Cloning of the cytochrome p450 reductase (crtR) gene and its involvement in the astaxanthin biosynthesis of Xanthophyllomyces dendrorhous

Background: The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin, a carotenoid with high commercial interest. The proposed biosynthetic route in this organism is isopentenylpyrophosphate (IPP) -> geranyleranyl pyrophosphate (GGPP) -> phytoene -> lycopene -> beta-carotene -> astaxanthin. Recently, it has been published that the conversion of beta-carotene into astaxanthin requires only one enzyme, astaxanthin synthase or CrtS, encoded by crtS gene. This enzyme belongs to the cytochrome P450 protein family. Results: In this work, a crtR gene was isolated from X. dendrorhous yeast, which encodes a cytochrome P450 reductase (CPR) that provides CrtS with the necessary electrons for substrate oxygenation. We determined the structural organization of the crtR gene and its location in the yeast electrophoretic karyotype. Two transformants, CBSTr and T13, were obtained by deleting the crtR gene and inserting a hygromycin B resistance cassette. The carotenoid composition of the transformants was altered in relation to the wild type strain. CBSTr forms yellow colonies because it is unable to produce astaxanthin, hence accumulating beta-carotene. T13 forms pale colonies because its astaxanthin content is reduced and its beta-carotene content is increased. Conclusion: In addition to the crtS gene, X. dendrorhous requires a novel gene, crtR, for the conversion of beta-carotene to astaxanthin.This work was supported by U. de Chile ENL 08/06 and: MECESUP UCH 0106 by graduate scholarships to JA and MN, Fundación María Ghilardi by a graduate scholarship to CL, CONICYT by a graduate scholarship to AM