KNOCKOUT OF STEROL DESATURASE GENES BY SOMATIC TRANSFORMATION OF TETRAHYMENA THERMOPHYLA

ALEJANDRO NUSBLAT; ANTONIO UTTARO; CLARA NUDEL

Rosario, Santa Fe, Argentina

Congreso; SAIB XLII Reunion Anual; 2006

SAIB

The non-pathogenic ciliate Tetrahymena has a unique set of desaturases including C5, C7 and C22 sterol desaturases. This set of desaturases has potential for biotechnology purposes, particularly the C7 sterol desaturase, as it may be used for decreasing cholesterol content in foodstuffs of animal origin with simultaneous enrichment in pro vitamin D3. Previous in vitro characterization of C7 and C22 cholesterol desaturases established unambiguously oxygen, cytochrome b5 and NADH requirements.  In order to identify the genes encoding for these desaturases, a somatic knockout out approach was selected. As a first attempt the sequences 51.m00243, 270.m00036 and 124.m00110 were chosen (TIGR Data base). The constructs were made by overlapping PCR, using flanking regions inserted into a neo 3 cassette, which confers paromomycin resistance, under a cadmiun inducible metallothionein (MTT) promoter. Around 20 transformants for each sequence were obtained by somatic transformation on T. thermophila CU 428 strain. After phenotypic assortment the sterol pattern of the mutants grown in different sterols were analyzed. One of the mutants (270.m00036 KO) showed the expected pattern of sterols corresponding to a KO of a C-5 sterol desaturase.  By this way the first sterol desaturase gene could be identified in ciliates.