a b s t r a c t
Rice endosperm is devoid of carotenoids because the initial biosynthetic steps are absent. The early
carotenogenesis reactions were constituted through co-transformation of endosperm-derived rice callus
with phytoene synthase and phytoene desaturase transgenes. Subsequent steps in the pathway such as
cyclization and hydroxylation reactions were catalyzed by endogenous rice enzymes in the endosperm.
The carotenoid pathway was extended further by including a bacterial ketolase gene able to form astaxanthin,
a high value carotenoid which is not a typical plant carotenoid. In addition to astaxanthin and
precursors, a carotenoid accumulated in the transgenic callus which did not fit into the pathway to astaxanthin.
This was subsequently identified as 4-keto-a-carotene by HPLC co-chromatography, chemical
modification, mass spectrometry and the reconstruction of its biosynthesis pathway in Escherichia coli.
We postulate that this keto carotenoid is formed from a-carotene which accumulates by combined reactions
of the heterologous gene products and endogenous rice endosperm cyclization reactions.