Chapter 26
Modulation of Activity of a Carotenoid Pathway Through
the Use of the TeT-on Regulatory System: Application
in the Fungus Fusarium fujikuroi
Julia Marente, Pedro Ortega, Javier Pardo-Medina, Javier Avalos,
and M. Carmen Limo ´ n
Abstract
Carotenoids are widespread pigments in photosynthetic species, but they are also found in nonphotosynthetic microorganisms, such as bacteria and fungi. The amenability of fungi to genetic studies have made
some fungal species advantageous models in the study of the genetics and biochemistry of carotenoid
biosynthesis, while others have been used for biotechnological carotenoid production. The availability of
molecular techniques that allow modulating the expression of target genes is a powerful tool in the
manipulation of carotenoid synthesis. An example of an adjustable gene expression is based on the
tetracycline-controlled transcriptional activation system, known as Tet-on. We describe here the material
and protocols for the construction of a Tet-on regulated gene, its introduction in the filamentous fungus
F. fujikuroi, and its use to modulate the expression of a negative regulator of carotenoid biosynthesis.
Key words Tet-on, Doxycycline, Neurosporaxanthin, Xanthophyll, Fusarium fujikuroi
1 Introduction
Some fungi are important sources of information on the genetics
and biochemistry of the genes and enzymes involved in microbial
carotenogenesis. Outstanding examples are Phycomyces blakesleeanus and Mucor circinelloides for the synthesis of β-carotene and
Neurospora crassa and Fusarium fujikuroi for the synthesis of the
carboxylic apocarotenoid acid neurosporaxanthin [1, 2]. Other
fungi are used for the biotechnological production of some carotenoids, as the filamentous fungus Blakeslea trispora for β-carotene
or lycopene, and the yeast Xhanthophyllomyces dendrorhous, formerly Phaffia rhodozyma, for the xanthophyll astaxanthin [3].
The ascomycetous fungus F. fujikuroi has been widely used as a
model of carotenoid production in fungi [4]. This species produces
a mixture of carotenoids, with neurosporaxanthin as the major
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_26,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
343
Modulation of Activity of a Carotenoid Pathway Through
the Use of the TeT-on Regulatory System: Application
in the Fungus Fusarium fujikuroi
Julia Marente, Pedro Ortega, Javier Pardo-Medina, Javier Avalos,
and M. Carmen Limo ´ n
Abstract
Carotenoids are widespread pigments in photosynthetic species, but they are also found in nonphotosynthetic microorganisms, such as bacteria and fungi. The amenability of fungi to genetic studies have made
some fungal species advantageous models in the study of the genetics and biochemistry of carotenoid
biosynthesis, while others have been used for biotechnological carotenoid production. The availability of
molecular techniques that allow modulating the expression of target genes is a powerful tool in the
manipulation of carotenoid synthesis. An example of an adjustable gene expression is based on the
tetracycline-controlled transcriptional activation system, known as Tet-on. We describe here the material
and protocols for the construction of a Tet-on regulated gene, its introduction in the filamentous fungus
F. fujikuroi, and its use to modulate the expression of a negative regulator of carotenoid biosynthesis.
Key words Tet-on, Doxycycline, Neurosporaxanthin, Xanthophyll, Fusarium fujikuroi
1 Introduction
Some fungi are important sources of information on the genetics
and biochemistry of the genes and enzymes involved in microbial
carotenogenesis. Outstanding examples are Phycomyces blakesleeanus and Mucor circinelloides for the synthesis of β-carotene and
Neurospora crassa and Fusarium fujikuroi for the synthesis of the
carboxylic apocarotenoid acid neurosporaxanthin [1, 2]. Other
fungi are used for the biotechnological production of some carotenoids, as the filamentous fungus Blakeslea trispora for β-carotene
or lycopene, and the yeast Xhanthophyllomyces dendrorhous, formerly Phaffia rhodozyma, for the xanthophyll astaxanthin [3].
The ascomycetous fungus F. fujikuroi has been widely used as a
model of carotenoid production in fungi [4]. This species produces
a mixture of carotenoids, with neurosporaxanthin as the major
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_26,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
343