Carotenoids Production: A Healthy and Profitable Industry.

Carotenoids relevance as natural pigments is mainly due to their uses as colorants, feed supplements, nutraceuticals and for medical, cosmetic, and biotechnological purposes. Since they have putative health beneficial effects, the demand and market of carotenoids are growing significantly. There is a diversity of natural and synthetic carotenoids, but only a few of them are commercially produced, including carotenes (ß-carotene and lycopene) and xanthophylls (astaxanthin, canthaxanthin, lutein, zeaxanthin, and capsanthin). Some biotechnological processes for carotenoids production were established some years ago, but new strains and technologies are being developed nowadays for carotenoids widely in demand. This chapter shows a revision of the main carotenoids from a commercial point of view.

Scale-Up of Phytosterols Bioconversion into Androstenedione.

Phytosterols, generated as a by-product of vegetable oils or wood pulp, contain the cyclopentane-perhydro-phenanthrene nucleus, and can be converted into steroid intermediates by removing the C17 side chain. This chapter shows the scale-up, from flask to fermentor, of the phytosterols bioconversion into 4-androstene-3,17-dione (androstenedione; AD) with Mycobacterium neoaurum B-3805. Due to the fact that phytosterols and AD are nearly insoluble in water, two-phase systems and the use of chemically modified cyclodextrins have been described as methods to solve it. Here we use a water-oil two-phase system that allows for the bioconversion of up to 20 g/L of phytosterols into AD in 20 L fermentor.

Metabolic engineering of Mucor circinelloides for zeaxanthin production.

Mucor circinelloides is a ß-carotene producing zygomycete amenable to metabolic engineering using molecular tools. The crtS gene of the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous encodes the enzymatic activities ß-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 ß-cryptoxanthin and zeaxanthin overproducing transformants. These two xanthophylls are hydroxylated compounds from ß-carotene. These results show that the crtS gene is involved in the conversion of ß-carotene into xanthophylls, being potentially useful to engineer carotenoid pathways.

The NADP-dependent glutamate dehydrogenase gene from the astaxanthin producer Xanthophyllomyces dendrorhous: use of Its promoter for controlled gene expression.

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase (GDH) activity from Xanthophyllomyces dendrorhous has been cloned and characterized, and its promoter used for controlled gene expression in this red-pigmented heterobasidiomycetous yeast. We determined the nucleotide sequence of a 4701 bp DNA genomic fragment, showing an open reading frame of 1871 bp interrupted by five introns with fungal consensus splice-site junctions. The predicted protein (455 amino acids; 49 kDa) revealed high identity to GDHs, especially to those from the fungi Cryptococcus neoformans (70%), Sclerotinia sclerotiorum (66%), and several species of Aspergillus (66-67%). Gene phylogenies support the grouping of X. dendrorhous GDH close to those from the majority of the filamentous fungi. The promoter region of the gdhA gene (PgdhA) contains a TATA-like box and two large pyrimidine stretches. The use of PgdhA for gene expression was validated by electrotransformation of X. dendrorhous using an in-frame fusion with the hygromycin resistance gene (hygR) as a reporter. X. dendrorhous transformants were able to grow in YEME complex medium and in Czapek minimal medium supplemented with 50 microg/ml hygromycin, but gene expression in Czapek medium was repressed when using ammonium acetate as a nitrogen source. PgdhA is a valuable tool for controlled gene expression in Basidiomycetes.