Phototrophy by antenna-containing rhodopsin pumps in aquatic environments.

Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps3 from marine photoheterotrophs have thus far failed4-6. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.

Chlorophyll to zeaxanthin energy transfer in nonphotochemical quenching: An exciton annihilation-free transient absorption study.

Zeaxanthin (Zea) is a key component in the energy-dependent, rapidly reversible, nonphotochemical quenching process (qE) that regulates photosynthetic light harvesting. Previous transient absorption (TA) studies suggested that Zea can participate in direct quenching via chlorophyll (Chl) to Zea energy transfer. However, the contamination of intrinsic exciton-exciton annihilation (EEA) makes the assignment of TA signal ambiguous. In this study, we present EEA-free TA data using Nicotiana benthamiana thylakoid membranes, including the wild type and three NPQ mutants (npq1, npq4, and lut2) generated by CRISPR/Cas9 mutagenesis. The results show a strong correlation between excitation energy transfer from excited Chl Qy to Zea S1 and the xanthophyll cycle during qE activation. Notably, a Lut S1 signal is absent in the npq1 thylakoids which lack zeaxanthin. Additionally, the fifth-order response analysis shows a reduction in the exciton diffusion length (LD) from 62 ± 6 nm to 43 ± 3 nm under high light illumination, consistent with the reduced range of exciton motion being a key aspect of plants' response to excess light.

Modulation of xanthophyll cycle impacts biomass productivity in the marine microalga Nannochloropsis.

Life on earth depends on photosynthetic primary producers that exploit sunlight to fix CO2 into biomass. Approximately half of global primary production is associated with microalgae living in aquatic environments. Microalgae also represent a promising source of biomass to complement crop cultivation, and they could contribute to the development of a more sustainable bioeconomy. Photosynthetic organisms evolved multiple mechanisms involved in the regulation of photosynthesis to respond to highly variable environmental conditions. While essential to avoid photodamage, regulation of photosynthesis results in dissipation of absorbed light energy, generating a complex trade-off between protection from stress and light-use efficiency. This work investigates the impact of the xanthophyll cycle, the light-induced reversible conversion of violaxanthin into zeaxanthin, on the protection from excess light and on biomass productivity in the marine microalgae of the genus Nannochloropsis. Zeaxanthin is shown to have an essential role in protection from excess light, contributing to the induction of nonphotochemical quenching and scavenging of reactive oxygen species. On the contrary, the overexpression of zeaxanthin epoxidase enables a faster reconversion of zeaxanthin to violaxanthin that is shown to be advantageous for biomass productivity in dense cultures in photobioreactors. These results demonstrate that zeaxanthin accumulation is critical to respond to strong illumination, but it may lead to unnecessary energy losses in light-limiting conditions and accelerating its reconversion to violaxanthin provides an advantage for biomass productivity in microalgae.

Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

Zeaxanthin is required for eyespot formation and phototaxis in Euglena gracilis.

The eyespot apparatus is an organelle that forms carotenoid-rich globules in diverse flagellated microalgae and functions in phototaxis. The euglenophytes have structurally and functionally distinct eyespot apparatuses from chlorophytes. ß-Carotene is the most abundant pigment detected in chlorophytes' eyespots, while xanthophylls such as zeaxanthin and diadinoxanthin have been suggested to function in euglenophytes' eyespots. Here, we investigated the association between carotenoid composition and eyespot formation via pathway-scale mutagenesis using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing in the euglenophyte Euglena gracilis. Lycopene cyclase (lcy) mutants exhibited sole lycopene accumulation, defective red eyespots, and phototactic insensitivity. Conversely, ß-carotene hydroxylase (cytochrome P450 97h1, cyp97h1) mutants accumulated ß-carotene and its hydroxylated products ß-cryptoxanthin and zeaxanthin and formed phototactic eyespot apparatuses, while cyp97h1 cyp97f2 double mutants were deficient in ß-carotene hydroxylation and mostly lacked functional eyespots. Thus, zeaxanthin is required for the stable formation of functional eyespots in E. gracilis, highlighting evolutionary differences between euglenophytes and chlorophytes in the metabolic regulation of photoreactive organelle formation.

Zeaxanthin impairs angiogenesis and tumor growth of glioblastoma: An in vitro and in vivo study.

OBJECTIVES: To investigate the therapeutic effects of Zeaxanthin (Zea), one of the oxidized xanthophyll carotenoids belonging to the isoprenoids, on inhibiting the angiogenesis and tumor growth of glioblastoma (GBM) via an in vitro and in vivo study. METHODS: The effects of Zea on the proliferation, adhesion, migration and invasion of human GBM cell lines were detected by cell proliferation assay, cell adhesion assay and Transwell assay. The effect of Zea on angiogenesis was detected by rat aortic ring assay and human umbilical vein endothelial cells (HUVEC) in vitro tube formation assay. The effects of Zea on PARP, Caspase 3 and VEGFR2 phosphorylation as well as VEGFR2's downstream signaling pathway were detected by Western blot. The in vivo human GBM xenograft mouse model was employed to study the therapeutic efficacy of Zea. RESULTS: Zea impaired the proliferation, adhesion, migration and invasion of U87 and U251 cells as well as HUVECs. Rat aortic ring experiments displayed Zea significantly inhibited angiogenesis during VEGF-induced microvascular germination. In vitro and in vivo vascular experiments verified that Zea inhibited VEGF-induced HUVEC proliferation and capillary-like tube formation. Additionally, Zea induced GBM cells apoptosis via increasing the expression of cleaved PARP and Caspase 3. In HUVECs and U251 GBM cells, Zea down-regulated VEGF-induced activation of the VEGFR2 kinase pathway. Meanwhile the expression of p-AKT, p-ERK, p-STAT3 and FAK were all attenuated in U251 cells. Moreover, the effects of Zea on GBM cells proliferation could be blocked by VEGFR2 kinase inhibitor SU5408. These results suggest that Zea may hinder GBM angiogenesis and tumor growth through down-regulating a cascade of oncogenic signaling pathways, both through the inhibition of angiogenesis and the anti-tumor mechanism of a direct cytotoxic effect. Besides, Zea inhibits GBM angiogenesis and tumor growth exemplified through a xenograft mouse model in vivo. CONCLUSION: Zea impairs angiogenesis and tumor growth of GBM both in vitro and in vivo. It can be declared that Zea is a potential valuable anticancer candidate for the future treatment strategy of GBM.

Lutimonas zeaxanthinifaciens sp. nov., a zeaxanthin-producing marine bacterium isolated from coastal sediment.

A Gram-stain-negative, yellow-pigmented, strictly aerobic, non-flagellated, motile by gliding, rod-shaped bacterium, designated strain YSD2104T, was isolated from a coastal sediment sample collected from the southeastern part of the Yellow Sea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain YSD2104T was closely related to three type strains, Lutimonas vermicola IMCC1616T (97.4 %), Lutimonas saemankumensis SMK-142T (96.9 %), and Lutimonas halocynthiae RSS3-C1T (96.8 %). Strain YSD2104T has a single circular chromosome of 3.54 Mbp with a DNA G+C content of 38.3 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain YSD2104T and the three type strains (L. vermicola IMCC1616 T, L. saemankumensis SMK-142T, and L. halocynthiae RSS3-C1T) were 74.0, 86.2 and 73.6 %, and 17.9, 30.3 and 17.8 %, respectively. Growth was observed at 20-30 °C (optimum, 30 °C), at pH 6.5-8.5 (optimum, pH 7.0), and with NaCl concentrations of 1.5-3.5 % (optimum, 2.5 %). The major carotenoid was zeaxanthin, and flexirubin-type pigment was not produced. The major respiratory quinone was menaquinone-6. The major fatty acids (>10 %) were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), and summed feature 9 (iso-C17 : 1 ω9c and/or 10-methyl C16 : 0). The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, two unidentified aminolipids, and eight unidentified lipids. Conclusively, based on this polyphasic approach, we classified strain YSD2104T (=KCTC 102008T=JCM 36287T) as representing a novel species of the genus Lutimonas and proposed the name Lutimonas zeaxanthinifaciens sp. nov.

Complete microbial synthesis of crocetin and crocins from glycerol in Escherichia coli.

BACKGROUND: Crocin, a glycosylated apocarotenoid pigment predominantly found in saffron, has garnered significant interest in the field of biotechnology for its bioactive properties. Traditional production of crocins and their aglycone, crocetin, typically involves extraction from crocin-producing plants. This study aimed to develop an alternative biosynthetic method for these compounds by engineering the metabolic pathways of zeaxanthin, crocetin, and crocin in Escherichia coli strains. RESULTS: Employing a series of genetic modifications and the strategic overexpression of key enzymes, we successfully established a complete microbial pathway for synthesizing crocetin and four glycosylated derivatives of crocetin, utilizing glycerol as the primary carbon source. The overexpression of zeaxanthin cleavage dioxygenase and a novel variant of crocetin dialdehyde dehydrogenase resulted in a notable yield of crocetin (34.77 ± 1.03 mg/L). Further optimization involved the overexpression of new types of crocetin and crocin-2 glycosyltransferases, facilitating the production of crocin-1 (6.29 ± 0.19 mg/L), crocin-2 (5.29 ± 0.24 mg/L), crocin-3 (1.48 ± 0.10 mg/L), and crocin-4 (2.72 ± 0.13 mg/L). CONCLUSIONS: This investigation introduces a pioneering and integrated microbial synthesis method for generating crocin and its derivatives, employing glycerol as a sustainable carbon feedstock. The substantial yields achieved highlight the commercial potential of microbial-derived crocins as an eco-friendly alternative to plant extraction methods. The development of these microbial processes not only broadens the scope for crocin production but also suggests significant implications for the exploitation of bioengineered compounds in pharmaceutical and food industries.

Carotenoid pattern intake and relation to metabolic status, risk and syndrome, and its components - divergent findings from the ORISCAV-LUX-2 survey.

Carotenoids are generally associated with health-beneficial effects; however, their intake patterns related to the metabolic syndrome (MetS) and its components remain controversial. This cross-sectional study investigated associations between dietary intakes of individual carotenoids, fruits and vegetables, and the MetS and its components. Dietary intakes of 1346 participants of the Observation des Risques et de la Santé Cardio-Vasculaire au Luxembourg (ORISCAV-LUX-2) study were investigated by a 174-item FFQ, and carotenoid intake was determined by linking findings using mainly the USDA food databases. Components of MetS and complementary variables, including anthropometric (BMI, waist circumferences and waist:hip ratio) and biological parameters (TAG, HDL-cholesterol, fasting blood glucose and blood pressure), were measured. Logistic (for MetS) and linear multivariable regression models (including assessing MetS as scores) adjusted for various confounders were created. α-and ß-Carotene, as well as lutein + zeaxanthin, were inversely associated with MetS (also when it was measured on a continuous scale), reducing the odds for MetS by up to 48 %. However, lycopene, phytoene and phytofluene were rather positively associated with MetS scores and its components, though these adverse effects disappeared, at least for lycopene, when controlling for intakes of tomato-based convenience foods, in line with indicating a rather unhealthy/westernised diet. All these associations remained significant when including fruits and vegetables as confounders, suggesting that carotenoids were related to MetS independently from effects within fruits and vegetables. Thus, a high intake of carotenoids was bidirectionally associated with MetS, its severity, risk and its components, depending on the type of carotenoid. Future investigations are warranted to explore the inverse role that tomato-based carotenoids appear to suggest in relation to the MetS.

Combined high light and salt stress enhances accumulation of PsbS and zeaxanthin in Chlamydomonas reinhardtii.

The performance and acclimation strategies of Chlamydomonas reinhardtii under stress conditions are typically studied in response to single stress factors. Under natural conditions, however, organisms rarely face only one stressor at a time. Here, we investigated the impact of combined salt and high light stress on the photoprotective response of C. reinhardtii. Compared to the single stress factors, the combination of both stressors decreased the photosynthetic performance, while the activation of energy dissipation remained unaffected. However, the PsbS protein was strongly accumulated and the conversion of violaxanthin to zeaxanthin was enhanced. These results support an important photoprotective function of PsbS and zeaxanthin independently of energy dissipation under combined salt and high light stress in C. reinhardtii.

Do red and yellow autumn leaves make use of different photoprotective strategies during autumn senescence?

Our goal was to determine whether anthocyanin-producing species (red) use different photoprotective strategies to cope with excess light during fall senescence compared with non-anthocyanin-producing species (yellow). In a previous study, we found that a yellow species retained the photoprotective PsbS protein in late autumn, while a red species did not. Specifically, we tested the hypothesis that red species make less use of zeaxanthin and PsbS-mediated thermal dissipation, as they rely on anthocyanins for photoprotection. We monitored four red (Acer ginnala, Rhus typhnia, Parenthocissus quinquefolia, Viburnum dentatum) and four yellow species (Acer negundo, Ostrya virginiana, Vitis riparia, Zanthoxylum americanum) throughout autumn senescence and analyzed pigments, protein content, and chlorophyll fluorescence. We found yellow species retained the PsbS protein at higher levels, and had higher dark retention of zeaxanthin in late autumn relative to red species. All species retained lutein and the pool of xanthophyll cycle pigments in higher amounts than other carotenoids in late autumn. Our data support the hypothesis that red species use anthocyanins as a photoprotective strategy during autumn senescence, and therefore make less use of PsbS and zeaxanthin-mediated thermal dissipation. We also found species-specific variation in the particular combination of photoprotective strategies used.

Lutein, zeaxanthin, and meso-zeaxanthin supplementation attenuates inflammatory cytokines and markers of oxidative cardiovascular processes in humans.

BACKGROUND AND AIMS: Systemic inflammation and oxidation are primary contributors to the development of atherosclerosis. Oxidation of low-density lipoprotein (LDL) particles within the vascular endothelium has been hypothesized to be an initial step in the formation of atherosclerotic plaques, with inflammatory cytokines serving as the signaling mechanism for concomitant macrophage activation. Supplementation with the antioxidative macular xanthophylls (lutein [L], zeaxanthin [Z], and meso-zeaxanthin [MZ]) has been shown to aid in the reduction of inflammatory physiologic responses; therefore, we hypothesized that in our study population, supplementation with these xanthophylls would facilitate a systemic reduction in markers of inflammation and cardiovascular lipid oxidation. METHODS AND RESULTS: In this double-blind placebo-controlled supplementation study, participants were randomly allocated to receive the active intervention containing L (10 mg) + MZ (10 mg) + Z (2 mg) or placebo (containing sunflower oil). Serum concentrations of carotenoids (assessed by HPLC), inflammatory cytokines (IL-6, IL-1ß, TNF-α) and oxidized LDL (OxLDL; by solid-phase sandwich ELISA) were measured at baseline and at 6-months. Results showed that over the supplementation period, compared to placebo, the active group demonstrated statistically significant increases in serum concentrations of L, Z, & MZ (p < 0.05), reductions in inflammatory cytokines IL-1ß (p < 0.001) and TNF-α (p = 0.003), as well as a corresponding reduction in serum OxLDL (p = 0.009). CONCLUSIONS: Our data show that L, Z, & MZ supplementation results in decreased serum IL-1ß, TNF-α, and OxLDL. This suggests that these carotenoids are acting systemically to attenuate oxidative lipid products and inflammation, thus reducing their contribution to atherosclerotic plaque formation.

Associations of serum carotenoids with all-cause and cardiovascular mortality in adults with MAFLD.

BACKGROUND AND AIMS: The associations between serum carotenoids and mortality are contradictory in various metabolic-associated diseases. This study aimed to examine the associations of five major serum carotenoids with mortality among adults with metabolic dysfunction-associated fatty liver disease (MAFLD). METHODS AND RESULTS: This analysis included 3040 individuals with MAFLD from the Third National Health and Nutrition Examination Survey (NHANES III). All-cause and cardiovascular mortality were ascertained by linkage to the National Death Index through December 31, 2019. Cox proportional hazards regression models were employed to estimate hazard ratios (HRs) and 95% confidence intervals (CIs), and restricted cubic spline (RCS) analyses were performed to assess the linearity of the associations. During a follow-up period of 826,547 person-years, 1325 all-cause and 429 cardiovascular deaths occurred. For all-cause mortality, compared with those in the lowest quartiles, the multivariable-adjusted HRs (95% CIs) in the highest quartiles were 0.63 (0.49-0.81) for α-carotene; 0.65 (0.52-0.80) for ß-carotene; 0.64 (0.51-0.81) for ß-cryptoxanthin; 0.73 (0.56-0.95) for lycopene; and 0.69 (0.52-0.91) for lutein/zeaxanthin. For cardiovascular mortality, the multivariable-adjusted HRs (95% CIs) in the highest quartiles were 0.51 (0.33-0.78) for α-carotene; 0.54 (0.35-0.82) for ß-carotene; 0.52 (0.34-0.80) for ß-cryptoxanthin; 0.63 (0.44-0.90) for lycopene; and 0.62 (0.39-0.99) for lutein/zeaxanthin. Besides, serum α-carotene, ß-cryptoxanthin, and lycopene exhibited linear correlations with all-cause mortality in MAFLD adults, and four serum carotenoids, except ß-carotene, were linearly correlated with cardiovascular mortality. CONCLUSIONS: Lower serum α-carotene, ß-carotene, ß-cryptoxanthin, lycopene, and lutein/zeaxanthin concentrations were associated with higher risk of all-cause and cardiovascular mortality in US adults with MAFLD.

Association between dietary carotenoid intake and breast cancer risk: a case-control study among Korean women.

The association between dietary carotenoids and breast cancer (BC) risks were inconsistent. Therefore, this study investigated the association between dietary carotenoid and BC risks among Korean women. We recruited participants from the National Cancer Centre of Korea. Odds ratios and 95% confidence intervals were calculated with a logistic regression model. There was an inverse association between dietary carotenoid subclasses and BC risks; in particular, a higher intake of ß-carotene and lutein/zeaxanthin was associated with reduced BC risks. After subgroup analysis with estrogen receptor (ER)/progesterone receptor (PR) status, there was similar trend among ER-/PR- women. We further investigated which foods contribute to the carotenoid intake. A higher intake of radish leaves, kale, and bracken was associated with lowered BC risks. Accordingly, dietary carotenoid, particularly ß-carotene and lutein/zeaxanthin, appears to be associated with a lower risk of BC among Korean women.

Zeaxanthin and Lutein Ameliorate Alzheimer's Disease-like Pathology: Modulation of Insulin Resistance, Neuroinflammation, and Acetylcholinesterase Activity in an Amyloid-ß Rat Model.

Alzheimer's disease (AD) is characterized by impaired insulin/insulin-like growth factor-1 signaling in the hippocampus. Zeaxanthin and lutein, known for their antioxidant and anti-inflammatory properties, have been reported to protect against brain damage and cognitive decline. However, their mechanisms related to insulin signaling in AD remain unclear. This study investigated the efficacy and mechanisms of zeaxanthin, lutein, and resveratrol in modulating an AD-like pathology in an amyloid-ß rat model. Rats were administered hippocampal infusions of 3.6 nmol/day amyloid-ß (Aß)(25-35) for 14 days to induce AD-like memory deficits (AD-CON). Normal control rats received Aß(35-25) (Normal-CON). All rats had a high-fat diet. Daily, AD rats consumed 200 mg/kg body weight of zeaxanthin (AD-ZXT), lutein (AD-LTN), and resveratrol (AD-RVT; positive-control) or resistant dextrin as a placebo (AD-CON) for eight weeks. The AD-CON rats exhibited a higher Aß deposition, attenuated hippocampal insulin signaling (reduced phosphorylation of protein kinase B [pAkt] and glycogen synthase kinase-3ß [pGSK-3ß]), increased neuroinflammation, elevated acetylcholinesterase activity, and memory deficits compared to the Normal-CON group. They also showed systemic insulin resistance and high hepatic glucose output. Zeaxanthin and lutein prevented memory impairment more effectively than the positive-control resveratrol by suppressing acetylcholinesterase activity, lipid peroxidation, and pro-inflammatory cytokines (TNF-α, IL-1ß). They also potentiated hippocampal insulin signaling and increased brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CTNF) mRNA expression to levels comparable to the Normal-CON rats. Additionally, zeaxanthin and lutein improved glucose disposal, reduced hepatic glucose output, and normalized insulin secretion patterns. In conclusion, zeaxanthin and lutein supplementation at doses equivalent to 1.5-2.0 g daily in humans may have practical implications for preventing or slowing human AD progression by reducing neuroinflammation and maintaining systemic and central glucose homeostasis, showing promise even when compared to the established neuroprotective compound resveratrol. However, further clinical trials are needed to evaluate their efficacy and safety in human populations.

The Na+ /Ca2+ antiporter slr0681 affects carotenoid production in Synechocystis sp. PCC 6803 under high-light stress.

BACKGROUND: Carotenoids play key roles in photosynthesis and are widely used in foods as natural pigments, antioxidants, and health-promoting compounds. Enhancing carotenoid production in microalgae via biotechnology has become an important area of research. RESULTS: We knocked out the Na+ /Ca2+ antiporter gene slr0681 in Synechocystis sp. PCC 6803 via homologous recombination and evaluated the effects on carotenoid production under normal (NL) and high-light (HL) conditions. On day 7 of NL treatment in calcium ion (Ca2+ )-free medium, the cell density of Δslr0681 decreased by 29% compared to the wild type (WT). After 8 days of HL treatment, the total carotenoid contents decreased by 35% in Δslr0681, and the contents of individual carotenoids were altered: myxoxanthophyll, echinenone, and ß-carotene contents increased by 10%, 50%, and 40%, respectively, while zeaxanthin contents decreased by ~40% in Δslr0681 versus the WT. The expression patterns of carotenoid metabolic pathway genes also differed: ipi expression increased by 1.2- to 8.5-fold, whereas crtO and crtR expression decreased by ~90% and 60%, respectively, in ∆slr0681 versus the WT. In addition, in ∆slr0681, the expression level of psaB (encoding a photosystem I structural protein) doubled, whereas the expression levels of the photosystem II genes psbA2 and psbD decreased by ~53% and 84%, respectively, compared to the WT. CONCLUSION: These findings suggest that slr0681 plays important roles in regulating carotenoid biosynthesis and structuring of the photosystems in Synechocystis sp. This study provides a theoretical basis for the genetic engineering of microalgae photosystems to increase their economic benefits and lays the foundation for developing microalgae germplasm resources with high carotenoid contents. © 2023 Society of Chemical Industry.

Microbial chassis as the platform for production of dihydroxy xanthophyll-based carotenoids: an overview of recent advances in biomanufacturing.

Physiological and environmental cues prompt microbes to synthesize diverse carotenoids, including dihydroxy xanthophylls, facilitating their adaptation and survival. Lutein and its isomeric counterpart, zeaxanthin, are notable dihydroxy xanthophylls with bioactive properties such as antioxidative, anti-inflammatory, anticancer, and neuroprotective effects, particularly beneficial for human ocular health. However, global natural resources for co-producing lutein and zeaxanthin are scarce, with zeaxanthin lacking commercial sources, unlike lutein sourced from marigold plants and microalgae. Traditionally, dihydroxy xanthophyll production primarily relies on petrochemical synthetic routes, with limited biological sourcing reported. Nonetheless, microbiological synthesis presents promising avenues as a commercial source, albeit challenged by low dihydroxy xanthophyll yield at high cell density. Strategies involving optimization of physical and chemical parameters are essential to achieve high-quality dihydroxy xanthophyll products. This overview briefly discusses dihydroxy xanthophyll biosynthesis and highlights recent advancements, discoveries, and industrial benefits of lutein and zeaxanthin production from microorganisms as alternative biofactories.

ASTER-B regulates mitochondrial carotenoid transport and homeostasis.

The scavenger receptor class B type 1 (SR-B1) facilitates uptake of cholesterol and carotenoids into the plasma membrane (PM) of mammalian cells. Downstream of SR-B1, ASTER-B protein mediates the nonvesicular transport of cholesterol to mitochondria for steroidogenesis. Mitochondria also are the place for the processing of carotenoids into diapocarotenoids by ß-carotene oxygenase-2. However, the role of these lipid transport proteins in carotenoid metabolism has not yet been established. Herein, we showed that the recombinant StART-like lipid-binding domain of ASTER-A and B preferentially binds oxygenated carotenoids such as zeaxanthin. We established a novel carotenoid uptake assay and demonstrated that ASTER-B expressing A549 cells transport zeaxanthin to mitochondria. In contrast, the pure hydrocarbon ß-carotene is not transported to the organelles, consistent with its metabolic processing to vitamin A in the cytosol by ß-carotene oxygenase-1. Depletion of the PM from cholesterol by methyl-ß-cyclodextrin treatment enhanced zeaxanthin but not ß-carotene transport to mitochondria. Loss-of-function assays by siRNA in A549 cells and the absence of zeaxanthin accumulation in mitochondria of ARPE19 cells confirmed the pivotal role of ASTER-B in this process. Together, our study in human cell lines established ASTER-B protein as key player in nonvesicular transport of zeaxanthin to mitochondria and elucidated the molecular basis of compartmentalization of the metabolism of nonprovitamin A and provitamin A carotenoids in mammalian cells.

The Amount of Zeaxanthin Epoxidase But Not the Amount of Violaxanthin De-Epoxidase Is a Critical Determinant of Zeaxanthin Accumulation in Arabidopsis thaliana and Nicotiana tabacum.

The generation of violaxanthin (Vx) de-epoxidase (VDE), photosystem II subunit S (PsbS) and zeaxanthin (Zx) epoxidase (ZEP) (VPZ) lines, which simultaneously overexpress VDE, PsbS and ZEP, has been successfully used to accelerate the kinetics of the induction and relaxation of non-photochemical quenching (NPQ). Here, we studied the impact of the overexpression of VDE and ZEP on the conversion of the xanthophyll cycle pigments in VPZ lines of Arabidopsis thaliana and Nicotiana tabacum. The protein amount of both VDE and ZEP was determined to be increased to about 3- to 5-fold levels of wild-type (WT) plants for both species. Compared to WT plants, the conversion of Vx to Zx, and hence VDE activity, was only marginally accelerated in VPZ lines, whereas the conversion of Zx to Vx, and thus ZEP activity, was strongly increased in VPZ lines. This indicates that the amount of ZEP but not the amount of VDE is a critical determinant of the equilibrium of the de-epoxidation state of xanthophyll cycle pigments under saturating light conditions. Comparing the two steps of epoxidation, particularly the second step (antheraxanthin to Vx) was found to be accelerated in VPZ lines, implying that the intermediate Ax is released into the membrane during epoxidation by ZEP.

Generation and physiological characterization of genome-edited Nicotiana benthamiana plants containing zeaxanthin as the only leaf xanthophyll.

MAIN CONCLUSION: Simultaneous genome editing of the two homeologous LCYe and ZEP genes of Nicotiana benthamiana results in plants in which all xanthophylls are replaced by zeaxanthin. Plant carotenoids act both as photoreceptors and photoprotectants in photosynthesis and as precursors of apocarotenoids, which include signaling molecules such as abscisic acid (ABA). As dietary components, the xanthophylls lutein and zeaxanthin have photoprotective functions in the human macula. We developed transient and stable combinatorial genome editing methods, followed by direct LC-MS screening for zeaxanthin accumulation, for the simultaneous genome editing of the two homeologous Lycopene Epsilon Cyclase (LCYe) and the two Zeaxanthin Epoxidase (ZEP) genes present in the allopolyploid Nicotiana benthamiana genome. Editing of the four genes resulted in plants in which all leaf xanthophylls were substituted by zeaxanthin, but with different ABA levels and growth habits, depending on the severity of the ZEP1 mutation. In high-zeaxanthin lines, the abundance of the major photosystem II antenna LHCII was reduced with respect to wild-type plants and the LHCII trimeric state became unstable upon thylakoid solubilization. Consistent with the depletion in LHCII, edited plants underwent a compensatory increase in PSII/PSI ratios and a loss of the large-size PSII supercomplexes, while the level of PSI-LHCI supercomplex was unaffected. Reduced activity of the photoprotective mechanism NPQ was shown in high-zeaxanthin plants, while PSII photoinhibition was similar for all genotypes upon exposure to excess light, consistent with the antioxidant and photoprotective role of zeaxanthin in vivo.