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Identification of the gene responsible for torulene cleavage in the Neurospora carotenoid pathway

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Abstract

Torulene, a C40 carotene, is the precursor of the end product of the Neurospora carotenoid pathway, the C35 xanthophyll neurosporaxanthin. Torulene is synthesized by the enzymes AL-2 and AL-1 from the precursor geranylgeranyl diphosphate and then cleaved by an unknown enzyme into the C35 apocarotenoid. In general, carotenoid cleavage reactions are catalyzed by carotenoid oxygenases. Using protein data bases, we identified two putative carotenoid oxygenases in Neurospora, named here CAO-1 and CAO-2. A search for novel mutants of the carotenoid pathway in this fungus allowed the identification of two torulene-accumulating strains, lacking neurosporaxanthin. Sequencing of the cao-2 gene in these strains revealed severe mutations, pointing to a role of CAO-2 in torulene cleavage. This was further supported by the identical phenotype found upon targeted disruption of cao-2. The biological function was confirmed by in vitro assays using the purified enzyme, which cleaved torulene to produce β-apo-4′-carotenal, the corresponding aldehyde of neurosporaxanthin. The specificity of CAO-2 was shown by the lack of γ-carotene-cleaving activity in vitro. As predicted for a structural gene of the carotenoid pathway, cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner. Our data demonstrate that CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin biosynthetic pathway.

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Acknowledgments

We thank Dr. T. J. Schmidhauser for laboratory facilities in the search of the color mutants, Dr. Hansgeorg Ernst (BASF) for providing the apocarotenoids, Dr. Jorge Mayer for proofreading the manuscript, and Dr. Peter Beyer for valuable discussions. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) Grant AL892-3, HarvestPlus (www.harvestplus.org) and the Spanish Government (Ministerio de Ciencia y Tecnología, projects BIO2003-01548 and BIO2006-01323).

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  1. Lorena Saelices

    Present address: Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Isla de la Cartuja, Universidad de Sevilla-CSIC, Av. Americo Vespucio s/n, Sevilla, 41092, Spain

  2. Loubna Youssar

    Present address: Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Apartado 1078, 41080, Sevilla, Spain

Authors and Affiliations

  1. Departamento de Genética, Universidad de Sevilla, Apartado 1095, 41080, Sevilla, Spain

    Lorena Saelices, Loubna Youssar & Javier Avalos

  2. Faculty of Biology, Albert-Ludwigs University of Freiburg, Schaenzlestr. 1, 79104, Freiburg, Germany

    Iris Holdermann & Salim Al-Babili

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  1. Lorena Saelices
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Correspondence to Javier Avalos.

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Communicated by J. Perez-Martin.

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Saelices, L., Youssar, L., Holdermann, I. et al. Identification of the gene responsible for torulene cleavage in the Neurospora carotenoid pathway. Mol Genet Genomics 278, 527–537 (2007). https://doi.org/10.1007/s00438-007-0269-2

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  • DOI: https://doi.org/10.1007/s00438-007-0269-2

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