Molecular characterization of phytoene desaturase (crti) gene from paracoccus bogoriensis

Abstract

Carotenoids are natural fat soluble isoprenoid pigments that occur widely in micro-organisms and plants. Increasing use of carotenoids as food colourant, health supplements, cosmetic additives and animal feeds has led to high demands in the global market. Commercially available carotenoids including β-carotene, astaxanthin and canthaxanthin, are produced by chemical synthesis, isolation from natural sources or by microbial fermentation. However these methods have limits, and there is therefore need to develop alternate methods for caroteinoid production. The elucidation of the carotenoid biosynthetic pathway at molecular level holds the promise of providing a toolbox of carotenogenic genes that can be used to engineer micro-organisms for carotenoid production. The biosynthesis of carotenoids involves several steps catalysed by enzymes encoded by carotenogenic genes. Conversion of phytoene to lycopene is the rate limiting step in carotenoid biosynthesis. It is catalyzed by phytoene desaturases (CrtI). Lycopene, a key intermediate in synthesis of xanthophylls such as astaxanthin, is formed from phytoene in four desaturation steps. This study thus aimed at characterizing the phytoene desaturase (CrtI) gene from Paracoccus bogoriensis, an essential gene in the astaxanthin biosynthetic pathway. The CrtI gene was isolated by extracting DNA from a 16 hour culture of Paracoccus bogoriensis. PCR was carried out using CrtI specific primers, the amplicon cloned in pGMET Easy vector and sequenced using Big dye chain termination method. A partial sequence of 1221 base pairs was sequenced. The sequence was translated to a protein sequence in molecular toolkit. Both sequences were then analysed by Phylogenetic tree building using MrBayes program and protein modeling using I-TASSER and COFACTOR to assess the evolution of this gene and to identify CrtI binding sites. Phytoene desaturase from Paracoccus bogoriensis was shown to have evolved from a common ancestor with other Phytoene desaturases. P. bogoriensis phytoene desaturase have highly conserved regions which are putative dinucleotide binding motif βαβ fold in the N-terminal and a signature at the C terminus. P. bogoriensis CrtI amino acid sequence in the C-terminus appears to have diverged slightly from other carotenoid producing bacteria, but perform a similar function. Superimposition of the 3D structure from P. bogoriensis CrtI on homologues from Paracoccus N81106, Xanthobacter autrophicus and Bradyrhizobium sp. ORS278 indicated that the CrtI structure from Paracoccus bogoriensis was different from its homologues from other genus of bacteria. However, Paracoccus N81106, Xanthobacter autrophicus and Bradyrhizobium sp.ORS278 CrtI 3D protein structures revealed structures with similar binding sites, indicating the 3 genus have a common CrtI type for desaturation, even though they produce different xanthophylls.