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 (beta-LCY) is the key enzyme that modifies the linear lycopene molecule into cyclic beta-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, beta-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-beta from Ficus carica (Lyc-beta Fc), which codes for the enzyme lycopene beta-cyclase (beta-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. beta-LCY activity was revealed by heterologous complementation analysis, with lycopene being converted to beta-carotene as a result of the enzyme's action. The beta-LCY activity of the expressed protein was confirmed by high-performance liquid chromatography (HPLC) identification of the beta-carotene. The lycopene to beta-carotene conversion rate was 90%. The experiments carried out in this work showed that beta-LYC is the enzyme responsible for converting lycopene, an acyclic carotene, to beta-carotene, a bicyclic carotene in F. carica. Therefore, by cloning and expressing beta-LCY in E. coli, we have obtained a new gene for beta-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.