Directed Evolution of Selenite Tolerance in Escherichia coli

Selenium is a vital micromineral that must be consumed through diet and is essential to physiological processes in the human body and other organismal functions. Selenium is usually found in organisms as selenocysteine (Sec), the 21st genetically encoded amino acid. Sec is unique in that it is incorporated into proteins in response to the stop codon, UGA, which normally signals for the termination of protein synthesis. While Sec is essential for proper cell function, excess Sec results in misincorporation of Sec at cysteine (Cys) positions during protein synthesis and lead to toxicity. This misincorporation of Sec at Cys codons during translation is caused by the inability of cysteinyl-tRNA synthetase to distinguish between these two amino acids. Recent data has shown that a single amino acid variant present in the active site of cysteinyl-tRNA synthetase from selenium accumulating plants, reduces the toxic effects of selenite when introduced into the E. coli cysteinyl-tRNA synthetase. Although this mutant E. coli enzyme reduces the incorporation of Sec at Cys codons, it is still able to utilize Sec at a low level. To overcome the limitation and to gain insight into the mechanism of cysteinyl-tRNA synthetase specificity, we performed directed evolution experiments in E. coli to increase tolerance to selenite.