Abstract
Unique chemical and physical properties are introduced by inserting selenocysteine (Sec) at specific sites within proteins. Recombinant and facile production of eukaryotic selenoproteins would benefit from a yeast expression system; however, the selenoprotein biosynthetic pathway was lost in the evolution of the kingdom Fungi as it diverged from its eukaryotic relatives. Based on our previous development of efficient selenoprotein production in bacteria, we designed a novel Sec biosynthesis pathway in Saccharomyces cerevisiae using Aeromonas salmonicida translation components. S. cerevisiae tRNASer was mutated to resemble A. salmonicida tRNASec to allow recognition by S. cerevisiae seryl-tRNA synthetase as well as A. salmonicida selenocysteine synthase (SelA) and selenophosphate synthetase (SelD). Expression of these Sec pathway components was then combined with metabolic engineering of yeast to enable the production of active methionine sulfate reductase enzyme containing genetically encoded Sec. Our report is the first demonstration that yeast is capable of selenoprotein production by site-specific incorporation of Sec.
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