Design and experimental evaluation of a minimal, innocuous watermarking strategy to distinguish near-identical DNA and RNA sequences

The construction of powerful cell factories requires intensive and extensive remodelling of microbial genomes. Considering the rapidly increasing number of these synthetic biology endeavours, 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 thirteen 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 alter transcript abundance, protein activity and yeast physiology. 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.构建高性能细胞工厂需要对微生物基因组进行深度且广泛的重塑。随着这类合成生物学研究项目的数量快速增长，对能够区分合成基因拷贝与天然基因拷贝的DNA水印策略的需求日益迫切。尽管已有充分文献证实密码子使用偏好会影响翻译过程，且在真核生物中大概率影响mRNA稳定性，但目前鲜有定量研究探讨水印对热门模式生物与工业酿酒酵母（Saccharomyces cerevisiae）的转录、蛋白质表达及生理状态的影响。本研究以酿酒酵母（Saccharomyces cerevisiae）作为真核生物研究范式，设计、实施并通过实验验证了一套系统性DNA水印策略，该策略可在最小程度改变酵母生理状态的前提下完成DNA水印。研究团队采用此前发表的通路置换策略，在一株酿酒酵母菌株中同时对参与糖利用主要通路（即糖酵解与酒精发酵）的13个编码蛋白基因进行了水印处理。对这些天然经过密码子优化的高表达基因的密码子进行精准置换后，并未改变转录本丰度、蛋白质活性以及酵母的生理状态。markerQuant生物信息学方法可可靠地区分天然基因与水印基因及其转录本。此外，水印的存在使得可通过CRISPR/Cas基因组编辑选择性靶向天然基因拷贝，同时保留合成的水印变体不受影响。本研究提供了一套经过验证的、可用于酿酒酵母基因简单水印的策略。