Creation of a eukaryotic multiplexed site-specific inversion system and its application for metabolic engineering

Jieyi Li, Simiao Gong, Yuan Ma, Peiyan Han, Nan Wang, Zongheng Fu, Xinyi Zhang, Xinyang Huang, Tianyu Yang, Hanze Tong, Guang-Rong Zhao, Yi Wu () and Ying-Jin Yuan
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Jieyi Li: Tianjin University
Simiao Gong: Tianjin University
Yuan Ma: Tianjin University
Peiyan Han: Tianjin University
Nan Wang: Tianjin University
Zongheng Fu: Tianjin University
Xinyi Zhang: Tianjin University
Xinyang Huang: Tianjin University
Tianyu Yang: Tianjin University
Hanze Tong: Tianjin University
Guang-Rong Zhao: Tianjin University
Yi Wu: Tianjin University
Ying-Jin Yuan: Tianjin University

Nature Communications, 2025, vol. 16, issue 1, 1-11

Abstract: Abstract The site-specific recombination system is a versatile tool in genome engineering, enabling controlled DNA inversion or deletion at specific sites to generate genetic diversity. The multiplexed inversion system, which preferentially facilitates inversion at reverse-oriented sites rather than deletion at same-oriented sites, has not been found in eukaryotes. Here, we establish a multiplexed site-specific inversion system, Rci51-5/multi-sfxa101, in yeast. Firstly, we develop a high-throughput screening system based on the on/off transcriptional control of multiple markers by DNA inversion. After two rounds of progressively stringent directed evolution, a mutant Rci51-5 shows an ability of multisite inversion and a ~ 1000-fold increase in total inversion efficiency against the wild-type Rci derived from Salmonella typhimurium. Subsequently, we demonstrate that the Rci51-5/multi-sfxa101 system exhibits significantly lower deletion rate than the Cre/multi-loxP system. Using the synthetic metabolic pathway of β-carotene as an example, we illustrate that the system can effectively facilitate promoter substitution in the metabolic pathway, resulting in a more than 7-fold increase in the yield of β-carotene. In summary, we develop a multiplexed site-specific inversion system in eukaryotes, providing an approach to metabolic engineering and a tool for eukaryotic genome manipulation.

Date: 2025
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DOI: 10.1038/s41467-025-57206-w

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