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.
Publisher
University of Nairobi