C5(6) sterol desaturase from Tetrahymena thermophila: gene identification and knockout, sequence analysis and comparison to other C5(6) sterol desaturases.

ALEJANDRO D. NUSBLAT; SEBASTIÁN R NAJLE ; MARIELA L TOMAZIC; ANTONIO D UTTARO; CLARA B NUDEL

EUKARYOTIC CELL

American Society for Microbiology,

Año: 2009 vol. 8 p. 1287 - 1297

1535-9778

The gene coding for a C5(6) sterol desaturase in Tetrahymena thermophila, DES5A, has been identified by knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biobalistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with DES5A deletion mutant. KO270 converted only 6 % of the added sterol into the C5 unsaturated derivative, while wild type accumulated 10-fold higher amounts under similar conditions. The decreased desaturation activity is specific for the C5(6) position of lathosterol and cholestanol; other desaturations, namely C7(8) and C22(23), were not affected. Analysis by RT-PCR reveals that DES5A is transcribed both in the presence and absence of cholestanol in wild type cells whereas the transcribed gene was not detected in KO270. Growth of KO270 was undistinguishable from the wild type strain. Des5Ap resembles known C5(6) sterol desaturases, displaying the three typical histidine motifs, four hydrophobic transmembrane regions and two other highly conserved domains of unknown function. A phylogenetic analysis placed T. thermophilas enzyme and Paramecium orthologues, in a cluster together with functionally characterized C5 sterol desaturases from vertebrates, fungi and plants, although in a different branch.