

PhiReX: a programmable and red light-regulated protein expression switch for yeast

“…The highest fold induction (∼3.5) was achieved with the combinations of HY1 and PcyA, MTS-mHY1/MTS-mHY2, MTS-mHY1/MTS-PcyA, and MTS-mHmx1/MTS-mHY2 (Figure 5D ). In contrast to published data ( 28 , 29 ), targeting HY1 and PcyA to mitochondria did not gravely boost induction rates compared to cells expressing the two enzymes in the cytoplasm. Still, by combining native HY1 and PcyA we achieved robust expression and doubled the fold induction compared to RL_CT_GI without PCB supply.…”

“…The different expression cassettes were genome integrated into the his1-Δ200 locus of RL_CT_GI cells (Figure 5C ). Previous studies in mammalian cells ( 28 ) and Pichia pastoris ( 29 ) revealed optimized chromophore biosynthesis when the two biosynthesis enzymes were targeted to mitochondria, the location of heme biosynthesis in eukaryotes ( 45 ). Accordingly, we deleted the proteins’ native target sequences, if applicable, and replaced them by a yeast-derived mitochondrial targeting sequence (MTS) ( Supplementary Table S1 ).…”

“…Even though RL_CT_GI cells show light-induced yEGFP output also in the absence of PCB, the obtained fold induction (∼1.5) was not satisfying; this prompted us to engineer S. cerevisiae cells able to produce the chromophore by expressing the enzymes HO and BR which catalyze the conversion of heme to biliverdin, and of biliverdin to PCB or PϕB, respectively ( 27 ). The successful production of holo-phytochrome was already shown in bacteria, mammalian cells and the yeast Pichia pastoris ( 27 – 29 ), but not yet in S. cerevisiae . To change this, we integrated different combinations and versions of HOs and BRs into RL_CT_GI cells to detect the best chromophore-producing combination by measuring the reporter output (Figure 5B ).…”

New Constitutively Active Phytochromes Exhibit Light-Independent Signaling Activity

“…Full-length phytochrome cDNAs of wild-type AsphyA and the AsYVA mutant fused with a 10-amino acid streptavidin affinity-tag at the C terminus were cloned into pPIC3.5K (Invitrogen), and recombinant phytochrome proteins were prepared using the Pichia protein expression system (Invitrogen) and streptavidin affinity chromatography (IBA), as described previously (Kim et al, 2004;Shin et al, 2014Shin et al, , 2016. The QuickChange Site-Directed Mutagenesis System (Stratagene) was used to generate AsYVA with the mutagenic primers shown in Supplemental Table S1.…”

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).…”