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Eukaryotic Cell, August 2009, p. 1287-1297, Vol. 8, No. 8
1535-9778/09/$08.00+0     doi:10.1128/EC.00057-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

C-5(6) Sterol Desaturase from Tetrahymena thermophila: Gene Identification and Knockout, Sequence Analysis, and Comparison to Other C-5(6) Sterol Desaturases{triangledown}

Alejandro D. Nusblat,1* Sebastián R. Najle,2 Mariela L. Tomazic,1 Antonio D. Uttaro,2 and Clara B. Nudel1

Cátedra de Biotecnología y Microbiología Industrial, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina,1 Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Santa Fe, Argentina2

Received 20 February 2009/ Accepted 4 June 2009

The gene coding for a C-5(6) sterol desaturase in Tetrahymena thermophila, DES5A, has been identified by the knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biolistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with that of the DES5A deletion mutant. KO270 converted only 6% of the added sterol into the C-5 unsaturated derivative, while the wild type accumulated 10-fold larger amounts under similar conditions. The decreased desaturation activity is specific for the C-5(6) position of lathosterol and cholestanol; other desaturations, namely C-7(8) and C-22(23), were not affected. Analysis by reverse transcription-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. The growth of KO270 was undistinguishable from that of the wild-type strain. Des5Ap resembles known C-5(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. thermophila's enzyme and Paramecium orthologues in a cluster together with functionally characterized C-5 sterol desaturases from vertebrates, fungi, and plants, although in a different branch.

* Corresponding author. Mailing address: Cátedra de Biotecnología y Microbiología Industrial, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina. Phone: 54-11-4964-8269. Fax: 54-11-4964-8270. E-mail: anusblat{at}ffyb.uba.ar

{triangledown} Published ahead of print on 12 June 2009.

Eukaryotic Cell, August 2009, p. 1287-1297, Vol. 8, No. 8
1535-9778/09/$08.00+0     doi:10.1128/EC.00057-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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