Knockout of a C5 Sterol Desaturase Gene in Tetrahymena thermophila

M. L. TOMAZIC; A. D. NUSBLAT; C. B. NUDEL

Philadelphia, Pennsylvania, EEUU

Congreso; American Society for Microbiology, 109 General Meeting; 2009

American Society for Microbiology

Knockout of a C5 Sterol Desaturase Gene in Tetrahymena thermophilaAuthor Block M. L. Tomazic, A. D. Nusblat, C. B. Nudel;Sch. of Pharm. and Biochemistry, Univ. of Buenos Aires, Buenos Aires, ARGENTINA.Abstract:Background: The non-pathogenic ciliate T. thermophila has a unique set of sterol desaturases composed of Δ5,Δ7 and Δ22 activities (1). The enzymes have potential for biotechnology purposes, pacularly the C7 steroldesaturase, as it may be used for decreasing cholesterol content in foodstuffs of animal origin with simultaneousenrichment in pro vitamin D3 (2). Previous in vitro characterization of C7 and C22 cholesterol desaturasesestablished unambiguously their oxygen, cytochrome b5 and NADH requirements (3). In order to identify the genesencoding for these desaturases, a gene knockout out approach was selected. Methods: Target sequences wereselected using the Histidine conserved motif of sterol desaturases (TIGR Database) (4). From eight putativesequences, six displaying E 1.0e-7 were assayed: 51.m00243, 270.m00036, 124.m00110, 5m.00476, 36.m00199,59.m00274 and 156m.00077. Constructs were made by overlapping PCR, using the upstream and downstreamregions homologous to each gene flanking a Neo 3 cassette expressing paromomycin resistance under a cadmiuminducible metallothionein (MTT) promoter (5). Transformants were obtained by biolistic bombardment on T.thermophila CU 428 strain. After phenotypic assortment, mutants were grown with various sterols, i.e, lathosterol,cholestanol, to confirm their phenotype. Sterols were analyzed by HPLC. Results: One of the mutants(270.m00036 KO) showed a phenotype corresponding to a C5 sterol desaturase KO and no significant changes inits growth pattern. Conclusion: The mutation introduced by targeted knockout and subsequent phenotypicassortment generated cells with an almost complete replacement of the wild type gene in their macronuclei, asshown by expression studies. By this way the first sterol desaturase gene could be identified in ciliates.Acknowledgments/ References::The authors are grateful to Martin A. Gorovsky and Jody Bowen Department of Biology, University of Rochester,Rochester, NY 14627, USA and Dr. Marcus Hartmann and Dr. Ulrike Bockau, Cilian AG, Muenster, Germany fortheir help in the experimental design.It is also acknowledged the support of ASM International Fellowship for Latin America (2005) (A. Nusblat) and theArgentinean grants PICT 33408 and UBACYT B108 (University of Buenos Aires).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. Valcarce G, Munoz L, Nusblat A, Nudel C, Florin-Christensen J. 2001. The improvement of milk by cultivationwith ciliates. J Dairy Sci. 84(10):2136-43.3.3. Nusblat AD, Muñoz L, Valcarce GA, Nudel CB. 2005. Characterization and properties of cholesterol desaturasesfrom the ciliate Tetrahymena thermophila. J Eukaryot Microbiol. 52(1):61-7.4. Eisen JA, Coyne RS, Wu M, Wu D, Thiagarajan M, Wortman JR, Badger JH, Ren Q, Amedeo P, Jones KM,Tallon LJ, Delcher AL, Salzberg SL, Silva JC, Haas BJ, Majoros WH, Farzad M, Carlton JM, Smith RK Jr, Garg J,Pearlman RE, Karrer KM, Sun L, Manning G, Elde NC, Turkewitz AP, Asai DJ, Wilkes DE, Wang Y, Cai H, CollinsK, Stewart BA, Lee SR, Wilamowska K, Weinberg Z, Ruzzo WL, Wloga D, Gaertig J, Frankel J, Tsao CC, GorovskyMA, Keeling PJ, Waller RF, Patron NJ, Cherry JM, Stover NA, Krieger CJ, del Toro C, Ryder HF, Williamson SC,Barbeau RA, Hamilton EP, Orias E. 2006. Macronuclear genome sequence of the ciliate Tetrahymena thermophila,a model eukaryote. PLoS Biol. 4(9):e286.5. Shang Y, Song X, Bowen J, Corstanje R, Gao Y, Gaertig J, Gorovsky MA. 2002. A robust inducible-repressiblepromoter greatly facilitates gene knockouts, conditional expression, and overexpression of homologous andheterologous genes in Tetrahymena thermophila. Proc Natl Acad Sci. 99(6):3734-9.