porphyra-334 and mycosporine-2-glycine

Mycosporine-like amino acids (MAAs) are promising natural sunscreens mainly produced in marine organisms. Until now, metabolic engineering efforts to produce MAAs in heterologous hosts have mainly focused on shinorine production, and the low production levels are still not suitable for industrial applications. In this study, we successfully developed Saccharomyces cerevisiae strains that can efficiently produce various disubstituted MAAs, including shinorine, porphyra-334, and mycosporine-2-glycine (M2G), which are formed by conjugating serine, threonine, and glycine to mycosporine-glycine (MG), respectively. We first generated an MG-producing strain by multiple integration of the biosynthetic genes from cyanobacteria and applying metabolic engineering strategies to increase sedoheptulose-7-phosphate pool, a substrate for MG production. Next, five mysD genes from cyanobacteria, which encode D-Ala-D-Ala ligase homologues that conjugate an amino acid to MG, were introduced into the MG-producing strain to determine the substrate preference of each MysD enzyme. MysDs from Lyngbya sp., Nostoclinckia, and Euhalothece sp. showed high specificity toward serine, threonine, and glycine, resulting in efficient production of shinorine, porphyra-334, and M2G, respectively. This is the first report on the production of porphyra-334 and M2G in S. cerevisiae. Furthermore, we identified that the substrate specificity of MysD was determined by the omega loop region of 43-45 amino acids predicted based on its structural homology to a D-Ala-D-Ala ligase from Thermus thermophilus involved in peptidoglycan biosynthesis. The substrate specificities of two MysD enzymes were interchangeable by swapping the omega loop region. Using the engineered strain expressing mysD from Lyngbya sp. or N. linckia, up to 1.53 g/L shinorine or 1.21 g/L porphyra-334 was produced by fed-batch fermentation in a 5-L bioreactor, the highest titer reported so far. These results suggest that S. cerevisiae is a promising host for industrial production of different types of MAAs, providing a sustainable and eco-friendly alternative for the development of natural sunscreens.

Topics: Amino Acids; Cyanobacteria; Glycine; Saccharomyces cerevisiae; Serine; Sunscreening Agents; Threonine

Topics: Animals; Antioxidants; Catalase; Cyclohexanols; Cyclohexanones; Cyclohexylamines; Emulsions; Glycine; Glycosylation; Male; Mice; Mice, Inbred DBA; Skin; Superoxide Dismutase; Ultraviolet Rays; Up-Regulation

Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.

Topics: Cell Line; Chlamydomonas; Cyclohexanols; Cyclohexanones; Cyclohexylamines; Focal Adhesion Protein-Tyrosine Kinases; Glycine; Humans; Keratinocytes; Mitogen-Activated Protein Kinases; Porphyra; Signal Transduction; Wound Healing

The bio-accumulation of mycosporine-like amino acids (MAAs) is common in planktonic copepods that inhabit environments exposed to high levels of solar radiation. MAAs accumulation in copepods can be affected both by extrinsic (environmental) and intrinsic factors (local adaptation, genotype, etc.). Laboratory experiments were performed to study the bio-accumulation of MAAs in two geographically-isolated populations of Boeckella gracilipes from a mountain and a piedmont lake of North Patagonia. We performed two series of 10-day incubations of B. gracilipes from the different lakes applying two radiation conditions (PAR + UVR and darkness), at five different temperatures (5 to 20 °C) and providing a MAA-free flagellate as food. We assumed that differences in final MAAs concentrations between copepod populations should be exclusively due to environmental factors, and that any difference in the patterns of MAAs accumulation should exclusively arise from differences in MAAs concentration at the time of collection. MAAs concentration was three fold higher in B. gracilipes from Lake Verde than in copepods from the Lake Morenito. The MAAs suite was dominated (∼90%) by a combination of porphyra-334 and mycosporine-glycine in copepods from Lake Verde, and porphyra-334 and MAA-332 in those from Lake Morenito. Two exclusive MAA compounds were identified, mycosporine-glycine in copepods from Lake Verde and shinorine in the copepod population from Lake Morenito. Laboratory experiments showed that: (i) exposure to PAR + UVR stimulated the accumulation of MAAs in both copepod populations; (ii) temperature affected the response of MAAs and, remarkably, low temperatures stimulated MAAs accumulation even in dark incubations, (iii) the response to radiation and temperature in MAAs accumulation was more pronounced in the population with low initial MAAs than in the population with high initial MAAs concentrations. The differences in intrinsic factors between B. gracilipes populations, such as local adaptation to contrasting UV and temperature scenarios, among others, appear to play an important role in determining levels and patterns of MAAs accumulation in B. gracilipes.

Topics: Animals; Copepoda; Cyclohexanols; Cyclohexanones; Cyclohexylamines; Darkness; Environment; Food; Glycine; Lakes; South America; Species Specificity; Temperature; Ultraviolet Rays

Certain photosynthetic marine organisms have evolved mechanisms to counteract UV-radiation by synthesizing UV-absorbing compounds, such as mycosporine-like amino acids (MAAs). In this study, MAAs were separated from the extracts of marine green alga Chlamydomonas hedleyi using HPLC and were identified as porphyra-334, shinorine, and mycosporine-glycine (mycosporine-Gly), based on their retention times and maximum absorption wavelengths. Furthermore, their structures were confirmed by triple quadrupole MS/MS. Their roles as UV-absorbing compounds were investigated in the human fibroblast cell line HaCaT by analyzing the expression levels of genes associated with antioxidant activity, inflammation, and skin aging in response to UV irradiation. The mycosporine-Gly extract, but not the other MAAs, had strong antioxidant activity in the 2,2-diphenyl-1-picryhydrazyl (DPPH) assay. Furthermore, treatment with mycosporine-Gly resulted in a significant decrease in COX-2 mRNA levels, which are typically increased in response to inflammation in the skin, in a concentration-dependent manner. Additionally, in the presence of MAAs, the UV-suppressed genes, procollagen C proteinase enhancer (PCOLCE) and elastin, which are related to skin aging, had increased expression levels equal to those in UV-mock treated cells. Interestingly, the increased expression of involucrin after UV exposure was suppressed by treatment with the MAAs mycosporine-Gly and shinorine, but not porphyra-334. This is the first report investigating the biological activities of microalgae-derived MAAs in human cells.

Topics: Amino Acids; Anti-Inflammatory Agents; Cell Line; Chlamydomonas; Chlorophyta; Cyclohexanols; Cyclohexanones; Cyclohexylamines; Cyclooxygenase 2; Elastin; Extracellular Matrix Proteins; Fibroblasts; Glycine; Glycoproteins; Humans; Inflammation; Skin; Skin Aging; Ultraviolet Rays

The composition and abundance of mycosporine-like amino acids (MAAs) were investigated in the surface waters along a 13,000-km meridional transect (52° N to 45° S) in the Atlantic Ocean (Atlantic Meridional Transect programme: Cruise AMT 18: 4/10/2008-10/11/2008). MAAs were ubiquitous along the transect, although the composition of the MAAs was variable. Highest concentrations were in the far south (below 40° S; MAA >1 μg L(-1)) and in north subtropical equatorial region (NER: 0-25° N; MAA up to 0.8 μg L(-1)). Highest MAA relative to chlorophyll-a occurred in the NER (MAA/chl-a ratio between 2 and 5). MAA/chl-a significantly correlated with the preceding month's mean daily UV dose and with UV-B/UV-A. In the far south, high MAA concentrations coincided with high phytoplankton biomass, high nutrients and a deep mixed layer associated with the austral spring. Here, the phytoplankton community was dominated by micro- and nano-eukaryotes. At the NER, the high MAA/chl-a coincided with low nutrient concentrations, a shallow mixed layer depth (20-70 m) and to a lesser extent to a shallow nitracline (40-90 m). Here, the phytoplankton consisted primarily of picophytoplankton (0-0.2 μm), dominated by the pico-cyanobacteria Synechococcus sp. and Prochlorococcus sp. and by the nitrogen fixing filamentous cyanobacterium Trichodesmium. The low nitrate concentrations (<0.1 μmol L(-1)) at the NER suggest that nitrogen fixation was required for MAA production. Specific MAAs could not easily be assigned to particular groups of phytoplankton and we could not rule out the possibility that MAAs were associated with symbiotic cyanobacteria contained within heterotrophic dinoflagellates or diatoms.

Topics: Amino Acids; Atlantic Ocean; Cyanobacteria; Cyclohexanols; Cyclohexanones; Cyclohexylamines; Glycine; Nitrogen Fixation; Phytoplankton; Prochlorococcus; Seawater; Species Specificity

The mycosporine-like amino acid (MAA) profile of a rice-field cyanobacterium, Anabaena doliolum, was studied under PAR and PAR + UVR conditions. The high-performance liquid chromatographic analysis of water-soluble compounds reveals the biosynthesis of three MAAs, mycosporine-glycine (lambda (max) = 310 nm), porphyra-334 (lambda (max) = 334 nm) and shinorine (lambda (max) = 334 nm), with retention times of 4.1, 3.5 and 2.3 min, respectively. This is the first report for the occurrence of mycosporine-glycine and porphyra-334 in addition to shinorine in Anabaena strains studied so far. The results indicate that mycosporine-glycine (monosubstituted) acts as a precursor for the biosynthesis of the bisubstituted MAAs shinorine and porphyra-334. Mycosporine-glycine was under constitutive control while porphyra-334 and shinorine were induced by UV-B radiation, indicating the involvement of UV-regulated enzymes in the biotransformation of MAAs. It seems that A. doliolum is able to protect its cell machinery from UVR by synthesizing a complex set of MAAs and thus is able to survive successfully during the summer in its natural brightly lit habitats.

Topics: Anabaena; Chromatography, High Pressure Liquid; Cyclohexanols; Cyclohexanones; Cyclohexylamines; Glycine; Oryza; Plant Extracts; Spectrum Analysis; Ultraviolet Rays

Coral reef organisms living in mutualistic symbioses with phototrophic dinoflagellates are widespread in shallow UV-transparent waters. Maristentor dinoferus is a recently discovered species of marine benthic ciliate that hosts symbiotic dinoflagellates of the genus Symbiodinium. In this study, we tested this ciliate for the occurrence of mycosporine-like amino acids, a family of secondary metabolites that minimize damage from exposure to solar UV radiation by direct screening. Using high-performance liquid chromatography and liquid chromatography coupled to mass spectrometry, five mycosporine-like amino acids (shinorine, palythenic acid, palythine, mycosporine-2-glycine, and porphyra-334) were identified in aqueous methanolic extracts of the symbiosis. This is the first report of mycosporine-like amino acids in a marine ciliate.

Topics: Amino Acids; Animals; Chromatography, High Pressure Liquid; Chromatography, Liquid; Ciliophora; Cyclohexanols; Cyclohexanones; Cyclohexylamines; Glycine; Seawater; Spectrometry, Mass, Electrospray Ionization; Ultraviolet Rays