Sunita Chaudhary

Cloning of Δ12- and Δ6-desaturases from Mortierella alpina and recombinant production of γ-linolenic acid in Saccharomyces cerevisiae

Two cDNA clones with homology to known desaturase genes were isolated from the fungus Mortierella alpina. The open reading frame in one clone encoded 399 amino acids and exhibited Δ12-desaturase activity when expressed in Saccharomyces cerevisiae in the presence of endogenous fatty acid substrate oleic acid. The insert in another clone contained an open reading frame encoding 457 amino acids and exhibited Δ6-desaturase activity in S. cerevisiae in the presence of exogenous fatty acid substrate linoleic acid. Expression of the Δ12-desaturase gene under appropriate media and temperature conditions led to the production of linoleic acid at levels up to 25% of the total fatty acids in yeast. When linoleic acid was provided as an exogenous substrate to the yeast cultures expressing the Δ6-desaturase activity, the level of γ-linolenic acid reached 10% of the total yeast fatty acids. Co-expression of both the Δ6- and Δ12-desaturase cDNA resulted in the endogenous production of γ-linolenic acid. The yields of γ-linolenic acid reached as high as 8% of total fatty acids in yeast.

Identification of delta5-desaturase from Mortierella alpina by heterologous expression in bakers' yeast and canola

A DNA fragment with homology to Δ6-desaturases from borage and cyanobacteria was isolated after polymerase chain reaction amplification of Mortierella alpina cDNA with oligonucleotide primers corresponding to the conserved regions of known Δ6-desaturase genes. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 446 amino acids from a M. alpina library. Expression of this open reading frame from an inducible promoter in Saccharomyces cerevisiae in the presence of various substrates revealed that the recombinant product had Δ5-desaturase activity. The effects of growth and induction conditions as well as host strain on activity of the recombinant Δ5-desaturase in S. cerevisiae were evaluated. Expression of the M. alpina Δ5-desaturase cDNA in transgenic canola seeds resulted in the production of taxoleic acid (Δ5,9–18:2) and pinolenic acid (Δ5,9,12–18:3), which are the Δ5-desaturation products of oleic and linoleic acids, respectively.

Distinct TFIID complexes mediate the effect of different transcriptional activators.

Multiple chromatographically separable complexes containing the TATA binding protein (TBP), which exhibit different functional properties, exist in HeLa cells. At least three distinct subpopulations of such complexes can be functionally defined as TFIID since they function with RNA polymerase II. Using a partially reconstituted HeLa cell in vitro transcription system and immunoprecipitation with a monoclonal antibody directed against TBP, we show that stimulation of transcription by the chimeric activators GAL‐VP16, GAL‐TEF‐1 and GAL‐ER(EF) requires the presence of factors which are tightly associated with these TFIID complexes. Moreover, the activity of GAL‐TEF‐1 appears to be mediated by at least two chromatographically distinct populations of TFIID. The factor(s) associated with one of these populations is also required for the activity of GAL‐ER (EF) and GAL‐VP16, while the factor(s) associated with the other population functions selectively with GAL‐TEF‐1. These two TFIID populations are composed of both common and unique TBP associated factors (TAFs).

Genetic Engineering of Seed Oil Fatty Acid Composition

Polyunsaturated fatty acids (PUFAs) are important components of infant as well as adult nutrition due to their roles as both structural elements of cell membranes and as precursors to eicosanoids, a family of potent biological effectors such as prostaglandins and prostacyclins. PUFAs have been reported to be of clinical significance in fetal growth and development, cardiovascular disease, skin disease and certain inflammatory conditions (Das et al 1988). There are two main families of PUFAs, n−6 and n−3, in which the n-designation refers to the position of the last double bond relative to the methyl end of the molecule. The n−6 and n−3 PUFAs are derived from linoleic acid (LA; 18∶2n−6) or alpha-linolenic acid (18∶3n−3), respectively, via an alternating series of desaturation and two-carbon elongation steps. The same enzymes are believed to catalyze reactions in both pathways although this has not been shown for most systems. In the n−6 pathway, γ-linolenic acid (GLA; 18∶3n−6) is formed from LA by the action of a Δ6-desaturase. GLA is elongated to produce dihomo-γ-linolenic acid (DGLA; 20∶3n−6), which then undergoes Δ5-desaturation to produce arachidonic acid (ARA; 20∶4n−6). A similar series of reactions in the n−3 pathway results in the production of eicosapentaenoic acid (EPA; 20∶5n−3).