Comparative Study of Phycoerythrobilin Synthases for Fine-Tuning Photosynthetic Light-Harvesting Complexes, Phycobilisomes

Comparative Study of Phycoerythrobilin Synthases for Fine-Tuning Photosynthetic Light-Harvesting Complexes, Phycobilisomes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comparative Study of Phycoerythrobilin Synthases for Fine-Tuning Photosynthetic Light-Harvesting Complexes, Phycobilisomes Mizuho Sato, Mai Watanabe, Kaisei Maeda, Kaori Nimura-Matsune, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8355607/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 3 You are reading this latest preprint version Abstract Phycobilisomes serve as the major light-harvesting antenna complexes in cyanobacteria; they capture light and transfer excitation energy to the photosystems, and their spectral and functional properties are primarily determined by the types of bilin chromophores attached to phycobiliproteins. We previously revealed that the properties of phycobilisomes can be modulated by altering the biosynthesis of these bilin chromophores. In this study, we introduced two distinct phycoerythrobilin (PEB) biosynthetic enzymes, pcyX and pebS , into cyanobacterium Synechococcus elongatus PCC 7942 and successfully enabled PEB incorporation into the native phycobilisome. PebS exhibited high PEB synthesis activity comparable to the canonical PebA-PebB pathway, whereas PcyX showed lower activity, enabling fine-tuning of PEB accumulation. Consistent with previous findings, moderate PEB supply enhanced growth under green light; notably, PcyX expression further promoted growth by providing a more balanced pigment supply. Transcriptome analysis confirmed comparable expression of pebA - pebB , pcyX , and pebS , and revealed that increased PEB accumulation triggered broad metabolic remodeling: genes for glucose metabolism, hydrogenase complexes, potassium transporters, and regulatory factors including sigma factors were upregulated, suggesting a shift toward dissipation of excess reducing power. These results indicate that natural diversity among bilin synthases can be exploited to engineer phycobilisome composition and redox homeostasis in cyanobacteria. Biological sciences/Biochemistry Biological sciences/Biophysics Biological sciences/Plant sciences Phycobilisome Cyanobacteria Phycoerythrin Phycocyanin Photosynthesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Photosynthesis, the process by which light energy is converted into chemical energy, is fundamental to sustaining life on Earth. Throughout evolution, photosynthetic organisms have developed diverse and optimized light-harvesting complexes to efficiently capture light energy and transfer excitation energy to the photosynthetic reaction centers within their ecological niches. Phycobilisomes (PBSs), found in cyanobacteria, eukaryotic red algae, and glaucophytes, are peripheral light-harvesting complexes attached to the stromal side of the thylakoid membrane. PBSs enhance photosynthetic efficiency by capturing light in the green to orange spectral regions, which are poorly absorbed by chlorophyll, and transferring this energy to photosystems for conversion into chemical energy 1 , 2 . Structurally, PBSs consist of rod and core subcomplexes composed mainly of phycobiliproteins and linker polypeptides. The rods comprise disk-like trimers, namely (αβ) 3 , which may further assemble as a hexamer ([αβ] 3 ) 2 composed of several types of phycobiliproteins, such as phycocyanins (PCs), phycoerythrins (PEs) and phycoerythrocyanins (PECs) 3 , 4 , 5 . The rods vary depending on the organism and can be made of PC only or a combination of PC and other phycobiliproteins such as PEs and PECs. The PBS core consists of a cylindrical structure composed of allophycocyanin (APC) with six PCBs per monomer. All units (disks within the rod and rod-to-core) are connected by linker proteins. Each of the phycobiliprotein contains linear tetrapyrrole chromophores known as bilins that are covalently bound to cysteine residues within the apoproteins. The spectral properties of PBSs are primarily determined by the type and ratio of these bilins—such as phycocyanobilin (PCB), phycoerythrobilin (PEB), phycourobilin (PUB), and phycoviolobilin (PVB)—that define the wavelength of light absorption. The biosynthesis of bilins is catalyzed by ferredoxin-dependent bilin reductases (FDBRs), including PcyA, PebA, PebB, PebS, and PcyX 6 , 7 , 8 , 9 , 10 . PcyA catalyzes the conversion of biliverdin IXα to PCB, whereas PebA and PebB function sequentially to produce PEB (Fig. 1 ). PebS and PcyX are single-component FDBRs that catalyze both reduction steps (Fig. 1 ). These enzymes have been identified not only in cyanobacteria but also in cyanophages and metagenomic datasets, suggesting that nature employs diverse enzymatic routes to fine-tune chromophore biosynthesis and adapt light-harvesting to different spectral environments. Previous studies have demonstrated that the spectral properties of PBSs can be modified by engineering the biosynthetic pathways of bilins 11 , 12 , 13 . However, achieving precise control over bilin composition and incorporation remains challenging. In Synechococcus elongatus 7942 ( Synechococcus 7942), heterologous production of phycoerythrobilin (PEB) has been reported to inhibit cell growth 13 . In cyanobacteria, bilins are known to participate in signal transduction by binding to bilin-binding photoreceptors known as cyanobacteriochromes (CBCRs) 14 , 15 . These photoreceptors contain a GAF domain that covalently binds bilins and mediate light sensing and downstream signal transduction. For instance, in Synechococcus elongatus UTEX 3055, PixJ, a bilin-binding photoreceptor, has been shown to regulate the expression of phototaxis-related genes 16 . Although Synechococcus 7942 lacks motility and does not exhibit phototaxis, introducing PEB biosynthesis into this strain, which natively lacks PEB, could potentially interfere with endogenous signaling networks. Nevertheless, the physiological consequences of such perturbations remain poorly understood. In this study, we introduced PcyX and PebS into Synechococcus elongatus PCC 7942 and systematically compared their contributions to PEB synthesis and PBS assembly. Transcriptome analysis revealed that the expression levels of the introduced pebA - pebB , pcyX , and pebS genes were comparable, yet the cellular responses to varying PEB accumulation differed. These included marked changes in genes related to glucose metabolism, hydrogenase complexes, potassium transporters, and regulatory factors such as sigma factors and circadian rhythm components, suggesting that cyanobacterial cells actively adjust redox balance and metabolic flow in response to bilin levels. Collectively, our findings reveal distinct activities between PcyX and PebS and demonstrate how these differences can be exploited to modulate light-harvesting capacity and promote growth under green-light conditions. RESULTS Construction of cyanobacterial strains with controlled PEB levels Three types of ferredoxin-dependent bilin reductases (FDBRs) have been identified to date, each reported to exhibit distinct activities in E. coli cells 6 , 17 , 18 . To compare their activities directly in cyanobacteria, we constructed a series of Synechococcus elongatus PCC 7942 strains expressing different FDBRs. The pebA–pebB operon, pcyX , and pebS were placed under the IPTG-inducible trc promoter and integrated into a neutral site (NS) on the Synechococcus 7942 chromosome. The resulting strains were designated Synechococcus 7942 AB1, X1, and S1, respectively (Fig. 2 A). Following IPTG induction, all three strains exhibited a brownish coloration in a concentration-dependent manner (Fig. 2 B), although the extent of this change differed among strains. In particular, the X1 strain showed visibly weaker coloration compared with AB1 and S1 under the same conditions. Absorption spectra revealed a decrease in the 625-nm peak associated with phycocyanin (PC) and an increase in the 560-nm peak associated with phycoerythrobilin (PEB), both of which varied with IPTG concentration (Fig. 2 C). Based on absorbance at 560 nm, the apparent activity of PcyX was lower than that of PebA–PebB and PebS, and was estimated to be less than one-tenth under the tested culture conditions. Comparison of PBS complexes under low PEB induction In a previous study, we demonstrated that the accumulation of small amounts of PEB in PBS complexes altered the PBS properties of Synechococcus 7942 toward a green-light–adapted type 13 . To examine whether similar changes occur in the newly constructed strains, each strain was cultured with 5 µM IPTG, and the size and properties of PBS complexes were analyzed (Fig. 3 A). Cell extracts from the wild type (WT) and the three FDBR-expressing strains were subjected to sucrose density gradient (SDG) centrifugation to separate intact PBS complexes from their components. In the AB1 and S1 strains, the fraction corresponding to full-length PBS complexes, typically detected at the bottom of the gradient, shifted toward an upper position (Fig. 3 A, solid line), indicating a reduction in complex size. In addition, red-colored fractions appeared in the upper region of the gradient (Fig. 3 A, dashed line), consistent with disassembled phycobiliproteins reported previously 13 . No notable differences in growth were observed under white-light conditions (Fig. 3 B), suggesting that the reduction in PBS complex size does not substantially affect cell proliferation under these culture conditions. The excitation spectra of 685-nm fluorescence emitted by APC in PBS cores showed that the PBS complexes from the FDBR-expressing strains (Fig. 3 A, solid line) exhibited an additional peak near 560 nm, corresponding to PE, in addition to the 620-nm peak associated with PC. The 560-nm peak was most pronounced in AB1, followed by S1 and X1, consistent with the cellular absorption spectra (Fig. 2 C). These observations indicate that PEB accumulates within the PBS complexes under low-level FDBR induction and that partial degradation and size reduction of the PBS complexes occur in the AB1 and S1 strains. The relative levels of the 560-nm peak further suggest that the apparent activities of the FDBRs in Synechococcus 7942 follow the order PebA–PebB > PebS > PcyX. To investigate the basis of the size reduction in PEB-containing PBS complexes, proteins from WT and AB1 PBS fractions were fluorescently labeled and analyzed by SDS–PAGE (Supplementary Figure S1 A). Comparison of the fluorescence intensities of rod–rod and rod–core linker proteins showed a reduction in the 30-kDa rod–rod linker protein relative to other linker proteins in AB1 (Figure S1 B). As the 30-kDa rod–rod linker has been reported to occupy the outermost position in the PBS rod and to be among the first components affected by changes in light conditions 19 , 20 , these results suggest that PEB accumulation may promote dissociation of the outer rod disc, contributing to PBS size reduction. Since PEB has been reported to bind to apoproteins of PCB-type PBSs 11 , 12 , 13 , 21 , we next examined the phycobiliproteins under conditions of PEB accumulation. Protein extracts were prepared from cells cultured with 5 µM IPTG for FDBR induction and analyzed by SDS–PAGE. Two colored bands were observed (Fig. 4 A), corresponding to the lower- and higher-molecular-weight disc components CpcA and CpcB, respectively. In the AB1 and S1 strains, both the CpcA and CpcB bands exhibited a red coloration, whereas in the X1 strain, red coloration was detected only in the CpcA band. Proteins from each band were extracted, and their absorption spectra were compared with those of the WT. In AB1 and S1, both the CpcA and CpcB extracts showed a shift toward shorter wavelengths, while in X1, a shift was observed only for CpcA (Fig. 4 B). The CpcA bands appeared visually redder than the corresponding CpcB bands, and the absorption peak of the CpcA extracts was markedly higher in all three FDBR-expressing strains. These results suggest that, at the chromophore-binding sites of CpcA, PEB is bound preferentially over PCB, consistent with previous reports 11 , 13 . Growth advantage of FDBR-expressing strain under green light Because the PBSs of the FDBR-expressing strains accumulated PEB and were able to absorb green light, we next evaluated whether this conferred a growth advantage under green-light illumination. The growth of the AB1 and X1 strains was compared with that of the WT. Under white-light conditions, no notable differences were observed among the strains (Fig. 3 B). In contrast, under green-light conditions, both AB1 and X1 exhibited faster growth than the WT (Fig. 5 A). Analysis of growth rates based on the slopes of the growth curves indicated that X1 showed a significantly higher growth rate than WT (Fig. 5 B). These results indicate that PEB accumulation is associated with enhanced proliferation under green light, and further suggest that proliferation is promoted when the PBS complex is maintained in a nearly complete state, consistent with our previous observations 13 . Effects of PEB accumulation on gene expression Because bilins function not only in light harvesting but also in the regulation of gene expression through binding to CBCR photoreceptors, changes in bilin metabolism are expected to elicit broad transcriptional responses. To evaluate the effects of PEB accumulation on gene expression in Synechococcus 7942, we performed RNA-seq analysis using the WT strain and three FDBR-expressing strains. Total RNA was extracted from cultures grown under white light for 4 days in the presence of 5 µM IPTG (for strains AB1, S1, and X1) and subjected to sequencing analysis. The TPM (transcripts per million) values, fold ratios, and FDR-adjusted p-values for the WT and each FDBR-expressing strain are summarized in Supplementary Table S1 A, and the resulting volcano plots are shown in Fig. 6 A (without ORF IDs) and Supplementary Figure S2 (with ORF IDs). Among the three FDBR-expressing strains, the expression levels of the heterologously introduced pebA – pebB , pcyX , and pebS genes in Synechococcus 7942 were comparable (Fig. 6 B), indicating that the observed differences among strains are attributable to enzymatic activity rather than expression level. Relative to the WT strain, no significant changes were detected in the expression of genes encoding components of PSI, PSII, or the phycobilisome. However, 39 genes were commonly upregulated and 6 were commonly downregulated across all three FDBR-expressing strains (Supplementary Figure S3 and Supplementary Table S1 B). The commonly downregulated genes did not clearly cluster into a specific functional category, whereas the commonly upregulated genes included those associated with glucose metabolism, hydrogenase activity, potassium transport, and regulatory factors, such as sigma factors and circadian clock–related genes (Fig. 6 B). Importantly, the magnitude of these transcriptional changes was highest in AB1, moderate in S1, and lowest in X1, demonstrating that the extent of gene expression remodeling positively correlates with PEB accumulation levels (Fig. 2 C, Fig. 3 C). These findings indicate that increased PEB levels exert a stronger influence on transcriptional responses, leading to broader remodeling of cellular metabolism. We observed a coordinated upregulation of genes involved in glucose metabolism—including 6-phosphogluconate dehydrogenase (6PG; Synpcc7942_0039), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Synpcc7942_0245), D-fructose 1,6-bisphosphatase (FBPase; Synpcc7942_2335), glucose-6-phosphate dehydrogenase (G6PDH; Synpcc7942_2334), pyruvate:ferredoxin (flavodoxin) oxidoreductase (PFOR; Synpcc7942_2384), and glycogen/α-glucan phosphorylase (Synpcc7942_0244)—together with genes encoding the NAD-reducing hydrogenase HoxS subunits (Synpcc7942_2551, 2555–2557). Because these genes are associated with cellular redox metabolism, their upregulation may reflect a cellular response to changes in redox balance, in which increased metabolic capacity could help utilize excess reducing equivalents and contribute to adjustment of the NADPH/NADH state and carbon flux distribution 22 , 23 . In addition, the induction of genes encoding a K⁺ transport system (Synpcc7942_1668–1671) in the FDBR-expressing strains suggests that PEB accumulation is accompanied by transcriptional changes in ion transport pathways, potentially reflecting a response related to osmotic or ionic homeostasis 24 . We also detected increased expression of several genes associated with transcriptional, post-transcriptional, and translational regulation in response to PEB accumulation. The group 2 sigma factor SigC (Synpcc7942_1849) and the RNA-binding protein Rbp1 (Synpcc7942_1999), both known to respond to darkness and cold stress 25 , 26 , 27 , showed elevated transcript levels. In addition, the genes encoding the circadian clock components KaiB (Synpcc7942_1217) and KaiC (Synpcc7942_1216) were upregulated in AB1 and S1, the strains with higher PEB levels, consistent with their roles in broad transcriptional regulation 28 . The gene encoding the translation elongation factor EF-G (Synpcc7942_2082) was also increased, aligning with previous reports that translation elongation factors contribute to PSII repair under oxidative stress conditions 29 . Overall, these observations indicate that PEB accumulation is accompanied by widespread changes in regulatory gene expression, potentially influencing cellular physiology through multiple regulatory layers. DISCUSSION In summary, our results clarify the relative behavior of the three FDBRs within cyanobacterial cells. When expressed under identical conditions, their apparent activities followed the order PebA–PebB > PebS > PcyX. Given that PebA–PebB are broadly conserved among cyanobacteria 10 , this may reflect a higher degree of compatibility with the native cellular environment compared with PebS or PcyX (Figs. 2 and 3 ). By contrast, PcyX—originating from metagenome-derived sequence 6 , 9 —exhibited lower activity, which may allow more gradual modulation of PEB levels. Our findings further indicate that effective adaptation of PBSs toward green-light utilization in Synechococcus 7942 requires PEB accumulation at levels that maintain PBS integrity, and that PcyX can serve as a useful means to achieve such controlled accumulation (Fig. 5 ). Finally, the observation that PebS alone supported PEB accumulation comparable to PebA–PebB highlights that both PebS and PcyX may represent practical tools for future synthetic biology applications. This study also offers new insight into the interaction between PEB and PBS complex in Synechococcus 7942. In agreement with previous reports 11 , 12 , 13 , our results support that CpcA has a higher propensity to associate with PEB than CpcB within cyanobacterial cells. Under conditions that induced modest PEB accumulation in the AB1 and S1 strains (5 µM IPTG), PEB binding was detected not only in CpcA but also in CpcB, indicating that incorporation into CpcB can occur once intracellular PEB levels exceed a certain threshold. Notably, this condition was accompanied by a reduction in PBS complex size, consistent with dissociation of the outermost rod disc. Although the mechanistic sequence remains unclear, these observations suggest that altered bilin occupancy may influence PBS stability and organization. Collectively, these findings refine our understanding of PBS assembly by highlighting the interplay between bilin loading, phycobiliprotein, and linker-dependent structural maintenance. Furthermore, the intracellular state during PEB accumulation was investigated through transcriptome analysis. Although FDBRs consume electrons from reduced ferredoxin, expression of heterologous FDBRs unexpectedly resulted in transcriptomic signatures indicative of an over-reduced state, likely due to altered electron distribution. Significant changes in gene expression were observed, including upregulation of genes involved in glucose metabolism, HoxS hydrogenase components, and potassium transporters, correlating with PEB accumulation levels. Additionally, fluctuations were noted in the expression of proteins related to gene expression regulation, such as SigC, Rbp1, EF-G, and KaiBC. These results suggest that these genes may contribute to mitigating the abnormal intracellular redox state induced by PEB accumulation. Further in-depth analysis of the transcriptome data is expected to provide insights into the mechanisms underlying PBS complex disassembly induced by PEB accumulation and to guide the engineering of PBS complexes better adapted to green light. In addition, bilin may influence regulatory pathways beyond redox homeostasis. One possibility is the modulation of gene expression through binding to the GAF domain of CBCR-type photoreceptors 14 , 15 . In Synechococcus 7942, two proteins with pigment-binding GAF domains have been identified: the PixJ homologue (Synpcc7942_0858) and a protein (Synpcc7942_2534) containing both a GAF domain and a GGDEF cyclic-di-GMP synthesis motif. In the closely related species Synechococcus 3055, PixJ functions in phototaxis 16 ; however, Synechococcus 7942 does not exhibit phototaxis, and no significant changes in phototaxis-related gene expression were observed even under conditions of PEB accumulation. This suggests that bilin-mediated signaling in Synechococcus 7942 may operate through regulatory mechanisms distinct from phototaxis, potentially linking bilin accumulation to broader cellular responses rather than behavioral outputs. Together, these findings point to a previously unknown connection between bilin biosynthesis, redox balance, and cellular signaling, highlighting the need to disentangle both the metabolic and regulatory effects of PEB accumulation to enable the rational engineering of PBS functionality in the future. METHODS Except for the transcriptome analysis described below, all other experiments were performed largely as described in our previous study 13 , with minor modifications. Culture conditions for cyanobacteria The cyanobacterium Synechococcus 7942 WT strain and its derivatives were grown photoautotrophically at 30°C under continuous WL illumination (60 µmol photons m − 2 ·s − 1 ). Cells were cultured in a modified BG-11 medium containing double the usual amount of sodium nitrate (final concentration = 35.3 mM) and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–KOH (pH 8.2) with continuous bubbling of 2% CO 2 . When required, spectinomycinwas added to the medium at a final concentration of 40 µg·mL − 1 . Growth experiments under GL conditions were performed using a Multi-Cultivator MC1000-OD MIX (Photon Systems Instruments, Czech Republic) with continuous GL illuminations (530 nm; 60 µmol photons m − 2 ·s − 1 ) and bubbling of ambient air. After pre-incubation on a BG-11 agar plates for 1 week, cells were harvested and inoculated into liquid BG-11 medium containing IPTG at an initial OD 720 of 0.05, followed by cultivation for 1 week. Growth was monitored by measuring OD 720 over 7 days (168 h) and approximate growth curves were constructed from these values. All measurements were performed in triplicate, and growth rates were compared based on the slope of the growth curves. Statistical significance was evaluated using a paired t -test in Microsoft Excel. Strain construction DNA fragments were amplified using KOD One DNA polymerase (TOYOBO, Japan) and subcloned into the vector plasmids using the In-Fusion HD Cloning Kit (TaKaRa, Japan). Codon-optimized FDBR genes, pcyX and pebS , which were designed and synthesized by Eurofins Genomics (Ebersberg, Germany), were amplified using specific primer sets (F1/R2 for pcyX and F3/R4 for pebS , Supplementary Table S2 and S3). The amplified fragments were cloned into the pBNS vector, which had been amplified using the primer set (F5/R6), generating the plasmids pBNS-pcyX and pBNS-pebS, respectively. To examine the effects of PEB synthesis using an identical expression construct, the pebA - pebB operon in Synechococcus sp. PCC 7803 was amplified using primer set F7 and R8 and cloned into the same vector backbone to generate pBNS-pebAB. These plasmids were introduced into Synechococcus 7942, and spectinomycin-resistant transformants were obtained. Successful transformation of strains with plasmids was confirmed by PCR amplification using specific primer sets (F9/R10) and the resulting strain was named Synechococcus 7942 X1, S1, and AB1, respectively. Analysis of phycobiliproteins Cells were harvested and resuspended in 100 µL of A buffer (10% glycerol, 100 mM NaCl, 20 mM HEPES–NaOH; pH 7.5) and then disrupted with glass beads using a bead beater (Micro Smash MS-100R, TOMY Seiko Co., Tokyo, Japan). After the centrifugation at 20,000× g for 1 min at 18°C, 60 µL of the supernatant was transferred to a fresh tube and mixed with 240 µL of acetone (final concentration = 80%) to remove chlorophyll and carotenoids from the cell lysates. The mixture was centrifuged again at 20,000× g for 1 min, yielding pellets enriched in phycobiliproteins. Bilins binding to apoproteins was analyzed by SDS-PAGE. The phycobilin pellets were resuspended and 20 µL of each sample was mixed with loading buffer (final concentration = 0.0625 Tris-HCl, 10% glycerol, 2% SDS, and 0.01% bromophenol blue) and separated on a 15% (w/v) polyacrylamide gel. After electrophoresis, gel bands were excised and extracted using ATTOPREP MF (ATTO, Tokyo, Japan), and the absorption spectra of the extracted phycobiliproteins were measured. Isolation of PBSs by SDG centrifugation Synechococcus 7942 cultures (OD 750 = 1.0; 30 mL) were harvested by centrifugation at 3,000× g for 10 min at 25°C. The cell pellets were washed once with 1 mL of 0.6 M potassium phosphate (KP) buffer (pH 7.0), centrifuged again under the same conditions, and stored at − 80°C until analysis. For PBS isolation, the cells were thawed, washed twice with 0.6 M KP buffer, and resuspended in 0.6 mL of the same buffer. Cells were disrupted by vortexing with glass beads, and PBS complexes were extracted from thylakoid membranes by incubation with Triton X-100 (final concentration = 2%) for 15 min with gentle voltexing. After centrifugation at 20,000 × g for 20 min at 18°C, 200 µL of the supernatant was loaded onto linear sucrose density gradients (10–50% sucrose in 0.6 M KP buffer) prepared in 14 × 89 mm open-top thinwall ultraclear tubes (Beckman Coulter, CA, USA) using a Gradient Master. The gradients were centrifuged at 154,300 × g for 18 h at 18°C using SW41Ti rotor in an Optima XE-90 ultracentrifuge (Beckman Coulter). Spectrometry Absorption spectra of whole cells, isolated phycobiliproteins, and PBS complexes were measured at 25°C using a spectrophotometer (UV-1800, Shimadzu, Japan) or a multimode plate reader (VICTOR Nivo, PerkinElmer). Fluorescence excitation spectra were measured with emission monitored at 685 nm to detect APC fluorescence using a spectrophotometer (FP-8200, JASCO, Japan). Transcriptome analysis Cells were inoculated into liquid BG-11 medium at an initial optical density (O.D. 750 ) of 0.05, supplemented with 5 µM IPTG, and cultivated for 4 days under continuous illumination at 60 µmol photons m⁻² s⁻¹ with 2% CO₂ bubbling. Total RNA was extracted from exponentially growing cultures of Synechococcus 7942 wild-type and engineered strains expressing pebA – pebB (AB1), pcyX (X1), or pebS (S1) as described previously 30 . Ribosomal RNA was removed using NEBNext rRNA Depletion Kit (Bacteria) (New England Biolabs). Sequencing libraries with an average insert size of approximately 200 bp were prepared according to the manufacturer's instructions using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). A total of 12 libraries (three biological replicates per strains) were sequenced on the Illumina NextSeq 1000 platform. Raw reads were trimmed and quality-filtered using CLC Genomics Workbench ver. 25.0.2 (QIAGEN, Hilden, Germany). Trimmed reads were mapped to the all genes in Synechococcus 7942 (accession number: CP000100, CP000101) as well as to plasmids carrying FDBR (pBNS_pebAB, pBNS_pebS, and pBNS_pcyX, Supporting information). Gene expression levels were normalized and quantified as transcripts per million (TPM). Differentially expressed genes (DEGs) were identified by pairwise comparisons between WT and each engineered strain (WT vs AB1, WT vs X1, and WT vs S1). Venn diagrams were generated to visualize the overlap of DEGs among comparisons, and volcano plots were used to display the magnitude and statistical significance of expression changes. TPM values of selected genes were plotted to compare expression patterns among the strains. The sequencing data underlying this study are available in the DDBJ Sequence Read Archive (DRA/SRA) under accession numbers DRR803588–DRR803599, within BioProject PRJDB39746. Abbreviations PBS phycobilisome PC phycocyanin PE phycoerythrin PEC phycoerythrocyanin APC allophycocyanin FDBR ferredoxin-dependent bilin reductase PCB phycocyanobilin PEB phycoerythrobilin PSII photosystem II PSI photosystem I NS neutral site IPTG isopropyl ß-D-1-thiogalactopyranoside SDG sucrose density gradient WL white light GL green light Declarations AUTHOR INFORMATION Corresponding Author Satoru Watanabe - Department of Bioscience, Tokyo University of Agriculture , Tokyo 156-8502, Japan ; orcid.org/0000-0001-7456-5053; Phone: +81-3-5477-2375; Email: [email protected] Authors Mizuho Sato - Department of Bioscience, Tokyo University of Agriculture , Tokyo 156-8502, Japan Mai Watanabe - Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan Kaisei Maeda - Laboratory for Chemistry and Life Science, Institute of Integrated Research, Institute of Science Tokyo, Yokohama 226-8503 , Japan Kaori Nimura-Matsune - Department of Bioscience, Tokyo University of Agriculture , Tokyo 156-8502, Japan Masahiko Ikeuchi - G raduate School of Arts and Sciences, University of Tokyo, Tokyo 153-0041, Japan Rei Narikawa - Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan Author contributions R.N. and S.W. designed the concept and the experiments of this study; M.S., K.N., K.M. and M.W. performed the experiments; M.W., K.N., R.N., M.I. and S.W. analyzed the data; M.W., R.N., M.I. and S.W. wrote the manuscript. ACKNOWLEDGMENTS We are grateful to Prof. Taku Chibazakura and Prof. Kei Asai for their valuable comments on the concept of this study. DATA AVILABILITY The RNA-seq data generated in this study have been deposited in the DRA/SRA database at the DNA Data Bank of Japan under accession numbers DRR803588-DRR803599 (BioProject PRJDB39746). All other data supporting the findings of this study are available from the corresponding authors upon reasonable request. FUNDINGS This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan to SW (23H02130, 24H00869, and 24H00871), New Energy and Industrial Technology Development Organization (NEDO, JPNP17005) (to S.W.) , and Cooperative Research Grant of the Genome Research for BioResource from NODAI Genome Research Center, Tokyo University of Agriculture to M.W. References Adir, N., Bar-Zvi, S. & Harris, D. The amazing phycobilisome. Biochim. Biophys. Acta Bioenerg . 1861 , 148047 (2020). Grossman, A. R., Schaefer, M. R., Chiang, G. G. & Collier, J. L. The phycobilisome, a light-harvesting complex responsive to environmental conditions. Microbiol. Rev. 57 , 725–749 (1993). Dominguez-Martin, M. A. et al. Structures of a phycobilisome in light-harvesting and photoprotected states. Nature 609 , 835–845 (2022). Hirose, Y. et al. Diverse Chromatic Acclimation Processes Regulating Phycoerythrocyanin and Rod-Shaped Phycobilisome in Cyanobacteria. Mol. Plant. 12 , 715–725 (2019). Sanchez-Baracaldo, P., Bianchini, G., Di Cesare, A., Callieri, C. & Chrismas, N. A. M. Insights Into the Evolution of Picocyanobacteria and Phycoerythrin Genes (mpeBA and cpeBA). Front. Microbiol. 10 , 45 (2019). Dammeyer, T., Bagby, S. C., Sullivan, M. B., Chisholm, S. W. & Frankenberg-Dinkel, N. Efficient phage-mediated pigment biosynthesis in oceanic cyanobacteria. Curr. Biol. 18 , 442–448 (2008). Frankenberg, N., Lagarias, J. C. & Phycocyanobilin Ferredoxin Oxidoreductase ofAnabaena sp. PCC 7120: BIOCHEMICAL AND SPECTROSCOPIC CHARACTERIZATION. J. Biol. Chem. 278 , 9219–9226 (2003). Frankenberg-Dinkel, N. & Terry, M. J. Synthesis and role of bilins in photosynthetic organisms. In: Tetrapyrroles: Birth, life and death ). Springer (2009). Ledermann, B., Beja, O. & Frankenberg-Dinkel, N. New biosynthetic pathway for pink pigments from uncultured oceanic viruses. Environ. Microbiol. 18 , 4337–4347 (2016). Rockwell, N. C., Martin, S. S. & Lagarias, J. C. Elucidating the origins of phycocyanobilin biosynthesis and phycobiliproteins. Proc. Natl. Acad. Sci. U S A . 120 , e2300770120 (2023). Alvey, R. M., Biswas, A., Schluchter, W. M. & Bryant, D. A. Effects of modified Phycobilin biosynthesis in the Cyanobacterium Synechococcus sp. Strain PCC 7002. J. Bacteriol. 193 , 1663–1671 (2011). Heck, S. et al. Expanding the toolbox for phycobiliprotein assembly: phycoerythrobilin biosynthesis in Synechocystis. Physiol. Plant. 176 , e14137 (2024). Sato, M. et al. Functional Modification of Cyanobacterial Phycobiliprotein and Phycobilisomes through Bilin Metabolism Control. ACS Synth. Biol. 13 , 2391–2401 (2024). Ikeuchi, M. & Ishizuka, T. Cyanobacteriochromes: a new superfamily of tetrapyrrole-binding photoreceptors in cyanobacteria. Photochem. Photobiol Sci. 7 , 1159–1167 (2008). Rockwell, N. C., Lagarias, J. C. & Cyanobacteriochromes A Rainbow of Photoreceptors. Annu. Rev. Microbiol. 78 , 61–81 (2024). Yang, Y. et al. Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus. Proceedings of the National Academy of Sciences 115, E12378-E12387 (2018). Dammeyer, T. & Frankenberg-Dinkel, N. Insights into phycoerythrobilin biosynthesis point toward metabolic channeling. J. Biol. Chem. 281 , 27081–27089 (2006). Ledermann, B. et al. Evolution and molecular mechanism of four‐electron reducing ferredoxin‐dependent bilin reductases from oceanic phages. FEBS J. 285 , 339–356 (2018). Kalla, R., Bhalerao, R. P. & Gustafsson, P. Regulation of phycobilisome rod proteins and mRNA at different light intensities in the cyanobacterium Synechococcus 6301. Gene 126, 77–83 (1993). Zheng, Z. et al. The structure of phycobilisome with a bicylindrical core from the cyanobacterium Synechococcus elongatus PCC 7942. bioRxiv 04.28.650843, (2025). (2025). Alvey, R. M., Biswas, A., Schluchter, W. M. & Bryant, D. A. Attachment of noncognate chromophores to CpcA of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 by heterologous expression in Escherichia coli. Biochemistry 50 , 4890–4902 (2011). Cano, M. et al. Glycogen synthesis and metabolite overflow contribute to energy balancing in cyanobacteria. Cell. Rep. 23 , 667–672 (2018). Eckert, C. et al. Genetic analysis of the Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 reveals subunit roles in association, assembly, maturation, and function. J. Biol. Chem. 287 , 43502–43515 (2012). Hagemann, M. Molecular biology of cyanobacterial salt acclimation. FEMS Microbiol. Rev. 35 , 87–123 (2011). Hayashi, R., Sugita, C. & Sugita, M. The 5′ untranslated region of the rbp1 mRNA is required for translation of its mRNA under low temperatures in the cyanobacterium Synechococcus elongatus. Arch. Microbiol. 199 , 37–44 (2017). Imamura, S. & Asayama, M. Sigma factors for cyanobacterial transcription. Gene regulation and systems biology 3, GRSB. S (2009). (2090). Osanai, T., Ikeuchi, M. & Tanaka, K. Group 2 sigma factors in cyanobacteria. Physiol. Plant. 133 , 490–506 (2008). Ito, H. et al. Cyanobacterial daily life with Kai-based circadian and diurnal genome-wide transcriptional control in Synechococcus elongatus. Proceedings of the National Academy of Sciences 106, 14168–14173 (2009). Nishiyama, Y., Jimbo, H. & Murata, N. Elongation factors regulate the repair of photosystem II oxido-reductively. Trends Plant. Science , (2025). Watanabe, S. et al. Regulation of RNase E during the UV stress response in the cyanobacterium Synechocystis sp. PCC 6803. Mlife 2, 43–57 (2023). Additional Declarations No competing interests reported. Supplementary Files PcyXPebSAdditional251214.pdf Cite Share Download PDF Status: Published Journal Publication published 27 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 22 Dec, 2025 Submission checks completed at journal 18 Dec, 2025 First submitted to journal 18 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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03:24:35","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19213,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/7b0807a77fa0c95d6fb4eefd.png"},{"id":98726049,"identity":"82785be1-19af-4b96-a51b-b5fba8ab71a2","added_by":"auto","created_at":"2025-12-22 03:24:32","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31140,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/a26102fe02273a2beeb775d6.png"},{"id":98726042,"identity":"5ea10621-25b7-4302-bb98-ebdb7dd9b970","added_by":"auto","created_at":"2025-12-22 03:24:31","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114641,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/85e824aa6c9928cab79f40e7.png"},{"id":98726056,"identity":"11c8e994-ad64-402d-8b26-0dd330d1a6e6","added_by":"auto","created_at":"2025-12-22 03:24:33","extension":"xml","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94863,"visible":true,"origin":"","legend":"","description":"","filename":"268732b0f12d4182bb79bfddfa3cda2c1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/962469fd32005abae0171d46.xml"},{"id":98726077,"identity":"643bb9a9-b552-419c-a4fa-815007cac6ea","added_by":"auto","created_at":"2025-12-22 03:24:34","extension":"html","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107793,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/ccbf6caf6072dbae1df093a0.html"},{"id":98777138,"identity":"2e2e35a7-f14f-4f23-b070-349b581b11c3","added_by":"auto","created_at":"2025-12-22 12:25:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":347725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePCB and PEB metabolic pathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic overview of phycobilin biosynthesis from heme. In \u003cem\u003eSynechococcus\u003c/em\u003e 7942, biliverdin IXα can be further converted to phycocyanobilin by the ferredoxin-dependent bilin reductase PcyA. Some cyanobacteria possess PebA and PebB in addition to PcyA, which convert biliverdin IXα to PEB in a two-step reaction. Meanwhile, PcyX and PebS, found in metagenome and phage, are known to synthesize PEB independently.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/6764755fb643b2ad9146ebc6.jpg"},{"id":98726041,"identity":"af89d3f3-e76a-4078-a50c-47ceb8c01172","added_by":"auto","created_at":"2025-12-22 03:24:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1425365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of FDBR-expressing strains allowing control of PEB levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of the genomic construct of \u003cem\u003eSynechococcus \u003c/em\u003e7942 strains expressing FDBRs (\u003cem\u003epebA\u003c/em\u003e-\u003cem\u003epebB\u003c/em\u003e, \u003cem\u003epcyX\u003c/em\u003e, and \u003cem\u003epebS\u003c/em\u003e) in an IPTG-dependent manner. The genes encoding FDBR were placed under the IPTG-dependent \u003cem\u003etrc\u003c/em\u003e promoter and introduced into the neutral site 1 (NS1) of \u003cem\u003eSynechococcus \u003c/em\u003e7942 along with the spectinomycin resistance gene (\u003cem\u003eaad\u003c/em\u003e) and \u003cem\u003elacI\u003c/em\u003e. (B) Effect of PEB synthase expression. After culturing PEB synthase-expressing and wild-type strains for one week, the strains were diluted to OD\u003csub\u003e750\u003c/sub\u003e = 0.5 and further cultivated under white light (60 µmol photons m\u003csup\u003e−2\u003c/sup\u003e·s\u003csup\u003e−1\u003c/sup\u003e) with and without IPTG (C) Absorption spectrum of the cultures. The spectra were normalized at 680 nm. Closed arrowhead: peak at 560 nm and open arrowhead: peak at 625 nm. AB1, \u003cem\u003epebA\u003c/em\u003e and \u003cem\u003epebB\u003c/em\u003e co-expressing strain; X1, \u003cem\u003epcyX\u003c/em\u003e expressing strain; S1, \u003cem\u003epebS\u003c/em\u003e expressing strain; WT, wild type; IPTG, isopropyl ß-D-1-thiogalactopyranoside.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/3581cb230c849da67c3ea297.jpg"},{"id":98726043,"identity":"a9334df4-8174-49c4-ac31-827ffa50551a","added_by":"auto","created_at":"2025-12-22 03:24:31","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":675280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the properties of phycobilisome (PBS) complexes fractionated by sucrose density gradient (SDG) centrifugation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePBS fractions were isolated from cell extracts of cultured cells expressing FDBR, and their properties were compared. (A) Images of culture (upper) and PBS fraction after SDG centrifugation (lower). (B) Growth curve under white light (60 µmol photons m\u003csup\u003e−2\u003c/sup\u003e·s\u003csup\u003e−1\u003c/sup\u003e) with and without 5 µM IPTG. (C) Excitation spectra of the PBS fractions emitting allophycocyanin fluorescence (685 nm). Spectra of the bottom fraction containing mature PBS (bold line in A) normalized at 630 nm. Closed arrowheads: peak at 560 nm.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/ffa228861bb9c2f829effd82.jpg"},{"id":98726067,"identity":"99caadc5-6502-48b9-a90f-b1fd9ca23b32","added_by":"auto","created_at":"2025-12-22 03:24:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":559418,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding of PEB to apoproteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cultures were grown under white light with 5 µM IPTG and used for the analysis. (A) Coomassie brilliant blue-stained gel images of phycobiliproteins. The arrows indicate low- and high-molecular-weight phycobiliproteins, which primarily contain the phycocyanin α subunit (CpcA) and β subunit (CpcB), respectively; thus, the low-molecular-weight fraction is labelled CpcA and the high-molecular-weight fraction is labelled CpcB. (C) Absorption spectra of phycobiliproteins extracted gel shown in B. Each set of spectra was normalized to 584 nm.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/cf84993e10fef6c31301d550.jpg"},{"id":98777905,"identity":"e812b3d1-45e5-4d4a-bbb6-7810a389e530","added_by":"auto","created_at":"2025-12-22 12:28:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1141668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePromotion of growth under green light conditions through FDBR expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Growth curves of WT, X1 and PEB1 cultures. (B) Comparison of growth rate estimated based on the slopes of the growth curves. For statistical evaluation, \u003cem\u003ep\u003c/em\u003e-values were calculated using the paired \u003cem\u003et\u003c/em\u003e‐test in Microsoft Excel, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. (WT and X1)\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/6c53b5fba07a0f4c84bb4e21.jpg"},{"id":98726044,"identity":"09e60788-df16-4b64-a4aa-603fd478ddd1","added_by":"auto","created_at":"2025-12-22 03:24:31","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1547131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative transcriptomic analysis of Synechococcus elongatus PCC 7942 expressing different FDBRs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults of the comparison between WT and FDBR-expressing strains (AB1, X1, and S1). After 4 days cultivation under white light with 5 µM IPTG (for PEB expressing strain) and CO\u003csub\u003e2\u003c/sub\u003e bubbling, the cells were harvested and subjected for the RNAseq analysis. (A) Volcano plot of significantly deferentially expressed genes. Upregulated and downregulated genes are depicted in red and blue, respectively (log\u003csub\u003e2 \u003c/sub\u003eFC ≥ │1│, FDR \u003cem\u003ep\u003c/em\u003e ≤ 0.1). The X-axis corresponds to log\u003csup\u003e2\u003c/sup\u003e fold change, and the Y-axis displays −log\u003csup\u003e10\u003c/sup\u003e of an adjusted \u003cem\u003ep\u003c/em\u003e-value of FDR. (B) The expression levels of genes showing a significantly increased expression compared to the wild-type strain. Data were extracted from Supplementary Table S1 and presented in the graph.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/0ba56f9cb0047614c10d1fb9.jpg"},{"id":108437786,"identity":"6830ecd6-c57c-4a13-996e-077b58752035","added_by":"auto","created_at":"2026-05-04 16:03:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6036620,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/daab6d84-7202-4624-aa30-55619c4e513c.pdf"},{"id":98726047,"identity":"ae30c2ab-a68e-4591-8362-d5d1888718f5","added_by":"auto","created_at":"2025-12-22 03:24:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1316469,"visible":true,"origin":"","legend":"","description":"","filename":"PcyXPebSAdditional251214.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8355607/v1/0b1f2d739aa3e99aeb741a5f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Study of Phycoerythrobilin Synthases for Fine-Tuning Photosynthetic Light-Harvesting Complexes, Phycobilisomes","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePhotosynthesis, the process by which light energy is converted into chemical energy, is fundamental to sustaining life on Earth. Throughout evolution, photosynthetic organisms have developed diverse and optimized light-harvesting complexes to efficiently capture light energy and transfer excitation energy to the photosynthetic reaction centers within their ecological niches. Phycobilisomes (PBSs), found in cyanobacteria, eukaryotic red algae, and glaucophytes, are peripheral light-harvesting complexes attached to the stromal side of the thylakoid membrane. PBSs enhance photosynthetic efficiency by capturing light in the green to orange spectral regions, which are poorly absorbed by chlorophyll, and transferring this energy to photosystems for conversion into chemical energy \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStructurally, PBSs consist of rod and core subcomplexes composed mainly of phycobiliproteins and linker polypeptides. The rods comprise disk-like trimers, namely (αβ)\u003csub\u003e3\u003c/sub\u003e, which may further assemble as a hexamer ([αβ]\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e composed of several types of phycobiliproteins, such as phycocyanins (PCs), phycoerythrins (PEs) and phycoerythrocyanins (PECs) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The rods vary depending on the organism and can be made of PC only or a combination of PC and other phycobiliproteins such as PEs and PECs. The PBS core consists of a cylindrical structure composed of allophycocyanin (APC) with six PCBs per monomer. All units (disks within the rod and rod-to-core) are connected by linker proteins.\u003c/p\u003e \u003cp\u003eEach of the phycobiliprotein contains linear tetrapyrrole chromophores known as bilins that are covalently bound to cysteine residues within the apoproteins. The spectral properties of PBSs are primarily determined by the type and ratio of these bilins\u0026mdash;such as phycocyanobilin (PCB), phycoerythrobilin (PEB), phycourobilin (PUB), and phycoviolobilin (PVB)\u0026mdash;that define the wavelength of light absorption. The biosynthesis of bilins is catalyzed by ferredoxin-dependent bilin reductases (FDBRs), including PcyA, PebA, PebB, PebS, and PcyX \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. PcyA catalyzes the conversion of biliverdin IXα to PCB, whereas PebA and PebB function sequentially to produce PEB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). PebS and PcyX are single-component FDBRs that catalyze both reduction steps (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These enzymes have been identified not only in cyanobacteria but also in cyanophages and metagenomic datasets, suggesting that nature employs diverse enzymatic routes to fine-tune chromophore biosynthesis and adapt light-harvesting to different spectral environments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that the spectral properties of PBSs can be modified by engineering the biosynthetic pathways of bilins \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, achieving precise control over bilin composition and incorporation remains challenging. In \u003cem\u003eSynechococcus elongatus\u003c/em\u003e 7942 (\u003cem\u003eSynechococcus\u003c/em\u003e 7942), heterologous production of phycoerythrobilin (PEB) has been reported to inhibit cell growth \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In cyanobacteria, bilins are known to participate in signal transduction by binding to bilin-binding photoreceptors known as cyanobacteriochromes (CBCRs) \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These photoreceptors contain a GAF domain that covalently binds bilins and mediate light sensing and downstream signal transduction. For instance, in \u003cem\u003eSynechococcus elongatus\u003c/em\u003e UTEX 3055, PixJ, a bilin-binding photoreceptor, has been shown to regulate the expression of phototaxis-related genes \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Although \u003cem\u003eSynechococcus\u003c/em\u003e 7942 lacks motility and does not exhibit phototaxis, introducing PEB biosynthesis into this strain, which natively lacks PEB, could potentially interfere with endogenous signaling networks. Nevertheless, the physiological consequences of such perturbations remain poorly understood.\u003c/p\u003e \u003cp\u003eIn this study, we introduced PcyX and PebS into \u003cem\u003eSynechococcus elongatus\u003c/em\u003e PCC 7942 and systematically compared their contributions to PEB synthesis and PBS assembly. Transcriptome analysis revealed that the expression levels of the introduced \u003cem\u003epebA\u003c/em\u003e-\u003cem\u003epebB\u003c/em\u003e, \u003cem\u003epcyX\u003c/em\u003e, and \u003cem\u003epebS\u003c/em\u003e genes were comparable, yet the cellular responses to varying PEB accumulation differed. These included marked changes in genes related to glucose metabolism, hydrogenase complexes, potassium transporters, and regulatory factors such as sigma factors and circadian rhythm components, suggesting that cyanobacterial cells actively adjust redox balance and metabolic flow in response to bilin levels. Collectively, our findings reveal distinct activities between PcyX and PebS and demonstrate how these differences can be exploited to modulate light-harvesting capacity and promote growth under green-light conditions.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of cyanobacterial strains with controlled PEB levels\u003c/h2\u003e \u003cp\u003eThree types of ferredoxin-dependent bilin reductases (FDBRs) have been identified to date, each reported to exhibit distinct activities in \u003cem\u003eE. coli\u003c/em\u003e cells \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. To compare their activities directly in cyanobacteria, we constructed a series of \u003cem\u003eSynechococcus elongatus\u003c/em\u003e PCC 7942 strains expressing different FDBRs. The \u003cem\u003epebA\u0026ndash;pebB\u003c/em\u003e operon, \u003cem\u003epcyX\u003c/em\u003e, and \u003cem\u003epebS\u003c/em\u003e were placed under the IPTG-inducible \u003cem\u003etrc\u003c/em\u003e promoter and integrated into a neutral site (NS) on the \u003cem\u003eSynechococcus\u003c/em\u003e 7942 chromosome. The resulting strains were designated \u003cem\u003eSynechococcus\u003c/em\u003e 7942 AB1, X1, and S1, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing IPTG induction, all three strains exhibited a brownish coloration in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), although the extent of this change differed among strains. In particular, the X1 strain showed visibly weaker coloration compared with AB1 and S1 under the same conditions. Absorption spectra revealed a decrease in the 625-nm peak associated with phycocyanin (PC) and an increase in the 560-nm peak associated with phycoerythrobilin (PEB), both of which varied with IPTG concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Based on absorbance at 560 nm, the apparent activity of PcyX was lower than that of PebA\u0026ndash;PebB and PebS, and was estimated to be less than one-tenth under the tested culture conditions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparison of PBS complexes under low PEB induction\u003c/h3\u003e\n\u003cp\u003eIn a previous study, we demonstrated that the accumulation of small amounts of PEB in PBS complexes altered the PBS properties of \u003cem\u003eSynechococcus\u003c/em\u003e 7942 toward a green-light\u0026ndash;adapted type \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. To examine whether similar changes occur in the newly constructed strains, each strain was cultured with 5 \u0026micro;M IPTG, and the size and properties of PBS complexes were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Cell extracts from the wild type (WT) and the three FDBR-expressing strains were subjected to sucrose density gradient (SDG) centrifugation to separate intact PBS complexes from their components. In the AB1 and S1 strains, the fraction corresponding to full-length PBS complexes, typically detected at the bottom of the gradient, shifted toward an upper position (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, solid line), indicating a reduction in complex size. In addition, red-colored fractions appeared in the upper region of the gradient (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, dashed line), consistent with disassembled phycobiliproteins reported previously \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. No notable differences in growth were observed under white-light conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), suggesting that the reduction in PBS complex size does not substantially affect cell proliferation under these culture conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe excitation spectra of 685-nm fluorescence emitted by APC in PBS cores showed that the PBS complexes from the FDBR-expressing strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, solid line) exhibited an additional peak near 560 nm, corresponding to PE, in addition to the 620-nm peak associated with PC. The 560-nm peak was most pronounced in AB1, followed by S1 and X1, consistent with the cellular absorption spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These observations indicate that PEB accumulates within the PBS complexes under low-level FDBR induction and that partial degradation and size reduction of the PBS complexes occur in the AB1 and S1 strains. The relative levels of the 560-nm peak further suggest that the apparent activities of the FDBRs in \u003cem\u003eSynechococcus\u003c/em\u003e 7942 follow the order PebA\u0026ndash;PebB\u0026thinsp;\u0026gt;\u0026thinsp;PebS\u0026thinsp;\u0026gt;\u0026thinsp;PcyX.\u003c/p\u003e \u003cp\u003eTo investigate the basis of the size reduction in PEB-containing PBS complexes, proteins from WT and AB1 PBS fractions were fluorescently labeled and analyzed by SDS\u0026ndash;PAGE (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Comparison of the fluorescence intensities of rod\u0026ndash;rod and rod\u0026ndash;core linker proteins showed a reduction in the 30-kDa rod\u0026ndash;rod linker protein relative to other linker proteins in AB1 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). As the 30-kDa rod\u0026ndash;rod linker has been reported to occupy the outermost position in the PBS rod and to be among the first components affected by changes in light conditions \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, these results suggest that PEB accumulation may promote dissociation of the outer rod disc, contributing to PBS size reduction.\u003c/p\u003e \u003cp\u003eSince PEB has been reported to bind to apoproteins of PCB-type PBSs \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, we next examined the phycobiliproteins under conditions of PEB accumulation. Protein extracts were prepared from cells cultured with 5 \u0026micro;M IPTG for FDBR induction and analyzed by SDS\u0026ndash;PAGE. Two colored bands were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), corresponding to the lower- and higher-molecular-weight disc components CpcA and CpcB, respectively. In the AB1 and S1 strains, both the CpcA and CpcB bands exhibited a red coloration, whereas in the X1 strain, red coloration was detected only in the CpcA band.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProteins from each band were extracted, and their absorption spectra were compared with those of the WT. In AB1 and S1, both the CpcA and CpcB extracts showed a shift toward shorter wavelengths, while in X1, a shift was observed only for CpcA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The CpcA bands appeared visually redder than the corresponding CpcB bands, and the absorption peak of the CpcA extracts was markedly higher in all three FDBR-expressing strains. These results suggest that, at the chromophore-binding sites of CpcA, PEB is bound preferentially over PCB, consistent with previous reports \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eGrowth advantage of FDBR-expressing strain under green light\u003c/h3\u003e\n\u003cp\u003eBecause the PBSs of the FDBR-expressing strains accumulated PEB and were able to absorb green light, we next evaluated whether this conferred a growth advantage under green-light illumination. The growth of the AB1 and X1 strains was compared with that of the WT. Under white-light conditions, no notable differences were observed among the strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, under green-light conditions, both AB1 and X1 exhibited faster growth than the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Analysis of growth rates based on the slopes of the growth curves indicated that X1 showed a significantly higher growth rate than WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These results indicate that PEB accumulation is associated with enhanced proliferation under green light, and further suggest that proliferation is promoted when the PBS complex is maintained in a nearly complete state, consistent with our previous observations \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEffects of PEB accumulation on gene expression\u003c/h3\u003e\n\u003cp\u003eBecause bilins function not only in light harvesting but also in the regulation of gene expression through binding to CBCR photoreceptors, changes in bilin metabolism are expected to elicit broad transcriptional responses. To evaluate the effects of PEB accumulation on gene expression in \u003cem\u003eSynechococcus\u003c/em\u003e 7942, we performed RNA-seq analysis using the WT strain and three FDBR-expressing strains. Total RNA was extracted from cultures grown under white light for 4 days in the presence of 5 \u0026micro;M IPTG (for strains AB1, S1, and X1) and subjected to sequencing analysis. The TPM (transcripts per million) values, fold ratios, and FDR-adjusted p-values for the WT and each FDBR-expressing strain are summarized in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, and the resulting volcano plots are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA (without ORF IDs) and Supplementary Figure S2 (with ORF IDs).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the three FDBR-expressing strains, the expression levels of the heterologously introduced \u003cem\u003epebA\u003c/em\u003e\u0026ndash;\u003cem\u003epebB\u003c/em\u003e, \u003cem\u003epcyX\u003c/em\u003e, and \u003cem\u003epebS\u003c/em\u003e genes in \u003cem\u003eSynechococcus\u003c/em\u003e 7942 were comparable (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), indicating that the observed differences among strains are attributable to enzymatic activity rather than expression level. Relative to the WT strain, no significant changes were detected in the expression of genes encoding components of PSI, PSII, or the phycobilisome. However, 39 genes were commonly upregulated and 6 were commonly downregulated across all three FDBR-expressing strains (Supplementary Figure S3 and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). The commonly downregulated genes did not clearly cluster into a specific functional category, whereas the commonly upregulated genes included those associated with glucose metabolism, hydrogenase activity, potassium transport, and regulatory factors, such as sigma factors and circadian clock\u0026ndash;related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Importantly, the magnitude of these transcriptional changes was highest in AB1, moderate in S1, and lowest in X1, demonstrating that the extent of gene expression remodeling positively correlates with PEB accumulation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These findings indicate that increased PEB levels exert a stronger influence on transcriptional responses, leading to broader remodeling of cellular metabolism.\u003c/p\u003e \u003cp\u003eWe observed a coordinated upregulation of genes involved in glucose metabolism\u0026mdash;including 6-phosphogluconate dehydrogenase (6PG; Synpcc7942_0039), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Synpcc7942_0245), D-fructose 1,6-bisphosphatase (FBPase; Synpcc7942_2335), glucose-6-phosphate dehydrogenase (G6PDH; Synpcc7942_2334), pyruvate:ferredoxin (flavodoxin) oxidoreductase (PFOR; Synpcc7942_2384), and glycogen/α-glucan phosphorylase (Synpcc7942_0244)\u0026mdash;together with genes encoding the NAD-reducing hydrogenase HoxS subunits (Synpcc7942_2551, 2555\u0026ndash;2557). Because these genes are associated with cellular redox metabolism, their upregulation may reflect a cellular response to changes in redox balance, in which increased metabolic capacity could help utilize excess reducing equivalents and contribute to adjustment of the NADPH/NADH state and carbon flux distribution \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In addition, the induction of genes encoding a K⁺ transport system (Synpcc7942_1668\u0026ndash;1671) in the FDBR-expressing strains suggests that PEB accumulation is accompanied by transcriptional changes in ion transport pathways, potentially reflecting a response related to osmotic or ionic homeostasis \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe also detected increased expression of several genes associated with transcriptional, post-transcriptional, and translational regulation in response to PEB accumulation. The group 2 sigma factor SigC (Synpcc7942_1849) and the RNA-binding protein Rbp1 (Synpcc7942_1999), both known to respond to darkness and cold stress \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, showed elevated transcript levels. In addition, the genes encoding the circadian clock components KaiB (Synpcc7942_1217) and KaiC (Synpcc7942_1216) were upregulated in AB1 and S1, the strains with higher PEB levels, consistent with their roles in broad transcriptional regulation \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The gene encoding the translation elongation factor EF-G (Synpcc7942_2082) was also increased, aligning with previous reports that translation elongation factors contribute to PSII repair under oxidative stress conditions \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Overall, these observations indicate that PEB accumulation is accompanied by widespread changes in regulatory gene expression, potentially influencing cellular physiology through multiple regulatory layers.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn summary, our results clarify the relative behavior of the three FDBRs within cyanobacterial cells. When expressed under identical conditions, their apparent activities followed the order PebA\u0026ndash;PebB\u0026thinsp;\u0026gt;\u0026thinsp;PebS\u0026thinsp;\u0026gt;\u0026thinsp;PcyX. Given that PebA\u0026ndash;PebB are broadly conserved among cyanobacteria \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, this may reflect a higher degree of compatibility with the native cellular environment compared with PebS or PcyX (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). By contrast, PcyX\u0026mdash;originating from metagenome-derived sequence \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e\u0026mdash;exhibited lower activity, which may allow more gradual modulation of PEB levels. Our findings further indicate that effective adaptation of PBSs toward green-light utilization in \u003cem\u003eSynechococcus\u003c/em\u003e 7942 requires PEB accumulation at levels that maintain PBS integrity, and that PcyX can serve as a useful means to achieve such controlled accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Finally, the observation that PebS alone supported PEB accumulation comparable to PebA\u0026ndash;PebB highlights that both PebS and PcyX may represent practical tools for future synthetic biology applications.\u003c/p\u003e \u003cp\u003eThis study also offers new insight into the interaction between PEB and PBS complex in \u003cem\u003eSynechococcus\u003c/em\u003e 7942. In agreement with previous reports\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, our results support that CpcA has a higher propensity to associate with PEB than CpcB within cyanobacterial cells. Under conditions that induced modest PEB accumulation in the AB1 and S1 strains (5 \u0026micro;M IPTG), PEB binding was detected not only in CpcA but also in CpcB, indicating that incorporation into CpcB can occur once intracellular PEB levels exceed a certain threshold. Notably, this condition was accompanied by a reduction in PBS complex size, consistent with dissociation of the outermost rod disc. Although the mechanistic sequence remains unclear, these observations suggest that altered bilin occupancy may influence PBS stability and organization. Collectively, these findings refine our understanding of PBS assembly by highlighting the interplay between bilin loading, phycobiliprotein, and linker-dependent structural maintenance.\u003c/p\u003e \u003cp\u003eFurthermore, the intracellular state during PEB accumulation was investigated through transcriptome analysis. Although FDBRs consume electrons from reduced ferredoxin, expression of heterologous FDBRs unexpectedly resulted in transcriptomic signatures indicative of an over-reduced state, likely due to altered electron distribution. Significant changes in gene expression were observed, including upregulation of genes involved in glucose metabolism, HoxS hydrogenase components, and potassium transporters, correlating with PEB accumulation levels. Additionally, fluctuations were noted in the expression of proteins related to gene expression regulation, such as SigC, Rbp1, EF-G, and KaiBC. These results suggest that these genes may contribute to mitigating the abnormal intracellular redox state induced by PEB accumulation. Further in-depth analysis of the transcriptome data is expected to provide insights into the mechanisms underlying PBS complex disassembly induced by PEB accumulation and to guide the engineering of PBS complexes better adapted to green light.\u003c/p\u003e \u003cp\u003eIn addition, bilin may influence regulatory pathways beyond redox homeostasis. One possibility is the modulation of gene expression through binding to the GAF domain of CBCR-type photoreceptors \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eSynechococcus\u003c/em\u003e 7942, two proteins with pigment-binding GAF domains have been identified: the PixJ homologue (Synpcc7942_0858) and a protein (Synpcc7942_2534) containing both a GAF domain and a GGDEF cyclic-di-GMP synthesis motif. In the closely related species \u003cem\u003eSynechococcus\u003c/em\u003e 3055, PixJ functions in phototaxis \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e; however, \u003cem\u003eSynechococcus\u003c/em\u003e 7942 does not exhibit phototaxis, and no significant changes in phototaxis-related gene expression were observed even under conditions of PEB accumulation. This suggests that bilin-mediated signaling in \u003cem\u003eSynechococcus\u003c/em\u003e 7942 may operate through regulatory mechanisms distinct from phototaxis, potentially linking bilin accumulation to broader cellular responses rather than behavioral outputs.\u003c/p\u003e \u003cp\u003eTogether, these findings point to a previously unknown connection between bilin biosynthesis, redox balance, and cellular signaling, highlighting the need to disentangle both the metabolic and regulatory effects of PEB accumulation to enable the rational engineering of PBS functionality in the future.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003cp\u003eExcept for the transcriptome analysis described below, all other experiments were performed largely as described in our previous study \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, with minor modifications.\u003c/p\u003e\n \u003cp\u003eCulture conditions for cyanobacteria\u003c/p\u003e\n \u003cp\u003eThe cyanobacterium \u003cem\u003eSynechococcus\u003c/em\u003e 7942 WT strain and its derivatives were grown photoautotrophically at 30\u0026deg;C under continuous WL illumination (60 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Cells were cultured in a modified BG-11 medium containing double the usual amount of sodium nitrate (final concentration\u0026thinsp;=\u0026thinsp;35.3 mM) and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)\u0026ndash;KOH (pH 8.2) with continuous bubbling of 2% CO\u003csub\u003e2\u003c/sub\u003e. When required, spectinomycinwas added to the medium at a final concentration of 40 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eGrowth experiments under GL conditions were performed using a Multi-Cultivator MC1000-OD MIX (Photon Systems Instruments, Czech Republic) with continuous GL illuminations (530 nm; 60 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and bubbling of ambient air. After pre-incubation on a BG-11 agar plates for 1 week, cells were harvested and inoculated into liquid BG-11 medium containing IPTG at an initial OD\u003csub\u003e720\u003c/sub\u003e of 0.05, followed by cultivation for 1 week. Growth was monitored by measuring OD\u003csub\u003e720\u003c/sub\u003e over 7 days (168 h) and approximate growth curves were constructed from these values. All measurements were performed in triplicate, and growth rates were compared based on the slope of the growth curves. Statistical significance was evaluated using a paired \u003cem\u003et\u003c/em\u003e-test in Microsoft Excel.\u003c/p\u003e\n \u003cp\u003eStrain construction\u003c/p\u003e\n \u003cp\u003eDNA fragments were amplified using KOD One DNA polymerase (TOYOBO, Japan) and subcloned into the vector plasmids using the In-Fusion HD Cloning Kit (TaKaRa, Japan). Codon-optimized FDBR genes, \u003cem\u003epcyX\u003c/em\u003e and \u003cem\u003epebS\u003c/em\u003e, which were designed and synthesized by Eurofins Genomics (Ebersberg, Germany), were amplified using specific primer sets (F1/R2 for \u003cem\u003epcyX\u003c/em\u003e and F3/R4 for \u003cem\u003epebS\u003c/em\u003e, Supplementary Table S2 and S3). The amplified fragments were cloned into the pBNS vector, which had been amplified using the primer set (F5/R6), generating the plasmids pBNS-pcyX and pBNS-pebS, respectively. To examine the effects of PEB synthesis using an identical expression construct, the \u003cem\u003epebA\u003c/em\u003e-\u003cem\u003epebB\u003c/em\u003e operon in \u003cem\u003eSynechococcus\u003c/em\u003e sp. PCC 7803 was amplified using primer set F7 and R8 and cloned into the same vector backbone to generate pBNS-pebAB. These plasmids were introduced into \u003cem\u003eSynechococcus\u003c/em\u003e 7942, and spectinomycin-resistant transformants were obtained. Successful transformation of strains with plasmids was confirmed by PCR amplification using specific primer sets (F9/R10) and the resulting strain was named \u003cem\u003eSynechococcus\u003c/em\u003e 7942 X1, S1, and AB1, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAnalysis of phycobiliproteins\u003c/h3\u003e\n\u003cp\u003eCells were harvested and resuspended in 100 \u0026micro;L of A buffer (10% glycerol, 100 mM NaCl, 20 mM HEPES\u0026ndash;NaOH; pH 7.5) and then disrupted with glass beads using a bead beater (Micro Smash MS-100R, TOMY Seiko Co., Tokyo, Japan). After the centrifugation at 20,000\u0026times; \u003cem\u003eg\u003c/em\u003e for 1 min at 18\u0026deg;C, 60 \u0026micro;L of the supernatant was transferred to a fresh tube and mixed with 240 \u0026micro;L of acetone (final concentration\u0026thinsp;=\u0026thinsp;80%) to remove chlorophyll and carotenoids from the cell lysates. The mixture was centrifuged again at 20,000\u0026times; \u003cem\u003eg\u003c/em\u003e for 1 min, yielding pellets enriched in phycobiliproteins. Bilins binding to apoproteins was analyzed by SDS-PAGE. The phycobilin pellets were resuspended and 20 \u0026micro;L of each sample was mixed with loading buffer (final concentration\u0026thinsp;=\u0026thinsp;0.0625 Tris-HCl, 10% glycerol, 2% SDS, and 0.01% bromophenol blue) and separated on a 15% (w/v) polyacrylamide gel. After electrophoresis, gel bands were excised and extracted using ATTOPREP MF (ATTO, Tokyo, Japan), and the absorption spectra of the extracted phycobiliproteins were measured.\u003c/p\u003e\n\u003ch3\u003eIsolation of PBSs by SDG centrifugation\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eSynechococcus\u003c/em\u003e 7942 cultures (OD\u003csub\u003e750\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0; 30 mL) were harvested by centrifugation at 3,000\u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at 25\u0026deg;C. The cell pellets were washed once with 1 mL of 0.6 M potassium phosphate (KP) buffer (pH 7.0), centrifuged again under the same conditions, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. For PBS isolation, the cells were thawed, washed twice with 0.6 M KP buffer, and resuspended in 0.6 mL of the same buffer. Cells were disrupted by vortexing with glass beads, and PBS complexes were extracted from thylakoid membranes by incubation with Triton X-100 (final concentration\u0026thinsp;=\u0026thinsp;2%) for 15 min with gentle voltexing. After centrifugation at 20,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 min at 18\u0026deg;C, 200 \u0026micro;L of the supernatant was loaded onto linear sucrose density gradients (10\u0026ndash;50% sucrose in 0.6 M KP buffer) prepared in 14 \u0026times; 89 mm open-top thinwall ultraclear tubes (Beckman Coulter, CA, USA) using a Gradient Master. The gradients were centrifuged at 154,300 \u0026times; \u003cem\u003eg\u003c/em\u003e for 18 h at 18\u0026deg;C using SW41Ti rotor in an Optima XE-90 ultracentrifuge (Beckman Coulter).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eSpectrometry\u003c/h2\u003e\n \u003cp\u003eAbsorption spectra of whole cells, isolated phycobiliproteins, and PBS complexes were measured at 25\u0026deg;C using a spectrophotometer (UV-1800, Shimadzu, Japan) or a multimode plate reader (VICTOR Nivo, PerkinElmer). Fluorescence excitation spectra were measured with emission monitored at 685 nm to detect APC fluorescence using a spectrophotometer (FP-8200, JASCO, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eTranscriptome analysis\u003c/h2\u003e\n \u003cp\u003eCells were inoculated into liquid BG-11 medium at an initial optical density (O.D. \u003csub\u003e750\u003c/sub\u003e) of 0.05, supplemented with 5 \u0026micro;M IPTG, and cultivated for 4 days under continuous illumination at 60 \u0026micro;mol photons m⁻\u0026sup2; s⁻\u0026sup1; with 2% CO₂ bubbling. Total RNA was extracted from exponentially growing cultures of \u003cem\u003eSynechococcus\u003c/em\u003e 7942 wild-type and engineered strains expressing \u003cem\u003epebA\u003c/em\u003e\u0026ndash;\u003cem\u003epebB\u003c/em\u003e (AB1), \u003cem\u003epcyX\u003c/em\u003e (X1), or \u003cem\u003epebS\u003c/em\u003e (S1) as described previously \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Ribosomal RNA was removed using NEBNext rRNA Depletion Kit (Bacteria) (New England Biolabs). Sequencing libraries with an average insert size of approximately 200 bp were prepared according to the manufacturer\u0026apos;s instructions using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). A total of 12 libraries (three biological replicates per strains) were sequenced on the Illumina NextSeq 1000 platform. Raw reads were trimmed and quality-filtered using CLC Genomics Workbench ver. 25.0.2 (QIAGEN, Hilden, Germany). Trimmed reads were mapped to the all genes in \u003cem\u003eSynechococcus\u003c/em\u003e 7942 (accession number: CP000100, CP000101) as well as to plasmids carrying FDBR (pBNS_pebAB, pBNS_pebS, and pBNS_pcyX, Supporting information). Gene expression levels were normalized and quantified as transcripts per million (TPM). Differentially expressed genes (DEGs) were identified by pairwise comparisons between WT and each engineered strain (WT vs AB1, WT vs X1, and WT vs S1). Venn diagrams were generated to visualize the overlap of DEGs among comparisons, and volcano plots were used to display the magnitude and statistical significance of expression changes. TPM values of selected genes were plotted to compare expression patterns among the strains. The sequencing data underlying this study are available in the DDBJ Sequence Read Archive (DRA/SRA) under accession numbers DRR803588\u0026ndash;DRR803599, within BioProject PRJDB39746.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003cbr\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephycobilisome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephycocyanin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephycoerythrin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephycoerythrocyanin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAPC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eallophycocyanin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFDBR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eferredoxin-dependent bilin reductase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephycocyanobilin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePEB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephycoerythrobilin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSII\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephotosystem II\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephotosystem I\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneutral site\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPTG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eisopropyl \u0026szlig;-D-1-thiogalactopyranoside\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esucrose density gradient\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewhite light\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egreen light\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSatoru Watanabe -\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eDepartment of Bioscience,\u003c/em\u003e\u003cem\u003e\u0026nbsp;Tokyo University of Agriculture\u003c/em\u003e\u003cem\u003e, Tokyo 156-8502, Japan\u003c/em\u003e; orcid.org/0000-0001-7456-5053; Phone: +81-3-5477-2375; Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMizuho Sato - \u003cem\u003eDepartment of Bioscience,\u003c/em\u003e\u003cem\u003e\u0026nbsp;Tokyo University of Agriculture\u003c/em\u003e\u003cem\u003e, Tokyo 156-8502, Japan\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMai Watanabe - \u003cem\u003eDepartment of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eKaisei Maeda - \u003cem\u003eLaboratory for Chemistry and Life Science,\u0026nbsp;\u003c/em\u003e\u003cem\u003eInstitute of Integrated Research, Institute of Science Tokyo, Yokohama\u0026nbsp;\u003c/em\u003e\u003cem\u003e226-8503\u003c/em\u003e\u003cem\u003e, Japan\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eKaori Nimura-Matsune - \u003cem\u003eDepartment of Bioscience,\u003c/em\u003e\u003cem\u003e\u0026nbsp;Tokyo University of Agriculture\u003c/em\u003e\u003cem\u003e, Tokyo 156-8502, Japan\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMasahiko Ikeuchi -\u0026nbsp;G\u003cem\u003eraduate School of Arts and Sciences, University of Tokyo, Tokyo 153-0041, Japan\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRei Narikawa - \u003cem\u003eDepartment of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.N. and S.W. designed the concept and the experiments of this study; M.S., K.N., K.M. and M.W. performed the experiments; M.W., K.N., R.N., M.I. and S.W. analyzed the data; M.W., R.N., M.I. and S.W. wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Prof. Taku Chibazakura and Prof. Kei Asai for their valuable comments on the concept of this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-seq data generated in this study have been deposited in the DRA/SRA database at the DNA Data Bank of Japan under accession numbers DRR803588-DRR803599 (BioProject PRJDB39746). All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDINGS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan to SW (23H02130, 24H00869, and 24H00871), New Energy and Industrial Technology Development Organization (NEDO, JPNP17005) (to S.W.) , and Cooperative Research Grant of the Genome Research for BioResource from NODAI Genome Research Center, Tokyo University of Agriculture to M.W.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdir, N., Bar-Zvi, S. \u0026amp; Harris, D. The amazing phycobilisome. \u003cem\u003eBiochim. Biophys. Acta Bioenerg\u003c/em\u003e. \u003cb\u003e1861\u003c/b\u003e, 148047 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrossman, A. R., Schaefer, M. R., Chiang, G. G. \u0026amp; Collier, J. L. The phycobilisome, a light-harvesting complex responsive to environmental conditions. \u003cem\u003eMicrobiol. Rev.\u003c/em\u003e \u003cb\u003e57\u003c/b\u003e, 725\u0026ndash;749 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDominguez-Martin, M. A. et al. Structures of a phycobilisome in light-harvesting and photoprotected states. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e609\u003c/b\u003e, 835\u0026ndash;845 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirose, Y. et al. Diverse Chromatic Acclimation Processes Regulating Phycoerythrocyanin and Rod-Shaped Phycobilisome in Cyanobacteria. \u003cem\u003eMol. Plant.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 715\u0026ndash;725 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanchez-Baracaldo, P., Bianchini, G., Di Cesare, A., Callieri, C. \u0026amp; Chrismas, N. A. M. Insights Into the Evolution of Picocyanobacteria and Phycoerythrin Genes (mpeBA and cpeBA). \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 45 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDammeyer, T., Bagby, S. C., Sullivan, M. B., Chisholm, S. W. \u0026amp; Frankenberg-Dinkel, N. Efficient phage-mediated pigment biosynthesis in oceanic cyanobacteria. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 442\u0026ndash;448 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrankenberg, N., Lagarias, J. C. \u0026amp; Phycocyanobilin Ferredoxin Oxidoreductase ofAnabaena sp. PCC 7120: BIOCHEMICAL AND SPECTROSCOPIC CHARACTERIZATION. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e278\u003c/b\u003e, 9219\u0026ndash;9226 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrankenberg-Dinkel, N. \u0026amp; Terry, M. J. Synthesis and role of bilins in photosynthetic organisms. In: \u003cem\u003eTetrapyrroles: Birth, life and death\u003c/em\u003e). Springer (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLedermann, B., Beja, O. \u0026amp; Frankenberg-Dinkel, N. New biosynthetic pathway for pink pigments from uncultured oceanic viruses. \u003cem\u003eEnviron. Microbiol.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 4337\u0026ndash;4347 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRockwell, N. C., Martin, S. S. \u0026amp; Lagarias, J. C. Elucidating the origins of phycocyanobilin biosynthesis and phycobiliproteins. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e120\u003c/b\u003e, e2300770120 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlvey, R. M., Biswas, A., Schluchter, W. M. \u0026amp; Bryant, D. A. Effects of modified Phycobilin biosynthesis in the Cyanobacterium Synechococcus sp. Strain PCC 7002. \u003cem\u003eJ. Bacteriol.\u003c/em\u003e \u003cb\u003e193\u003c/b\u003e, 1663\u0026ndash;1671 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeck, S. et al. Expanding the toolbox for phycobiliprotein assembly: phycoerythrobilin biosynthesis in Synechocystis. \u003cem\u003ePhysiol. Plant.\u003c/em\u003e \u003cb\u003e176\u003c/b\u003e, e14137 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato, M. et al. Functional Modification of Cyanobacterial Phycobiliprotein and Phycobilisomes through Bilin Metabolism Control. \u003cem\u003eACS Synth. Biol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 2391\u0026ndash;2401 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIkeuchi, M. \u0026amp; Ishizuka, T. Cyanobacteriochromes: a new superfamily of tetrapyrrole-binding photoreceptors in cyanobacteria. \u003cem\u003ePhotochem. Photobiol Sci.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 1159\u0026ndash;1167 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRockwell, N. C., Lagarias, J. C. \u0026amp; Cyanobacteriochromes A Rainbow of Photoreceptors. \u003cem\u003eAnnu. Rev. Microbiol.\u003c/em\u003e \u003cb\u003e78\u003c/b\u003e, 61\u0026ndash;81 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, Y. et al. Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 115, E12378-E12387 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDammeyer, T. \u0026amp; Frankenberg-Dinkel, N. Insights into phycoerythrobilin biosynthesis point toward metabolic channeling. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e281\u003c/b\u003e, 27081\u0026ndash;27089 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLedermann, B. et al. Evolution and molecular mechanism of four‐electron reducing ferredoxin‐dependent bilin reductases from oceanic phages. \u003cem\u003eFEBS J.\u003c/em\u003e \u003cb\u003e285\u003c/b\u003e, 339\u0026ndash;356 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalla, R., Bhalerao, R. P. \u0026amp; Gustafsson, P. Regulation of phycobilisome rod proteins and mRNA at different light intensities in the cyanobacterium Synechococcus 6301. \u003cem\u003eGene\u003c/em\u003e 126, 77\u0026ndash;83 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, Z. et al. The structure of phycobilisome with a bicylindrical core from the cyanobacterium Synechococcus elongatus PCC 7942. \u003cem\u003ebioRxiv\u003c/em\u003e 04.28.650843, (2025). (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlvey, R. M., Biswas, A., Schluchter, W. M. \u0026amp; Bryant, D. A. Attachment of noncognate chromophores to CpcA of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 by heterologous expression in Escherichia coli. \u003cem\u003eBiochemistry\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 4890\u0026ndash;4902 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCano, M. et al. Glycogen synthesis and metabolite overflow contribute to energy balancing in cyanobacteria. \u003cem\u003eCell. Rep.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 667\u0026ndash;672 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEckert, C. et al. Genetic analysis of the Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 reveals subunit roles in association, assembly, maturation, and function. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e287\u003c/b\u003e, 43502\u0026ndash;43515 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHagemann, M. Molecular biology of cyanobacterial salt acclimation. \u003cem\u003eFEMS Microbiol. Rev.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e, 87\u0026ndash;123 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayashi, R., Sugita, C. \u0026amp; Sugita, M. The 5\u0026prime; untranslated region of the rbp1 mRNA is required for translation of its mRNA under low temperatures in the cyanobacterium Synechococcus elongatus. \u003cem\u003eArch. Microbiol.\u003c/em\u003e \u003cb\u003e199\u003c/b\u003e, 37\u0026ndash;44 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImamura, S. \u0026amp; Asayama, M. Sigma factors for cyanobacterial transcription. \u003cem\u003eGene regulation and systems biology\u003c/em\u003e 3, GRSB. S (2009). (2090).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsanai, T., Ikeuchi, M. \u0026amp; Tanaka, K. Group 2 sigma factors in cyanobacteria. \u003cem\u003ePhysiol. Plant.\u003c/em\u003e \u003cb\u003e133\u003c/b\u003e, 490\u0026ndash;506 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIto, H. et al. Cyanobacterial daily life with Kai-based circadian and diurnal genome-wide transcriptional control in Synechococcus elongatus. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 106, 14168\u0026ndash;14173 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishiyama, Y., Jimbo, H. \u0026amp; Murata, N. Elongation factors regulate the repair of photosystem II oxido-reductively. \u003cem\u003eTrends Plant. Science\u003c/em\u003e, (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatanabe, S. et al. Regulation of RNase E during the UV stress response in the cyanobacterium Synechocystis sp. PCC 6803. \u003cem\u003eMlife\u003c/em\u003e 2, 43\u0026ndash;57 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Phycobilisome, Cyanobacteria, Phycoerythrin, Phycocyanin, Photosynthesis","lastPublishedDoi":"10.21203/rs.3.rs-8355607/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8355607/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhycobilisomes serve as the major light-harvesting antenna complexes in cyanobacteria; they capture light and transfer excitation energy to the photosystems, and their spectral and functional properties are primarily determined by the types of bilin chromophores attached to phycobiliproteins. We previously revealed that the properties of phycobilisomes can be modulated by altering the biosynthesis of these bilin chromophores. In this study, we introduced two distinct phycoerythrobilin (PEB) biosynthetic enzymes, \u003cem\u003epcyX\u003c/em\u003e and \u003cem\u003epebS\u003c/em\u003e, into cyanobacterium \u003cem\u003eSynechococcus elongatus\u003c/em\u003e PCC 7942 and successfully enabled PEB incorporation into the native phycobilisome. PebS exhibited high PEB synthesis activity comparable to the canonical PebA-PebB pathway, whereas PcyX showed lower activity, enabling fine-tuning of PEB accumulation. Consistent with previous findings, moderate PEB supply enhanced growth under green light; notably, PcyX expression further promoted growth by providing a more balanced pigment supply. Transcriptome analysis confirmed comparable expression of \u003cem\u003epebA\u003c/em\u003e-\u003cem\u003epebB\u003c/em\u003e, \u003cem\u003epcyX\u003c/em\u003e, and \u003cem\u003epebS\u003c/em\u003e, and revealed that increased PEB accumulation triggered broad metabolic remodeling: genes for glucose metabolism, hydrogenase complexes, potassium transporters, and regulatory factors including sigma factors were upregulated, suggesting a shift toward dissipation of excess reducing power. These results indicate that natural diversity among bilin synthases can be exploited to engineer phycobilisome composition and redox homeostasis in cyanobacteria.\u003c/p\u003e","manuscriptTitle":"Comparative Study of Phycoerythrobilin Synthases for Fine-Tuning Photosynthetic Light-Harvesting Complexes, Phycobilisomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 03:24:21","doi":"10.21203/rs.3.rs-8355607/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-23T03:46:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-18T08:59:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-18T08:43:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52ea2513-5309-4ad5-bbb4-1aa7190c3749","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":59871565,"name":"Biological sciences/Biochemistry"},{"id":59871566,"name":"Biological sciences/Biophysics"},{"id":59871567,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-05-04T16:02:04+00:00","versionOfRecord":{"articleIdentity":"rs-8355607","link":"https://doi.org/10.1038/s41598-026-50582-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-27 15:57:54","publishedOnDateReadable":"April 27th, 2026"},"versionCreatedAt":"2025-12-22 03:24:21","video":"","vorDoi":"10.1038/s41598-026-50582-3","vorDoiUrl":"https://doi.org/10.1038/s41598-026-50582-3","workflowStages":[]},"version":"v1","identity":"rs-8355607","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8355607","identity":"rs-8355607","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}