title: Structure and function characterization of the phytoene desaturase related to the lutein biosynthesis in Chlorella protothecoides CS-41.
abstract: Phytoene desaturase is the key enzyme involved in the biosynthesis pathway of lutein. The unicellular microalga, Chlorella protothecoides CS-41, had been selected for the heterotrophic production of high concentrations of lutein. In this study, a cDNA copy of the pds gene from C. protothecoides was obtained using the rapid amplification of cDNA ends (RACE) technique. Phylogenetic analysis of the deduced amino acid sequence revealed that the phytoene desaturases derived from the algal family. Expression of the pds gene in Escherichia coli produced a single protein of 61 kDa. The PDS activity of the expressed protein was confirmed by the production of ζ-carotene as the result from the action of the enzyme's desaturation activity, which was identified by high-performance liquid chromatography and heterologous complementation analysis. Using random and site-directed mutagenesis, a single amino acid mutation (N144D) was identified and confirmed. This mutant encodes an inactive enzyme, which implies that amino acid 144 is crutial to the activity of the PDS enzyme. Therefore, by gene cloning and expression in prokaryotic cells, the gene for ζ-carotene production or as part of the biosynthetic pathway of lutein had been characterized from Chlorella protothecoides CS-41.
authors.affiliation: Department of Food Science & Technology, MOST-USDA Joint Research Center for Food Safety and Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
authors.forename: Meiya
authors.initials: M
authors.lastname: Li
authors.forename: Zhibing
authors.initials: Z
authors.lastname: Gan
authors.forename: Yan
authors.initials: Y
authors.lastname: Cui
authors.forename: Chunlei
authors.initials: C
authors.lastname: Shi
authors.forename: Xianming
authors.initials: X
authors.lastname: Shi
mesh_terms: D000595:Amino Acid Sequence; D002708:Chlorella / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D014975:Lutein / Q000096:biosynthesis / Q000235:genetics*; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D024321:zeta Carotene / Q000378:metabolism
chemical_list: D024321:zeta Carotene; D010088:Oxidoreductases; C022398:phytoene dehydrogenase; D014975:Lutein