SummaryPhycobilisomes are large light‐harvesting complexes attached to the stromal side of thylakoids in cyanobacteria and red algae. They can be remodeled or degraded in response to changing light and nutritional status. Both the core and the peripheral rods of phycobilisomes contain biliproteins. During biliprotein biosynthesis, open‐chain tetrapyrrole chromophores are attached covalently to the apoproteins by dedicated lyases. Another set of non‐bleaching (Nb) proteins has been implicated in phycobilisome degradation, among them NblA and NblB. We report in vitro experiments with lyases, biliproteins and NblA/B which imply that the situation is more complex than currently discussed: lyases can also detach the chromophores and NblA and NblB can modulate lyase‐catalyzed binding and detachment of chromophores in a complex fashion. We show: (i) NblA and NblB can interfere with chromophorylation as well as chromophore detachment of phycobiliprotein, they are generally inhibitors but in some cases enhance the reaction; (ii) NblA and NblB promote dissociation of whole phycobilisomes, cores and, in particular, allophycocyanin trimers; (iii) while NblA and NblB do not interact with each other, both interact with lyases, apo‐ and holo‐biliproteins; (iv) they promote synergistically the lyase‐catalyzed chromophorylation of the β‐subunit of the major rod component, CPC; and (v) they modulate lyase‐catalyzed and lyase‐independent chromophore transfers among biliproteins, with the core protein, ApcF, the rod protein, CpcA, and sensory biliproteins (phytochromes, cyanobacteriochromes) acting as potential traps. The results indicate that NblA/B can cooperate with lyases in remodeling the phycobilisomes to balance the metabolic requirements of acclimating their light‐harvesting capacity without straining the overall metabolic economy of the cell.

Cyanobacteriochromes (CBCRs) are biliprotein photoreceptors that only exist in cyanobacteria and have a broad spectral response range from ultra-violet to far-red. The red/green-type CBCRs can show red/green reversible photoconversion via a covalently bound phycocyanobilin (PCB). In recent years, several CBCRs binding with not only PCB but also biliverdin (BV) have been discovered, which raises the possibility of CBCRs being applied as optogenetic tools. Through molecular modification, we hope to engineer BV-binding CBCRs responsive to the near-infrared spectral region (650–900 nm), of which the red/green type of CBCRs are suitable resources for experimentation. Here, we use Slr1393g3 (the third GAF domain of a red/green photoswitching CBCR from Synechocystis sp. PCC 6803) as a template to perform such molecular evolution using both random mutagenesis and site-directed mutagenesis. After several rounds of random mutagenesis, we obtained several BV-binding variants of Slr1393g3. These BV-binding variants have a maximal absorbance at ̃690 nm and a fluorescence at ̃720 nm. Additionally, some of them have remarkable photochromicity between a far-red light-absorbing state and a red light-absorbing state. Based on the primary amino acid sequence and structural models, the Phe474 surrounding ring D of BV is thought as a crucial site for chromophore selectivity.