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Asthaxanthin synthase in Xhanthophyllomyces

A gene (ast) from Xanthophyllomyces encoding a cytochrome P450, described in a F. Hoffman La Roche-owned patent (EP 1 035 206 A1). The gene product was able to convert b-carotene to astaxanthin after the introduction of the gene in a b-carotene accumulating mutant of X. dendrorhous. Based on information from patent literature, the ast gene of Xanthophyllomyces strain CBS 6938 (our model strain), including the promoter and terminator regions, was isolated by PCR techniques. Furthermore, an ast cDNA copy was isolated from a Xanthophyllomyces cDNA library, also by PCR. Expression of the ast gene in a b-carotene accumulating Xanthophyllomyces strain (PR-1-104) restored astaxanthin production, indicating that the gene is functionally expressed.

The ast gene was disrupted by insertional inactivation. A fragment of the ast gene was cloned into a X. dendrorhous vector that contained a G418 selection marker. The linearized vector was transferred to X. dendrorhous by means of electroporation.
By over-expression of the homologous carotenogenic ast gene in strain(s) CBS 6938 (and PR-1-104), an increase in astaxanthin content was observed. Recently, strains have been constructed that over-express a combination of two carotenogenic genes in order to further increase the astaxanthin (or other carotenoid) content. These genes are crtE, crtYB, crtI and ast.

Comparison of carotenoid levels, especially intermediates, of wild and crt gene knock-out strains, showed no increase of these intermediates in the knock-out strains. This indicates that the end product astaxanthin (or its precursor b-carotene) does not exert a feed back regulatory effect on the carotenogenic enzymes.

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Johan VAN DEN BERG, (Head of Fungal Genomics)
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