|O'Quin, Jami - U OF NEW ORLEANS, LA|
|Mullen, Robert - U OF GUELPH, ONTARIO, CAN|
Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 1, 2008
Publication Date: January 10, 2009
Citation: O'Quin, J.B., Mullen, R.T., Dyer, J.M. 2009. Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells. Applied Microbiology and Biotechnology. 83:117-125. Interpretive Summary: Omega-3 fatty acids are important components of human nutrition that are essential for proper functioning of the human brain and eyes, and for the proper development of newborn babies. They are also used in some industrial applications such as drying agents in inks, dyes, coatings and resins. There is significant interest in developing new and sustainable sources of omega-3 fatty acids, and one possibility is to produce these types of valuable fatty acids in a microbe such as common baker’s yeast. Plants are known to contain many of the genes involved in production of omega-3 fatty acids, but expression of plant genes in yeast cells can be difficult, and accumulation of the desired omega-3 fatty acids is generally low. Here we describe a method that can be used to increase the amounts of fatty acids by up to 4-fold. The method involves a simple manipulation of the plant gene sequence that allows the plant gene to function much more efficiently in yeasts. The method was able to increase the amounts of fatty acids produced by three different plant genes expressed in yeast, demonstrating that the technique is broadly applicable. This technology will be valuable not only to those scientists who are interested in studying the functional aspects of plant genes, but also to those who are interested in developing microbial sources of high value omega-3 fatty acid products.
Technical Abstract: Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared towards the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically-expressed enzymes. Here we show that the addition of an N-terminal epitope tag sequence (either Myc or hemagglutinin) to oleate desaturase (FAD2) or omega-3 linoleate desaturase (FAD3) enzymes from plants, which catalyze consecutive reactions in the production of long chain omega-3 fatty acids, significantly increases their activity up to 4-fold when expressed in yeast cells. Quantitative protein blotting using an antibody specific for native FAD2 revealed that the steady-state amount of the epitope-tagged FAD2 protein was also approximately 4-fold higher than that of its untagged counterpart, demonstrating a direct relationship between the epitope tag-induced increase in enzyme amount and fatty acid product formation. Protein half-life and RNA blotting experiments indicated that the half-lives and mRNA content of the tagged and untagged FAD2 proteins were essentially the same, suggesting that the epitope tags increased protein abundance by improving translational efficiency. Taken together, these results indicate that the addition of an epitope tag sequence to a plant FAD not only provides a useful means for protein immunodetection using highly-specific, commercially-available antibodies, but that it also significantly increases FAD activity and the production of polyunsaturated fatty acids in yeast cells.