Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line.

Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol) with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzyme DES7 is encoded by the gene TTHERM_00310640, identified with RNAi and gene knock-out experiments, but has not yet been heterologously expressed actively in any organism. A model derived from its amino acid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The protein has catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in the synthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity, we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line, identified and measured the steroid metabolites formed, and extended the actual knowledge on its localization. We found that the accumulation of 7-dehydrocholesterol could be increased 16-40-fold in Spodopterafrugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7. The protein was detected in the microsomal fraction, in accordance with previous reports. Although the electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown, we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite of the large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that there is significant functional conservation of the electron donors, since the ciliate's sterol desaturase can function in the context of the insect electron transport system. The results achieved demonstrate that DES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insect cell line.

Incomplete sterols and hopanoids pathways in ciliates: gene loss and acquisition during evolution as a source of biosynthetic genes.

Polycyclic triterpenoids, such as sterols and hopanoids, are essential components of plasmatic membrane in eukaryotic organisms. Although it is generally assumed that ciliates do not synthesize sterols, and many of them are indeed auxotrophic, a large set of annotated genomic sequences and experimental data from recently studied organisms indicate that they can carry putative genes and respond to the presence/absence of precursors in various ways. The pre-squalene pathway, for instance, is largely present in all sequenced ciliates except in Ichthyophthirius multifiliis; although Paramecium tetraurelia lacks the squalene synthase and Oxytricha trifallax the squalene hopene synthase, in addition to the former. On the other hand, the post-squalene pathway, requiring oxygen in several steps, is mostly incomplete in all ciliates analyzed. Nevertheless, a number of predicted genes, with high sequence similarity to C-4 methyl oxidase/s, C-14 demethylase, C-5 and C-7 desaturases and C-24 reductase of sterols are found in Tetrahymena and Paramecium, and scattered in other Stichotrichia ciliates. Moreover, several of these sequences are present in multiples paralogs, like the C-7 desaturase in Paramecium, that carries six versions of the only one present in Tetrahymena. The phylogenetic analyses suggest a mixed origin for the genes involved in the biosynthesis of sterols and surrogates in this phylum; while the genes encoding enzymes of the pre-squalene pathway are most likely of bacterial origin, those involved in the post-squalene pathway, including the processing of sterols obtained from the environment, may have been partially retained or acquired indistinctly from lower eukaryotes or prokaryotes. This particular combination of diverse gene/s acquisition patterns allows for survival in conditions of poor oxygen availability, in which tetrahymanol and other hopanoids may be advantageous, but also conditions of excess oxygen availability and abundant sterols, in which the latter are preferentially phagocyte, and/or transformed. Furthermore, the possibility that some of the genes involved in sterol metabolism may have another biological function in the most studied ciliate T. thermophila, was also explored.

The cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae.

Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b(5), Cyt b(5) reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes.