Elements of the C-terminal tail of a C-terminal domain homolog of the Orange Carotenoid Protein determining xanthophyll uptake from liposomes.

Carotenoids perform multifaceted roles in life ranging from coloration over light harvesting to photoprotection. The Orange Carotenoid Protein (OCP), a light-driven photoswitch involved in cyanobacterial photoprotection, accommodates a ketocarotenoid vital for its function. OCP extracts its ketocarotenoid directly from membranes, or accepts it from homologs of its C-terminal domain (CTDH). The CTDH from Anabaena (AnaCTDH) was shown to be important for carotenoid transfer and delivery from/to membranes. The C-terminal tail of AnaCTDH is a critical structural element likely serving as a gatekeeper and facilitator of carotenoid uptake from membranes. We investigated the impact of amino acid substitutions within the AnaCTDH-CTT on echinenone and canthaxanthin uptake from DOPC and DMPG liposomes. The transfer rate was uniformly reduced for substitutions of Arg-137 and Arg-138 to Gln or Ala, and depended on the lipid type, indicating a weaker interaction particularly with the lipid head group. Our results further suggest that Glu-132 has a membrane-anchoring effect on the PC lipids, specifically at the choline motif as inferred from the strongly different effects of the CTT variants on the extraction from the two liposome types. The substitution of Pro-130 by Gly suggests that the CTT is perpendicular to both the membrane and the main AnaCTDH protein during carotenoid extraction. Finally, the simultaneous mutation of Leu-133, Leu-134 and Leu-136 for alanines showed that the hydrophobicity of the CTT is crucial for carotenoid uptake. Since some substitutions accelerated carotenoid transfer into AnaCTDH while others slowed it down, carotenoprotein properties can be engineered toward the requirements of applications.

Induced Chirality in Canthaxanthin Aggregates Reveals Multiple Levels of Supramolecular Organization.

Carotenoids tend to form supramolecular aggregates via non-covalent interactions where the chirality of individual molecules is amplified to the macroscopic level. We show that this can also be achieved for non-chiral carotenoid monomers interacting with polysaccharides. The chirality induction in canthaxanthin (CAX), caused by heparin (HP) and hyaluronic acid (HA), was monitored by chiroptical spectroscopy. Electronic circular dichroism (ECD) and Raman optical activity (ROA) spectra indicated the presence of multiple carotenoid formations, such as H- and J-type aggregates. This is consistent with molecular dynamics (MD) and density functional theory (DFT) simulations of the supramolecular structures and their spectroscopic response.

Improvement of Carotenoids' Production by Increasing the Activity of Beta-Carotene Ketolase with Different Strategies.

Canthaxanthin is an important antioxidant with wide application prospects, and ß-carotene ketolase is the key enzyme involved in the biosynthesis of canthaxanthin. However, the challenge for the soluble expression of ß-carotene ketolase is that it hinders the large-scale production of carotenoids such as canthaxanthin and astaxanthin. Hence, this study employed several strategies aiming to improve the soluble expression of ß-carotene ketolase and its activity, including selecting optimal expression vectors, screening induction temperatures, adding soluble expression tags, and adding a molecular chaperone. Results showed that all these strategies can improve the soluble expression and activity of ß-carotene ketolase in Escherichia coli. In particular, the production of soluble ß-carotene ketolase was increased 8 times, with a commercial molecular chaperon of pG-KJE8, leading to a 1.16-fold enhancement in the canthaxanthin production from ß-carotene. Interestingly, pG-KJE8 could also enhance the soluble expression of ß-carotene ketolase derived from eukaryotic microalgae. Further research showed that the production of canthaxanthin and echinenone was significantly improved by as many as 30.77 times when the pG-KJE8 was added, indicating the molecular chaperone performed differently among different ß-carotene ketolase. This study not only laid a foundation for further research on the improvement of ß-carotene ketolase activity but also provided new ideas for the improvement of carotenoid production.

Canthaxanthin Mitigates Cardiovascular Senescence in Vitro and in Vivo.

BACKGROUND: The number of older people in the world is increasing year by year; studies have shown that more than 90% of cardiovascular disease occurs in the older people population, indicating that aging is one of the major risks involved in the development of cardiovascular disease. Therefore, retarding the development of cardiac aging is an important strategy to prevent aging-related cardiovascular diseases. METHODS: In the current study, we examined the anti-cardiovascular aging potential of canthaxanthin in vitro and in vivo experiments. For this, a model of cardiomyocyte senescence induced by D-galactose was established, which was used to investigate the canthaxanthin's effect on cardiac premature aging. RESULTS: We found that canthaxanthin obviously mitigated the cardiomyocyte senescence in vitro. Further mechanistic studies revealed that canthaxanthin seems to alleviate cardiomyocyte senescence by regulating the autophagy process. Furthermore, the effects of canthaxanthin on cardiovascular senescence were further evaluated. We also observed that canthaxanthin mitigated cardiac aging and fibrosis in the aged mice model. CONCLUSIONS: To sum up, the current work showed that canthaxanthin could obviously alleviate cardiac premature aging, indicating that canthaxanthin could be used as a biologically active molecule for the treatment of cardiac aging and fibrosis.

Lipid composition and properties affect protein-mediated carotenoid uptake efficiency from membranes.

Carotenoids are pigments of diverse functions ranging from coloration over light-harvesting to photoprotection. Yet, the number of carotenoid-binding proteins, which mobilize these pigments in physiological media, is limited, and the mechanisms of carotenoid mobilization are still not well understood. The same applies for the determinants of carotenoid uptake from membranes into carotenoproteins, especially regarding the dependence on the chemical properties of membrane lipids. Here, we investigate xanthophyll uptake capacity and kinetics of a paradigmatic carotenoid-binding protein, the homolog of the Orange Carotenoid Protein's C-terminal domain from Anabaena sp. PCC 7120 (AnaCTDH), using liposomes formed from defined lipid species and loaded with canthaxanthin (CAN) and echinenone (ECN), respectively. Phospholipids with different chain length and degree of saturation were investigated. The composition of carotenoid-loaded liposomes directly affected the incorporation yield and storage ratio of CAN and ECN as well as the rate of carotenoid uptake by AnaCTDH. Generally, saturated PC lipids were identified as unsuitable, and a high phase transition temperature of the lipids negatively affected the carotenoid incorporation and storage yield. For efficient carotenoid transfer, the velocity increases with increasing chain length or membrane thickness. An average transfer yield of 93 % and 43 % were obtained for the formation of AnaCTDH(CAN) and AnaCTDH(ECN) holoproteins, respectively. In summary, the most suitable lipids for the formation of AnaCTDH(CAN/ECN) holoproteins by carotenoid transfer from artificial liposomes are phosphatidylcholine (18:1) and phosphatidylglycerol (14:0). Thus, these two lipids provide the best conditions for further investigation of lipid-protein interaction and the carotenoid uptake process.

Dietary Canthaxanthin Supplementation Promotes the Laying Rate and Follicular Development of Huaixiang Hens.

Canthaxanthin(CX) is a ketocarotenoid, which is widely used in poultry production as a lipophilic antioxidant. Huaixiang chickens are a local breed in China famous for their excellent meat quality; improving their laying rate via nutritional regulation has attracted extensive attention. The aim of this study was to evaluate the effects of dietary CX on the laying rate and follicular development in Huaixiang hens. A total of 180 Huaixiang hens were randomly divided into five groups with six replicates, and six chickens per replication. The control group (CON) were fed a basal diet, and the treatment group (NT) were fed a basal diet supplemented with 4, 6, 8 and 10 mg/kg CX. All chickens were 26 weeks old, living at an average environmental temperature of 25 ± 2 °C with a relative humidity of 65-75%. The results showed that supplementing the CX improved the laying rate and large white follicles (LWF) number (p < 0.05) and increased the concentration of reproductive hormones (LH, FSH, E2 and Prog) (p < 0.05), and the basal diet supplemented with 6 mg/kg CX worked best. Moreover, CX could increase the activities of antioxidant enzymes SOD and GSH-Px (p < 0.05) and reduce the content of the lipid peroxidation product MDA in Huaixiang chickens (p < 0.05); again, 6 mg/kg CX was best. In conclusion, dietary CX had positive effects on the laying rate, ovarian structure, reproductive hormone secretion, follicle development, and the antioxidant capacity of Huaixiang hens, and 6 mg/kg CX was recommended to be added to the diet of Huaixiang chickens.

Microbial canthaxanthin: an orange-red keto carotenoid with potential pharmaceutical applications.

Canthaxanthin is an orange-red keto-carotenoid that occurs naturally and is also manufactured by synthetic methods for regular applications. In nature, canthaxanthin mainly exists in microbes such as different bacterial species, fungi, and algae, as well as in animals such as crustaceans, certain fishes, and birds. However, the amount of canthaxanthin produced in these organisms varies significantly. Additionally, the compound can be generated from genetically modified organisms using genetic engineering techniques Canthaxanthin finds extensive application as an additive in animal feed, in the pharmaceutical industry, as a coloring agent for various food products, and in cosmetics. It has powerful antioxidant properties and plays a role in lipid metabolism, neuroprotection, and immunomodulation. This article gives an extensive insight into the structure and methods of synthesis of canthaxanthin along with its various newly discovered sources identified so far. The significant applications of canthaxanthin, particularly its role in pharmaceuticals, are critically evaluated. Furthermore, the article discusses future aspects and challenges associated with canthaxanthin production and regulation.

Coproduction of Phase-Separated Carotenoids and ß-Farnesene as a Yeast Biomass Valorization Strategy.

Valorization, the process whereby waste materials are converted into more valuable products, is rarely practiced in industrial fermentation. We developed a model valorization system whereby Saccharomyces cerevisiae that had previously been engineered to produce high concentrations (>100 g/L) of extracellular ß-farnesene was further engineered to simultaneously produce intracellular carotenoids, both products being isoprenoids. Thus, a single fermentation generates two valuable products, namely, ß-farnesene in the liquid phase and carotenoids in the solid biomass phase. Initial attempts to produce high levels of canthaxanthin (a ketocarotenoid used extensively in animal feed) in a ß-farnesene production strain negatively impacted both biomass growth and ß-farnesene production. A refined approach used a promoter titration strategy to reduce ß-carotene production to a level that had minimal impact on growth and ß-farnesene production in fed-batch fermentations and then engineered the resulting strain to produce canthaxanthin. Further optimization of canthaxanthin coproduction used a bioprospecting approach to identify ketolase enzymes that maximized conversion of ß-carotene to canthaxanthin. Finally, we demonstrated that ß-carotene is not present in the extracellular ß-farnesene at a significant concentration and that which is present can be removed by a simple distillation, indicating that ß-farnesene (the primary fermentation product) purity is unaffected by coproduction of carotenoids.

Color-Tuning Mechanism of the Lit Form of Orange Carotenoid Protein.

Orange carotenoid protein (OCP) of photosynthetic cyanobacteria binds to ketocarotenoids noncovalently and absorbs excess light to protect the host organism from light-induced oxidative damage. Herein, we found that mutating valine 40 in the α3 helix of Gloeocapsa sp. PCC 7513 (GlOCP1) resulted in blue- or red-shifts of 6-20 nm in the absorption maxima of the lit forms. We analyzed the origins of absorption maxima shifts by integrating X-ray crystallography, homology modeling, molecular dynamics simulations, and hybrid quantum mechanics/molecular mechanics calculations. Our analysis suggested that the single residue mutations alter the polar environment surrounding the bound canthaxanthin, thereby modulating the degree of charge transfer in the photoexcited state of the chromophore. Our integrated investigations reveal the mechanism of color adaptation specific to OCPs and suggest a design principle for color-specific photoswitches.

Dietary supplementation of spirulina and canthaxanthin boosts laying performance, lipid profile in blood and egg yolk, hatchability, and semen quality of chickens.

This study examined the impacts of dietary spirulina (SP) and canthaxanthin (CX) supplementation to laying hens on reproductive performance and cock's semen quality evaluation. Three hundred and sixty of indigenous Sinai chickens (300 hens + 60 cocks), 48-weeks-old, were randomly divided into five treatments; a control and four supplemented (0.6 g SP mg/kg, 0.8 g SP mg/kg, 6 mg CX/kg diet, and 8 mg CX/kg diet) with six replications of 10 females and 1 male per treatment for laying trial and 1 male for semen evaluation. The animals were fed with experimental diets for 12 weeks. Supplementing 6 mg CX/kg diet led to an increase in the final body weight and egg production traits, while 8 mg CX/kg diet resulted in an improving feed conversion ratio. Supplementation of 0.6 g SP mg/kg or 6.0 mg CX/kg diet resulted in the highest egg production. The heaviest egg weight and egg mass were induced by 0.8 g SP mg/kg. Eggs laid from hens fed 0.6 g SP/kg diet had a higher concentration of cholesterol than those fed 0.8 g SP/kg and those fed 6.0 and 8.0 mg CX/kg. Cocks fed 6 mg CX/kg and those fed 0.8 g SP/kg diet had the greatest sperm concentration. The supplementation of 0.8 g SP mg/kg diet or both 6 and 8 mg CX/kg diet enhanced hatchability. So, dietary spirulina (0.8 mg/kg) and canthaxanthin (6 mg/kg) supplementation to Sinai laying hens and cocks could be used to improve productive and reproductive and performance.

Improvement of Astaxanthin Production in Aurantiochytrium limacinum by Overexpression of the Beta-Carotene Hydroxylase Gene.

Aurantiochytrium limacinum is a heterotrophic eukaryotic microorganism that can accumulate high levels of commercial products such as astaxanthin and docosahexaenoic acid. Due to its rapid growth and relatively simple extraction method, A. limacinum is considered a promising astaxanthin resource to replace the conventional microalgal production. However, the astaxanthin biosynthetic process in A. limacinum remains incompletely understood, especially in those catalysed by ß-carotene hydroxylase (CrtZ) and ketolase. In this study, we overexpressed a crtZ candidate gene to increase astaxanthin production and expand our understanding of the conversion from beta-carotene to astaxanthin. The resultant transformant AlcrtZ#10 cultivated for 5 days showed a significant increase in astaxanthin production per culture (2.8-fold) and per cell (4.5-fold) compared with that of the wild-type strain. Strikingly, longer light exposure increased astaxanthin production and decreased the beta-carotene content in the wild-type strain, suggesting that light exposure duration is important for astaxanthin production in A. limacinum. Among several predicted intermediates, furthermore, the cantaxanthin produced from ß-carotene by ketolase activity were enhanced in the transformant AlcrtZ#10. Although the further investigation is needed, this result suggested that the main route of astaxanthin was via cantaxanthin. Thus, our findings will be valuable not only for its application, but also for understanding the astaxanthin biosynthetic process in A. limacinum.

The role of the local environment on the structural heterogeneity of carotenoid ß-ionone rings.

Our analysis of the X-ray crystal structure of canthaxanthin (CAN) showed that its ketolated ß-ionone rings can adopt two energetically equal, but structurally distinct puckers. Quantum chemistry calculations revealed that the potential energy surface of the ß-ionone ring rotation over the plane of the conjugated π-system in carotenoids depends on the pucker state of the ß-ring. Considering different pucker states and ß-ionone ring rotation, we found six separate local minima on the potential energy surface defining the geometry of the keto-ß-ionone ring-two cis and one trans orientation for each of two pucker states. We observed a small difference in energy and no difference in relative orientation for the cis-minima, but a pronounced difference for the position of trans-minimum in alternative pucker configurations. An energetic advantage of ß-ionone ring rotation from a specific pucker type can reach up to 8 kJ/mol ([Formula: see text]). In addition, we performed the simulation of linear absorption of CAN in hexane and in a unit cell of the CAN crystal. The electronic energies of [Formula: see text] transition were estimated both for the CAN monomer and in the CAN crystal. The difference between them reached [Formula: see text], which roughly corresponds to the energy gap between A and B pucker states predicted by theoretical estimations. Finally, we have discussed the importance of such effects for biological systems whose local environment determines conformational mobility, and optical/functional characteristics of carotenoid.

Canthaxanthin Attenuates the Vascular Aging or Endothelial Cell Senescence by Inhibiting Inflammation and Oxidative Stress in Mice.

BACKGROUND: Vascular endothelial dysfunction is an early phenotype of aging-related vascular dysfunction. Delaying vascular aging and preventing cardiovascular disease are major public health problems that urgently need to be solved. Scientists have studied various drugs to prevent the occurrence and progress of cardiovascular disease, but progress has been slow. Here, the antisenescence and anti-endothelial damage of canthaxanthin (CX, which is an active molecule from food) has been studied. METHODS: This study was performed by adding CX to a model of cell senescence and oxidative damage induced by hydrogen peroxide. Cellular senescence markers (e.g., p16, p21, and p53) and oxidative damage markers (e.g., reactive oxygen species, nitric oxide, malondialdehyde, superoxide dismutase) were evaluated by the enzyme-linked immunosorbent assay, laser scanning confocal microscopy, and Western blotting. RESULTS: We found that CX downregulated the expression level of senescence-associated molecules, and significantly reduced the oxidative damage of vascular endothelial cells. These observations showed that CX effectively alleviated the senescence of vascular endothelial cells. Furthermore, CX treatment reduced the expression levels of interleukin-6 (IL-6), tumor necrosis factor alpha, and IL-1ß. Finally, in vivo, CX significantly alleviated vascular senescence. CONCLUSIONS: The current study shows that CX has potential application value for treating vascular aging or endothelial cell senescence.

Production of Carotenoids and Phospholipids by Thraustochytrium sp. in Batch and Repeated-Batch Culture.

The carotenogenic thraustochytrid Thraustochytrium sp. RT2316-16 was grown in batch and repeated-batch cultures using different feeds containing glucose, or glycerol, and yeast extract, for the production of lipids, phospholipids and carotenoids. RT2316-16 produced canthaxanthin, astaxanthin and ß-carotene. The effects of biotin, ascorbic acid, light and temperature were evaluated in some of the experiments. In 2-day-old batch cultures, the combined mass percentage of eicosapentaenoic acid and docosahexaenoic acid in total lipids was between 16.5% (glycerol-based medium in the dark; biomass concentration = 4.2 ± 1.1 g L-1) and 42.6% (glucose-based medium under light; biomass concentration = 3.3 ± 0.1 g L-1), decreasing to 3.8% and 6.1%, respectively, after day 4. In repeated-batch cultures, the total lipids in the biomass increased after glucose or glycerol was fed alone, whereas the total carotenoids (168 ± 7 µg g-1 dry weight (DW)) and phospholipids in the biomass increased after feeding with yeast extract. The biomass with the highest content of phospholipids (28.7 ± 4.3 mg g-1 DW) was obtained using a feed medium formulated with glycerol, yeast extract and ascorbic acid. Glycerol was the best carbon source for the production of a biomass enriched with total lipids (467 ± 45 mg g-1 DW). The composition of carotenoids depended strongly on the composition of the feed. Repeated-batch cultures fed with yeast extract contained canthaxanthin as the main carotenoid, whereas in the cultures fed only with glucose, the biomass contained mainly ß-carotene.

Canthaxanthin shows anti-liver aging and anti-liver fibrosis effects by down-regulating inflammation and oxidative stress in vivo and in vitro.

The elderly population is growing rapidly all over the world. The aging population has brought great medical pressure to the society. It is found that aging is one of the pathogenic factors of liver fibrosis and liver cancer. Therefore, it is very important to explore functional foods with anti-aging, anti-fibrosis and anti-liver cancer effect. Therefore, in this work, we studied the potential effects of Canthaxanthin on liver aging, liver fibrosis and liver cancer. Firstly, we established the aging modelof liver cells by using H2O2. On this basis, the anti-aging effect of Canthaxanthin was analyzed, and the results showed that Canthaxanthin could significantly alleviate the aging of liver cells through Sa-ß-Gal staining and analysis of the expression of aging related markers. In vivo, aged mice wereused as the animal model for studying the effect of anti-aging of Canthaxanthin. The results showed that Canthaxanthin could significantly alleviate the aging of liver in vivo. Further study show that Canthaxanthin may alleviatethe aging of liver cells by regulating SIRT6; Secondly, we evaluated the effect of Canthaxanthin on liver fibrosis. A model of liver fibrosis was established by CCl4. Masson and Sirius red staining showed that Canthaxanthin could significantly reduce the fibrosis area. Additionally, the level of liver inflammation was also reduced; Thirdly, the effect of Canthaxanthin on hepatoma cells has also been investigated. The resultsshowed that Canthaxanthin could promote the apoptosis of hepatoma cells in vivo and in vitro. To sum up, these results show that canthaxanthin can significantly alleviate liver aging and fibrosis, and Canthaxanthin can also promote the apoptosis of liver cancer cells, indicating that Canthaxanthin can be used as a potential drug or health food for the treatment of liveraging related diseases.

The effects of canthaxanthin microencapsulation on yolk color and canthaxanthin deposition in egg yolk of laying hens.

Canthaxanthin is widely used as a feed additive to improve skin and yolk color in poultry. It is insoluble in water and sensitive to oxidation, so commercial canthaxanthin is often microencapsulated with wall materials to improve its solubility and stability. The objective of this study was to evaluate the effects of canthaxanthin microencapsulation on yolk color and canthaxanthin deposition in egg yolk of laying hens. A total of 288 Hyline Brown laying hens (48 wk of age) were allocated to 4 groups with 6 replicates of 12 hens each, and fed a basal diet or the basal diet supplemented with 5 mg/kg canthaxanthin microencapsulated with modified starch (CMMS), gelatin (CMG), and sodium lignosulfonate (CMSL), respectively. Canthaxanthin supplementation did not affect laying performance of hens, but improved (P < 0.05) yolk color of fresh, fried, boiled, and stored (4 and 25°C) eggs. The improvement of yolk color of fresh eggs was greatest in the CMSL group and least in the CMG group (P < 0.05). Both CMMS and CMSL resulted in higher (P < 0.05) yolk canthaxanthin concentration than CMG. The CMSL resulted in higher (P < 0.05) yolk color score of fried eggs than CMMS and CMG and higher (P < 0.05) yolk color score of boiled eggs than CMG, but no difference was observed in stored eggs among three canthaxanthin groups. In conclusion, CMMS and CMSL were more effective in yolk pigmentation than CMG, and CMSL was slightly better than CMMS.

A Fast-Growing Oleaginous Strain of Coelastrella Capable of Astaxanthin and Canthaxanthin Accumulation in Phototrophy and Heterotrophy.

Considering the importance of microalgae as a promising feedstock for the production of both low- and high-value products, such as lipids and pigments, it is desirable to isolate strains which simultaneously accumulate these two types of products and grow in various conditions in order to widen their biotechnological applicability. A novel freshwater strain from the genus Coelastrella was isolated in Belgium. Compared to other Coelastrella species, the isolate presented rapid growth in phototrophy, dividing 3.5 times per day at a light intensity of 400 µmol·m-2·s-1 and 5% CO2. In addition, nitrogen depletion was associated with the accumulation of astaxanthin, canthaxanthin, and fatty acids, which reached ~30% of dry weight, and a majority of SFAs and MUFAs, which are good precursors for biodiesel. This strain also accumulated astaxanthin and canthaxanthin in heterotrophy. Although the content was very low in this latter condition, it is an interesting feature considering the biotechnological potential of the microalgal heterotrophic growth. Thus, due to its rapid growth in the light, its carotenogenesis, and its fatty acids characteristics, the newly identified Coelastrella strain could be considered as a potential candidate for biorefinery purposes of both low- and high-values products.

Coelastrella terrestris for Adonixanthin Production: Physiological Characterization and Evaluation of Secondary Carotenoid Productivity.

A novel strain of Coelastrella terrestris (Chlorophyta) was collected from red mucilage in a glacier foreland in Iceland. Its morphology showed characteristic single, ellipsoidal cells with apical wart-like wall thickenings. Physiological characterization revealed the presence of the rare keto-carotenoid adonixanthin, as well as high levels of unsaturated fatty acids of up to 85%. Initial screening experiments with different carbon sources for accelerated mixotrophic biomass growth were done. Consequently, a scale up to 1.25 L stirred photobioreactor cultivations yielded a maximum of 1.96 mg·L-1 adonixanthin in free and esterified forms. It could be shown that supplementing acetate to the medium increased the volumetric productivity after entering the nitrogen limitation phase compared to autotrophic control cultures. This study describes a promising way of biotechnological adonixanthin production using Coelastrella terrestris.

Canthaxanthin Biofabrication, Loading in Green Phospholipid Vesicles and Evaluation of In Vitro Protection of Cells and Promotion of Their Monolayer Regeneration.

In the present study, canthaxanthin was produced by biofermentation from Dietzia natronolimnaea HS-1 (D. natronolimnaea) and was loaded in phospholipid vesicles prepared with natural component using an easy and low dissipative method. Indeed, glycerosomes, hyalurosomes, and glycerohyalurosomes were prepared by direct hydration of both phosphatidylcholine and the biotechnological canthaxanthin, avoiding the use of organic solvents. Vesicles were sized from 63 nm to 87 nm and highly negatively charged. They entrapped a high number of the biomolecules and were stable on storage. Canthaxanthin-loaded vesicles incubated with fibroblasts did not affect their viability, proving to be highly biocompatible and capable of inhibiting the death of fibroblasts stressed with hydrogen peroxide. They reduced the nitric oxide expression in macrophages treated with lipopolysaccharides. Moreover, they favoured the cell migration in an in vitro lesion model. Results confirmed the health-promoting potential of canthaxanthin in skin cells, which is potentiated by its suitable loading in phospholipid vesicles, thus suggesting the possible use of these natural bioformulations in both skin protection and regeneration, thanks to the potent antioxidant, anti-inflammatory and antiageing effects of canthaxanthin.

Construction of Canthaxanthin-Producing Yeast by Combining Spatiotemporal Regulation and Pleiotropic Drug Resistance Engineering.

The ketocarotenoid canthaxanthin has important applications in the feed industry. Its biosynthesis using microbial cell factories is an attractive alternative to the current chemical synthesis route. Canthaxanthin-producing Saccharomyces cerevisiae was constructed by introducing the ß-carotene ketolase variant OBKTM29 into a ß-carotene producer. Subcellular re-localization of OBKTM29 was explored, together with copy number adjustment both in the cytoplasm and on the periplasmic membrane, to accelerate the conversion of ß-carotene to canthaxanthin. Moreover, pleiotropic drug resistance (PDR) regulators Pdr1 and Pdr3 were overexpressed to improve the stress tolerance of the yeast strain, leading to obviously enhanced canthaxanthin production. The synthetic pathway was then regulated by a temperature-responsive GAL system to separate product synthesis from cell growth. Finally, 1.44 g/L canthaxanthin was harvested in fed-batch fermentation. This work demonstrated the power of spatial and temporal regulation and the efficiency of PDR engineering in heterologous biosynthesis.