A deeper understanding of the spontaneous derepression of the URA3 gene in MaV203 Saccharomyces cerevisiae and its implications for protein engineering and the reverse two-hybrid system.

Within the field of protein-based biomaterials, the need exists for both covalent and oriented bioconjugation strategies for improved performance. Such bioconjugation reactions can be facilitated by engineering proteins with chemically activated amino acids at strategically chosen sites. The incorporation of these unnatural amino acids (uAAs) can be achieved by using the nonsense suppression technique. This requires an aminoacyl-tRNA-synthetase (aaRS) that exclusively recognizes the uAA and loads it to the corresponding tRNA. Appropriate (aaRS) mutants can be found through reverse engineering using the Saccharomyces cerevisiae strain MaV203. This strain contains a counterselectable, Gal4p-inducible SPAL10::URA3 fusion and deletions in the endogenous GAL80 and GAL4 genes. Therefore, it has been used extensively for the screening of aaRS mutant libraries. It is generally assumed that the SPAL10 promoter actively represses the URA3 gene in the absence of Gal4p, resulting in MaV203 cells with a Ura- phenotype. The current contribution reveals that in a small fraction of MaV203 cells, a basal expression of the URA3 gene occurs. The unexpected URA3 expression is reported for the first time, and the nature of the mutation causing this expression was identified as a spontaneous recessive mutation in a single gene of a protein involved in the repression of the SPAL10 promoter. The basal URA3 expression causes aaRS mutants to be missed, which affects the outcome of the library screening. It is demonstrated that the use of diploid cells can circumvent the MaV203 Ura+ phenotype, allowing for an optimization of S. cerevisiae library screening.