Ketolase Gene Research Articles

Novel β-carotene ketolases from non-photosynthetic bacteria for canthaxanthin synthesis

We reported previously that the Rhodococcus erythropolis strain AN12 synthesizes the monocyclic carotenoids 4-keto gamma-carotene and gamma-carotene. We also identified a novel lycopene beta-monocyclase in this strain. Here we report the identification of the rest of the carotenoid synthesis genes in AN12. Two of these showed apparent homology to putative phytoene dehydrogenases. Analysis of Rhodococcus knockout mutants suggested that one of them ( crtI) encodes a phytoene dehydrogenase, whereas the other ( crtO) encodes a beta-carotene ketolase. Expression of the beta-carotene ketolase gene in an Escherichia coli strain which accumulates beta-carotene resulted in the production of canthaxanthin. In vitro assays using a crude extract of the E. coli strain expressing the crtO gene confirmed its ketolase activity. A crtO homologue (DR0093) from Deinococcus radiodurans R1 was also shown to encode a beta-carotene ketolase, despite its sequence homology to phytoene dehydrogenases. The Rhodococcus and Deinococcus CrtO ketolases both catalyze the symmetric addition of two keto groups to beta-carotene to produce canthaxanthin. Even though this activity is similar to the CrtW-type of ketolase activity, the CrtO ketolases show no significant sequence homology to CrtW-type ketolases. The presence of six conserved regions may be a signature for the CrtO-type of beta-carotene ketolases.

Cloning of two carotenoid ketolase genes from Nostoc punctiforme for the heterologous production of canthaxanthin and astaxanthin.

For the heterologous synthesis of keto-carotenoids such as astaxanthin, two carotenoid ketolase genes crtW38 and crtW148, were cloned from the cyanobacterium, Nostoc punctiforme PCC 73102 and functionally characterized. Upon expression in Escherichia coli, both genes mediated the conversion of beta-carotene to canthaxanthin. However in a zeaxanthin-producing E. coli, only the gene product of crtW148 introduced 4-keto groups into the 3,3'-dihydroxy carotenoid zeaxanthin yielding astaxanthin. The gene product of crtW38 was unable to catalyze this reaction. Both ketolases differ in their interaction with a hydroxylase in the biosynthetic pathway from beta-carotene to astaxanthin.

Expression of a Ketolase Gene Mediates the Synthesis of Canthaxanthin in Synechococcus Leading to Tolerance Against Photoinhibition, Pigment Degradation and UV-B Sensitivity of Photosynthesis¶

The potential of ketocarotenoids to protect the photosynthetic apparatus from damage caused by excess light and UV-B radiation was assessed. Therefore, the cyanobacterium Synechococcus was transformed with a foreign beta-carotene ketolase gene under a strong promoter leading to the accumulation of canthaxanthin. This diketo carotenoid is absent in the original strain. Most of the newly formed canthaxanthin was located in the thylakoid membranes. The endogenous beta-carotene hydroxylase was unable to interact with the ketolase. Therefore, only traces of astaxanthin were found. The transformant was treated with strong light (500 or 1200 mumol m-2 s-1) and with UV-B radiation. In contrast to a nontransformed strain the overall photosynthesis, measured as oxygen evolution, was protected from inhibition by light of 500 mumol m-2 s-1 and UV-B radiation of 6.8 W m-2. Furthermore, degradation in the light of chlorophyll and carotenoids at an irradiance of 1200 mumol m-2 s-1, which was substantial in the nontransformed control, was prevented. These results indicate that in situ canthaxanthin, which is formed at the expense of zeaxanthin and replaces this hydroxy carotenoid within the photosynthetic apparatus, is a better protectant against solar radiation. These findings are discussed on the basis of the in vitro properties such as inactivating peroxyl radicals, quenching of singlet oxygen and oxidation stability of these different carotenoid structures.

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

High level expression of the functional β-carotene ketolase gene bkt from Haematococcus pluvialis occurred in Escherichia coli transformants producing β-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 β-carotene is canthaxanthin (4,4′-diketo-β-carotene) with the 4-monoketo derivative echinenone as an intermediate. A reaction sequence for the formation of astaxanthin from β-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.