Nature Communications (Oct 2023)

A robust yeast biocontainment system with two-layered regulation switch dependent on unnatural amino acid

  • Tiantian 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

DOI
https://doi.org/10.1038/s41467-023-42358-4
Journal volume & issue
Vol. 14, no. 1
pp. 1 – 13

Abstract

Read online

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.