Phycocyanobilin Research Articles

Optimization of Phycocyanobilin Synthesis in E. coli BL21: Biotechnological Insights and Challenges for Scalable Production.

Phycocyanobilin (PCB) is a small chromophore found in certain phycobiliproteins, such as phycocyanins (PCs) and allophycocyanins (APCs). PCB, along with other phycobilins (PBs) and intermediates such as biliverdin (BV) or phycoerythrobilin (PEB), is attracting increasing biotechnological interest due to its fluorescent and medicinal properties that allow potential applications in biomedicine and the food industry. This study aims to optimize PCB synthesis in Escherichia coli BL21 (DE3) and scale the process to a pre-industrial level. Parameters such as optimal temperature, inducer concentration, initial OD600, and stirring speed were analyzed in shake flask cultures to maximize PCB production. The best results were obtained at a temperature of 28 °C, an IPTG concentration of 0.1 mM, an initial OD600 of 0.5, and an orbital shaking speed of 260 rpm. Furthermore, the optimized protocol was scaled up into a 2 L bioreactor batch, achieving a maximum PCB concentration of 3.8 mg/L. Analysis of the results revealed that biosynthesis of exogenous PBs in Escherichia coli BL21 (DE3) is highly dependent on the metabolic burden of the host. Several scenarios, such as too rapid growth, high inducer concentration, or mechanical stress, can advance entry into the stationary phase. That progressively halts pigment synthesis, leading, in some cases, to its excretion into the growth media and ultimately triggering rapid degradation by the host. These conclusions provide a promising protocol for scalable PCB production and highlight the main biotechnological challenges to increase the yields of the process.

Red/green cyanobacteriochromes acquire isomerization from phycocyanobilin to phycoviolobilin.

Cyanobacteriochromes (CBCRs) are unique cyanobacteria-specific photoreceptors that share a distant relation with phytochromes. Most CBCRs contain conserved cysteine residues known as canonical Cys, while some CBCRs have additional cysteine residues called second Cys within the DXCF motif, leading to their classification as DXCF CBCRs. They typically undergo a process where they incorporate phycocyanobilin (PCB) and subsequently isomerize it to phycoviolobilin (PVB). Conversely, CBCRs with conserved Trp residues and without the second Cys are called extended red/green (XRG) CBCRs. Typical XRG CBCRs bind PCB without undergoing PCB-to-PVB isomerization, displaying red/green reversible photoconversion, and there are also atypical CBCRs that exhibit diverse photoconversions. We discovered novel XRG CBCRs with Cys residue instead of the conserved Trp residue. These novel XRG CBCRs exhibited the ability to isomerize PCB to PVB, displaying green/teal reversible photoconversion. Through sequence- and structure-based comparisons coupled with mutagenesis experiments, we identified three amino acid residues, including the Cys residue, crucial for facilitating PCB-to-PVB isomerization. This research expands our understanding of the diversity of XRG CBCRs, highlighting the remarkable molecular plasticity of CBCRs.

Functional Modification of Cyanobacterial Phycobiliprotein and Phycobilisomes through Bilin Metabolism Control.

Phycobilisomes (PBSs) are light-harvesting antenna complexes in cyanobacteria that adapt to diverse light environments through the use of phycobiliproteins within the PBS structures. Freshwater cyanobacteria, such as Synechococcus elongatus PCC 7942, thrive under red light because of the presence of phycocyanin (PC) and its chromophore, phycocyanobilin (PCB), in the PBS. Cyanobacteria in shorter-wavelength light environments such as green light, employ phycoerythrin paired with phycoerythrobilin (PEB) along with PC in the PBS. Synthetic biology studies have shown that PEB production can be achieved by expression of the heterologous PEB synthases 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA) and PEB:ferredoxin oxidoreductase (PebB), leading to PEB accumulation and cellular browning. This approach is genetically unstable, and the properties of the resulting PEB-bound PBS complexes remain uncharacterized. In this study, we engineered a novel strain of Synechococcus 7942 PEB1 with finely tuned control of PEB biosynthesis. PEB1 exhibited a reversible change in the color of the culture from green to brown and pink based on PebA and PebB induction levels. High induction led to complete PCB-to-PEB substitution, causing the disassembly of the PBS rod complex. In contrast, low induction levels of PebA and PebB resulted in the formation of a stable chimeric PBS complex with partial PCB-to-PEB substitution. This acclimation enabled efficient light harvesting in the green spectrum and energy transfer to the photosynthetic reaction center. These findings, which improve our understanding of PBS and highlight the structural importance of the bilin composition, provide a foundation for future studies on PBS adaptation in bioengineering, synthetic biology, and renewable energy.

Phytochromobilin Binding and Specific Amino Acid Residues Near The Chromophore Contribute To Orange Light Perception By The Dualchrome Phytochrome Region.

A novel photoreceptor dualchrome 1 (DUC1), containing a fused structure of cryptochrome and phytochrome, was discovered in the marine green alga Pycnococcus provasolli. The DUC1 phytochrome region (PpDUC1-N) binds to the bilin (linear tetrapyrrole) chromophores, phytochromobilin (PΦB) or phycocyanobilin (PCB), and reversibly photoconverts between the orange-absorbing dark-adapted state and the far-red-absorbing photoproduct state. This contrasts with typical phytochromes, which photoconvert between the red-absorbing dark-adapted and far-red-absorbing photoproduct states. In this study, we examined the molecular mechanism of PpDUC1-N to sense orange light by identifying the chromophore species synthesized by P. provasolli and the amino acid residues within the PpDUC1-N responsible for sensing orange light in the dark-adapted state. We focused on the PcyA homolog of P. provasolli (PpPcyA). Coexpression with the photoreceptors followed by an enzymatic assay revealed that PpPcyA synthesized PCB. Next, we focused on the PpDUC1-N GAF domain responsible for chromophore binding and light sensing. Ten amino acid residues were selected as the mutagenesis target near the chromophore. Replacement of these residues with those conserved in typical phytochromes revealed that three mutations (F290Y/M304S/L353M) resulted in a 23-nm red-shift in the dark-adapted state. Finally, we combined these constructs to obtain the PΦB-binding F290Y/M304S/L353M mutant and a 38-nm red-shift was observed compared with the PCB-binding wild-type PpDUC1. The binding chromophore species and the key residues near the chromophore contribute to blue-shifted orange light sensing in the dark-adapted state of the PpDUC1-N.

Teal-light absorbing cyanobacterial phytochrome superfamily provides insights into the diverse mechanisms of spectral tuning and facilitates the engineering of photoreceptors for optogenetic tools

Cyanobacteriochromes (CBCRs) are distinctive tetrapyrrole (bilin)-binding photoreceptors exclusively found in cyanobacteria. Unlike canonical phytochromes, CBCRs require only a GAF (cGMP-phosphodiesterase/adenylate cyclase/FhlA) domain for autolyase activity to form a bilin adduct via a Cys residue and cis-trans photoisomerization. Apart from the canonical Cys, which attaches covalently to C31 in the A-ring of the bilin, some GAF domains of CBCRs contain a second-Cys in the Asp-Xaa-Cys-Phe (DXCF) motif, responsible for isomerization of phycocyanobilin (PCB) to phycoviolobilin (PVB) and/or for the formation of a reversible 2nd thioether linkage to the C10. Unlike green/teal-absorbing GAF proteins lacking ligation activity, the second-Cys in another teal-absorbing lineage (DXCF blue/teal group) exhibits both isomerization and ligation activity due to the presence of the Tyr instead of His next to the canonical Cys. Herein, we discovered an atypical CBCR GAF protein, Tpl7205g1, belonging to the DXCF blue/teal group, but having His instead of Tyr next to the first-Cys. Consistent with its subfamily, the second-Cys of Tpl7205g1 did not form a thioether linkage at C10 of PCB, showing only isomerization activity. Instead of forming 2nd thioether linkage, this novel GAF protein exhibits a pH-dependent photocycle between protonated 15Z and deprotonated 15E. Site-directed mutagenesis to the GAF scaffolds revealed its combined characteristics, including properties of teal-DXCF CBCRs and red/green-absorbing CBCRs (XRG CBCRs), suggesting itself as the evolutionary bridge between the two CBCR groups. Our study thus sheds light on the expanded spectral tuning characteristics of teal-light absorbing CBCRs and enhances feasibility of engineering these photoreceptors.

Fabrication and characterization of bovine serum albumin-phycocyanobilin conjugate: effect on antioxidant and ligand-binding properties.

Phycocyanobilin (PCB) is an open-chain blue tetrapyrrole chromophore of C-phycocyanin (C-PC), a major chromoprotein derived from the cyanobacterium Arthrospira platensis having numerous health-promoting effects. Relying on the ability of PCB to attach to the sulfhydryl group of proteins, we propose a new method for covalent attachment of PCB to bovine serum albumin (BSA) as a means of its functionalization. Traut's reagent (TR, 2-iminothiolane), modifying lysine residues, was used to optimize the introduction of sulfhydryl groups in BSA. A higher degree of BSA thiolation by TR induces more profound alterations of its structure, resulting in minor oligomerization and aggregation. A 50-fold molar excess of TR was found to be the optimal, balancing thiolation level and adverse effect on protein structure. PCB was covalently attached to newly introduced sulfhydryl groups at pH 9 at 20-fold PCB/BSA ratio. An increase in the TR/BSA molar ratio leads to increased efficiency of PCB conjugation with thiolated BSA. Compared to native BSA, BSA-PCB conjugate binds quercetin with similar affinity but has higher antioxidant activity and increased oxidative stability. PCB-modified BSA could serve as a stable, food-compatible carrier of bioactive PCB, but also bind other ligands that would be protected from oxidative damage due to the high antioxidant potential of covalently bound PCB. Thiolation by TR is, at the same time, a simple method for the covalent functionalization of virtually any protein by bioactive PCB or for obtaining PCB-based fluorescent probes. © 2024 Society of Chemical Industry.

Heterologous Expression of Phycocyanobilin in Escherichia coli and Determination of Its Antioxidant Capacity In Vitro.

Phycocyanobilin (PCB) is a blue pigment with antioxidant, anti-inflammatory, and anticancer properties. It is used in the medical and cosmetic industries. In this study, a high-expression plasmid, pET-30a-PCB, was constructed for expression of PCB in Escherichia coli BL21(DE3). The PCB was analyzed using UV-visible absorptionspectrum, MALDI-TOF-MS, and fluorescence spectra. The stability and half-life of PCB in different serum were determined. The yield of PCB was optimized through single-factor and orthogonal experiments. The optimal expression conditions were determined as a lactose concentration of 5mmol/L, an induction time of 8h, an induction temperature of 27°C, and an induction duration of 22h. PCB yield of 6.5mg/L was achieved and subsequently purified using nickel-affinity chromatography. The purified PCB was quantified indirectly using Hist-tag ELISA detection, and the concentration was 11.66μg/L. In the range of 0-33μg/mL, the total antioxidant capacity and reducing the capacity of PCB were stronger than Vitamin E (Ve), with 1,1-diphenyl-2-picrylhydrazil (DPPH) scavenging reaching up to 87.07%, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) free radical (ABTS) scavenging up to 100%, hydroxyl radicals (·OH) scavenging up to 64.19%, hydrogen peroxide (H2O2) scavenging up to 78.75%, This study provides theoretical evidence for PCB as a potent antioxidant.

Rational Design of Key Enzymes to Efficiently Synthesize Phycocyanobilin in Escherichia coli.

Phycocyanobilin (PCB) is a natural blue tetrapyrrole chromophore that is found in phycocyanin and plays an essential role in photosynthesis. Due to PCB's antioxidation, anti-inflammatory and anti-cancer properties, it has been utilized in the food, pharmaceutical and cosmetic industries. Currently, the extraction of PCB from Spirulina involves complex processes, which has led to increasing interest in the biosynthesis of PCB in Escherichia coli. However, the PCB titer remains low because of the poor activity of key enzymes and the insufficient precursor supply. Here, the synthesis of PCB was firstly improved by screening the optimal heme oxygenase (HO) from Thermosynechococcus elongatus BP-1(HOT) and PCB: ferredoxin oxidoreductase from Synechocystis sp. PCC6803 (PcyAS). In addition, based on a rational design and the infrared fluorescence method for high-throughput screening, the mutants of HOT(F29W/K166D) and PcyAS(D220G/H74M) with significantly higher activities were obtained. Furthermore, a DNA scaffold was applied in the assembly of HOT and PcyAS mutants to reduce the spatial barriers, and the heme supply was enhanced via the moderate overexpression of hemB and hemH, resulting in the highest PCB titer (184.20 mg/L) obtained in a 5 L fermenter. The strategies applied in this study lay the foundation for the industrial production of PCB and its heme derivatives.

Therapeutic effect of phycocyanin on chronic obstructive pulmonary disease in mice

IntroductionThe prevention and treatment of chronic obstructive pulmonary disease (COPD) is closely tied to antioxidation and anti-inflammation. Phycocyanin (PC) has numerous pharmacological effects, such as antioxidation and anti-inflammation. However, it remains unclear whether PC can play a therapeutic role in COPD. ObjectiveAs inflammation and oxidative stress can aggravate COPD, this study is to explore the effect of PC on COPD mice and its mechanisms. MethodsThe COPD mice model was established by exposing them to lipopolysaccharide (LPS) and cigarette smoke (CS); PC was administrated in a concentration of 50 mg/kg for 30 days. On the last day, lung function was measured, and bronchoalveolar lavage fluid (BALF) was obtained and classified for cells. Lung tissue pathological change was analyzed, and organ indices statistics were measured. Based on molecular docking, the mechanism was explored with Western blotting, immunohistochemical, and immunofluorescence in vivo and in vitro. ResultsPC significantly ameliorated the pulmonary function of COPD mice and reduced inflammation of the lung (p < 0.05), and hematoxylin and eosin (H&E) staining showed PC depressed lung inflammatory cell accumulation and emphysema. Periodic acid Schiff (PAS) and Masson staining revealed that PC retarded goblet cells metaplasia and collagen deposition (p < 0.05). In addition, in vivo PC regulated Heme oxygenase 1 (HO-1) (p < 0.05) and NAD(P)H dehydrogenase quinone 1 (NQO1) level (p < 0.01) in the lung, as well as NOX2 level in pulmonary macrophages. Molecular docking results indicate that phycocyanobilin (PCB) in PC had a good binding site in Keap1 and NOX2 proteins; the phycocyanobilin-bound phycocyanin peptide (PCB-PC-peptide) was obtained for further studies. In vitro, PCB-PC-peptide could depress the phospho-NF-E2-related factor 2 (p-Nrf2) and NQO1 protein expression in RAW264.7 cells induced by cigarette smoke extract (CSE) (p < 0.05). ConclusionPC exerts beneficial effects on COPD via anti-inflammatory and antioxidative stress, which may be achieved through PCB.

Phase Separation of Phytochrome B in HEK293T Cells.

In this method, we employed HEK293T cells to express the plant photoreceptor phytochrome B (phyB). Through the application of various treatments such as phycocyanobilin (PCB) supplementation, red light exposure, and temperature adjustments, the phyB proteins exhibited liquid-liquid phase separation, leading to the formation of biomolecular condensates. Here, we present a comprehensive description of the protein expression, cell treatment, and imaging capture procedures. This detailed guide provides step-by-step instructions on how to induce phase separation of phyB proteins in HEK293T cells. By utilizing this approach, researchers can investigate the physicochemical characteristics and dynamic formation process of phyB photobodies with precision.

Expanding the toolbox for phycobiliprotein assembly: phycoerythrobilin biosynthesis in Synechocystis

AbstractPhycobiliproteins (PBPs) play a vital role in light harvesting by cyanobacteria, which enables efficient utilization of photon energy for oxygenic photosynthesis. The PBPs carry phycobilins, open‐chain tetrapyrrole chromophores derived from heme. The structure and chromophore composition of PBPs is dependent on the organism's ecological niche. In cyanobacteria, these holo‐proteins typically form large, macromolecular antenna complexes called phycobilisomes (PBSs). The PBS of Synechocystis sp. PCC 6803 (hereafter Synechocystis) consists of allophycocyanin (APC) and phycocyanin (PC), which exclusively harbor phycocyanobilin (PCB) as a chromophore. Investigations into heterologous PBP biosynthesis in E. coli have proven limiting with respect to PBP assembly and their functional characterization. Consequently, we wanted to engineer a platform for the investigation of heterologously produced PBPs, focusing on unusual, phycoerythrobilin (PEB)‐containing light‐harvesting proteins called phycoerythrins (PEs) in Synechocystis. As a first step, a gene encoding for the synthesis of the natural cyanobacterial chromophore, PEB, was introduced into Synechocystis. We provide spectroscopic evidence for heterologous PEB formation and show covalent attachment of PEB to the α‐subunit of PC, CpcA, by HPLC and LC–MS/MS analyses. Fluorescence microscopy and PBS isolation demonstrate a cellular dispersal of PBPs with modified phycobilin content. However, these modifications have minor effects on physiological responses, as demonstrated by growth rates, oxygen evolution, nutrient accumulation, and PBP content analyses. As a result, Synechocystis demonstrates the capacity to efficiently manage PEB biosynthesis and therefore reflects a promising platform for both biochemical and physiological investigations of foreign and unusual PEs.

The effects of Phycocyanobilin on experimental arthritis involve the reduction in nociception and synovial neutrophil infiltration, inhibition of cytokine production, and modulation of the neuronal proteome.

The antinociceptive and pharmacological activities of C-Phycocyanin (C-PC) and Phycocyanobilin (PCB) in the context of inflammatory arthritis remain unexplored so far. In the present study, we aimed to assess the protective actions of these compounds in an experimental mice model that replicates key aspects of human rheumatoid arthritis. Antigen-induced arthritis (AIA) was established by intradermal injection of methylated bovine serum albumin in C57BL/6 mice, and one hour before the antigen challenge, either C-PC (2, 4, or 8 mg/kg) or PCB (0.1 or 1 mg/kg) were administered intraperitoneally. Proteome profiling was also conducted on glutamate-exposed SH-SY5Y neuronal cells to evaluate the PCB impact on this key signaling pathway associated with nociceptive neuronal sensitization. C-PC and PCB notably ameliorated hypernociception, synovial neutrophil infiltration, myeloperoxidase activity, and the periarticular cytokine concentration of IFN-γ, TNF-α, IL-17A, and IL-4 dose-dependently in AIA mice. In addition, 1 mg/kg PCB downregulated the gene expression for T-bet, RORγ, and IFN-γ in the popliteal lymph nodes, accompanied by a significant reduction in the pathological arthritic index of AIA mice. Noteworthy, neuronal proteome analysis revealed that PCB modulated biological processes such as pain, inflammation, and glutamatergic transmission, all of which are involved in arthritic pathology. These findings demonstrate the remarkable efficacy of PCB in alleviating the nociception and inflammation in the AIA mice model and shed new light on mechanisms underlying the PCB modulation of the neuronal proteome. This research work opens a new avenue to explore the translational potential of PCB in developing a therapeutic strategy for inflammation and pain in rheumatoid arthritis.

Phycocyanobilin is a new binding partner of human alpha-2-macroglobulin that protects the protein against oxidative stress

Under simulated physiological conditions, this study investigates the interaction between nutraceutical phycocyanobilin (PCB) and the universal anti-protease protein human alpha-2-macroglobulin (α2M). Extensive molecular docking analyses on multiple α2M conformations, spectroscopic techniques, and α2M activity assays were utilized to examine the complex formation. The results revealed that for every protein conformation, two high energy binding sites exist: the first, conformationally independent, at the interface region between two monomer chains and the second, conformationally dependent, in the pocket composed of amino acids from four distinct domains (TED, RBD, CUB, and MG2) of the single protein chain. Spectrofluorimetric measurements indicated a moderate affinity between α2M and PCB with a moderately high binding constant of 6.3 × 105 M−1 at 25 °C. The binding of PCB to α2M resulted in minor changes in the secondary structure content of α2M. Furthermore, PCB protected α2M from oxidation and preserved its anti-protease activity in the oxidative environment. These findings suggest that PCB binding could indirectly impact the body’s response to oxidative stress by influencing α2M's role in controlling enzyme activity during the inflammatory process. Communicated by Ramaswamy H. Sarma

On the Role of a Conserved Tryptophan in the Chromophore Pocket of Cyanobacteriochrome

The cyanobacteriochrome Slr1393 can be photoconverted between a red (Pr) and green absorbing form (Pg). The recently determined crystal structures of both states suggest a major movement of Trp496 from a stacking interaction with ring D of the phycocyanobilin (PCB) chromophore in Pr to a position outside the chromophore pocket in Pg. Here, we investigated the role of this amino acid during photoconversion in solution using engineered protein variants in which Trp496 was substituted by natural and non-natural amino acids. These variants and the native protein were studied by various spectroscopic techniques (UV–vis absorption, fluorescence, IR, NIR and UV resonance Raman) complemented by theoretical approaches. Trp496 is shown to affect the electronic transition of PCB and to be essential for the thermal equilibrium between Pr and an intermediate state O600. However, Trp496 is not required to stabilize the tilted orientation of ring D in Pr, and does not play a role in the secondary structure changes of Slr1393 during the Pr/Pg transition. The present results confirm the re-orientation of Trp496 upon Pr → Pg conversion, but do not provide evidence of a major change in the microenvironment of this residue. Structural models indicate the penetration of water molecules into the chromophore pocket in both Pr and Pg states and thus water-Trp contacts, which can readily account for the subtle spectral changes between Pr and Pg. Thus, we conclude that reorientation of Trp496 during the Pr-to-Pg photoconversion in solution is not associated with a major change in the dielectric environment in the two states.

Phycocyanobilin from Arthrospira platensis: A potential photodynamic anticancer agent

Phycocyanobilin (PCB), a tetrapyrrole chromophore compound of Phycocyanin (PC) from Arthrospira platensis. Previous studies have shown that it has anti-inflammatory and immunomodulatory biological activities. In this work, we found that it can be used as a new anticancer photosensitizer, which is almost non-toxic readily available and less costly. A rapid production process of PCB was developed, with a yield of 19.2 ± 1.1 mg/g. By using semi-preparative liquid chromatography and silica gel column chromatography, PCB was refined to a purity of 99.97 ± 0.02%. Nuclear magnetic resonance spectroscopy and mass spectrometry were used to determine the PCB structure. The optimized wavelength of the light source that caused PCB to produce reactive oxygen species (ROS) was 660 nm. PCB had an estimated active oxygen quantum yield of 0.1595, 14.5 times that of PC (0.0110). Meanwhile, PCB produced ROS in MCF-7 cells under light conditions. After the protein of PC was partially removed, the quantum yield of active oxygen in PCB is greatly improved, which was helpful to enhance its anticancer activity. The photodynamic anticancer activity of PCB (IC50:8.26 ± 1.19 μM) was significantly enhanced compared with PC (IC50:255.29 ± 1.72 μM). PCB (without light) had no significant anticancer activity in vitro, but it showed strong anticancer activity in vivo, which was equivalent to the activity of the positive drug group: Zinc-Phthalocyanine (ZnPc) with light. This may be because PCB had the effect of eliminating inflammation and improving immunity. PCB significantly enhanced its anticancer activity under light conditions due to the production of reactive oxygen species. The experimental results showed that PCB had dual anticancer effects of chemotherapy and phototherapy in vivo, and had potential to become a new anticancer photosensitizer.

On the evolution of the plant phytochrome chromophore biosynthesis.

Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria, and fungi. Land plant phytochromes use phytochromobilin (PΦB) as the bilin chromophore. Phytochromes of streptophyte algae, the clade within which land plants evolved, employ phycocyanobilin (PCB), leading to a more blue-shifted absorption spectrum. Both chromophores are synthesized by ferredoxin-dependent bilin reductases (FDBRs) starting from biliverdin IXα (BV). In cyanobacteria and chlorophyta, BV is reduced to PCB by the FDBR phycocyanobilin:ferredoxin oxidoreductase (PcyA), whereas, in land plants, BV is reduced to PФB by phytochromobilin synthase (HY2). However, phylogenetic studies suggested the absence of any ortholog of PcyA in streptophyte algae and the presence of only PФB biosynthesis-related genes (HY2). The HY2 of the streptophyte alga Klebsormidium nitens (formerly Klebsormidium flaccidum) has already indirectly been indicated to participate in PCB biosynthesis. Here, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in Escherichia coli. Employing anaerobic bilin reductase activity assays and coupled phytochrome assembly assays, we confirmed the product and identified intermediates of the reaction. Site-directed mutagenesis revealed 2 aspartate residues critical for catalysis. While it was not possible to convert KflaHY2 into a PΦB-producing enzyme by simply exchanging the catalytic pair, the biochemical investigation of 2 additional members of the HY2 lineage enabled us to define 2 distinct clades, the PCB-HY2 and the PΦB-HY2 clade. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.

Elucidating the origins of phycocyanobilin biosynthesis and phycobiliproteins

Terrestrial ecosystems and human societies depend on oxygenic photosynthesis, which began to reshape our atmosphere approximately 2.5 billion years ago. The earliest known organisms carrying out oxygenic photosynthesis are the cyanobacteria, which use large complexes of phycobiliproteins as light-harvesting antennae. Phycobiliproteins rely on phycocyanobilin (PCB), a linear tetrapyrrole (bilin) chromophore, as the light-harvesting pigment that transfers absorbed light energy from phycobilisomes to the chlorophyll-based photosynthetic apparatus. Cyanobacteria synthesize PCB from heme in two steps: A heme oxygenase converts heme into biliverdin IXα (BV), and the ferredoxin-dependent bilin reductase (FDBR) PcyA then converts BV into PCB. In the current work, we examine the origins of this pathway. We demonstrate that PcyA evolved from pre-PcyA proteins found in nonphotosynthetic bacteria and that pre-PcyA enzymes are active FDBRs that do not yield PCB. Pre-PcyA genes are associated with two gene clusters. Both clusters encode bilin-binding globin proteins, phycobiliprotein paralogs that we designate as BBAGs (bilin biosynthesis-associated globins). Some cyanobacteria also contain one such gene cluster, including a BBAG, two V4R proteins, and an iron-sulfur protein. Phylogenetic analysis shows that this cluster is descended from those associated with pre-PcyA proteins and that light-harvesting phycobiliproteins are also descended from BBAGs found in other bacteria. We propose that PcyA and phycobiliproteins originated in heterotrophic, nonphotosynthetic bacteria and were subsequently acquired by cyanobacteria.

Biological Advantage of the Arrangements of C-Phycocyanin Chromophores in Phycobilisome from the Electronic Energy Transfer Viewpoint

Abstract In the present study, we report the electronic energy transfer (EET) rate and the EET dynamics in the hexamer of C-phycocyanin (C-PC). The EET rate for the Förster theory was calculated by a quantum chemical method and a master equation was used to describe the dynamics. Our calculation results suggest that the natural arrangements of phycocyanobilin (PCB) chromophores α84, β84, and β155 in C-PC are cooperatively well-adjusted to achieve the shortest EET time-length. It is the appropriate regular periodicity of the intermolecular distances and intermolecular angles of the ground and the first excited transition dipole moments of PCBs α84, β84, and β155, i.e., the three-fold symmetry and stacking order of their layers found in terrestrial plants and algae, that are some of the most important requisites in achieving such a highly efficient EET in PBS.

Enhancement of phycocyanobilin biosynthesis in Escherichia coli by strengthening the supply of precursor and artificially self-assembly complex

Phycocyanobilin (PCB) is widely used in healthcare, food processing, and cosmetics. Escherichia coli is the common engineered bacterium used to produce PCB. However, it still suffers from low production level, precursor deficiency, and low catalytic efficiency. In this study, a highly efficient PCB-producing strain was created. First, chassis strains and enzyme sources were screened, and copy numbers were optimized, affording a PCB titer of 9.1 mg/L. Most importantly, the rate-limiting steps of the PCB biosynthetic pathway were determined, and the supply of precursors necessary for PCB synthesis was increased from endogenous sources, affording a titer of 21.4 mg/L. Then, the key enzymes for PCB synthesis, HO1 and PcyA, were assembled into a multi-enzyme complex using the short peptide tag RIAD-RIDD, and 23.5 mg/L of PCB was obtained. Finally, the basic conditions for PCB fermentation were initially determined in 250 mL shake flasks and a 5-L bioreactor to obtain higher titers of PCB. The final titer of PCB reached 147.0 mg/L, which is the highest reported titer of PCB so far. This research provided the foundation for the industrial production of PCB and its derivatives.

Optical Control of Cell Signaling with Red/Far-Red Light-Responsive Optogenetic Tools in Caenorhabditis elegans.

Optogenetic techniques have been intensively applied to the nematode Caenorhabditis elegans to investigate its neural functions. However, as most of these optogenetics are responsive to blue light and the animal exhibits avoidance behavior to blue light, the application of optogenetic tools responsive to longer wavelength light has been eagerly anticipated. In this study, we report the implementation in C.elegans of a phytochrome-based optogenetic tool that responds to red/near-infrared light and manipulates cell signaling. We first introduced the SynPCB system, which enabled us to synthesize phycocyanobilin (PCB), a chromophore for phytochrome, and confirmed the biosynthesis of PCB in neurons, muscles, and intestinal cells. We further confirmed that the amount of PCBs synthesized by the SynPCB system was sufficient for photoswitching of phytochrome B (PhyB)-phytochrome interacting factor 3 (PIF3). In addition, optogenetic elevation of intracellular Ca2+ levels in intestinal cells induced a defecation motor program. These SynPCB system and phytochrome-based optogenetic techniques would be of great value in elucidating the molecular mechanisms underlying C.elegans behaviors.