JosÃ© Miguel JM Araya-Garay, LucÃa L Feijoo-Siota, Patricia P Veiga-Crespo, TomÃ¡s GonzÃ¡lez TG Villa
Applied Microbiology and Biotechnology
Lycopene beta-cyclase (Î²-LCY) is the key enzyme that modifies the linear lycopene molecule into cyclic Î²-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, Î²-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-Î² from Ficus carica (Lyc-Î² Fc), which codes for the enzyme lycopene Î²-cyclase (Î²-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. Î²-LCY activity was revealed by heterologous complementation analysis, with lycopene being converted to Î²-carotene as a result of the enzyme's action. The Î²-LCY activity of the expressed protein was confirmed by high-performance liquid chromatography (HPLC) identification of the Î²-carotene. The lycopene to Î²-carotene conversion rate was 90%. The experiments carried out in this work showed that Î²-LYC is the enzyme responsible for converting lycopene, an acyclic carotene, to Î²-carotene, a bicyclic carotene in F. carica. Therefore, by cloning and expressing Î²-LCY in E. coli, we have obtained a new gene for Î²-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.
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