Carotenogenesis Enzymes Research Articles

A New 5'-UTR <i>LcyE </i>allele Correlates with Increased Expression of the Lycopine-ε-Cyclase Gene Determining the Flow of the β-ε Branch of the Carotenoid Biosynthesis Pathway in Maize

The color of Zea mays L. kernel is determined by the content and composition of carotenoids, including provitamin A, which is a product of the β-β (β-carotene, β-cryptoxanthin) and β-ε (α-carotene) branches of carotenogenesis. The ratio of the fluxes of the branches depends on the activity of the lycopene-ε-cyclase LcyE, which determines the β-ɛ branch. In this work, we analyzed allelic variants of the LcyE gene, which are potentially effective for increasing the biosynthesis of β-carotene, in 20 maize inbred lines of domestic selection, which differ in grain color. The 5'-UTR region of the LcyE gene were amplified and sequenced. Fragment analysis showed the presence of allele “2” in four lines and a new allele “5” in 16 lines. The polymorphism of the new allele “5” was characterized – four mononucleotide polymorphisms and two deletions. The comparison of cis-regulatory elements in the analyzed region of the 5'-UTR of alleles “2” and “5” revealed a difference in binding sites with transcription factors. Expression of the LcyE gene was determined in the leaves of two lines with the allele “2” and three lines with the allele “5”. A direct relationship was shown between the presence of the allele “5” and a decrease in gene expression: the level of gene transcription in the case of the allele “2” was 10–15 times higher than in the case of the allele “5”. It has been suggested that the presence of allele “5” of the LcyE gene in the maize genome correlates with a decrease or suppression of the LcyE expression and, with stable activity of other carotenogenesis enzymes, with grain color. The use of allele “5” donors in combination with the known dark yellow or orange color of the grain can be used in the breeding of maize with increased synthesis of provitamin A in the grain.

Proteomic and metabolomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous using different carbon sources.

BackgroundAstaxanthin is a potent antioxidant with increasing biotechnological interest. In Xanthophyllomyces dendrorhous, a natural source of this pigment, carotenogenesis is a complex process regulated through several mechanisms, including the carbon source. X. dendrorhous produces more astaxanthin when grown on a non-fermentable carbon source, while decreased astaxanthin production is observed in the presence of high glucose concentrations. In the present study, we used a comparative proteomic and metabolomic analysis to characterize the yeast response when cultured in minimal medium supplemented with glucose (fermentable) or succinate (non-fermentable).ResultsA total of 329 proteins were identified from the proteomic profiles, and most of these proteins were associated with carotenogenesis, lipid and carbohydrate metabolism, and redox and stress responses. The metabolite profiles revealed 92 metabolites primarily associated with glycolysis, the tricarboxylic acid cycle, amino acids, organic acids, sugars and phosphates. We determined the abundance of proteins and metabolites of the central pathways of yeast metabolism and examined the influence of these molecules on carotenogenesis.Similar to previous proteomic-stress response studies, we observed modulation of abundance from several redox, stress response, carbohydrate and lipid enzymes. Additionally, the accumulation of trehalose, absence of key ROS response enzymes, an increased abundance of the metabolites of the pentose phosphate pathway and tricarboxylic acid cycle suggested an association between the accumulation of astaxanthin and oxidative stress in the yeast. Moreover, we observed the increased abundance of late carotenogenesis enzymes during astaxanthin accumulation under succinate growth conditions.ConclusionsThe use of succinate as a carbon source in X. dendrorhous cultures increases the availability of acetyl-CoA for the astaxanthin production compared with glucose, likely reflecting the positive regulation of metabolic enzymes of the tricarboxylic acid and glyoxylate cycles. The high metabolite level generated in this pathway could increase the cellular respiration rate, producing reactive oxygen species, which induces carotenogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1484-6) contains supplementary material, which is available to authorized users.

Carotenoids in Rhodoplanes Species: Variation of Compositions and Substrate Specificity of Predicted Carotenogenesis Enzymes

Phototrophic bacteria necessarily contain carotenoids for photosynthesis, and accumulate unusual carotenoids in some cases. The carotenoids in all established species of Rhodoplanes (Rpl.), a representative of phototrophic genera, were identified using spectroscopic methods. The major carotenoid was spirilloxanthin in Rpl. roseus and Rpl. serenus, and rhodopin in "Rpl. cryptolactis". Rpl. elegans contained rhodopin, anhydrorhodovibrin, and spirilloxanthin. Rpl. pokkaliisoli contained not only rhodopin but also 1,1'-dihydroxylycopene and 3,4,3',4'-tetrahydrospirilloxanthin. These variations in carotenoid composition suggested that Rpl. roseus and Rpl. serenus had normal substrate specificity of the carotenogenesis enzymes of CrtC (acyclic carotene 1,2-hydratase), CrtD (acyclic carotenoid 3,4-desaturase), and CrtF (acyclic 1-hydroxycarotenoid methyltransferase). On the other hand, CrtC of Rpl. elegans, CrtD of "Rpl. cryptolactis", and CrtC, CrtD, and CrtF of Rpl. pokkaliisoli might have different characteristics from the usual activity of these normal enzymes in the normal spirilloxanthin pathway. These results suggest that the variation of carotenoids among the species of Rhodoplanes results from modified substrate specificity of the carotenogenesis enzymes involved.

Biosynthesis of the biomarker okenone: χ‐ring formation

Purple sulfur bacteria (PSB) mainly occur in anoxic aquatic and benthic environments, where they play important roles in cycling carbon and sulfur. Many PSB characteristically produce the unique keto-carotenoid, okenone, which is important not only for its light absorption and photoprotection properties but also because of its diagenesis product, okenane, which is a biomarker for ancient sediments derived from anoxic environments. The specific methylation pattern of the χ-ring of okenane is unlikely to be formed by diagenetic processes and should therefore reflect an enzymatic activity from okenone biosynthesis. This study describes two enzymes that produce the χ-ring of okenone, the only structural element of okenone preserved in okenane. Genes encoding enzymes of carotenogenesis were identified in the draft genome sequence of an okenone-producing PSB, Thiodictyon sp. strain CAD16. Two divergently transcribed genes encoded a CrtY-type lycopene cyclase and a CrtU/CruE-type γ-carotene desaturase/methyltransferase. Expression of crtY in Escherichia coli showed that this gene encoded a lycopene cyclase that produced γ-carotene as the only product. Although the sequence of the γ-carotene desaturase/methyltransferase was more similar to CrtU sequences of green sulfur bacteria than to CruE sequences of cyanobacteria, expression of the crtU gene in Chlorobaculum tepidum showed that the enzyme produced carotenoids with χ-rings rather than φ-rings. Phylogenetic analysis of the carotene desaturase/methyltransferases revealed that enzymes capable of converting β-rings to χ-rings have independently evolved at least two times. These results indicate that it probably will not be possible to deduce the activity of carotene desaturase/methyltransferases solely from sequence data.

Carotenoids in algae: distributions, biosyntheses and functions.

For photosynthesis, phototrophic organisms necessarily synthesize not only chlorophylls but also carotenoids. Many kinds of carotenoids are found in algae and, recently, taxonomic studies of algae have been developed. In this review, the relationship between the distribution of carotenoids and the phylogeny of oxygenic phototrophs in sea and fresh water, including cyanobacteria, red algae, brown algae and green algae, is summarized. These phototrophs contain division- or class-specific carotenoids, such as fucoxanthin, peridinin and siphonaxanthin. The distribution of α-carotene and its derivatives, such as lutein, loroxanthin and siphonaxanthin, are limited to divisions of Rhodophyta (macrophytic type), Cryptophyta, Euglenophyta, Chlorarachniophyta and Chlorophyta. In addition, carotenogenesis pathways are discussed based on the chemical structures of carotenoids and known characteristics of carotenogenesis enzymes in other organisms; genes and enzymes for carotenogenesis in algae are not yet known. Most carotenoids bind to membrane-bound pigment-protein complexes, such as reaction center, light-harvesting and cytochrome b6f complexes. Water-soluble peridinin-chlorophyll a-protein (PCP) and orange carotenoid protein (OCP) are also established. Some functions of carotenoids in photosynthesis are also briefly summarized.

Genus Specific Unusual Carotenoids in Purple Bacteria, Phaeospirillum and Roseospira: Structures and Biosyntheses

Phototrophic bacteria necessarily contain carotenoids for photosynthesis, and a few phototrophic purple bacteria accumulate unusual carotenoids. The carotenoids in the genera Phaeospirillum and Roseospira were identified using spectroscopic methods. All species of the genus Phaeospirillum contained characteristic polar carotenoids in addition to lycopene and hydroxylycopene (rhodopin); hydroxylycopene glucoside, dihydroxylycopene, and its mono- and/or diglucosides. From the structures of these carotenoids, their accumulation was suggested to be due to absence of CrtD (acyclic carotenoid C-3,4 desaturase) and to possession of glucosyltransferase. Species of the genus Roseospira have been reported to have unusual absorption spectra in acetone extract, and they were found to accumulate 3,4-didehydrorhodopin as a major carotenoid. This may be due to low activity of CrtF (acyclic 1-hydroxycarotenoid methyltransferase). The study concludes in identifying genus specific unusual carotenoids, which is probably due to characteristic nature of some carotenogenesis enzymes.

Roles of xanthophyll carotenoids in protection against photoinhibition and oxidative stress in the cyanobacterium Synechococcus sp. strain PCC 7002

Synechococcus sp. strain PCC 7002 is a robust, genetically tractable cyanobacterium that produces six different xanthophyll carotenoids (zeaxanthin, cryptoxanthin, myxoxanthophyll (myxol-2′-fucoside), echinenone, 3′-hydroxyechinenone, and synechoxanthin) and tolerates many environmental stresses, including high light intensities. Targeted mutations were introduced to block the branches of the carotenoid biosynthetic pathway leading to specific xanthophylls, and a mutant lacking all xanthophylls was constructed. Some of the mutants showed severe growth defects at high light intensities, and multi-locus mutants had somewhat lower chlorophyll contents and lower photosystem I levels. The results suggested that xanthophylls, particularly zeaxanthin and echinenone, might play regulatory roles in thylakoid biogenesis. Measurements of reactive oxygen (ROS) and nitrogen (RNS) species in the mutants showed that all xanthophylls participate in preventing ROS/RNS accumulation and that a mutant lacking all xanthophylls accumulated very high levels of ROS/RNS. Results from transcription profiling showed that mRNA levels for most genes encoding the enzymes of carotenogenesis are significantly more abundant after exposure to high light. These studies indicated that all xanthophylls contribute to protection against photo-oxidative stress.

Carotenoids and carotenogenesis in cyanobacteria: unique ketocarotenoids and carotenoid glycosides.

Cyanobacteria grow by photosynthesis, and necessarily contain chlorophyll and carotenoids, whose main functions are light harvesting and photoprotection. In this review, we discuss the carotenoids, carotenogenesis pathways, and characteristics of carotenogenesis enzymes and genes in some cyanobacteria, whose carotenogenesis enzymes have been functionally confirmed. In these cyanobacteria, various carotenoids have been identified, including the unique ketocarotenoids, echinenone and 4-ketomyxol; and the carotenoid glycosides, myxol glycosides and oscillol diglycosides. From these findings, certain carotenogenesis pathways can be proposed. The different compositions of carotenoids among these species might be due to the presence or absence of certain gene(s), or to different enzyme characteristics. For instance, two distinct beta-carotene ketolases, CrtO and CrtW, are properly used in two pathways depending on the species. One beta-carotene hydroxylase, CrtR, has been identified, and its substrate specificities vary across species. At present, functionally confirmed genes have been found in only a few species, and further studies are needed.

Phytochrome Regulation of Carotenoid Biosynthesis during Ripening of Tomato Fruit

The accumulation of carotenoids such as lycopene and beta-carotene greatly influences the quality of ripe tomato (Lycopersicon esculentum) fruit because cellular levels of these compounds determine the intensity of red color. As well, lycopene has anti-cancer properties and beta-carotene is a Vitamin A precursor. Recent work has demonstrated phytochrome regulation of the carotenoid pathway but the mechanism is not completely understood. This work investigates phytochrome regulation of 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and phytoene synthase (PSY), two key enzymes of carotenogenesis. A simple procedure for the assay of PSY from crude pericarp extracts was developed and mRNA levels of DXS and PSY1 genes were measured by relative RT-PCR. Discs from mature green tomatoes were ripened in total darkness, or in darkness interrupted by brief daily treatments of red light, or red light followed by far red light. After ten days of incubation, lycopene levels of red light-treated discs had reached ≈12 mg/100 g fresh weight; nearly a 50% increase over discs ripened in total darkness. This increase was not observed in discs treated with red light followed by far red light, demonstrating the red/far red reversibility (and thus phytochrome control) of carotenoid accumulation. Similar patterns of phytochrome control are observed for PSY activity but not for DXS and PSY1 transcript levels, suggesting the mechanism of control may be at the level of post-translational modification of PSY. Potential applications of this regulation of carotenoid accumulation will be discussed.

Characterization of unusual hydroxy- and ketocarotenoids in Rubrivivax gelatinosus: involvement of enzyme CrtF or CrtA.

Carotenoids are widely spread terpenoids found in photosynthetic organisms and a number of non-photosynthetic fungi and bacteria. The photosynthetic non-sulfur purple bacterium Rubrivivax gelatinosus produces carotenoids by both the spheroidene and the normal spirilloxanthin pathways. The characteristics of two carotenogenesis enzymes, spheroidene monooxygenase CrtA and O-methyltransferase CrtF, were investigated. Disruption of the corresponding genes by insertional mutagenesis affected carotenoid species in both pathways, and the genetic evidence indicated that both genes are involved in the two pathways. In these mutants, several unusual hydroxy- and ketocarotenoids were identified by spectroscopic and chemical methods. Moreover, the carotenoid analyses demonstrated that a large number of different carotenoid intermediates are accepted as substrates by the CrtA enzyme. The combined manipulation of crtF and crtA allowed new carotenoids to be produced and broadened the diversity of structurally different carotenoids synthesized by Rvi. gelatinosus. Methylated carotenoids, such as spheroidene and spirilloxanthin, are known to function as accessory pigments in the light-harvesting and reaction-center complexes of purple bacteria; the demethylated carotenoids described here were able to fulfill the same functions in the mutants.