Phycocyanin, which covalently binds phycocyanobilin chromophores, is not only a candidate fluorescent probe for biological imaging, but also a potential antioxidative agent for healthcare. Herein, a plasmid harboring two cassettes was constructed, with cpcB from Spirulina subsalsa in one cassette and the fusion gene cpcS::ho1::pcyA in the other, and then expressed in Escherichia coli. PCB-CpcB(C-82), a fluorescent phycocyanin β subunit, was biosynthesized in E. coli, exhibiting an absorption maximum at 620 nm and fluorescence emission maximum at 640 nm. When cpcS was replaced by cpcT, PCB-CpcB(C-153), another fluorescent phycocyanin β subunit, was produced, exhibiting an absorption maximum at 590 nm and fluorescence emission maximum at 620 nm. These two fluorescent biliproteins showed stronger scavenging activity toward hydroxyl and DPPH free radicals than apo-CpcB. The IC₅₀ values for hydroxyl radical scavenging by PCB-CpcB(C-82), PCB-CpcB(C-153), and apo-CpcB were 38.72 ± 2.48 µg/mL, 51.06 ± 6.74 µg/mL, and 81.82 ± 0.67 µg/mL, respectively, and the values for DPPH radical scavenging were 201.00 ± 5.86 µg/mL, 240.34 ± 4.03 µg/mL, and 352.93 ± 26.30 µg/mL, respectively. The comparative antioxidant capacities of the proteins were PCB-CpcB(C-82) > PCB-CpcB(C-153) > apo-CpcB, due to bilin binding. The two fluorescent biliproteins exhibited a significant effect on relieving the growth of E. coli cells injured by H₂O₂. The results of this study suggest that the fluorescent phycocyanin β subunits of S. subsalsa were reconstructed by one expression vector in E. coli, and could be developed as potential antioxidants.

Cyanobacteriochromes (CBCRs) are biliproteins for photoreception that are present in cyanobacteria. These proteins possess one or more unique cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that can covalently bind the linear tetrapyrrole (bilin). Light absorption triggers the photoisomerization of bilin between the 15Z and 15E photostates. The 15E photoproduct of some CBCR GAF domains can revert to the stable 15Z state in the absence of light. In some cases, this property makes these domains function as sensors of light intensity or as red/dark optogenetic switches. However, there have been few reports regarding the applicability of these fluorescent properties. Here, we report a red/green cyanobacteriochrome GAF domain from Spirulina subsalsa, designated SPI1085g3, which exhibited photoconversion from the red-absorbing dark state (Pr, λmax = 642 nm) to the orange-absorbing photoproduct state (Po, λmax = 590 nm), and exhibited moderate dark reversion (t_1/2 = 3.3 min) from the Po state to the Pr state. The SPI1085g3 Pr state exhibited intense red fluorescence (λmax = 662 nm), with a quantum yield of 0.14. The fluorescence was switched off by red light irradiation and increased in the dark. Replacement of Cys448 of SPI1085g3 with Ser resulted in a slightly improved fluorescence quantum yield and nearly 13-fold faster dark reversion (t_1/2 = 15.2 s) than that of the wild type. This novel red/dark-switchable fluorescent biliprotein expands the present repertoire and diversity of photoswitchable fluorescent protein candidates.

Su-Dan Jiang, Yi sheng, Xian-Jun Wu, Yong-Li Zhu, and Ping-Ping Li. J. Microbiol. Biotechnol. 2021;31:233-9. https://doi.org/10.4014/jmb.2009.09048

Cyanobacteriochromes (CBCRs) bind linear tetrapyrrole chromophores, mostly phycocyanobilin (PCB), and exhibit considerable spectral diversity with a high potential for biotechnological applications. Particular attention has been given to the conversion into intrinsic biliverdin (BV) incorporation due to the absence of PCB in mammalian cells. Our recent study discovered that a red/green CBCR of Spirulina subsalsa, SPI1085g3, was covalently attached to PCB and exhibited strong red fluorescence with a unique red/dark switch. In this study, we found that SPI1085g3 could be modestly chromophorylated with BV and absorb somewhat shifted (10 nm) red light, while the single C448S mutant could efficiently bind BV and exhibit unidirectional photoconversion and moderate dark reversion. The fluorescence in its dark-adapted state was switched off by red light, followed by a moderate recovery in the dark, and these were properties similar to those of PCB-binding SPI1085g3. Furthermore, by introducing the CY motif into the conserved CH motif for chromophore attachment, we developed another variant, C448S_CY, which showed increased BV-binding efficiency. As expected, C448S_CY had a significant enhancement in fluorescence quantum yield, reaching that of PCB-binding SPI1085g3 (0.14). These BV-binding CBCRs offer an improved platform for the development of unique photoswitchable fluorescent proteins compared with PCB-binding CBCRs.