Genome-wide engineering for synthetic auxotrophy biocontainment in yeast

Comprehensive safety measures need to be developed and put into place for the safe use of genetically engineered microorganisms for open-environment applications. Synthetic auxotrophy for non-canonical amino acids (ncAAs) can be a genetic safeguard, leveraging a translation system incorporating ncAAs at UAG codons. These stop codons are placed in the reading frame of essential genes such that in the presence of the ncAA functional essential protein is made through stop codon suppression, whereas its absence results in a truncated non-functional gene product. We have performed a large-scale screening for suitable proteins and positions for TAG codon placement to create synthetic auxotrophy in Saccharomyces cerevisiae, testing ~4000 edits across 397 essential proteins using a pooled, barcoded editing library. We determined barcode frequency changes over time when growing with and without the ncAA O-methyl-tyrosine, looking for barcodes being depleted without the ncAA, but maintained in its presence. This screening provided information about the suitability of essential genes, sites, and sequence context for this containment approach. We combined multiple TAG edits aiming to achieve stringent containment while maintaining high fitness in permissive condition, being most successful with introducing two select TAG edits in a single gene (PRP5), with an escape frequency of 2.3×10-5. Further, we layered a synthetic auxotrophy system with a single TAG edit with another containment system based on conditional essential protein stability, achieving extremely stringent containment (determined escape frequency 7.5×10-11) and nearly wild-type like growth in permissive medium. These findings lay the groundwork for reliable, high-stringency eukaryotic biocontainment systems that balance environmental safety with functional performance.