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The construction of powerful cell factories requires intensive and extensive remodelling of microbial genomes. Considering the rapidly increasing number of these synthetic biology endeavors, there is an increasing need for DNA watermarking strategies that enable the discrimination between synthetic and native gene copies. While it is well documented that codon usage can affect translation, and most likely mRNA stability in eukaryotes, remarkably few quantitative studies explore the impact of watermarking on transcription, protein expression, and physiology in the popular model and industrial yeast Saccharomyces cerevisiae . The present study, using S. cerevisiae as eukaryotic paradigm, designed, implemented, and experimentally validated a systematic strategy to watermark DNA with minimal alteration of yeast physiology. The 13 genes encoding proteins involved in the major pathway for sugar utilization ( i.e. , glycolysis and alcoholic fermentation) were simultaneously watermarked in a yeast strain using the previously published pathway swapping strategy. Carefully swapping codons of these naturally codon optimized, highly expressed genes, did not affect yeast physiology and did not alter transcript abundance, protein abundance, and protein activity besides a mild effect on Gpm1. The markerQuant bioinformatics method could reliably discriminate native from watermarked genes and transcripts. Furthermore, presence of watermarks enabled selective CRISPR/Cas genome editing, specifically targeting the native gene copy while leaving the synthetic, watermarked variant intact. This study offers a validated strategy to simply watermark genes in S. cerevisiae .

Design and Experimental Evaluation of a Minimal, Innocuous Watermarking Strategy to Distinguish Near-Identical DNA and RNA Sequences