Gene fusions for the directed modification of the carotenoid biosynthesis pathway in mucor circinelloides

Enrique A. Iturriaga, T. Papp, María Isabel Álvarez, Arturo P. Eslava

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Several fungal species, particularly some included in the Mucorales, have been used to develop fermentation processes for the production of β-carotene. Oxygenated derivatives of β-carotene are more valuable products, and the preference by the market of carotenoids from biological sources has increased the research in different carotenoid-producing organisms. We currently use Mucor circinelloides as a model organism to develop strains able to produce new, more valuable, and with an increased content of carotenoids. In this chapter we describe part of our efforts to construct active gene fusions which could advance in the diversification of carotenoid production by this fungus. The main carotenoid accumulated by M. circinelloides is β-carotene, although it has some hydroxylase activity and produces low amounts of zeaxanthin. Two enzymatic activities are required for the production of astaxanthin from β-carotene: a hydroxylase and a ketolase. We used the ctrW gene of Paracoccus sp. N81106, encoding a bacterial β-carotene ketolase, to construct gene fusions with two fungal genes essential for the modification of the pathway in M. circinelloides. First we fused it to the carRP gene of M. circinelloides, which is responsible for the phytoene synthase and lycopene cyclase activities in this fungus. The expected activity of this fusion gene would be the accumulation by M. circinelloides of canthaxanthin and probably some astaxanthin. A second construction was the fusion of the crtW gene of Paracoccus sp. to the crtS gene of Xanthophyllomyces dendrorhous, responsible for the synthesis of astaxanthin from β-carotene in this fungus, but which was shown to have only hydroxylase activity in M. circinelloides. The expected result in M. circinelloides transformants was the accumulation of astaxanthin. Here we describe a detailed and empirically tested protocol for the construction of these gene fusions.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Pages109-122
Number of pages14
Volume898
DOIs
Publication statusPublished - 2012

Publication series

NameMethods in Molecular Biology
Volume898
ISSN (Print)10643745

Fingerprint

Mucor
Gene Fusion
Carotenoids
Paracoccus
Mixed Function Oxygenases
Fungi
Mucorales
Fungal Genes
Canthaxanthin
Genes
Fermentation

Keywords

  • Astaxanthin
  • Gene fusions
  • Metabolic engineering
  • Mucor
  • Xanthophylls

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Iturriaga, E. A., Papp, T., Álvarez, M. I., & Eslava, A. P. (2012). Gene fusions for the directed modification of the carotenoid biosynthesis pathway in mucor circinelloides. In Methods in Molecular Biology (Vol. 898, pp. 109-122). (Methods in Molecular Biology; Vol. 898). https://doi.org/10.1007/978-1-61779-918-1_6

Gene fusions for the directed modification of the carotenoid biosynthesis pathway in mucor circinelloides. / Iturriaga, Enrique A.; Papp, T.; Álvarez, María Isabel; Eslava, Arturo P.

Methods in Molecular Biology. Vol. 898 2012. p. 109-122 (Methods in Molecular Biology; Vol. 898).

Research output: Chapter in Book/Report/Conference proceedingChapter

Iturriaga, EA, Papp, T, Álvarez, MI & Eslava, AP 2012, Gene fusions for the directed modification of the carotenoid biosynthesis pathway in mucor circinelloides. in Methods in Molecular Biology. vol. 898, Methods in Molecular Biology, vol. 898, pp. 109-122. https://doi.org/10.1007/978-1-61779-918-1_6
Iturriaga EA, Papp T, Álvarez MI, Eslava AP. Gene fusions for the directed modification of the carotenoid biosynthesis pathway in mucor circinelloides. In Methods in Molecular Biology. Vol. 898. 2012. p. 109-122. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-61779-918-1_6
Iturriaga, Enrique A. ; Papp, T. ; Álvarez, María Isabel ; Eslava, Arturo P. / Gene fusions for the directed modification of the carotenoid biosynthesis pathway in mucor circinelloides. Methods in Molecular Biology. Vol. 898 2012. pp. 109-122 (Methods in Molecular Biology).
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