Identification and expression of genes encoding carotenoid biosynthetic enzymes

Carotenoids are common isoprenoids in plants where they serve as accessory photosynthetic pigments and provide most of the yellow, orange and red coloration in both plants and animals. However, how carotenoid is synthesized and how the biosynthesis is regulated in plants is not well understood.; This dissertation deals with the identification and functional analysis of several carotenoid biosynthetic enzymes of plants and how carotenoid biosynthesis is regulated in a green alga, Haematococcus pluvialis, which can accumulate massive amounts of carotenoids under unfavorable growth conditions. First, a number of novel genes were cloned and characterized from Haematococcus and Arabidopsis. These include lycopene {dollar}beta{dollar}-cyclase, {dollar}epsilon{dollar}-cyclase, {dollar}beta{dollar}-carotene hydroxylase and isopentenyl pyrophosphate isomerases. It was found that {dollar}beta{dollar}-cyclase and {dollar}epsilon{dollar}-cyclase are related genes. {dollar}beta{dollar}-Cyclase can cyclize both ends of lycopene to {dollar}beta{dollar}-carotene whereas {dollar}epsilon{dollar}-cyclase can only cyclize one end of lycopene to form monocyclic {dollar}delta{dollar}-carotene. Plant {dollar}beta{dollar}-carotene hydroxylase is significantly longer than bacterial homologues and it catalyzes the formation of the xanthophyll zeaxanthin via {dollar}beta{dollar}-cryptoxanthin as the intermediate. Second, the cloned genes were used as molecular tools to study the regulation of carotenoid biosynthesis in Haematococcus. Two isopentenyl pyrophosphate isomerases (ipiHp1 and ipiHp2) were cloned from this unicellular organism. Our results showed that two isomerase genes were up-regulated by light and so was the gene encoding {dollar}beta{dollar}-carotene ketolase. Protein expression indicated that the two isomerases were differentially regulated, with an increased accumulation of the ipiHp2 gene product in cultures switched from 20 {dollar}mu{dollar}mol photon {dollar}rm msp{lcub}-2{rcub}ssp{lcub}-1{rcub}{dollar} to 150 {dollar}mu{dollar}mol photon {dollar}rm msp{lcub}-2{rcub}ssp{lcub}-1{rcub}.{dollar} Finally, the last chapter of this dissertation focuses on the cloning and characterization of genes involved in IPP biosynthesis in E. coli, because there is evidence that green algae synthesize isopentenyl pyrophosphate via a novel pathway. A number of mutants impaired in isopentenyl pyrophosphate synthesis were isolated and characterized, and a number of putative genes were also cloned from the IPP biosynthetic pathway.