Exogenous and endogenous optimization for efficient nonconical amino acids incorporation in Saccharomyces cerevisiae

Most therapeutic proteins and enzymes with precise noncanonical amino acids (ncAAs) modifications can only be expressed in eukaryotic systems, positioning Saccharomyces cerevisiae as a promising chassis cell. However, ncAA incorporation in S. cerevisiae is hindered by low efficiency and complex transcriptional regulation. In this study, we optimized exogenous factors by increasing the tRNALeu CUA copy from 1x to 2x and raising the OMeY concentration from 1 mM to 5 mM while reducing the cultivation time from 48 h to 24 h. This optimization enhanced the fluorescence intensity (FI) of EGFP_40UAG(Tyr40OMeY) from 3.17 % to 105.82 % of wild-type EGFP. Using SCRaMbLE-induced structural variations (SVs) in the synV genome, we identified two strains, yWJR104 and yWJR105, with improved OMeY incorporation efficiencies. These strains achieved FIs of 129.52 % and 125 % of wild-type EGFP, respectively, corresponding to 2.21-fold and 2.13-fold increases relative to the parental strain. Notably, this optimization revealed two novel genomic targets, YEL014C and YEL013W, whose deletion led to FIs of 114.52 % and 137.6 % of wild-type EGFP, respectively. Transcriptomic analysis suggested that deletion of YEL014C may enhance [PSI⁺] prion formation, which further improved ncAA incorporation efficiency. This study provides insights into ncAA regulatory mechanisms of S. cerevisiae, opening new avenues for optimizing ncAA incorporation in yeast.