Green and efficient method to acquire high-value phycobiliprotein from microalgal biomass involving deep eutectic solvent-based ultrasound-assisted extraction.

Phycoerythrin (PE) is a phycobiliprotein holding great potential as a high-value food colorant and medicine. Deep eutectic solvent (DES)-based ultrasound-assisted extraction (UAE) was applied to extract B-PE by disrupting the resistant polysaccharide cell wall of Porphyridium purpureum. The solubility of cell wall monomers in 31 DESs was predicted using COSMO-RS. Five glycerol-based DESs were tested for extraction, all of which showed significantly higher B-PE yields by up to 13.5 folds than water. The DES-dependent B-PE extraction efficiencies were proposedly associated with different cell disrupting capabilities and protein stabilizing effects of DESs. The DES-based UAE method could be considered green according to a metric assessment tool, AGREEprep. The crude extract containing DES was further subjected to aqueous two-phase system, two-step ammonium sulfate precipitation, and ultrafiltration processes. The final purified B-PE had a PE purity ratio of 3.60 and a PC purity ratio of 0.08, comparable to the purity of commercial products.

Monomeric Far-red and Near-infrared Fluorescent Biliproteins of Ultrahigh Brightness.

Far-red and near-infrared fluorescent proteins have regions of maximum transmission in most tissues and can be widely used as fluorescent biomarkers. We report that fluorescent phycobiliproteins originating from the phycobilisome core subunit ApcF2 can covalently bind biliverdin, named BDFPs. To further improve BDFPs, we conducted a series of studies. Firstly, we mutated K53Q and T144A of BDFPs to increase their effective brightness up to 190 % in vivo. Secondly, by homochromatic tandem fusion of high-brightness BDFPs to achieve monomerization, which increases the effective brightness by up to 180 % in vivo, and can effectively improve the labeling effect. By combining the above two approaches, the brightness of the tandem BDFPs was much improved compared with that of the previously reported fluorescent proteins in a similar spectral range. The tandem BDFPs were expressed stably while maintaining fluorescence in mammalian cells and Caenorhabditis elegans. They were also photostable and resistant to high temperature, low pH, and chemical denaturation. The tandem BDFPs advantages were proved in applications as biomarkers for imaging in super-resolution microscopy.

Structure of the antenna complex expressed during far-red light photoacclimation in Synechococcus sp. PCC 7335.

Far-red light photoacclimation, or FaRLiP, is a facultative response exhibited by some cyanobacteria that allows them to absorb and utilize lower energy light (700-800 nm) than the wavelengths typically used for oxygenic photosynthesis (400-700 nm). During this process, three essential components of the photosynthetic apparatus are altered: photosystem I, photosystem II, and the phycobilisome. In all three cases, at least some of the chromophores found in these pigment-protein complexes are replaced by chromophores that have red-shifted absorbance relative to the analogous complexes produced in visible light. Recent structural and spectroscopic studies have elucidated important features of the two photosystems when altered to absorb and utilize far-red light, but much less is understood about the modified phycobiliproteins made during FaRLiP. We used single-particle, cryo-EM to determine the molecular structure of a phycobiliprotein core complex comprising allophycocyanin variants that absorb far-red light during FaRLiP in the marine cyanobacterium Synechococcus sp. PCC 7335. The structure reveals the arrangement of the numerous red-shifted allophycocyanin variants and the probable locations of the chromophores that serve as the terminal emitters in this complex. It also suggests how energy is transferred to the photosystem II complexes produced during FaRLiP. The structure additionally allows comparisons with other previously studied allophycocyanins to gain insights into how phycocyanobilin chromophores can be tuned to absorb far-red light. These studies provide new insights into how far-red light is harvested and utilized during FaRLiP, a widespread cyanobacterial photoacclimation mechanism.

Molecular dissection of the soluble photosynthetic antenna from the cryptophyte alga Hemiselmis andersenii.

Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption from photosystems. However, it is unclear how the antenna transfers energy efficiently to these photosystems. We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein, each composed of two αß protomers but with different quaternary structures arising from a diverse α subunit family. We report crystal structures of the major phycobiliprotein from each spectrotype. Two-thirds of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors. These are supplemented by a newly discovered open-braced form (~15%), where an insertion in the α subunit produces ~10 nm absorbance red-shift. The final components (~15%) are closed forms with a long wavelength spectral feature due to substitution of a single chromophore. This chromophore is present on only one ß subunit where asymmetry is dictated by the corresponding α subunit. This chromophore creates spectral overlap with chlorophyll, thus bridging the energetic gap between the phycobiliprotein antenna and the photosystems. We propose that the macromolecular organization of the cryptophyte antenna consists of bulk open and open-braced forms that transfer excitations to photosystems via this bridging closed form phycobiliprotein.

The Unique Light-Harvesting System of the Algal Phycobilisome: Structure, Assembly Components, and Functions.

The phycobilisome (PBS) is the major light-harvesting apparatus in cyanobacteria and red algae. It is a large multi-subunit protein complex of several megadaltons that is found on the stromal side of thylakoid membranes in orderly arrays. Chromophore lyases catalyse the thioether bond between apoproteins and phycobilins of PBSs. Depending on the species, composition, spatial assembly, and, especially, the functional tuning of different phycobiliproteins mediated by linker proteins, PBSs can absorb light between 450 and 650 nm, making them efficient and versatile light-harvesting systems. However, basic research and technological innovations are needed, not only to understand their role in photosynthesis but also to realise the potential applications of PBSs. Crucial components including phycobiliproteins, phycobilins, and lyases together make the PBS an efficient light-harvesting system, and these provide a scheme to explore the heterologous synthesis of PBS. Focusing on these topics, this review describes the essential components needed for PBS assembly, the functional basis of PBS photosynthesis, and the applications of phycobiliproteins. Moreover, key technical challenges for heterologous biosynthesis of phycobiliproteins in chassis cells are discussed.

Infrared Signatures of Phycobilins within the Phycocyanin 645 Complex.

Aquatic photosynthetic organisms evolved to use a variety of light frequencies to perform photosynthesis. Phycobiliprotein phycocyanin 645 (PC645) is a light-harvesting complex in cryptophyte algae able to transfer the absorbed green solar light to other antennas with over 99% efficiency. The infrared signatures of the phycobilin pigments embedded in PC645 are difficult to access and could provide useful information to understand the mechanism behind the high efficiency of energy transfer in PC645. We use visible-pump IR-probe and two-dimensional electronic vibrational spectroscopy to study the dynamical evolution and assign the fingerprint mid-infrared signatures to each pigment in PC645. Here, we report the pigment-specific vibrational markers that enable us to track the spatial flow of excitation energy between the phycobilin pigment pairs. We speculate that two high-frequency modes (1588 and 1596 cm-1) are involved in the vibronic coupling leading to fast (

Who's your neighbor? Suggesting alternative assemblies of E/F type bilin lyases from crystal lattice analysis.

Attachment of bilins to phycobiliproteins is performed by dedicated lyases. In this issue of Structure, Kumarapperuma et al., 2022 present the structure of an E/F type lyase-isomerase that identifies the correct biological interface between active domains, suggesting that a previous E/F lyase misidentified the heterodimer structure from the crystal lattice.

Probing heavy metal binding to phycobiliproteins.

Blue-green algae, also known as cyanobacteria, contain some of the most efficient light-harvesting complexes known. These large, colourful complexes consist of phycobiliproteins which are extremely valuable in the cosmetics, food, nutraceutical and pharmaceutical industries. Additionally, the colourful and fluorescent properties of phycobiliproteins can be modulated by metal ions, making them highly attractive as heavy metal sensors and heavy metal scavengers. Although the overall quenching ability metal ions have on phycobiliproteins is known, the mechanism of heavy metal binding to phycobiliproteins is not fully understood, limiting their widespread quantitative applications. Here, we show using high-resolution native mass spectrometry that phycobiliprotein complexes bind metal ions in different manners. Through monitoring the binding equilibria and metal-binding stoichiometry, we show in particular copper and silver to have drastic, yet different effects on phycobiliprotein structure, both copper and silver modulate the overall complex properties. Together, the data reveals the mechanisms by which metal ions can modulate phycobiliprotein properties which can be used as a basis for the future design of metal-related phycobiliprotein applications.

Phycobiliprotein as fluorescent probe and photosensitizer: A systematic review.

Phycobiliprotein is a natural product with many biological activities in various seaweeds. Phycobiliproteins have been widely used for anti-oxidation, anti-tumor, anti-inflammatory and immune-enhancing activities as a functional factor. Phycobiliproteins with high purity are considerably more expensive than common. To provide with a systematic, deep and detailed information about those features of phycobiliproteins, we performed a relatively comprehensive analysis on structural composition, the application of phycobiliproteins in the fields of fluorescent probe and photodynamic therapy in this report.

Stability, kinetics, and application study of phycobiliprotein pigments extracted from red algae Gracilaria gracilis.

Phycobiliprotein (PBP) pigments were extracted from red algae Gracilaria gracilis through maceration in phosphate buffer using previously optimized conditions. The stability of PBPs in the extracts was assessed by monitoring the extracts at different pHs and temperatures for 10 days. Since phycoerythrin (PE) is the main PBP present in G. gracilis, PE content was spectroscopically determined and used as a response factor. Kinetic modeling was used to describe PE degradation under different ranges of T and pH. The pigment extracts presented higher stability at pH 6.9 and -20 °C. PE was semipurified by precipitation with ammonium sulphate 65% followed by dialysis against water until a purity index of 0.7. The pigment was successfully applied as colorant in pancakes and yogurts with a pigment concentration of 0.15%. This study highlights the potential of PE pigments extracted from G. gracilis for applications in food products. PRACTICAL APPLICATION: Phycobiliprotein pigments were extracted from red algae Gracilaria gracilis through maceration in phosphate buffer. The stability of the pigment was evaluated at different pHs and temperatures, presenting higher stability at neutral pH and low temperatures. The pigment was successfully applied as colorant in pancakes and yogurts with a low pigment concentration. This study highlights the potential of phycobiliprotein pigments extracted from G. gracilis for applications in food products.

Optimization of the Freezing-Thawing Method for Extracting Phycobiliproteins from Arthrospira sp.

The freezing-thawing method had been reported to be the best phycobiliprotein extraction technique. However, optimum parameters of this extraction method for Arthrospira sp. (one of the major phycobiliprotein sources) still remained unclear. Hence, this study aimed to optimize the freezing-thawing parameters of phycobiliprotein extraction in Arthrospira sp. (UPMC-A0087). The optimization of the freezing-thawing method was conducted using different solvents, biomass/solvent ratios, temperatures, time intervals and freezing-thawing cycles. The extracted phycobiliproteins were quantified using a spectrophotometric assay. Double distilled water (pH 7) with a 0.50% w/v biomass/solvent ratio was the most efficient solvent in extracting high concentrations and purity of phycobiliproteins from Arthrospira sp. In addition, the combination of freezing at -80 °C (2 h) and thawing at 25 °C (24 h) appeared to be the optimum temperature and extraction time to obtain the highest amount of phycobiliproteins. A minimum of one cycle of freezing and thawing was sufficient for extracting high concentrations of phycobiliproteins. The findings from this study could reduce the cost and labor needed for extracting high quality phycobiliproteins. It also allowed the harvesting of large amounts of valuable phycobiliproteins.

Improved stability of phycobiliprotein within liposome stabilized by polyethylene glycol adsorbed cellulose nanocrystals.

This study ascertained the stability of phycobiliprotein (PBP), a bioactive protein from Dulse (Palmaria palmata) loaded within liposomes and stabilized with polyethylene glycol (2000 and 4000 g/mol) and desulfated CNCs (DCs) containing adsorbed polyethylene glycol (DCs-2000 and DCs-4000). The effect of pH, temperature and illumination on the stability of PBP was investigated. Results showed that the temperature had the most significant (p < 0.05) effect on the fluorescence intensity of the PBP, accounting for up to 70% loss of the fluorescence intensity for PBP loaded liposome (PL), PL stabilized with PEG-2000 (PLP-2000) and PEG 4000 (PLP-4000) and PL stabilized with desulfated CNCs (DCs), however, 60% for the PL stabilized with PEG 2000 and PEG 4000 adsorbed CNCs (PLDCs-2000 and PLDCs-4000) at 60 °C compared to those stabilized at 4 °C. A further increase in temperature to 80 °C led to a complete loss of fluorescence. Operating at the extreme pH's of 1.0 and 11.0 resulted in a loss of 90% and 30% fluorescence intensity, respectively for PBP in solution, whereas, 20% and 2% loss was observed for PBP incorporated inside the liposomes. Regarding the effect of illumination, PLDCs-2000 and PLDCs-4000 were the most stable, retaining the fluorescence intensity of PBP up to 70% after 72 h of exposure. This is compared to 85% loss of fluorescence for PBP in solution. Furthermore, at pH of 1.0, there was an increase in average particle size for the PLDCs-2000 and PLDCs-4000 from 189 ± 3 & 206 ± 2 nm to 6464 ± 211 & 6698 ± 317 nm and a charge reversal in the zeta potential from -36 ± 1 & -34 ± 2 to +16 ± 3 & +14 ± 1. Confocal and optical microscopic images confirmed the coalescence of PBP loaded liposome and agglomeration PLDCs-2000 and PLDCs-4000 under acidic pH (<3.0). In contrast, changes in temperature from 4 °C to 100 °C and illumination as a function of time up to 72 h resulted in no change in liposome size and zeta potential.

Optimization of phycobiliprotein pigments extraction from red algae Gracilaria gracilis for substitution of synthetic food colorants.

The extraction of phycobiliprotein (PBP) pigments from red algae Gracilaria gracilis was optimized using maceration, ultrasound-assisted extraction (ultrasonic water bath and ultrasonic probe), high pressure-assisted extraction, and freeze-thaw. The experimental conditions, namely homogenization time (t1), buffer concentration (C), treatment time (t2), biomass: buffer ratio (R), and pressure (P), were optimized using Response Surface Methodology (RSM). The yield of phycoerythrin (PE) extracted, determined spectroscopically, was used as the response variable. Maceration was the most efficient extraction method yielding 3.6 mg PE/g biomass under the optimal conditions (t1 = t2 = 10 min; C = 0.1 M; R = 1:50). Scanning Electron Microscopy (SEM) analysis of the biomass before and after the cell disruption treatments revealed a more efficient cell wall rupture with maceration.

Phycobiliproteins from Arthrospira Platensis (Spirulina): A New Source of Peptides with Dipeptidyl Peptidase-IV Inhibitory Activity.

Arthrospira platensis (spirulina) is a cyanobacterium, which contains mainly two phycobiliproteins (PBP), i.e., C-phycocyanin (C-PC) and allophycocyanin (APC). In this study, PBP were hydrolyzed using trypsin, and the composition of the hydrolysate was characterized by HPLC-ESI-MS/MS. Furthermore, the potential anti-diabetic activity was assessed by using either biochemical or cellular techniques. Findings suggest that PBP peptides inhibit DPP-IV activity in vitro with a dose-response trend and an IC50 value falling in the range between 0.5 and 1.0 mg/mL. A lower inhibition of the DPP-IV activity expressed by Caco-2 cells was observed, which was explained by a secondary metabolic degradation exerted by the same cells.

Ultrasound-Assisted Extraction and Assessment of Biological Activity of Phycobiliprotein-Rich Aqueous Extracts from Wild Cyanobacteria (Aphanizomenon flos-aquae).

Cyanobacteria are photosynthetic microorganisms that are considered as an important source of bioactive metabolites, among which phycobiliproteins (PBPs) are a class of water-soluble macromolecules of cyanobacteria with a wide range of applications. Massive proliferation of cyanobacteria can lead to excessive surface water blooms, of which removal, as a management measure, should be prioritized. In this study, the utilization of wild cyanobacteria biomass (Aphanizomenon flos-aquae) for extraction of phycobiliproteins is reported. Extraction of phycobiliproteins by conventional methods, such as homogenization, freeze-thaw cycles, and solid-liquid extraction, were optimized prior to ultrasound-assisted extraction. Standardization of ultrasonication for different parameters, such as ultrasonication amplitude (38, 114, and 190 µm) and ultrasonication time (1, 5.5, and 10 min), was carried out using a central composite design and response surface methodology for each of the primary techniques. A substantial increase on the individual and total phycobiliprotein yields was observed after ultrasonic treatment. The highest total PBP yield (115.37 mg/g of dry weight) was observed with samples treated with a homogenizer (30 min, 30 °C, and 1 cycle) combined with ultrasound treatment (8.7 min at 179 µm). Moreover, in vitro antioxidant capacity was observed for the obtained extracts in the Folin-Ciocalteu and ABTS* + assays. In addition, a cytotoxic effect against C6 glioma cells was observed for A. flos-aquae PBPs. Conclusively, wild cyanobacteria could be considered as an alternative feedstock for recovery of PBPs.

Structure-Based Mutagenesis of Phycobiliprotein smURFP for Optoacoustic Imaging.

Photo- or optoacoustics (OA) imaging is increasingly being used as a non-invasive imaging method that can simultaneously reveal structure and function in deep tissue. However, the most frequent transgenic OA labels are current fluorescent proteins that are not optimized for OA imaging. Thus, they lack OA signal strength, and their absorption maxima are positioned at short wavelengths, thus giving small penetration depths and strong background signals. Here, we apply insights from our recent determination of the structure of the fluorescent phycobiliprotein smURFP to mutate a range of residues to promote the nonradiative decay pathway that generates the OA signal. We identified hydrophobic and aromatic substitutions within the chromophore-binding pocket that substantially increase the intensity of the OA signal and red-shift the absorption. Our results demonstrate the feasibility of structure-based mutagenesis to repurpose fluorescent probes for OA imaging, and they may provide structure-function insights for de novo engineering of transgenic OA probes.

Subunit pI Can Influence Protein Complex Dissociation Characteristics.

Mass spectrometry is frequently used to determine protein complex topology. By combining in-solution and gas-phase dissociation measurements, information can be indirectly inferred about the original composition of the protein complex. Although the mechanisms behind gas-phase complex dissociation are becoming more established, protein complex dissociation is not always predictable. Here, we looked into the effect of the protein subunits pI on complex dissociation. We chose two structurally similar, hexameric protein complexes that consist of a ring of alternating alpha and beta subunits. For one complex, allophycocyanin, the alpha and beta subunits are structurally similar, almost identical in mass, but have distinct pIs. In contrast, the other complex, phycoerythrin, is structural similar to allophycocyanin, yet the subunits have identical pIs. As predicted based on the structural arrangement, dissociation of phycoerythrin resulted in the observation of both the alpha and beta monomeric subunits in the mass spectrometer. However, for allophycocyanin, the results differed dramatically, with only the alpha monomeric subunit being detected upon gas-phase dissociation. Together, the results highlighted the importance of considering the isoelectric points of individual subunits within a protein complex when using tandem mass spectrometry data to elucidate protein complex topology.

Crystal structure of phycocyanin from heterocyst-forming filamentous cyanobacterium Nostoc sp. WR13.

Phycocyanin (PC) is the principal pigment protein in the light-harvesting antenna of cyanobacteria. Here the biochemical characterization and the 1.51 Šcrystal structure of PC from cyanobacterium Nostoc sp. WR13 (Nst-PC) is reported. The P63 crystal lattice is composed of the minimal biological entities of Nst-PC, the (αß)3 trimeric rings. The structure has been refined to R factor 11.5% (Rfree 15.4%) using anisotropic atomic B factors. A phylogenetic study shows that the α and ß chains of Nst-PC are significantly clustered in a distinct clade with Acaryochloris marina. The structure was examined to look for any significant differences between Nst-PC and PC from non-desert species. Only minor differences were found in the chromophore microenvironments. The tentative energy transfer pathways in Nst-PC were modeled based on simple structural considerations.

Very Bright Phycoerythrobilin Chromophore for Fluorescence Biolabeling.

Biliproteins have extended the spectral range of fluorescent proteins into the far-red (FR) and near-infrared (NIR) regions. These FR and NIR fluorescent proteins are suitable for the bioimaging of mammalian tissues and are indispensable for multiplex labeling. Their application, however, presents considerable challenges in increasing their brightness, while maintaining emission in FR regions and oligomerization of monomers. Two fluorescent biliprotein triads, termed BDFP1.2/1.6:3.3:1.2/1.6, are reported. In mammalian cells, these triads not only have extremely high brightness in the FR region, but also have monomeric oligomerization. The BDFP1.2 and BDFP1.6 domains covalently bind to biliverdin, which is accessible in most cells. The BDFP3.3 domain noncovalently binds phycoerythrobilin that is added externally. A new method of replacing phycoerythrobilin with proteolytically digested BDFP3.3 facilitates this labeling. BDFP3.3 has a very high fluorescence quantum yield of 66 %, with maximal absorbance at λ=608 nm and fluorescence at λ=619 nm. In BDFP1.2/1.6:3.3:1.2/1.6, the excitation energy that is absorbed in the red region by phycoerythrobilin in the BDFP3.3 domain is transferred to biliverdin in the two BDFP1.2 or BDFP1.6 domains and fluoresces at λ≈670 nm. The combination of BDFP3.3 and BDFP1.2/1.6:3.3:1.2/1.6 can realize dual-color labeling. Labeling various proteins by fusion to these new fluorescent biliproteins is demonstrated in prokaryotic and mammalian cells.

Phycobiliproteins from cyanobacteria: Chemistry and biotechnological applications.

Phycobiliproteins are a group of water soluble proteins with an associated chromophore, responsible for the light-harvesting in cyanobacteria. They are divided in four main types: phycoerythrin, phycocyanin, phycoerythrocyanin and allophycocyanin, and they are characterized according to their structure and light quality absorption. Phycobiliproteins from cyanobacteria have been described as potential bioactive compounds, and recognized as high-valued natural products for biotechnological applications. Moreover, phycobiliproteins have been associated to antioxidant, anticancer and anti-inflammatory capacities among others. Thus, in order to produce phycobiliproteins from cyanobacteria for industrial application, it is necessary to optimize the whole bioprocess, including the processing parameters (such as light, nitrogen and carbon source, pH, temperature and salinity) that affects the growth and phycobiliprotein accumulation, as well as the optimization of phycobiliproteins extraction and purification. The aim of this review is to give an overview of phycobiliproteins not only in terms of their chemistry, but also in terms of their biotechnological applicability and the advances and challenges in the production of such compounds.