Canthaxanthin substitution of β-carotene yields structurally stable yet spectrally robust photosystem I cores in Synechocystis sp. PCC 6803.

Carotenoids (Cars) are essential cofactors in photosystem I (PSI), where they stabilize the pigment-protein framework and support excitation-energy transfer and photoprotection. Although metabolic engineering has enabled the production of diverse keto-carotenoids (keto-Cars) across oxyphototrophs, the structural and photophysical consequences of installing a single engineered Car into purified PSI cores remain unclear. Here, we generated a Synechocystis sp. PCC 6803 strain expressing only the crtW gene (CrtW strain), which converts β-carotene to canthaxanthin, and isolated PSI trimers to evaluate how extensive β-carotene substitution influences PSI organization and photophysical properties. HPLC analysis showed no detectable β-carotene in the CrtW PSI, indicating that it was largely replaced by canthaxanthin together with a minor unidentified Car. SDS-PAGE and BN-PAGE showed that the polypeptide composition and trimeric architecture of the CrtW PSI were indistinguishable from those of the control complex. Room-temperature absorption spectra revealed pronounced Car-dependent differences between 450 and 570 nm, consistent with the altered pigment composition, whereas the chlorophyll (Chl) Qy band remained unchanged. Notably, 77-K fluorescence-emission profiles of the CrtW PSI overlapped almost perfectly with those of the control regardless of excitation wavelength, suggesting that the organization of the low-energy Chl network is preserved. These findings indicate that cyanobacterial PSI trimers can assemble and maintain photophysical integrity with canthaxanthin in place of β-carotene without perturbing their oligomeric organization. The CrtW PSI thus provides a clean and tractable platform for dissecting how Car chemical features shape pigment-protein interactions and for guiding rational pigment redesign in photosynthetic complexes.