A novel carotenoid, 4-keto-α-carotene, as an unexpected by-product during genetic engineering of carotenogenesis in rice callus.

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-α-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 α-carotene which accumulates by combined reactions of the heterologous gene products and endogenous rice endosperm cyclization reactions.