Photogeneration and quenching of singlet molecular oxygen by bacterial C40 carotenoids with long chain of conjugated double bonds.

Measurement of photosensitized luminescence of singlet oxygen has been applied to studies of singlet oxygen generation and quenching by C40 carotenoids (neurosporene, lycopene, rhodopin, and spirilloxanthin) with long chain of conjugated double bonds (CDB) using hexafluorobenzene as a solvent. It has been found that neurosporene, lycopene, and rhodopin are capable of the low efficient singlet oxygen generation in aerated solutions upon photoexcitation in the spectral region of their main absorption maxima. The quantum yield of this process was estimated to be (1.5-3.0) × 10-2. This value is near the singlet oxygen yields in solutions of ζ-carotene (7 CDB) and phytoene (3 CDB) and many-fold smaller than in solutions of phytofluene (5 CDB) (Ashikhmin et al. Biochemistry (Mosc) 85:773-780, https://doi.org/10.1134/S0006297920070056 , 2020, Biochemistry (Mosc) 87:1169-1178, 2022, https://doi.org/10.1134/S00062979221001082022 ). Photogeneration of singlet oxygen was not observed in spirilloxanthin solutions. A correlation was found between the singlet oxygen yields and the quantum yields and lifetimes of the fluorescence of the carotenoid molecules. All carotenoids were shown to be strong physical quenchers of singlet oxygen. The rate constants of 1O2 quenching by the carotenoids with long chain of CDB (9-13) were close to the rate constant of the diffusion-limited reactions ≈1010 M-1 s-1, being many-fold greater than the rate constants of 1O2 quenching by the carotenoids with the short chain of CDB (3-7) phytoene, phytofluene, and ζ-carotene studied in prior papers of our group (Ashikhmin et al. 2020, 2022). To our knowledge, the quenching rate constants of rhodopin and spirilloxanthin have been obtained in this paper for the first time. The mechanisms of 1O2 photogeneration by carotenoids in solution and in the LH2 complexes of photosynthetic cells, as well as the efficiencies of their protective action are discussed.

Beneficial effect of ζ-carotene-like compounds on acute UVB irradiation by alleviating inflammation and regulating intestinal flora.

ζ-Carotene is a key intermediate in the carotenoid pathway, but owing to its low content and difficulties in isolation, its application is restricted. In this study, three genes (pnCrtE, pnCrtB, and pnCrtP) in the carotenoid pathway of Antarctic moss were identified, recombined, and expressed in Escherichia coli (E. coli) BL21(DE3). The expression product was identified as one of the ζ-carotenes by UV absorbance spectrum, thin layer chromatography (TLC), and super-high-performance liquid chromatography-mass spectrum (UPLC-MS), and was called a ζ-carotene-like compound (CLC). Excessive exposure to ultraviolet B (UVB) irradiation is one of the main risk factors for skin photodamage. The purpose of this study was to investigate the preventive and therapeutic effects of CLC on UVB-induced skin photodamage in mice. In this paper, through histological examinations (hematoxylin-eosin, HE; Masson and TdT-mediated dUTP Nick-End Labeling, Tunel), biochemical index detection (reactive oxygen species, ROS; inflammatory factors; cyclobutyl pyrimidine dimers, CPDs and hyaluronic acid, HA), quantitative real time polymerase chain reaction (qRT-PCR), immunohistochemistry and intestinal content flora, etc., it is concluded that CLC has the potential to enhance skin antioxidant capacity by activating the nuclear transcription factor/antioxidant reaction element (Nrf2/ARE) pathway and also reduce skin inflammation and aging by inhibiting the mitogen-activated protein kinase (MAPK) pathway. Moreover, the regulation of intestinal flora may potentially mitigate skin damage induced by UVB radiation. This research not only developed a green and sustainable platform for the efficient synthesis of CLC but also laid a foundation for its application in functional food and medicine for skin resistance against UVB damage.

Natural overexpression of CAROTENOID CLEAVAGE DIOXYGENASE 4 in tomato alters carotenoid flux.

Carotenoids and apocarotenoids function as pigments and flavor volatiles in plants that enhance consumer appeal and offer health benefits. Tomato (Solanum lycopersicum.) fruit, especially those of wild species, exhibit a high degree of natural variation in carotenoid and apocarotenoid contents. Using positional cloning and an introgression line (IL) of Solanum habrochaites "LA1777', IL8A, we identified carotenoid cleavage dioxygenase 4 (CCD4) as the factor responsible for controlling the dark orange fruit color. CCD4b expression in ripe fruit of IL8A plants was ∼8,000 times greater than that in the wild type, presumably due to 5' cis-regulatory changes. The ShCCD4b-GFP fusion protein localized in the plastid. Phytoene, ζ-carotene, and neurosporene levels increased in ShCCD4b-overexpressing ripe fruit, whereas trans-lycopene, ß-carotene, and lutein levels were reduced, suggestive of feedback regulation in the carotenoid pathway by an unknown apocarotenoid. Solid-phase microextraction-gas chromatography-mass spectrometry analysis showed increased levels of geranylacetone and ß-ionone in ShCCD4b-overexpressing ripe fruit coupled with a ß-cyclocitral deficiency. In carotenoid-accumulating Escherichia coli strains, ShCCD4b cleaved both ζ-carotene and ß-carotene at the C9-C10 (C9'-C10') positions to produce geranylacetone and ß-ionone, respectively. Exogenous ß-cyclocitral decreased carotenoid synthesis in the ripening fruit of tomato and pepper (Capsicum annuum), suggesting feedback inhibition in the pathway. Our findings will be helpful for enhancing the aesthetic and nutritional value of tomato and for understanding the complex regulatory mechanisms of carotenoid and apocarotenoid biogenesis.

ζ-Carotene: Generation and Quenching of Singlet Oxygen, Comparison with Phytofluene.

It is known that C40 carotenoids with a short chain of conjugated double bonds (CDB) (5 and 7, respectively) are universal precursors in the biosynthesis of colored carotenoids in plant cells. Previously, using mainly stationary measurements of photosensitized phosphorescence of singlet oxygen (1O2), we discovered that phytofluene efficiently generates 1O2 in aerated solution and therefore, can serve as a source of the UV photodynamic stress in living cells [Ashikhmin et al., Biochemistry (Moscow), 2020, 85, 773]. In the present paper, by using novel pulsed light emitting diodes (LEDs), aerated hexafluorobenzene as a solvent and time-resolved measurements of 1O2 phosphorescence we confirmed that phytofluene efficiently photosensitizes 1O2 formation. The quantum yield of this process according to the novel experiments is about 0.4. An ability to generate 1O2 was also found in aerated solutions of ζ-carotene although the quantum yield of this process is 30-fold lower that in phytofluene solutions. Both carotenoids were found to quench 1O2 in the dark with the quenching rate constants equal to (3.6 ± 0.9)×107 and (2.1 ± 0.2)×108 M-1s-1, respectively. To our knowledge, the rate constant of 1O2 quenching by ζ-carotene has been reported in the present paper for the first time. It follows from the data obtained that the rate constants of 1O2 quenching by both carotenoids are much (by 2-3 orders of magnitude) smaller than the rate constant of the diffusion-limited biomolecular reactions. Hence, both carotenoids are weak protectors against 1O2 oxidative activity. It is more likely that they are potential promoters of photodynamic stress in living cells.

Subfunctionalization of D27 Isomerase Genes in Saffron.

Chromoplasts and chloroplasts contain carotenoid pigments as all-trans- and cis-isomers, which function as accessory light-harvesting pigments, antioxidant and photoprotective agents, and precursors of signaling molecules and plant hormones. The carotenoid pathway involves the participation of different carotenoid isomerases. Among them, D27 is a ß-carotene isomerase showing high specificity for the C9-C10 double bond catalyzing the interconversion of all-trans- into 9-cis-ß-carotene, the precursor of strigolactones. We have identified one D27 (CsD27-1) and two D27-like (CsD27-2 and CsD27-3) genes in saffron, with CsD27-1 and CsD27-3, clearly differing in their expression patterns; specifically, CsD27-1 was mainly expressed in the undeveloped stigma and roots, where it is induced by Rhizobium colonization. On the contrary, CsD27-2 and CsD27-3 were mainly expressed in leaves, with a preferential expression of CsD27-3 in this tissue. In vivo assays show that CsD27-1 catalyzes the isomerization of all-trans- to 9-cis-ß-carotene, and could be involved in the isomerization of zeaxanthin, while CsD27-3 catalyzes the isomerization of all-trans- to cis-ζ-carotene and all-trans- to cis-neurosporene. Our data show that CsD27-1 and CsD27-3 enzymes are both involved in carotenoid isomerization, with CsD27-1 being specific to chromoplast/amyloplast-containing tissue, and CsD27-3 more specific to chloroplast-containing tissues. Additionally, we show that CsD27-1 is co-expressed with CCD7 and CCD8 mycorrhized roots, whereas CsD27-3 is expressed at higher levels than CRTISO and Z-ISO and showed circadian regulation in leaves. Overall, our data extend the knowledge about carotenoid isomerization and their implications in several physiological and ecological processes.

Enzymatic isomerization of ζ-carotene mediated by the heme-containing isomerase Z-ISO.

Carotenoids are a large and diverse class of isoprenoid compounds synthesized by plants, algae, some bacteria, arthropods, and fungi. These pigments contribute to plant growth and survival by protecting plants from photooxidative stress and serving as precursors of plant hormones and other signaling compounds. In humans, carotenoids are essential components of the diet and contribute anti-oxidant and provitamin A activities. Carotenoids are synthesized in the membranes of plant plastids where phytoene is converted into all trans lycopene by a biosynthetic pathway that was only recently completed by the discovery of the new enzyme, 15-cis-ζ-carotene isomerase (Z-ISO), which controls carotenoid pathway flux to products necessary for plant development and function. Z-ISO catalysis of the cis to trans isomerization of the 15-cis double bond in 15-cis-ζ-carotene is mediated by a unique mechanism dependent on the redox-state of a heme b cofactor. This chapter describe methods for the functional analysis of Z-ISO, including complementation of Z-ISO in engineered E. coli, separation of Z-ISO enzyme substrate and products, ζ-carotene isomers, by high pressure liquid chromatography (HPLC), expression and purification of Z-ISO and in vitro enzymatic reactions.

Genomics-based strategies toward the identification of a Z-ISO carotenoid biosynthetic enzyme suitable for structural studies.

Over the past 20years, structural genomics efforts have proven enormously successful for the determination of integral membrane protein structures, particularly for those of prokaryotic origin. However, traditional genomic expansion screens have included up to hundreds of targets, necessitating the use of robotics and other automation not available to most laboratories. Moreover, such large-scale screens of eukaryotic targets are not easily performed at such a scale. To have broader appeal, traditional structural genomic approaches need to be modified and improved such that they are feasible for most laboratories and especially so for proteins from eukaryotic organisms. One such refinement, termed "microgenomic expansion," has been recently described. This approach improves the process of target selection by making target screening a two-step process, with a minimal number of targets tested at each step. Microgenomic expansion methods are applied here theoretically to a project that has the objective of acquiring a structure for the plant 15-cis-ζ-carotene isomerase, Z-ISO.

ClZISO mutation leads to photosensitive flesh in watermelon.

KEY MESSAGE: The mutation of ClZISO identified in EMS-induced watermelon leads to photosensitive flesh in watermelon. Watermelon (Citrullus lanatus) has a colorful flesh that attracts consumers and benefits human health. We developed an ethyl-methanesulfonate mutation library in red-fleshed line '302' to create new flesh color lines and found a yellow-fleshed mutant which accumulated ζ-carotene. The initial yellow color of this mutant can be photobleached within 10 min under intense sunlight. A long-term light-emitting diode (LED) light treatment turned flesh color from yellow to pink. We identified this unique variation as photosensitive flesh mutant ('psf'). Using bulked segregant analysis, we fine-mapped an EMS-induced G-A transversion in 'psf' which leads to a premature stop codon in 15-cis-ζ-carotene isomerase (ClZISO) gene. We detected that wild-type ClZISO is expressed in chromoplasts to catalyze the conversion of 9,15,9'-tri-cis-ζ-carotene to 9,9'-di-cis-ζ-carotene. The truncated ClZISOmu protein in psf lost this catalytic function. Light treatment can partially compensate ClZISOmu isomerase activity via photoisomerization in vitro and in vivo. Transcriptome analysis showed that most carotenoid biosynthesis genes in psf were downregulated. The dramatic increase of ABA content in flesh with fruit development was blocked in psf. This study explores the molecular mechanism of carotenoid biosynthesis in watermelon and provides a theoretical and technical basis for breeding different flesh color lines in watermelon.

Effect of Lumenato oral supplementation on plasma carotenoid levels and improvement of visual and experiential skin attributes.

BACKGROUND: Cellular metabolism and exposure to solar irradiation result in generation of free radicals which are destructive and can lead to premature aging. Antioxidants and free radical scavengers such as carotenoids successfully protect from these free radicals by quenching and neutralizing them thereby strengthening skin barrier which leads to improved skin moisturization, desquamation, and a more youthful look. This study was designed to evaluate the consumer-perceived efficacy of an oral supplement (Lumenato™) containing a mix of tomato carotenoids and oil-soluble vitamins in improving skin appearance after 12 weeks of supplement use. MATERIALS AND METHODS: Plasma levels of phytoene, phytofluene, zeta-carotene, and lycopene were quantitated before and after 1-, 2-, 3-, and 4-week administration of Lumenato by 24 healthy volunteers. Part II of the study addressed skin visual attributes as assessed by validated tools (questionnaires). A total of 60 females, aged 35 to 55 years, completed part II of the study. The subjects answered questionnaires pertaining to their assessment of skin appearance before and after 12 weeks of taking the supplement. RESULTS: There was a significant increase (p < 0.001) in plasma levels of phytoene, phytofluene, and zeta-carotene after 1- to 4-week treatment with Lumenato. After 12 weeks of using the supplement, the score of different skin parameters was reported to significantly improve (p < 0.001). Improvement was recorded in skin elasticity, firmness, brightness, skin tone, reduction in dark spots and periorbital dark circles, skin hydration, texture and fine lines and wrinkles. A significant (p < 0.001) improvement in overall skin condition after using the supplement was observed. The subjects noticed statistically significant (p < 0.001) improvement in skin elasticity, firmness, brightness, skin tone, reduction in dark spots and periorbital dark circles, skin hydration, texture and fine lines and wrinkles after 12 weeks of using the supplement. The overall skin condition also exhibited a significant improvement (p < 0.001). Self-assessed improvement of the face was identified at the first time point (4 weeks) and improved significantly (p < 0.001) for the 12 weeks of use. Interestingly, these improvements persisted even after treatment was stopped. CONCLUSION: Based on the confines and conditions of this study, the use of oral supplement containing a mix of tomato carotenoids significantly increased plasma levels of phytoene, phytofluene, and zeta-carotene, and continuous use resulted in improved facial skin attributes which were palpable by the consumers and continued even after treatment was stopped.

ζ-Carotene Isomerase Suppresses Tillering in Rice through the Coordinated Biosynthesis of Strigolactone and Abscisic Acid.

Rice tillering is an important agronomic trait affecting grain yield. Here, we identified a high-tillering mutant tillering20 (t20), which could be restored to the wild type by treatment with the strigolactone (SL) analog rac-GR24. T20 encodes a chloroplast ζ-carotene isomerase (Z-ISO), which is involved in the biosynthesis of carotenoids and their metabolites, SL and abscisic acid (ABA). The t20 mutant has reduced SL and ABA, raising the question of how SL and ABA biosynthesis is coordinated, and whether they have overlapping functions in tillering. We discovered that rac-GR24 stimulated T20 expression and enhanced all-trans-ß-carotene biosynthesis. Importantly, rac-GR24 also stimulated expression of Oryza sativa 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (OsNCED1) through induction of Oryza sativa HOMEOBOX12 (OsHOX12), promoting ABA biosynthesis in shoot base. On the other hand, ABA treatment significantly repressed SL biosynthesis and the ABA biosynthetic mutants displayed elevated SL biosynthesis. ABA treatment reduced the number of basal tillers in both t20 and wild-type plants. Furthermore, while ABA-deficient mutants aba1 and aba2 had the same number of basal tillers as wild type, they had more unproductive upper tillers at maturity. This work demonstrates complex interactions in the biosynthesis of carotenoid, SLs and ABA, and reveals a role for ABA in the regulation of rice tillering.

Oxygenic Phototrophs Need ζ-Carotene Isomerase (Z-ISO) for Carotene Synthesis: Functional Analysis in Arthrospira and Euglena.

For carotenogenesis, two biosynthetic pathways from phytoene to lycopene are known. Most bacteria and fungi require only phytoene desaturase (PDS, CrtI), whereas land plants require four enzymes: PDS (CrtP), ζ-carotene desaturase (ZDS, CrtQ), ζ-carotene isomerase (Z-ISO) and cis-carotene isomerase (CrtISO, CrtH). The gene encoding Z-ISO has been functionally identified in only two species, Arabidopsis thaliana and Zea mays, and has been little studied in other organisms. In this study, we found that the deduced amino acid sequences of Arthrospira Z-ISO and Euglena Z-ISO have 58% and 62% identity, respectively, with functional Z-ISO from Arabidopsis. We studied the function of Z-ISO genes from the cyanobacterium Arthrospira platensis and eukaryotic microalga Euglena gracilis. The Z-ISO genes of Arthrospira and Euglena were transformed into Escherichia coli strains that produced mainly 9,15,9'-tri-cis-ζ-carotene in darkness. In the resulting E. coli transformants cultured under darkness, 9,9'-di-cis-ζ-carotene was accumulated predominantly as Z-ISO in Arabidopsis. This indicates that the Z-ISO genes were involved in the isomerization of 9,15,9'-tri-cis-ζ-carotene to 9,9'-di-cis-ζ-carotene in darkness. This is the first functional analysis of Z-ISO as a ζ-carotene isomerase in cyanobacteria and eukaryotic microalgae. Green sulfur bacteria and Chloracidobacterium also use CrtP, CrtQ and CrtH for lycopene synthesis as cyanobacteria, but their genomes did not comprise Z-ISO genes. Consequently, Z-ISO is needed in oxygenic phototrophs, whereas it is not found in anoxygenic species.

Effect of Illumination Intensity and Inhibition of Carotenoid Biosynthesis on Assembly of Peripheral Light-Gathering Complexes in Purple Sulfur Bacteria C Allochromatium vinosum ATCC 17899.

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assemblage of different spectral types of LH2 complexes in a purple sulfur bacterium Allochromatium (Alc.) vinosum ATCC 17899 was studied. Under illumination of 1200 and 500 lx, the complexes B800-850 and B800-840 and B800-820 were assembled. While rhodopine was the major carotenoid in all spectral types of the LH2 complex, a certain- increase in the content of carotenoids with higher numbers of conjugated double bonds (anhydrorhodovibrin and didehydrorhodovibrin) was observed in the B800-820 complex. At 1200 lx, the cells grew slowly at diphe- nylamine (DPA) concentrations not exceeding 53 .iM, while at illumination intensity decreased to 500 Ix they could grow at 71 jiM DPA (DPA cells). Independent on illumination level, the inhibitor is supposed to impair the functioning of phytoine synthetase (resulting in a decrease in the total carotenoid content) and of phyto- ine desturase, which results in formation of neurosporene hydroxy derivatives and ;-carotene. In the cells grown at 500 lx, small amounts of spheroidene and.OH-spheroidene were detected. These carotenoids were originally found under conditions of carotenoid synthesis inhibition in bacteria with spirilloxanthin as the major carotenoid. Carotenoid content in the LH2 complexes isolated from the DPA cells was -15% of the control (without inhibition) for the B800-850 and -20%of the control for the B800-820 and B800-840 DPA complexes. Compared to the DPA pigment-containing membranes, the DPA complexes were enriched with -carotenoids due to- disintegration of some carotenoid-free complexes in the course of isolation. These results support the supposition that some of the B800-820, B800-840, and B800-850 complexes may be Assembled in the cells of Alc. vinosum ATCC 17899 without carotenoids. Comparison of the characteristics obtained for Alc. vinosum ATCC 17899 and the literature data on strain D of the same bacteria shows that they belong to two different strains, rather than to one as was previously supposed.

Control of carotenoid biosynthesis through a heme-based cis-trans isomerase.

Plants synthesize carotenoids, which are essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway for all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not known whether Z-ISO catalyzes isomerization alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-trans isomerization of the 15-15' carbon-carbon double bond in 9,15,9'-cis-ζ-carotene to produce the substrate required by the subsequent biosynthetic-pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large hydrophobic compound in a membrane was previously undescribed. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially uses a new chemical mechanism.

Enhanced bioavailability of lycopene when consumed as cis-isomers from tangerine compared to red tomato juice, a randomized, cross-over clinical trial.

SCOPE: Tangerine tomatoes (Solanum lycopersicum) are rich in tetra-cis-lycopene resulting from natural variation in carotenoid isomerase. Our objective was to compare the bioavailability of lycopene from tangerine to red tomato juice, and elucidate physical deposition forms of these isomers in tomatoes by light and electron microscopy. METHODS AND RESULTS: Following a randomized cross-over design, subjects (n = 11, 6 M/5 F) consumed two meals delivering 10 mg lycopene from tangerine (94% cis) or red tomato juice (10% cis). Blood was sampled over 12 h and triglyceride-rich lipoprotein fractions of plasma were isolated and analyzed using HPLC-DAD-MS/MS. Lycopene was crystalline in red tomato chromoplasts and globular in tangerine tomatoes. With tangerine tomato juice we observed a marked 8.5-fold increase in lycopene bioavailability compared to red tomato juice (p < 0.001). Fractional absorption was 47.70 ± 8.81% from tangerine and 4.98 ± 1.92% from red tomato juices. Large heterogeneity was observed among subjects. CONCLUSION: Lycopene is markedly more bioavailable from tangerine than from red tomato juice, consistent with a predominance of cis-lycopene isomers and presence in chromoplasts in a lipid dissolved globular state. These results justify using tangerine tomatoes as a lycopene source in studies examining the potential health benefits of lycopene-rich foods.

Dissection of tomato lycopene biosynthesis through virus-induced gene silencing.

Lycopene biosynthesis in tomato (Solanum lycopersicum) fruits has been proposed to proceed through a poly-cis pathway catalyzed by phytoene synthase (PSY), two desaturases (phytoene desaturase [PDS] and ζ-carotene desaturase [ZDS]), and two cis-trans isomerases (ζ-carotene isomerase [ZISO] and prolycopene isomerase [CrtISO]). The mechanism of action of these enzymes has been studied in Escherichia coli, but a systematic study of their in vivo function is lacking. We studied the function of nine candidate genes (PSY1, PSY2, PSY3, PDS, ZDS, ZISO, CrtISO, CrtISO-Like1, and CrtISO-Like2) using virus-induced gene silencing (VIGS) coupled to high-resolution liquid chromatography coupled with diode array detector and mass spectrometry, which allowed the identification and quantitation of 45 different carotenoid isomers, including linear xanthophylls. The data confirm the confinement of the VIGS signal to the silenced fruits and the similarity of the phenotypes of PSY1- and CrtISO-silenced fruits with those of the yellow flesh and tangerine mutants. Light was able to restore lycopene biosynthesis in ZISO-silenced fruits. Isomeric composition of fruits silenced at different metabolic steps suggested the existence of three functional units, comprising PSY1, PDS/ZISO, and ZDS/CrtISO, and responsible for the synthesis of 15-cis-phytoene, 9,9'-di-cis-ζ-carotene, and all-trans-lycopene, respectively. Silencing of a desaturase (PDS or ZDS) resulted in the induction of the isomerase in the same functional unit (ZISO or CrtISO, respectively). All-trans-ζ-carotene was detectable in nonsilenced fruits, greatly increased in ZDS-silenced ones, and disappeared in CrtISO-Like1-/CrtISO-Like2-silenced ones, suggesting the existence of a metabolic side branch, comprising this compound and initiated by the latter enzymes.

Contribution of two ζ-carotene desaturases to the poly-cis desaturation pathway in the cyanobacterium Nostoc PCC 7120.

The presence of two completely unrelated ζ-carotene desaturases CrtQa and CrtQb in some Nostoc strains is unique. CrtQb is the ζ-carotene desaturase, which was acquired by almost all cyanobacteria. The additional CrtQa can be regarded as an evolutionary relict of the CrtI desaturase present in non-photosynthetic bacteria. By reconstruction of the carotene desaturation pathway, we showed that both enzymes from Nostoc PCC 7120 were active. However, they differed in their preferred utilization of ζ-carotene Z isomers. CrtQa converted ζ-carotene isomers that were poorly metabolized by CrtQb. In this respect, CrtQa complemented the reactions of CrtQb, which is an advantage avoiding dead ends in the poly-cis desaturation pathway. In addition to ζ-carotene desaturation, CrtQa still possesses the Z to E isomerase function of the ancestral desaturase CrtI. Biochemical characterization showed that CrtQb is an enzyme with one molecule of tightly bound FAD and acts as a dehydrogenase transferring hydrogen to oxidized plastoquinone.

Structure and function characterization of the phytoene desaturase related to the lutein biosynthesis in Chlorella protothecoides CS-41.

Phytoene desaturase is the key enzyme involved in the biosynthesis pathway of lutein. The unicellular microalga, Chlorella protothecoides CS-41, had been selected for the heterotrophic production of high concentrations of lutein. In this study, a cDNA copy of the pds gene from C. protothecoides was obtained using the rapid amplification of cDNA ends (RACE) technique. Phylogenetic analysis of the deduced amino acid sequence revealed that the phytoene desaturases derived from the algal family. Expression of the pds gene in Escherichia coli produced a single protein of 61 kDa. The PDS activity of the expressed protein was confirmed by the production of ζ-carotene as the result from the action of the enzyme's desaturation activity, which was identified by high-performance liquid chromatography and heterologous complementation analysis. Using random and site-directed mutagenesis, a single amino acid mutation (N144D) was identified and confirmed. This mutant encodes an inactive enzyme, which implies that amino acid 144 is crutial to the activity of the PDS enzyme. Therefore, by gene cloning and expression in prokaryotic cells, the gene for ζ-carotene production or as part of the biosynthetic pathway of lutein had been characterized from Chlorella protothecoides CS-41.

Biosynthesis of fucoxanthin and diadinoxanthin and function of initial pathway genes in Phaeodactylum tricornutum.

The biosynthesis pathway to diadinoxanthin and fucoxanthin was elucidated in Phaeodactylum tricornutum by a combined approach involving metabolite analysis identification of gene function. For the initial steps leading to ß-carotene, putative genes were selected from the genomic database and the function of several of them identified by genetic pathway complementation in Escherichia coli. They included genes encoding a phytoene synthase, a phytoene desaturase, a ζ-carotene desaturase, and a lycopene ß-cyclase. Intermediates of the pathway beyond ß-carotene, present in trace amounts, were separated by TLC and identified as violaxanthin and neoxanthin in the enriched fraction. Neoxanthin is a branching point for the synthesis of both diadinoxanthin and fucoxanthin and the mechanisms for their formation were proposed. A single isomerization of one of the allenic double bounds in neoxanthin yields diadinoxanhin. Two reactions, hydroxylation at C8 in combination with a keto-enol tautomerization and acetylation of the 3'-HO group results in the formation of fucoxanthin.

Carotenoid content impacts flavor acceptability in tomato (Solanum lycopersicum).

BACKGROUND: Tomatoes contain high levels of several carotenoids including lycopene and ß-carotene. Beyond their functions as colorants and nutrients, carotenoids are precursors for important volatile flavor compounds. In order to assess the importance of apocarotenoid volatiles in flavor perception and acceptability, we conducted sensory evaluations of near-isogenic carotenoid biosynthetic mutants and their parent, Ailsa Craig. RESULTS: The carotenoid contents of these tomatoes were extremely low in the r mutant, increased in lycopene in old gold, and higher in tetra-cis-lycopene and ζ-carotene in tangerine. The volatiles derived from these carotenoids (ß-ionone, geranylacetone and 6-methyl-5-hepten-2-one) were proportionally altered relative to their precursors. Fruits were also analyzed for soluble solids, sugars, acids and flavor volatiles. Consumer panels rated the r mutant lowest for all sensory attributes, while Ailsa Craig was generally rated highest. Old gold and tangerine were rated intermediate in two of the three harvests. CONCLUSIONS: Several chemicals were negatively correlated with at least one of the hedonic scores while several others were positively correlated with tomato flavor acceptability. The results permitted identification of positive and negative interactions of volatiles with tomato flavor.