Expanding the genetic code of Saccharomyces cerevisiae with methionine analogues.

We replaced the single N-terminal methionine in heterologously expressed human Cu/Zn superoxide dismutase with the non-canonical methionine analogues homopropargylglycine and norleucine in the yeast Saccharomyces cerevisiae. Our non-canonical amino acid incorporation protocol involves a two-step procedure. In the first step, the methionine auxotrophic yeast cells are accumulated in synthetic medium containing methionine while the target protein production is shut off. After a short methionine depletion phase, the cells are transferred to inducing medium that contains the methionine analogue instead of methionine and target protein expression is switched on. The initially low level incorporation of approximately 12% could be elevated to 40% by increasing the non-canonical amino acid concentration in the medium by 10-fold. With this approach we were able to produce up to 5 mg substituted protein per litre of yeast culture.