The genes for geranylgeranyl diphosphate synthase (crtE) and phytoene synthase (crtB) from the epiphytic bacterium Erwinia uredovora and the phytoene desaturase gene from the photosynthetic bacterium Rhodobacter capsulatus (Rc‐crtI) were introduced into Escherichia coli, which resulted in the accumulation of the acyclic carotenoid, neurosporene. Further introduction of the lycopene cyclase gene from E. uredovora (crtY) or the higher plant Capsicum annuum (Icy) resulted in the production of a bicyclic carotenoid, 7,8‐dihydro‐β‐carotene, via monocyclic β‐zeacarotene. ζ‐Carotene was also found to be cyclized to bicyclic 7,8,7′,8′‐tetrahydro‐β‐carotene by the Erwinia cyclase. These results indicate that both lycopene cyclases can cyclize a 7,8‐dihydro‐ψ end group to a 7,8‐dihydro‐β end group, in addition to the usual cyclization of the ψ end group to the β end group. Furthermore, β‐carotene hydroxylase from Enwnia (CrtZ) was able to add a hydroxyl group to the 7,8‐dihydro‐β end group and the β end group.
Lycopene cyclase, an enzyme responsible for the formation of cyclic carotenoids from acyclic precursors has been purified to homogeneity in an active state. The Erwinia uredovora lycopene cyclase gene (crtY) was over-expressed in Escherichia coli. From this recombinant strain the enzyme was purified by immuno-affinity chromatography and its cyclization activity characterized as a two-step reaction in which both sides of the lycopene molecule are cyclized to beta-ionone rings with the monocyclic gamma-carotene as an intermediate. Furthermore, neurosporene as well as l-hydroxylycopene were cyclized to beta-zeacarotene and hydroxy-gamma-carotene respectively. In contrast, neither 1,1'- dihydroxylycopene nor the tetra-cis-prolycopene were accepted as substrates. The cofactors involved in the reaction were either NADH or NADPH. K(m) values were determined for lycopene and NADPH to be 1.8 microM and 2.5 mM respectively.
Summary
Six genes have been mapped and identified by hybridization or by carotenoid analysis of deletion mutants on a 9 kb chromosomal fragment originating from Erwinia herbicola Eho 10. These genes include crtB, E and I which have been formerly described for Rhodobacter, and three new ones: crtZ encoding lycopene cyclase, crtH for β‐carotene hydroxylase, and crtG for zeaxanthin glycosilase. Their arrangement on the plasmid has been elucidated.
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