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Romero F.,Leon Institute of Technology | Fernandez-Chimeno R.I.,Leon Institute of Technology | De La Fuente J.L.,Gadea Biopharma | Barredo J.-L.,Gadea Biopharma
Methods in Molecular Biology | Year: 2012

Carotenoids are important pigments produced by plants and many microorganisms, including fungi, microalgae, cyanobacteria, and bacteria. Marine actinomycetes are a group of bacteria that produce a variety of metabolites with economic potential. Here, we describe a general method of selecting marine actinomycetes as carotenoids' producers. The screening is carried out at two levels: the first one involves a quick selection of strains by visual color inspection, and the second consists in the analysis of the extracts by HPLC. The taxonomic analysis of the producing strains gives us an overview of the groups of actinomycetes in which carotenoids can be found. © 2012 Springer Science+Business Media, LLC.

Rodriguez-Saiz M.,Gadea Biopharma | De La Fuente J.-L.,Gadea Biopharma | Barredo J.-L.,Gadea Biopharma
Methods in Molecular Biology | Year: 2012

Mucor circinelloides is a β-carotene producing zygomycete amenable to metabolic engineering using molecular tools. The crtS gene of the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous encodes the enzymatic activities β-carotene hydroxylase and ketolase, allowing this yeast to produce the xanthophyll called astaxanthin. Here we describe the fermentation of X. dendrorhous in astaxanthin producing conditions to purify mRNA for the cloning of the cDNA from the crtS gene by RT-PCR. Further construction of an expression plasmid and transformation of M. circinelloides protoplasts allow the heterologous expression of the crtS cDNA in M. circinelloides to obtain β-cryptoxanthin and zeaxanthin overproducing transformants. These two xanthophylls are hydroxylated compounds from β-carotene. These results show that the crtS gene is involved in the conversion of β-carotene into xanthophylls, being potentially useful to engineer carotenoid pathways. © 2012 Springer Science+Business Media New York.

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