Metabolism of cyclic carotenoids: a model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts.

The biosynthetic pathway of cyclic carotenoid is known to be quantitatively and qualitatively different in the non-green plastids of Capsicum annuum fruits compared with chloroplasts. Here, the cloning is described of a novel cDNA from this organism, which encodes an enzyme catalyzing the cyclization of lycopene to beta-carotene when expressed in Escherichia coli. The corresponding gene is constitutively expressed during fruit development. Significant amino acid sequence identity was observed between this enzyme and capsanthin/capsorubin synthase which is involved in the synthesis of the species-specific red carotenoids of C. annuum fruits. The latter enzyme was found also to possess a lycopene beta-cyclase activity when expressed in E. coli. A model is proposed for the origin of the capsanthin/capsorubin synthase gene and the role of this enzyme, together with the newly cloned lycopene cyclase, in the specific re-channeling of linear carotenoids into beta-cyclic carotenoids in C. annuum ripening fruits.

Hugueney P ，Badillo A ，Chen HC ，Klein A ，Hirschberg J ，Camara B ，Kuntz M ... -  《PLANT JOURNAL》

Manipulation of Carotenoid Metabolic Flux by Lycopene Cyclization in Ripening Red Pepper ( Capsicum annuum var. conoides) Fruits.

Carotenoids are essential phytonutrients for the human body. Higher plants usually synthesize and accumulate carotenoids in their leaves, flowers, and fruits. Most carotenoids have either two β-rings on both ends or β- and ε-rings separately on two ends of their molecules and are synthesized from the acyclic lycopene as the precursor. Lycopene β- and ε-cyclases (LCYB and LCYE, respectively) catalyze the β- and ε-cyclization of lycopene, respectively, and regulate the metabolic flux from lycopene to its downstream β,β-branches (by LCYB alone) and β,ε-branches (by LCYE and LCYB). In this study, we identified and characterized genes for two LCYBs (CaLCYB1 and CaLCYB2), one LCYE (CaLCYE1), and a capsanthin/capsorubin synthase (CaCCS1) which is also able to β-cyclize lycopene from the red pepper ( Capsicum annuum var. conoides) genome. By quantifying transcript abundances of these genes and contents of different carotenoid components in ripening fruits, we observed a correlation between the induction of both CaLCYBs and the accumulation of carotenoids of the β,β-branch during ripening. Although capsanthin was accumulated in ripened fruits, our quantification demonstrated a strong induction of CaCCS1 at the breaker stage, together with the simultaneous repression of CaLCYE1 and the decrease of lutein content, suggesting the involvement of CaCCS1 in competing against CaLCYE1 for synthesizing carotenoids of the β,β-branch. Our results provide important information for future metabolic engineering studies to manipulate carotenoid biosynthesis and accumulation in fruits.

Wang Q ，Cao TJ ，Zheng H ，Zhou CF ，Wang Z ，Wang R ，Lu S ... -  《-》

Xanthophyll biosynthesis in chromoplasts: isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocarotenoid.

The late steps of carotenoid biosynthesis in plants involve the formation of xanthophylls. Little is known about the enzymology of these steps. This paper reports the purification to homogeneity of a xanthophyll biosynthetic enzyme from Capsicum annuum chromoplasts, which catalyzes the conversion of the ubiquitous 5,6-epoxycarotenoids, antheraxanthin and violaxanthin, into capsanthin and capsorubin, respectively. Owing to its bifunctionality, the name capsanthin-capsorubin synthase is proposed for this new enzyme. The purified enzyme is a monomer with a molecular mass of 50 kDa. Antibodies raised against this enzyme allowed the isolation of a full-length cDNA clone encoding a capsanthin capsorubin synthase high molecular weight precursor. The primary deduced structure reveals the presence of a consensus nucleotide binding site. The capsanthin-capsorubin synthase gene is specifically expressed during chromoplast development in fruits accumulating ketocarotenoids, but not in mutants impaired in this biosynthetic step.

Bouvier F ，Hugueney P ，d'Harlingue A ，Kuntz M ，Camara B ... -  《PLANT JOURNAL》

Characterization under quasi-native conditions of the capsanthin/capsorubin synthase from Capsicum annuum L.

Chromoplasts are typical plastids of fruits and flowers, deriving from chloroplasts through complex processes of re-organization and recycling. Since this transition leads to the production of reactive species, chromoplasts are characteristic sites for biosynthesis and accumulation of carotenoids and other antioxidants. Here, we have analysed the chromoplast membranes from Capsicum annuum L. fruits, finding a significant expression of the capsanthin/capsorubin synthase. This enzyme was isolated by a very mild procedure allowing its analyses under quasi-native conditions. The isolated complex appeared as a red coloured homo-trimer, suggesting the retention of at least one of the typical carotenoids from C. annuum. Moreover, the protein complex was co-purified with a non-proteinaceous fraction of carotenoid aggregates carrying a high molecular weight and separable only by Size Exclusion Chromatography. This last finding suggested a relationship between the carotenoids synthesis on chromoplast membranes, the presence, and storage of organised carotenoids aggregates typical for chromoplasts. Further MS analyses also provided important hints on the interactome network associated to the capsanthin/capsorubin synthase, confirming its functional relevance during ripening. Results are discussed in the frame of the primary role played by carotenoids in quenching the growing oxidative stress during fruits ripening.

Piano D ，Cocco E ，Guadalupi G ，Kalaji HM ，Kirkpatrick J ，Farci D ... -  《-》

cDNA cloning of a novel gene codifying for the enzyme lycopene β-cyclase from Ficus carica and its expression in Escherichia coli.

Lycopene beta-cyclase (β-LCY) is the key enzyme that modifies the linear lycopene molecule into cyclic β-carotene, an indispensable carotenoid of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Owing to its antioxidant activity, it is commercially used in the cosmetic and pharmaceutical industries, as well as an additive in foodstuffs. Therefore, β-carotene has a large share of the carotenoidic market. In this study, we used reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR to obtain and clone a cDNA copy of the gene Lyc-β from Ficus carica (Lyc-β Fc), which codes for the enzyme lycopene β-cyclase (β-LCY). Expression of this gene in Escherichia coli produced a single polypeptide of 56 kDa of weight, containing 496 amino acids, that was able to cycle both ends of the lycopene chain. Amino acid analysis revealed that the protein contained several conserved plant cyclase motifs. β-LCY activity was revealed by heterologous complementation analysis, with lycopene being converted to β-carotene as a result of the enzyme's action. The β-LCY activity of the expressed protein was confirmed by high-performance liquid chromatography (HPLC) identification of the β-carotene. The lycopene to β-carotene conversion rate was 90%. The experiments carried out in this work showed that β-LYC is the enzyme responsible for converting lycopene, an acyclic carotene, to β-carotene, a bicyclic carotene in F. carica. Therefore, by cloning and expressing β-LCY in E. coli, we have obtained a new gene for β-carotene production or as part of the biosynthetic pathway of astaxanthin. So far, this is the first and only gene of the carotenoid pathway identified in F. carica.

Araya-Garay JM ，Feijoo-Siota L ，Veiga-Crespo P ，Villa TG ... -  《-》