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.

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 (

Characterization of Molecular Diversity and Organization of Phycobilisomes in Thermophilic Cyanobacteria.

Thermophilic cyanobacteria are cosmopolitan and abundant in the thermal environment. Their light-harvesting complexes, phycobilisomes (PBS), are highly important in photosynthesis. To date, there is limited information on the PBS composition of thermophilic cyanobacteria whose habitats are challenging for survival. Herein, genome-based methods were used to investigate the molecular components of PBS in 19 well-described thermophilic cyanobacteria. These cyanobacteria are from the genera Leptolyngbya, Leptothermofonsia, Ocullathermofonsia, Thermoleptolyngbya, Trichothermofonsia, Synechococcus, Thermostichus, and Thermosynechococcus. According to the phycobiliprotein (PBP) composition of the rods, two pigment types are observed in these thermophiles. The amino acid sequence analysis of different PBP subunits suggests several highly conserved cysteine residues in these thermophiles. Certain amino acid contents in the PBP of thermophiles are significantly higher than their mesophilic counterparts, highlighting the potential roles of specific substitutions of amino acid in the adaptive thermostability of light-harvesting complexes in thermophilic cyanobacteria. Genes encoding PBS linker polypeptides vary among the thermophiles. Intriguingly, motifs in linker apcE indicate a photoacclimation of a far-red light by Leptolyngbya JSC-1, Leptothermofonsia E412, and Ocullathermofonsia A174. The composition pattern of phycobilin lyases is consistent among the thermophiles, except for Thermostichus strains that have extra homologs of cpcE, cpcF, and cpcT. In addition, phylogenetic analyses of genes coding for PBPs, linkers, and lyases suggest extensive genetic diversity among these thermophiles, which is further discussed with the domain analyses. Moreover, comparative genomic analysis suggests different genomic distributions of PBS-related genes among the thermophiles, indicating probably various regulations of expression. In summary, the comparative analysis elucidates distinct molecular components and organization of PBS in thermophilic cyanobacteria. These results provide insights into the PBS components of thermophilic cyanobacteria and fundamental knowledge for future research regarding structures, functions, and photosynthetic improvement.

Assessment of the Anticancer Potentials of the Free and Metal-Organic Framework (UiO-66) - Delivered Phycocyanobilin.

Phycocyanin (C-PC) is a constitutive chromoprotein of Arthrospira platensis, which exhibits promising efficacy against different types of cancer. In this study, we cleaved C-PC's chromophore phycocyanobilin (PCB) and demonstrated its ability as an anti-cancer drug for Colorectal cancer (CRC). PCB displayed an anti-cancer effect for CRC (HT-29) cells with IC50 of 108 µg/ml. Assessing the transcripts levels of some biomarkers revealed that the PCB caused an upregulation in the anti-metastatic gene NME1 level and downregulation of the COX-2 level. The flow cytometric results showed the effect of PCB on the arrest of the cell cycle's G1 phase. In addition, we successfully synthesized the UiO-66 (Zr-MOF). We incorporated the PCB into UiO-66 nanoparticles with a loading percentage of 46 %. Assessment of the cytotoxic effects of UiO-66@PCB showed a 2-fold improvement in the IC50 compared to the free PCB. In conclusion, we have shown that PCB displayed a promising potential as an anti-cancer agent. Yet, it is considered a safe and natural substance that can help to mitigate cancer spread and symptoms. In the meantime, UiO-66 can be used as a safe nano-delivery tool for PCB.

The Cyanobacterial "Nutraceutical" Phycocyanobilin Inhibits Cysteine Protease Legumain.

The blue biliprotein phycocyanin, produced by photo-autotrophic cyanobacteria including spirulina (Arthrospira) and marketed as a natural food supplement or "nutraceutical," is reported to have anti-inflammatory, antioxidant, immunomodulatory, and anticancer activity. These diverse biological activities have been specifically attributed to the phycocyanin chromophore, phycocyanobilin (PCB). However, the mechanism of action of PCB and the molecular targets responsible for the beneficial properties of PCB are not well understood. We have developed a procedure to rapidly cleave the PCB pigment from phycocyanin by ethanolysis and then characterized it as an electrophilic natural product that interacts covalently with thiol nucleophiles but lacks any appreciable cytotoxicity or antibacterial activity against common pathogens and gut microbes. We then designed alkyne-bearing PCB probes for use in chemical proteomics target deconvolution studies. Target identification and validation revealed the cysteine protease legumain (also known as asparaginyl endopeptidase, AEP) to be a target of PCB. Inhibition of this target may account for PCB's diverse reported biological activities.

Photosynthesis in Plants and Algae.

Photosynthesis is the basis of almost all life on Earth. In addition to providing energy, plants and algae provide a plethora of secondary substances useful in the treatment of a number of illnesses including a wide array of cancer maladies. The first organisms on Earth used chemosynthesis for their energy needs. Photosynthetic bacteria utilize one of two different photosystems whereas cyanobacteria, eukaryotic algae and plants combine two photosystems in a linear electron transport chain. Accessory pigments such as various chlorophylls, carotenoids and phycobilins absorb the energy of impinging photons and funnel it to the reaction centers (P680 in photosystem II and P700 in photosystem I). Water is split photochemically, electrons are transported to reduce NADPH, oxygen is discarded as waste product, and protons accumulate inside the thylakoid vesicles in the chloroplasts. The resulting electrochemical gradient across the membrane is used to drive an ATPase. The produced ATP and NADPH+H+ are utilized in the Calvin cycle to fix CO2 and to produce fructose.

Monitoring effect of nickel, copper, and zinc on growth and photosynthetic pigments of Spirulina platensis with suitability investigation in Idku Lake.

Owing to the increase of pollutant sources in oceans, seas, and lakes, there is an expected effect on growth and metabolism of planktonic algae which are considered primary producers in the ecosystem. Therefore, it becomes urgent to carry out laboratory studies to test to what extent these pollutants can affect the growth of algae which is necessary as a food for marine fishes. Spirulina is considered the most important algal species due to its high nutritional value for humans and animals. Therefore, this work investigated the effect of different concentrations of Ni2+, Zn2+, and Cu2+ metal ion pollutants on growth of the blue-green alga Spirulina platensis. EC50 was identified to be around 2 mg/l for the three heavy metals. The suitability of Idku Lake for Spirulina platensis growth was investigated using multi-criteria spatial modeling integrated with remotely sensed data processing. Spatial distribution maps of turbidity, water nutrients, and phytoplankton were the input criteria used to assess Idku Lake's suitability. The results obtained proved that low concentrations of the tested heavy metals stimulated growth and pigment fractions (chlorophyll a, carotenoids, and total phycobilins content) but to different degrees. The inhibitory effect was more prominent in the case of copper ions than zinc and nickel ions with all concentrations used. The overall suitability map of Spirulina platensis in Idku Lake showed that the whole lake is suitable for growth and proliferation except for the northwestern corner due to the high salinity levels. The present paper helps to understand the behavior of algae responding to environmental pollution, which supports environmental planners with the necessary baseline for investigating the fate of pollutants and the potential risk.

The Bioactivities of Phycocyanobilin from Spirulina.

Phycocyanobilin (PCB) is a linear open-chain tetrapyrrole chromophore that captures and senses light and a variety of biological activities, such as anti-oxidation, anti-cancer, and anti-inflammatory. In this paper, the biological activities of PCB are reviewed, and the related mechanism of PCB and its latest application in disease treatment are introduced. PCB can resist oxidation by scavenging free radicals, inhibiting the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and delaying the activity of antioxidant enzymes. In addition, PCB can also be used as an excellent anti-inflammatory agent to reduce the proinflammatory factors IL-6 and IFN-γ and to up-regulate the production of anti-inflammatory cytokine IL-10 by inhibiting the inflammatory signal pathways NF-κB and mitogen-activated protein kinase (MAPK). Due to the above biological activities of phycocyanobilin PCB, it is expected to become a new effective drug for treating various diseases, such as COVID-19 complications, atherosclerosis, multiple sclerosis (MS), and ischaemic stroke (IS).

Efficient Synthesis of Phycocyanobilin by Combinatorial Metabolic Engineering in Escherichia coli.

Phycocyanobilin (PCB) is a kind of light-harvesting pigment which naturally exists in algae and plays important roles in absorbing and transferring energy. Based on its antioxidant and optical properties, PCB has been applied in food, medicine, and cosmetics. Currently, PCB is mainly extracted from Spirulina through complicated steps; thus, the biosynthesis of PCB in Escherichia coli has attracted more attention. However, due to the lower catalytic efficiency of synthetic enzymes and the deficiency of precursors and cofactors, the titer of PCB remains at a low level. Here, we report the efficient synthesis of PCB by the expression of heme oxygenase-1 from Thermosynechococcus elongatus and PCB: ferredoxin oxidoreductase (PcyA) from Synechocystis sp. using a high-copy number plasmid with an inducible T7lac promoter and the assembly of these two enzymes at a suitable ratio of 2:1 with DNA scaffolds. Additionally, the synthesis of PCB was further enhanced by direct supplementation of 5-aminolevulinic acid (ALA), moderate overexpression of key enzymes in the heme biosynthetic pathway (hemB and hemH), and accelerated cycle of cofactors (NADPH) through the expression of NAD+ kinase and the addition of a reducing agent. Finally, based on the optimal conditions (Modified R medium with 200 mg/L ALA, 20 mg/L FeSO4·7H2O, and 5 g/L vitamin C induced by 0.8 mM isopropylthio-ß-galactoside at 30 °C), the highest reported titer of PCB (28.32 mg/L) was obtained at the fermenter level by feeding glucose and FeSO4·7H2O. The strategies applied in this study will be useful for the synthesis of other natural pigments and PCB or heme derivatives in E. coli.

Identification of significant residues for intermediate accumulation in phycocyanobilin synthesis.

Phycocyanobilin, the primary pigment of both light perception and light-harvesting in cyanobacteria, is synthesized from biliverdin IXα (BV) through intermediate 181, 182-dihydrobiliverdin (181, 182-DHBV) by a phycocyanobilin:ferredoxin oxidoreductase (PcyA). In our previous study, we discovered two PcyA homologs (AmPcyAc and AmPcyAp) derived from Acaryochloris marina MBIC 11017 (A. marina) that exceptionally uses chlorophyll d as the primary photosynthetic pigment, absorbing longer wavelength far-red light than chlorophyll a, the photosynthetic pigment found in most cyanobacteria. Biochemical characterization of the two PcyA homologs identified functional diversification of these two enzymes: AmPcyAc provides 181, 182-DHBV, and PCB to the cyanobacteriochrome (CBCR) photoreceptors, whereas, AmPcyAp specifically provides PCB to the light-harvesting phycobilisome subunit. In this study, we focused on the residues necessary for 181, 182-DHBV supply to the CBCR photoreceptors by AmPcyAc. Based on the SyPcyA structure, we concentrated on the 30 residues that constitute the substrate-binding pocket. Among them, we discovered that Leu151 and Val225 in AmPcyAc were both substituted with isoleucine. During the enzymatic reaction, the SyPcyA variant molecule, possessing V225I and L151I replacements, accumulates the 181, 182-DHBV and supplies it to a CBCR molecule derived from A. marina. It is worth noting that the substitution of Val225 with isoleucine was specifically conserved among the Acaryochloris genus. Collectively, we propose that the specific evolution of PcyA among the Acaryochloris genus may correlate with the acquisition of Chl. d synthetic ability and growth in long-wavelength far-red light environments.

Phycocyanin ameliorates mouse colitis via phycocyanobilin-dependent antioxidant and anti-inflammatory protection of the intestinal epithelial barrier.

Phycocyanin is a typical microalgal active compound with antioxidant and anti-inflammatory efficacy, and the pigment moiety phycocyanobilin has been recently proposed as its active structural component. Here, to explore the structural basis for phycocyanin's intestinal protective action, we evaluated the therapeutic effects and mechanism of action of phycocyanin and phycocyanobilin in dextran sodium sulphate (DSS)-induced colitis mice and in Caco-2 and RAW 264.7 cells. Phycocyanobilin was obtained by solvothermal alcoholysis of phycocyanin and characterized by spectroscopy and mass spectrometry methods. Phycocyanin, phycocyanobilin and a positive drug mesalazine were intragastrically administered to C57BL/6 mice daily for 7 days during and after 4-day DSS exposure. Clinical signs and colon histopathology revealed that phycocyanin and phycocyanobilin had an equivalent anti-colitis efficacy that was even superior to mesalazine. Based on biochemical analysis of colonic tight junction proteins, mucus compositions and goblet cells, and colonic and peripheral proinflammatory cytokines, phycocyanin and phycocyanobilin displayed equivalent intestinal epithelial barrier-protecting and anti-inflammatory potential that was evidently superior to that of mesalazine. Flow cytometry analysis of phycocyanobilin fluorescence in Caco-2 cells unveiled a similar uptake efficacy of phycocyanin and phycocyanobilin by intestinal epithelial cells. According to lactic dehydrogenase release, 2',7'-dichlorodihydrofluorescein fluorescence and methylthiazolyldiphenyl-tetrazolium bromide assay in Caco-2 cells, phycocyanin and phycocyanobilin could equally and effectively protect the intestinal epithelial barrier from oxidant-induced disruption. Phycocyanin and phycocyanobilin also showed equivalent anti-inflammatory effects in tumor necrosis factor-α-stimulated Caco-2 cells and in lipopolysaccharides- and tumor necrosis factor-α-activated RAW264.7 cells. Overall, our results demonstrate the phycocyanobilin-dependent anti-colitis role of phycocyanin via antioxidant and anti-inflammatory mechanisms.

Red fluorescent protein from cyanobacteriochrome chromophorylated with phycocyanobilin and biliverdin.

Cyanobacteriochromes are the extended family of phytochrome photosensors characterized in cyanobacteria. Alr1966g2C56A is a cyanobacteriochrome mutant of Alr1966g2 in Nostoc sp. PCC 7120 from freshwater. In this paper, we truncated ten residues in the N-terminus and ten residues in the C-terminus of Alr1966g2C56A and obtained truncated Alr1966g2C46A, termed as Alr1966g2C46A-tr. Alr1966g2C46A-tr binded covalently not only phycocyanobilin but also biliverdin via Cys74 of the conserved CH motif, and showed a significant improvement in binding-PCB efficiency in E. coli, compared with that of untruncated Alr1966g2C56A. We also captured a persistent red fluorescence of Alr1966g2C46A-tr-PCB or Alr1966g2C46A-tr-BV expressed in live E. coli. Thus, Alr1966g2C46A-tr was suitable for the stable red fluorescent probe as a starting material.

The role of photosynthesis related pigments in light harvesting, photoprotection and enhancement of photosynthetic yield in planta.

Photosynthetic pigments are an integral and vital part of all photosynthetic machinery and are present in different types and abundances throughout the photosynthetic apparatus. Chlorophyll, carotenoids and phycobilins are the prime photosynthetic pigments which facilitate efficient light absorption in plants, algae, and cyanobacteria. The chlorophyll family plays a vital role in light harvesting by absorbing light at different wavelengths and allowing photosynthetic organisms to adapt to different environments, either in the long-term or during transient changes in light. Carotenoids play diverse roles in photosynthesis, including light capture and as crucial antioxidants to reduce photodamage and photoinhibition. In the marine habitat, phycobilins capture a wide spectrum of light and have allowed cyanobacteria and red algae to colonise deep waters where other frequencies of light are attenuated by the water column. In this review, we discuss the potential strategies that photosynthetic pigments provide, coupled with development of molecular biological techniques, to improve crop yields through enhanced light harvesting, increased photoprotection and improved photosynthetic efficiency.

Near-infrared imaging in fission yeast using a genetically encoded phycocyanobilin biosynthesis system.

Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with near-infrared excitation and emission maxima. Unlike the other conventional fluorescent proteins, iRFP requires biliverdin (BV) as a chromophore. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and that biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV and therefore did not show any iRFP fluorescence. The brightness of iRFP-PCB was higher than that of iRFP-BV both in vitro and in fission yeast. We introduced SynPCB2.1, a PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids carrying the iRFP-fused marker proteins together with SynPCB2.1. These tools not only enable the easy use of multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms. This article has an associated First Person interview with the first author of the paper.

Phycobilin heterologous production from the Rhodophyta Porphyridium cruentum.

Phycobiliproteins are colored, active molecules with potential use in different industries. They are the union of proteins and bilins (Chromophores). The primary source of phycobiliproteins is algae; however, the traditional algae culture has production restrictions. The production in bacterial models can be a more efficient alternative to produce these molecules. However, the lack of knowledge in some steps of the phycobiliprotein metabolic pathway limits this alternative. Porphyridium cruentum is a single cell red alga with a high phycobiliprotein content. Its protein sequences were the basis for phycobilin production in this study. In this study, we cloned and characterized enzymes presumably involved in the chromophore production of P. cruentum. Using sequences obtained from its transcriptome, we characterized two cDNA sequences predicted to code respectively for a ferredoxin-dependent bilin reductase and a bilin lyase-isomerase. We expressed these enzymes in Escherichia coli to obtain in vivo evidence of their enzymatic activity on the substrate biliverdin IXα. Lastly, we analyzed them using thin-layer chromatography, spectrophotometry, and fluorescence spectroscopy. These experiments provided evidence of bilin modification. The expressed bilin lyase-isomerase did not show significant activity over the biliverdin molecule. On the contrary, the expressed ferredoxin-dependent bilin reductase showed activity over the biliverdin.

The impact of chromophore choice on the assembly kinetics and primary photochemistry of a red/green cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are bi-stable photoreceptor proteins with high potential for biotechnological applications. Most of these proteins utilize phycocyanobilin (PCB) as a light-sensing co-factor, which is unique to cyanobacteria, but some variants also incorporate biliverdin (BV). The latter are of particular interest for biotechnology due to the natural abundance and red-shifted absorption of BV. Here, AmI-g2 was investigated, a CBCR capable of binding both PCB and BV. The assembly kinetics and primary photochemistry of AmI-g2 with both chromophores were studied in vitro. The assembly reaction with PCB is roughly 10× faster than BV, and the formation of a non-covalent intermediate was identified as the rate-limiting step in the case of BV. This step is fast for PCB, where the formation of the covalent thioether bond between AmI-g2 and PCB becomes rate-limiting. The photochemical quantum yields of the forward and backward reactions of AmI-g2 were estimated and discussed in the context of homologous CBCRs.

Artificial complementary chromatic acclimation gene expression system in Escherichia coli.

BACKGROUND: The development of multiple gene expression systems, especially those based on the physical signals, such as multiple color light irradiations, is challenging. Complementary chromatic acclimation (CCA), a photoreversible process that facilitates the control of cellular expression using light of different wavelengths in cyanobacteria, is one example. In this study, an artificial CCA systems, inspired by type III CCA light-regulated gene expression, was designed by employing a single photosensor system, the CcaS/CcaR green light gene expression system derived from Synechocystis sp. PCC6803, combined with G-box (the regulator recognized by activated CcaR), the cognate cpcG2 promoter, and the constitutively transcribed promoter, the PtrcΔLacO promoter. RESULTS: One G-box was inserted upstream of the cpcG2 promoter and a reporter gene, the rfp gene (green light-induced gene expression), and the other G-box was inserted between the PtrcΔLacO promoter and a reporter gene, the bfp gene (red light-induced gene expression). The Escherichia coli transformants with plasmid-encoded genes were evaluated at the transcriptional and translational levels under red or green light illumination. Under green light illumination, the transcription and translation of the rfp gene were observed, whereas the expression of the bfp gene was repressed. Under red light illumination, the transcription and translation of the bfp gene were observed, whereas the expression of the rfp gene was repressed. During the red and green light exposure cycles at every 6 h, BFP expression increased under red light exposure while RFP expression was repressed, and RFP expression increased under green light exposure while BFP expression was repressed. CONCLUSION: An artificial CCA system was developed to realize a multiple gene expression system, which was regulated by two colors, red and green lights, using a single photosensor system, the CcaS/CcaR system derived from Synechocystis sp. PCC6803, in E. coli. The artificial CCA system functioned repeatedly during red and green light exposure cycles. These results demonstrate the potential application of this CCA gene expression system for the production of multiple metabolites in a variety of microorganisms, such as cyanobacteria.

Synergy of production of value-added bioplastic, astaxanthin and phycobilin co-products and Direct Green 6 textile dye remediation in Spirulina platensis.

Phyco-remediation of dyestuffs in textile wastewaters is of economic, industrial, and environmental importance. We evaluated the remediation of the textile dye, Direct Green 6 (DG6), by Spirulina platensis, and investigated the novel possibility that DG6 treatment enhances production of the biopolymer, polyhydroxybutyrate (PHB). We showed that both live and dead cells of Spirulina were capable of DG6 remediation, but live cells could be re-used with no loss of remediation efficiency. Furthermore, DG6 remediation by live cells resulted in increased algal biomass and trichome lengths, and stimulated production of valuable metabolites, including PHB, antioxidants, carbohydrates and pigments (phycobilins and astaxanthin). We determined the optimal conditions for DG6 remediation and an artificial neural network (ANN) accurately modeled the experimental data and predicted the concentration of dye as the most and algal turbidity as the least important parameters for DG6 removal efficiency. A DG6 concentration of 60 mg L-1 resulted in the highest simultaneous co-production of PHB (12.7 ± 1.7% DW) and increase of astaxanthin (194%), carotenoids (50%), phenol (51%), carbohydrates (27%) total phycobilin (43%), together with the enhancement of biomass and trichome lengths (95%). Oxidative stress indices and enzyme activities such as peroxidases and laccase (involved in dye removal/antioxidant functions) were also increased by dye dosage. On the basis of our results, we propose that S. platensis may use DG6 dye as a nitrogen/carbon source for co-accumulation of valuable bioplastic and metabolites.

Hydrogen-Bond Network Determines the Early Photoisomerization Processes of Cph1 and AnPixJ Phytochromes.

Phytochrome proteins are light receptors that play a pivotal role in regulating the life cycles of plants and microorganisms. Intriguingly, while cyanobacterial phytochrome Cph1 and cyanobacteriochrome AnPixJ use the same phycocyanobilin (PCB) chromophore to absorb light, their excited-state behavior is very different. We employ multiscale calculations to rationalize the different early photoisomerization mechanisms of PCB in Cph1 and AnPixJ. We found that their electronic S1 , T1 , and S0 potential minima exhibit distinct geometric and electronic structures due to different hydrogen bond networks with the protein environment. These specific interactions influence the S1 electronic structures along the photoisomerization paths, ultimately leading to internal conversion in Cph1 but intersystem crossing in AnPixJ. This explains why the excited-state relaxation in AnPixJ is much slower (ca. 100 ns) than in Cph1 (ca. 30 ps). Further, we predict that efficient internal conversion in AnPixJ can be achieved upon protonating the carboxylic group that interacts with PCB.