title: cDNA cloning of a novel gene codifying for the enzyme lycopene β-cyclase from Ficus carica and its expression in Escherichia coli.
abstract: 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.
authors.affiliation: Department of Microbiology, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
authors.forename: José Miguel
authors.initials: JM
authors.lastname: Araya-Garay
authors.forename: Lucía
authors.initials: L
authors.lastname: Feijoo-Siota
authors.forename: Patricia
authors.initials: P
authors.lastname: Veiga-Crespo
authors.forename: Tomás González
authors.initials: TG
authors.lastname: Villa
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D053898:Biosynthetic Pathways; D002338:Carotenoids / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D030681:Ficus / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D019753:Intramolecular Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000077276:Lycopene; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019207:beta Carotene / Q000032:analysis
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D019207:beta Carotene; D002338:Carotenoids; D019753:Intramolecular Lyases; C070925:lycopene cyclase-isomerase; D000077276:Lycopene