A robust yeast biocontainment system with two-layered regulation switch dependent on unnatural amino acid
Tian-Tian Chang, Weichao Ding, Shirui Yan, Yun Wang, Haoling Zhang, Yu Zhang, Zhi Ping, Huiming Zhang, Yijian Huang, Jiahui Zhang, Dan Wang, Wenwei Zhang, Xun Xu, Yue Shen, Xian Fu
Abstract
Abstract Synthetic auxotrophy in which cell viability depends on the presence of an unnatural amino acid (unAA) provides a powerful strategy to restrict unwanted propagation of genetically modified organisms (GMOs) in open environments and potentially prevent industrial espionage. Here, we describe a generic approach for robust biocontainment of budding yeast dependent on unAA. By understanding escape mechanisms, we specifically optimize our strategies by introducing designed “immunity” to the generation of amber-suppressor tRNAs and developing the transcriptional- and translational-based biocontainment switch. We further develop a fitness-oriented screening method to easily obtain multiplex safeguard strains that exhibit robust growth and undetectable escape frequency (<~10 −9 ) on solid media for 14 days. Finally, we show that employing our multiplex safeguard system could restrict the proliferation of strains of interest in a real fermentation scenario, highlighting the great potential of our yeast biocontainment strategy to protect the industrial proprietary strains.