Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a β-carotene 3-hydroxylase/4-ketolase

Abstract

A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a beta-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the beta-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this beta-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from beta-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of beta-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both beta-ionone ends.

Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli

“…Therefore, we assume that a specific in vivo environment may account for the unusual activity of PAZ in E. coli. It is worth noting that CrtZ has a relatively broad substrate preference (26) and is bifunctional as a hydroxylase/ ketolase (9). Detailed structural analysis of these compounds is under way.…”

“…This combinatorial approach basically relies on the functional coordination/complementation of pathway enzymes from different sources in a heterologous host (2,8,9). Among the carotenogenic microorganisms (2,8), Pantoea (formerly Enterobacter) is a well-known carotenogenic bacterium that produces zeaxanthin and its derivatives (8).…”

Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize

“…SD212, which appears to be more efficient in ketolating 3-hydroxy-␤-ionone end groups (39). Alternatively, it might be possible to use the astaxanthin synthase (ASY) gene from Xanthophyllomyces dendrohous, a multifunctional enzyme catalyzing all steps from ␤-carotene to astaxanthin formation by the oxygenation of carbons 3 and 4 (40). The reaction involves the 4-ketolation of ␤-carotene followed by 3-hydroxylation without the accumulation of 3-hydroxy intermediates.…”

“…Three different HPLC systems were used at flow rates of 1 mL/min. For ketocarotenoid samples, a Hypersil HyPurity Elite C18 5 column was used with a mobile phase of acetonitrile/methanol/2-propanol (85:10:5 vol/vol/vol) and a column temperature of 32°C (40). All other samples were separated on a Nucleosil C18 3 column with the same mobile phase at 25°C.…”

Metabolic Engineering of Saccharomyces cerevisiae for Astaxanthin Production and Oxidative Stress Tolerance

“…Finally, ␤-carotene is converted to astaxanthin via four steps in which two keto and hydroxy groups are added to each ring by ␤-carotene ketolase encoded by crtW and ␤-carotene hydroxylase encoded by crtZ, respectively. In contrast, a single X. dendrorhous gene, crtYB, encodes a bifunctional enzyme phytoene synthase/lycopene cyclase (30), and crtS encodes astaxanthin synthase, catalyzing ketolation and hydroxylation of ␤-carotene (22). These findings suggest that only three genes are required for astaxanthin production from GGPP in X. dendrorhous.…”