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Identification of the first C24-sterol deethylase gene in Tetrahymena thermophila A. D. Nusblat1, M. L. Tomazic1, S. R. Najle2, M. A. Rinaldi1, A. D. Uttaro2 and C. B. Nudel1. 1Cátedra de Biotecnología y Microbiología Industrial, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina. 2Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Santa Fe, Argentina. Background: Ciliates, as well as nematodes and insects, lack a full metabolic pathway for sterol biosynthesis. Nevertheless, they can perform several enzymatic reactions on the sterol moiety, such as dehydration, reduction, oxidation, demethylation or desaturation at specific positions. In particular Tetrahymena thermophila desaturates sterols at positions C5(6), C7(8) and C22(23) and removes the C24 ethyl group in C29 sterols (1). Characterization of sterol desaturases have revealed some typical features, such as their oxygen, Cytochrome b5 and NAD(P)H dependence (2). In addition, gene identification of the first C5 desaturase in this organism has recently been achieved (3). The dealkylation reaction, on the other hand, has never been metabolically or genetically characterized, nor has it been reported in any other organism. Using a gene knockout out approach followed by phenotypic assortment, the sequence encoding a C24 deethylase gene was identified in Tetrahymena. Methods: Putative target sequences were selected in the TIGR Database (www.Tigr.org) using sterol desaturases as query, based on the conserved Histidine motif typical for non-heme Cyt b5 dependent enzymes. From seven putative sequences retrieved, six of them, displaying E<1.0e-7 were assayed: TTHERM_ 00438800, THERM_ 00758950, TTHERM_ 00077800, TTHERM_ 00348230, TTHERM_ 00487050 and TTHERM_ 00876970. Constructs were made by overlapping PCR, using the upstream and downstream regions homologous to each gene, flanking a neo3 cassette expressing paromomycin resistance under a cadmium inducible metallothionein (MTT) promoter (4). Transformants were obtained by biolistic bombardment on T. thermophila CU428 strain. After phenotypic assortment, mutants were grown with stigmasterol, and clones impaired in deethylase activity were selected. Sterol identification was performed by GC-MS and HPLC-MS analysis. Results: One of the mutants (TTHERM_00438800 KO) showed a phenotype corresponding to a C24-sterol deethylase KO, as shown by the sterol composition of the extracted lipid fraction. Growth of the mutant with various sterols, such as cholesterol, cholestanol had no consequences on growth rate or biomass yield, except for stigmasterol; with this sterol both parameters were clearly diminished, thus indicating that the ethyl group in position C24 clearly impairs the normal performance of the ciliate. Conclusion: The mutation introduced by targeted knockout and subsequent phenotypic assortment generated cells with a complete replacement of the wild type gene in their macronuclei. GC-MS and HPLC-MS analysis confirmed the expected sterols profile for the mutant KO. By this way the first 24-sterol deethylase gene was identified in an organism. References: 1. Conner RL, Mallory FB, Landrey JR, Iyengar CW. 1969. The conversion of cholesterol to delta-5,7,22-cholestatrien-3-beta-ol by Tetrahymena pyriformis. J Biol Chem. 244(9):2325-33.2. 2. Nusblat AD, Muñoz L, Valcarce GA, Nudel CB. 2005. Characterization and properties of cholesterol desaturases from the ciliate Tetrahymena thermophila. J Eukaryot Microbiol. 52(1):61-7. 3. Nusblat AD, Najle SR, Tomazic ML, Uttaro AD, and Nudel CB. C5(6) sterol desaturase from Tetrahymena thermophila: gene identification and knockout, sequence analysis and comparison to other C5(6) sterol desaturases. Eukaryotic Cell (In revision) . 4. Shang Y, Song X, Bowen J, Corstanje R, Gao Y, Gaertig J, Gorovsky MA. 2002. A robust inducible-repressible promoter greatly facilitates gene knockouts, conditional expression, and overexpression of homologous and heterologous genes in Tetrahymena thermophila. Proc Natl Acad Sci. 99(6):3734-9.

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