Identification of Desiccation Stress-Inducible Antioxidative and Antiglycative Ultraviolet-Absorbing Oxylipins, Saclipin A and Saclipin B, in an Edible Cyanobacterium Aphanothece sacrum.

Aphanothece sacrum, a freshwater cyanobacterium, is an edible cyanobacterial strain. We identified two compounds belonging to the oxylipin family that possess UV-absorbing abilities and accumulate in the dried sample of A. sacrum. The compounds, named saclipin A and saclipin B, exhibited strong UV-absorption properties with the absorption maxima at 316 and 319 nm, respectively, and the molar extinction coefficients of 26,454 and 30,555 M-1 cm-1, respectively. The chemical structures of saclipins A and B have been elucidated, revealing that they have an all-E and a 12Z isomeric relationship within the triene structure. The saclipins could be isomerized by photoirradiation, with the cis-form saclipin B proving to be more stable in methanol, ethanol, or acetonitrile. Under drought stress conditions, the accumulation of saclipins A and B in A. sacrum was found to be increased 20- and 10-fold, respectively. Purified saclipins from A. sacrum showed biocompatibility and valuable bioactivities. Specifically, saclipins exhibited radical scavenging activity, maintaining their activity even 40 min after the reaction began. Additionally, they demonstrated inhibitory activity against glycation of elastin and collagen, which are constituents of dermal tissue. Notably, saclipins showed higher activity than the well-known glycation inhibitor aminoguanidine against collagen glycation.

Active role of the protein translation machinery in protecting against stress tolerance in Synechococcus elongatus PCC7942.

In vivo protein synthesis is crucial for all domains of life. It is accomplished through translational machinery, and a key step is the translocation of tRNA-mRNA by elongation factor G (EF-G). Genome-based analysis revealed two EF-G encoding genes (S0885 and S2082) in the freshwater cyanobacterium model Synechococcus elongatus PCC7942. S0885 is the essential EF-G gene for photosynthesis. We generated a strain of S. elongatus PCC7942 that overexpressed S0885 (OX-S0885) to identify EF-G functionality. RT-PCR and Western blot analyses revealed increased transcriptional and translational levels in OX-S0885 at 10.5-13.5 and 2.0-3.0 fold, respectively. Overexpression of S0885 led to an increase in specific growth rate. Additionally, polysome-to-monosome ratio (P/M) and RNA-to-protein ratio (R/P) were elevated in OX-S0885 compared with the empty vector. Interestingly, R/P in OX-S0885 was retained at more than 70% under oxidative stress while R/P in the empty vector was severely depleted, suggesting the maintenance of translation. Thus, S0885 appeared to be the important target of oxidative stress because it was protected by the stress response system to maintain its function. These results suggest that cyanobacterial EF-G has a primary function in translation and an unrelated activity during stress conditions. These findings support the substantial role of EF-G in the formation and maintenance of cellular protein formation, and in the protection of the global translational mechanism under oxidative stress condition.

Halotolerance mechanisms in salt­tolerant cyanobacteria.

Cyanobacteria are ubiquitously distributed in nature and are the most abundant photoautotrophs on Earth. Their long evolutionary history reveals that cyanobacteria have a remarkable capacity and strong adaptive tendencies to thrive in a variety of conditions. Thus, they can survive successfully, especially in harsh environmental conditions such as salty environments, high radiation, or extreme temperatures. Among others, salt stress because of excessive salt accumulation in salty environments, is the most common abiotic stress in nature and hampers agricultural growth and productivity worldwide. These detrimental effects point to the importance of understanding the molecular mechanisms underlying the salt stress response. While it is generally accepted that the stress response mechanism is a complex network, fewer efforts have been made to represent it as a network. Substantial evidence revealed that salt-tolerant cyanobacteria have evolved genomic specific mechanisms and high adaptability in response to environmental changes. For example, extended gene families and/or clusters of genes encoding proteins involved in the adaptation to high salinity have been collectively reported. This chapter focuses on recent advances and provides an overview of the molecular basis of halotolerance mechanisms in salt­tolerant cyanobacteria as well as multiple regulatory pathways. We elaborate on the major protective mechanisms, molecular mechanisms associated with halotolerance, and the global transcriptional landscape to provide a gateway to uncover gene regulation principles. Both knowledge and omics approaches are utilized in this chapter to decipher the mechanistic insights into halotolerance. Collectively, this chapter would have a profound impact on providing a comprehensive understanding of halotolerance in salt­tolerant cyanobacteria.

Halotolerance, stress mechanisms, and circadian clock of salt-tolerant cyanobacteria.

Cyanobacteria harbor a high level of physiological flexibility, which enables them to reside in virtually all available environmental niches, including extreme environments. In this review, we summarize the recent advancements in stress mechanisms of salt-tolerant (a.k.a. halotolerant) cyanobacteria. Omics approaches have been extensively employed in recent years to decipher mechanisms of halotolerance and to understand the relevance of halotolerance-associated gene regulatory networks. The vast knowledge from genome mining disclosed that halotolerant cyanobacteria possess extended gene families and/or clusters, encoding enzymes that synthesize unique osmoprotectants, including glycine betaine (GB), betaine derivatives, and mycosporine-like amino acids (MAAs). Comprehensive transcriptomic analyses were conducted using Halothece sp. PCC7418 (hereafter referred to as Halothece), a cyanobacterium that exhibits remarkable halotolerance. These studies revealed a specific transcriptional response when Halothece was subjected to salt stress, whereas salt and osmotic stresses were found to share a common transcriptomic response. Transcriptome and metabolite analyses of Halothece illustrated a complex dynamic relationship between the biosyntheses of osmoprotectants, as well as corresponding and ancillary pathways. Lastly, novel insights highlight the relationship between the molecular regulation of the circadian rhythm and salt stress tolerance. Since the circadian rhythm of gene expression was distorted under salt stress, halotolerant cyanobacteria may prioritize the adaptation to salt stress by attenuation of circadian rhythmicity. KEY POINTS: • Recent advancements in the understanding of stress mechanisms in halotolerant cyanobacteria are described based on omics analyses. • Transcriptome and metabolite analyses of Halothece illustrated a complex dynamic relationship between the biosyntheses of osmoprotectants, as well as corresponding and ancillary pathways. • Since salt stress affects the molecular regulation among clock-related proteins, salt stress may attenuate circadian rhythmicity.

Global transcriptome analyses and regulatory mechanisms in Halothece sp. PCC 7418 exposed to abiotic stresses.

Halotolerant species are of interest since they occur naturally in environments with excess toxic ions. The cyanobacterium Halothece sp. PCC 7418 (hereafter referred to as Halothece) exhibits remarkable halotolerance and was used to examine stress-responsive regulatory mechanisms. The effects of five different stimuli on Halothece transcriptomes were examined using RNA sequencing. In response to diverse stresses, there were both common and stress-specific transcriptional responses. A common upregulated gene set under all stresses consisted of nine differentially expressed genes (DEGs). We also found that osmotic stress elicited the largest set of DEGs. Salt- and osmotic-responsive regulatory mechanisms shared common pathways. DEGs that were upregulated under salt stress encoded proteins involved in photosynthesis and related machineries. Furthermore, DEGs encoding two-component system (TCS) factors, transcriptional factors, scaffolds for protein-protein interactions, transporters, protein turnover factors, and lipid biosynthesis enzymes were also identified under salt stress. Notably, one-carbon (1C) metabolism factors, glycine betaine (GB) synthesis enzymes, and GB transporters were upregulated under salt stress. Metabolic analyses revealed that GB accumulated under salt stress, while mycosporine-2-glycine (M2G) accumulated under salt or osmotic stress. None of the nutrient starvations induced GB nor M2G accumulation. These results suggested that GB and M2G are two osmoprotectants that contribute to halotolerance. Based on our results, we proposed regulatory mechanisms that are crucial for halotolerance, which are coordinated with the GB, M2G, 1C, amino acid, and central carbon interlinking metabolic pathways. 1C metabolism directly fulfills the high metabolite requirements for halotolerance together with the ancillary role of several metabolic pathways.Key Points• Global transcriptome surveys together with molecular and metabolite analyses provide insights into regulatory networks that are crucial for halotolerance• Regulatory networks that are crucial for halotolerance coordinated with the two key osmoprotectants, one carbon, amino acid, and central carbon interlinking metabolic pathways• The findings have translational relevance in genomic and transcriptomic mechanisms of halotolerance.

Global transcriptional and circadian regulation in a halotolerant cyanobacterium Halothece sp. PCC7418.

Substantial evidence has been accumulated about the molecular basis underlying halotolerance; however, insights into the regulatory networks for relevant genes and mechanisms of their interplay remain elusive. Here, we present a comprehensive transcriptome investigation, using RNA sequencing, of specific metabolic pathways and networks in a halotolerant cyanobacterium, Halothece sp. PCC7418, including the circadian rhythm profile. Dissecting the transcriptome presented the intracellular regulation of gene expressions, which was linked with ion homeostasis, protein homeostasis, biosynthesis of compatible solutes, and signal transduction, for adaptations to high-salinity environments. The efficient production and distribution of energy were also implicated in this acclimation process. Furthermore, we found that high-salinity environments had a dramatic effect on the global transcriptional expression regulated by the circadian clock. Our findings can provide a comprehensive transcriptome for elucidating the molecular mechanisms underlying halotolerance in cyanobacteria.

Oral Supplementation with Z-Isomer-Rich Astaxanthin Inhibits Ultraviolet Light-Induced Skin Damage in Guinea Pigs.

The effect of oral supplementation with astaxanthin of different Z-isomer ratios on ultraviolet (UV) light-induced skin damage in guinea pigs was investigated. Astaxanthin with a high Z-isomer content was prepared from the all-E-isomer via thermal isomerization. Intact (all-E)-astaxanthin and the prepared Z-isomer-rich astaxanthin were suspended in soybean oil and fed to guinea pigs for three weeks. The UV-light irradiation was applied to the dorsal skin on the seventh day after the start of the test diet supplementation, and skin parameters, such as elasticity, transepidermal water loss (TEWL), and pigmentation (melanin and erythema values), were evaluated. The accumulation of astaxanthin in the dorsal skin was almost the same after consumption of the all-E-isomer-rich astaxanthin diet (E-AST-D; total Z-isomer ratio = 3.2%) and the Z-isomer-rich astaxanthin diet (Z-AST-D; total Z-isomer ratio = 84.4%); however, the total Z-isomer ratio of astaxanthin in the skin was higher in the case of the Z-AST-D supplementation. Both diets inhibited UV light-induced skin-damaging effects, such as the reduction in elasticity and the increase in TEWL level. Between E-AST-D and Z-AST-D, Z-AST-D showed better skin-protective ability against UV-light exposure than E-AST-D, which might be because of the greater UV-light-shielding ability of astaxanthin Z-isomers than the all-E-isomer. Furthermore, supplementation with Z-AST-D resulted in a greater reduction in skin pigmentation caused by astaxanthin accumulation compared to that of E-AST-D. This study indicates that dietary astaxanthin accumulates in the skin and appears to prevent UV light-induced skin damage, and the Z-isomers are more potent oral sunscreen agents than the all-E-isomer.

Molecular and functional insights into glutathione S-transferase genes associated with salt stress in Halothece sp. PCC7418.

Evolution and function of glutathione S-transferase (GST) in primordial oxygenic phototrophs such as cyanobacteria are poorly understood. In this study, we identified and functionally characterized the GST gene family in the halotolerant cyanobacterium Halothece sp. PCC7418. Four putative Halothece-GSTs had very low homology, which implies evolutionary divergence. Of these, H0647, H0729 and H3557 were differentially expressed by oxidative stress whereas H3557 was highly and specifically upregulated under salt stress. In vitro analysis revealed that the recombinant H3557 exhibited GST activity toward 1-chloro-2, 4-dinitrobenzene (CDNB) and glutathione (GSH). H3557 displayed a broad range of activity at pH 6.5-10.5. Kinetic parameters showed the apparent Km for CDNB and GSH was 0.14 and 0.75 mM, respectively. H3557 remained catalytically active in the presence of NaCl. Structural modelling supported that H3557 is salt-adaptive enzyme with highly acidic residues on the protein surface. The vital function of H3557 in heterologous expression system was evaluated. The H3557-expressing cells were more tolerant to H2 O2 -induced oxidative stress compared with other GST-expressing cells and conferred salt tolerance. Taken together, the findings of this study provide insights into the molecular and cellular functions of GST in cyanobacteria, particularly under salt stress, which is less understood compared with other species.

Protective effects of mycosporine-like amino acid-containing emulsions on UV-treated mouse ear tissue from the viewpoints of antioxidation and antiglycation.

Mycosporine-like amino acids (MAAs) are promising natural antioxidative compounds with cosmetic applications for the prevention of skin aging. In this study, we evaluated the protective effects of natural resources-derived MAA-containing emulsions on mouse ear tissue exposed to UV irradiation. DBA/2CrSlc male mice were irradiated by UV light at 120 mJ/cm2/day for 9 days. MAA-containing emulsions were prepared using mycosporine-2-glycine (M2G), shinorine (SHI), or porphyra-334 (P334) and applied to mice ears at a dose of 50 mg/ear/day. After that, collected ear skin tissues were subjected to the observation of melanocytes, investigation for antioxidative stress markers, and measurement of advanced glycation-end products (AGEs). In addition, the antiglycative effects of MAAs were investigated in vitro. MAA-containing emulsions prepared in this study upregulated the activities of total superoxide dismutase (SOD) and catalase (CAT) in mouse ear tissue exposed to UV irradiation. Increased accumulation of copper/zinc (Cu/Zn) -SOD and/or CAT was also found in mouse ear tissue on which M2G- or P334-containing emulsion had been applied. Furthermore, P334 exhibited an antiglycative effect on elastin in vitro. Although MAA-containing emulsions have antioxidative effects as well as in vitro antiglycation, a protective effect by the accumulation of AGEs in mice ears exposed to UV was not observed. Thus, application of MAA-containing emulsions stimulated or protected the expression of antioxidant-associated proteins, thereby leading to upregulation of antioxidative activities in mouse ear skin samples tissues under UV irradiation. Additional optimization of MAA-containing emulsions, including composition, process, and dosage should be considered for further improvement of efficacy.

Insights into the phylogeny and transcriptional response of serine proteases in a halotolerant cyanobacterium Halothece sp. PCC7418.

Serine proteases are a class of versatile proteolytic enzymes. They are necessary for protein catabolism, intracellular amino acid turnover, and regulation of proteins involved in diverse molecular and cellular processes across taxa. In this study, bioinformatic analyses revealed a significantly large number of serine proteases in the halotolerant cyanobacterium Halothece sp. PCC7418 (hereafter referred to as Halothece 7418) compared to the model freshwater cyanobacterium Synechococcus elongatus PCC7942 (hereafter referred to as S. elongatus 7942). The cyanobacterial serine proteases are likely derived from different linages since no conserved motifs were detected. The presence of highly diverse serine proteases in Halothece 7418 implicated an evolutionary-mediated modification of several proteases, which may play numerous physiological roles. We also examined the gene expression patterns of 34 serine protease encoding genes in Halothece 7418 exposed to salt stress. Our results revealed that several serine protease genes were drastically up-regulated under salt with high concentration but remained unchanged under salt with low concentration. All four clp genes (H1996, H1997, H0950, and H3375) and H3553 gene (which encodes a putative HtrA protease) were significantly induced upon salt stress. These responses support the roles of the housekeeping pathways in both the degradation of damaged proteins induced by salt stress and regulation of proteins involved in the molecular recovery from salt stress. Since serine proteases share several biochemical features and physiological functions, the results from this study provide an insight into diversification of serine proteases in cyanobacteria. Further, these results will increase our understanding of several mechanisms at the subcellular level.

Evaluation and improvement of storage stability of astaxanthin isomers in oils and fats.

Astaxanthin Z-isomers potentially have greater bioavailability and biological activity than (all-E)-astaxanthin. However, the stability of the Z-isomers is lower than the all-E-isomer, which is a serious problem affecting its practical use. In this study, we investigated the impacts of different suspension media (oils and fats) and additives on astaxanthin isomer stability and identified suitable ones for astaxanthin stabilization. The evaluations showed that several vegetable oils and antioxidants significantly improved astaxanthin isomer stability, e.g., when soybean and sunflower oils were used as the suspension medium, astaxanthin isomers were hardly degraded; however the total Z-isomer ratio decreased from ~80% to ~50% during 6-week storage at 30 °C. Moreover, it was revealed that (9Z)-astaxanthin showed higher stability than the 13Z- and 15Z-isomers. Hence, to maintain astaxanthin concentration and the Z-isomer ratio over long periods, it is important to use suitable suspension mediums and antioxidants, and select a Z-isomerization method that increases (9Z)-astaxanthin ratio.

Synergistic Effects of Food Ingredients and Vegetable Oils on Thermal Isomerization of Lycopene.

Recent investigations have demonstrated that some food ingredients and vegetable oils, such as onion, garlic, and sesame oil, enhanced thermal Z-isomerization of (all-E)-lycopene in tomatoes. However, the synergistic effects of these ingredients and oils have not yet been investigated. This study aims at clarifying how the combined use of lycopene Z-isomerization-promoting food ingredients and vegetable oils impacts thermal Z-isomerization of (all-E)-lycopene in tomato puree. Apart from a few exceptions, when olive oil was used as a reaction medium, the combined use of garlic, cabbage, broccoli, shiitake mushroom, and makonbu improved the total Z-isomer ratio of lycopene after heating compared to the separate use of the tested ingredients. However, when onion was used together with the other ingredients, the Z-isomer ratio significantly decreased compared to its individual use. Moreover, when garlic, cabbage, broccoli, shiitake mushroom, and makonbu were used with sesame and mustard oils, that exhibit higher Z-isomerizationpromoting effect than that of olive oil, the lycopene Z-isomerization reaction was further enhanced. However, when onion was combined with these oils, the Z-isomer ratio decreased compared to that measured upon the combined use of onion with olive oil. Our results on these synergistic effects are not only important for the food and drink manufacturing industries but also for daily home cooking.

Antioxidative and Antiglycative Properties of Mycosporine-Like Amino Acids-Containing Aqueous Extracts Derived from Edible Terrestrial Cyanobacteria.

The terrestrial filamentous cyanobacterium, Nostoc commune, has been used as a food source in many countries, especially countries in Asia. In this study, N. commune-derived aqueous extracts were evaluated with regard to their antioxidative and antiglycative properties. The antioxidative activity was significantly higher in N. commune colonies isolated from the field than in extracts from colonies cultured in the laboratory. The antioxidative compound content of extracts, including phenolic compounds and phycobiliproteins, was correlated with their antioxidative power. In addition, two mycosporine-like amino acids (MAAs), specifically detected in colonies isolated from the field, were purified. In addition to assessing their antioxidative properties, the antiglycative activity of these MAAs was also assessed. Their inhibitory effects on glycation-dependent protein cross-linking might contribute to the antiglycative power of the extract prepared from field colonies. Taken together, the results from this study revealed that N. commune may have beneficial properties for functional food applications, both by preventing oxidative stress and suppressing the formation of advanced glycation end-products.

Morphological plasticity of hyperelongated cells caused by overexpression of translation elongation factor P in Synechococcus elongatus PCC7942.

Translation elongation factors (EFs) are proteins that play important roles during the elongation stage of protein synthesis. In prokaryotes, at least four EFs function in repetitive reactions (EF-Tu, EF-Ts, EF-G, and EF-P). EF-P plays a vital role in the specialized translation of consecutive proline amino acid motifs. It was also recently recognized that EF-P acts throughout translation elongation. Here, we demonstrated for the first time that cell division and morphology are intimately linked to the control of EF-P in the model cyanobacterium Synechococcus elongatus PCC7942. We constructed the overexpression of a wild-type gene product for EF-P (Synpcc7942_2565) as a tool to identify EF-P functionality. The overexpression of EF-P resulted in the morphological plasticity of hyperelongated cells. During the stationary phase, EF-P overexpressors displayed cell lengths of 150 µm or longer, approximately 35 times longer than the control. Total cellular protein and amino acid content were also increased in overexpressors. To explore the molecular mechanisms underlying hyperelongation, gene expression analysis was performed. The results revealed that cell division genes, including ftn6, minD, mreB, mreC, and ftsZ, were modulated in overexpressors. Strikingly, ftn6 was severely down-regulated. Little is known regarding EF-P in prokaryotic photosynthetic organisms. Our results suggest that cyanobacterial EF-P participates in the acceleration of protein synthesis and also regulates cell division processes. These findings suggest new ways to modify translation and metabolism in cyanobacteria. Phenotypic and metabolic alterations caused by overexpressing EF-P may also be beneficial for applications such as low-cost, green molecular factories. KEY POINTS: • Cell division and cell morphology in the cyanobacterium Synechococcus elongatus PCC7942 are closely linked with the control of translation elongation factor P (EF-P). • Overexpression of EF-P leads to morphological plasticity in hyperelongated cells. • Cyanobacterial EF-P is involved in the acceleration of protein synthesis and the regulation of cell division processes.

The Effects of Salts and Osmoprotectants on Enzyme Activities of Fructose-1,6-biphosphate Aldolases in a Halotolerant Cyanobacterium, Halothece sp. PCC 7418.

The halotolerant cyanobacterium, Halothece sp. PCC 7418, possesses two classes of fructose-1,6-bisphosphate aldolase (FBA): H2846 and H2847. Though class I (CI)-FBA H2846 is thought to be associated with salt tolerance, the regulatory mechanisms, molecular characteristics, and expression profiles between H2846 and class II (CII)-FBA H2847 have scarcely been investigated. Here, we show that the accumulation of the H2846 protein is highly responsive to both up- and down-shock with NaCl, whereas H2847 is constitutively expressed. The activity of CI- and CII-FBA in cyanobacterial extracts is correlated with the accumulation patterns of H2846 and H2847, respectively. In addition, it was found that these activities were inhibited by NaCl and KCl, with CII-FBA activity strikingly inhibited. It was also found that the CI-FBA activity of recombinant H2846 was hindered by salts and that this hindrance could be moderated by the addition of glycine betaine (GB), whereas no moderation occurred with other potential osmoprotectant molecules (proline, sucrose, and glycerol). In addition, a phylogenetic analysis showed that CI-FBAs with higher similarities to H2846 tended to be distributed among potential GB-synthesizing cyanobacteria. Taken together, our results provide insights into the independent evolution of the CI- and CII-FBA gene families, which show distinct expression profiles and functions following salt stress.

The evolutionarily conserved HtrA is associated with stress tolerance and protein homeostasis in the halotolerant cyanobacterium Halothece sp. PCC7418.

The HtrA protein family represents an important class of serine proteases that are widely distributed across taxa. These evolutionarily conserved proteins are crucial for survival and function as monitors of protein synthesis during various stresses. Here, we performed gene expression analysis of the entire set of putative serine protease genes in Halothece sp. PCC7418 under salt stress conditions. The gene-encoding HtrA2 (H3553) was highly upregulated. This gene was cloned and functionally characterized, and its sub-cellular localization was determined. The recombinant H3553 protein (rH3553) displayed a pH optimum of 8.0, remained stable at 45 °C, and its proteolytic activity was not affected by salts. H3553 completely degraded the unfolded model protein, ß-casein. In contrast, the folded model substrates (lysozyme or BSA) were not degraded by rH3553. Denaturation of BSA at a high temperature significantly increased its degradation by rH3553. H3553 was detected in the soluble protein fraction as well as the plasma membrane and thylakoid membrane fractions. Interestingly, the majority of H3553 was present in the plasma membrane under salt and heat stress conditions. Thus, H3553 resides in multiple sub-cellular locations and its localization drastically changes after exposure to stresses. Taken together, H3553 underpins protein quality-control process and is involved in the response and adaptation to salinity and heat stresses.

Isomerization of Commercially Important Carotenoids (Lycopene, ß-Carotene, and Astaxanthin) by Natural Catalysts: Isothiocyanates and Polysulfides.

Effects of natural catalysts, isothiocyanates and polysulfides, on Z-isomerization and decomposition of (all-E)-carotenoids (lycopene, ß-carotene, and astaxanthin) after heat treatment were investigated. When isothiocyanates were added to (all-E)-carotenoid solutions and heated, Z-isomerization and decomposition of carotenoids were enhanced and the degree differed depending on the isothiocyanate type. Interestingly, when polysulfides were applied in the same manner, in addition to promoting the Z-isomerization reaction, they markedly improved the thermal stability of carotenoids. Successively, we investigated the reaction characteristics of allyl isothiocyanate (AITC) and diallyl disulfide (DADS) using (all-E)-lycopene; that is, effects of the amount added, solvent used, and reaction temperature and time, as well as the combination use on Z-isomerization and decomposition of lycopene, were investigated. With increases in the amount added and reaction temperature and time, Z-isomerization of lycopene was promoted for both catalysts. The high-temperature treatment tests clearly showed that AITC induced thermal decomposition of lycopene, whereas DADS improved the lycopene stability. Moreover, the simultaneous use of AITC and DADS resulted in a synergetic effect on the Z-isomerization efficiency.

Enhanced Lipid Production and Molecular Dynamics under Salinity Stress in Green Microalga Chlamydomonas reinhardtii (137C).

Microalgal lipids are a source of valuable nutritional ingredients in biotechnological industries, and are precursors to biodiesel production. Here, the effects of salt-induced stresses, including NaCl, KCl, and LiCl stresses, on the production of lipid in green microalga Chlamydomonas reinhardtii (137c) were investigated. NaCl stress dramatically increased saturated fatty acids (SFAs), which accounted for 70.2% of the fatty acid methyl ester (FAMEs) under stress. In contrary, KCl stress led to a slight increase in SFAs (47.05%) with the remaining being polyunsaturated fatty acids (PUFAs) (45.77%). RT-PCR analysis revealed that the genes involved in FA biosynthesis, such as PDH2, ACCase, MAT and KAS2, were up-regulated by NaCl-induced stress. Conversely, the genes responsible for the Kennedy pathway were suppressed. The alteration of FA homeostasis was further assessed by overexpressing MAT, the enzyme responsible for the production of malonyl-ACP, a key building block for FA biosynthesis, in the cyanobacterium Synechococcus elongatus PCC 7942. Intracellular FA composition was affected, with a predominant synthesis of SFAs in transformed cells. Owing to the diversity and relative abundance of SFAs, monounsaturated fatty acid (MUFAs) and PUFAs enable the feasibility of using microorganisms as a source of microalgal lipids or valuable nutritional ingredients; salt-induced stress and expression of MAT are useful in providing precursors for enhanced lipid production.

A class I fructose-1,6-bisphosphate aldolase is associated with salt stress tolerance in a halotolerant cyanobacterium Halothece sp. PCC 7418.

Fructose-1,6-bisphosphate aldolase (FBA) is a key metabolic enzyme, which is involved in glycolysis, gluconeogenesis and the Calvin cycle. The distinct physiological roles of FBAs in various organisms have been reported; however, in cyanobacteria, the functional characterization of FBAs and investigation of the intracellular dynamics of FBAs largely remains unknown. Here, we utilized a two-step chromatographic technique to identify a class I FBA (CI-FBA), which we named H2846. H2846 was induced by salt stress in the halotolerant cyanobacterium Halothece sp. PCC 7418 (hereafter referred to as Halothece 7418). Phylogenetic analysis showed that H2846-like CI-FBAs existed mainly in cyanobacterial species that inhabit hypersaline environments. Subcellular fractionation revealed that H2846 localized in the cytosolic and periplasmic spaces and size-exclusion chromatography suggested that H2846 formed a homohexamer. The CI-FBA activity of recombinant H2846-mediated cleavage of fructose bisphosphate (FBP) was characterized using a coupled enzymatic assay. This analysis allowed us to determine the Km and Vmax values of recombinant H2846, which were then compared to previously reported Km and Vmax values of several FBAs. Our data suggested that H2846 was likely responsible for the salt stress-induced CI-FBA activity from the total soluble protein extracts derived from Halothece 7418 cells. Moreover, heterologous expression of H2846 but not H2847, a class II FBA (CII-FBA), conferred salt stress tolerance to the salt-sensitive freshwater cyanobacterium, Synechococcus elongatus PCC 7942, which only contains the CII-FBA, S1443. S. elongatus PCC 7942 with a S1443 gene deletion was complemented by H2847 expression, but was not complemented by expression of H2846. Taken together, these results indicate the functional differences between two distinct sets of FBAs in cyanobacteria. H2846 is an active CI-FBA that contributes to the mechanism of salt stress tolerance in Halothece 7418.

Improved Carotenoid Processing with Sustainable Solvents Utilizing Z-Isomerization-Induced Alteration in Physicochemical Properties: A Review and Future Directions.

Carotenoids-natural fat-soluble pigments-have attracted considerable attention because of their potential to prevent of various diseases, such as cancer and arteriosclerosis, and their strong antioxidant capacity. They have many geometric isomers due to the presence of numerous conjugated double bonds in the molecule. However, in plants, most carotenoids are present in the all-E-configuration. (all-E)-Carotenoids are characterized by high crystallinity as well as low solubility in safe and sustainable solvents, such as ethanol and supercritical CO2 (SC-CO2). Thus, these properties result in the decreased efficiency of carotenoid processing, such as extraction and emulsification, using such sustainable solvents. On the other hand, Z-isomerization of carotenoids induces alteration in physicochemical properties, i.e., the solubility of carotenoids dramatically improves and they change from a "crystalline state" to an "oily (amorphous) state". For example, the solubility in ethanol of lycopene Z-isomers is more than 4000 times higher than the all-E-isomer. Recently, improvement of carotenoid processing efficiency utilizing these changes has attracted attention. Namely, it is possible to markedly improve carotenoid processing using safe and sustainable solvents, which had previously been difficult to put into practical use due to the low efficiency. The objective of this paper is to review the effect of Z-isomerization on the physicochemical properties of carotenoids and its application to carotenoid processing, such as extraction, micronization, and emulsification, using sustainable solvents. Moreover, aspects of Z-isomerization methods for carotenoids and functional difference, such as bioavailability and antioxidant capacity, between isomers are also included in this review.