Modification of plant and yeast lipids by heterologous expression of protist, algal, and animal desaturases

Modification of lipids by expression of transgenes to alter levels of endogenous fatty acids, and/or to produce novel fatty acids has long been a goal of plant researchers. In this attempt, the identification of desaturases and other fatty acid modification enzymes that can act in heterologous systems was undertaken. Tetrahymena species Tetrahymena pyriformis and Tetrahymena thermophila have three endogenous fatty acid desaturases, a Delta6, a Delta9, and a Delta12. To examine the activity of Tetrahymena enzymes, Delta6 and Delta9 desaturases were cloned from a T. thermophila cDNA library and modified for expression in heterologous systems. Alignment of these proteins with known desaturases revealed homology seen in conserved domains of these proteins. Desaturation was not seen with the Delta9 desaturase and limited desaturation was seen with the Delta6. However, desaturation was increased 10-fold when the Delta6 desaturase was co-expressed with a cytochrome b5 from Arabidopsis thaliana. Further analysis of the desaturases of Tetrahymena involved the cloning of the Delta12 desaturase homologs of both T. pyriformis and T. thermophila which could account for the Delta14 desaturation of 20:1(11) to 20:2(11,14) seen in Tetrahymena. Following the cloning of these desaturase cDNAs, the codons were altered to allow for expression in three heterologous expression systems. Desaturation was not seen in any of the systems until the codons were optimized and a small amount of Delta12 desaturation was seen in yeast. With the overall goal of modification of plant lipids by expression of desaturases two other cDNAs were introduced into Arabidopsis, a Delta8 from E. gracilis, and a Delta9 from C. elegans. Expression of the Delta8 with a seed-specific promoter resulted in the desaturation of 20:1(11) to 20:2(8,11) in Arabidopsis seeds. The Delta9 from C. elegans was introduced into Arabidopsis seeds plants to reduce the amount of 16:0 saturated fatty acid. Expression of this desaturase with a seed-specific promoter resulted in a reduction of 16:0 from 10% to around 3%. Overall this project highlights the issue that can arise from heterologous expression of desaturases but also the results that can be obtained in alteration of endogenous fatty acids by desaturase expression.