Expression in Escherichia coli and properties of the carotene ketolase from Haematococcus pluvialis

High level expression of the functional beta-carotene ketolase gene bkt from Haematococcus pluvialis occurred in Escherichia coli transformants producing beta-carotene or zeaxanthin as a result of the presence of additional carotenoid genes from Erwinia uredovora. Requirement of molecular oxygen for the insertion of the keto group was demonstrated. The final product of this two-step ketolase reaction from beta-carotene is canthaxanthin (4,4'-diketo-beta-carotene) with the 4-monoketo derivative echinenone as an intermediate. A reaction sequence for the formation of astaxanthin from beta-carotene was established based on kinetic data on astaxanthin formation in E. coli transformants carrying the hydroxylase gene crtZ from Erwinia along with bkt. We conclude that the carotenoids zeaxanthin and adonixanthin which accumulate in addition to astaxanthin in this transformant are products of side reactions rather than direct precursors of astaxanthin. The possible mechanisms for the formation of the keto derivatives are discussed.