IDEAS home Printed from https://ideas.repec.org/a/nat/natcom/v16y2025i1d10.1038_s41467-025-57206-w.html
   My bibliography  Save this article

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

Author

Listed:
  • Jieyi Li

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Simiao Gong

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Yuan Ma

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Peiyan Han

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Nan Wang

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Zongheng Fu

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Xinyi Zhang

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Xinyang Huang

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Tianyu Yang

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Hanze Tong

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Guang-Rong Zhao

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Yi Wu

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Ying-Jin Yuan

    (Tianjin University
    Tianjin University
    Tianjin University)

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.

Suggested Citation

  • 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 & Ying-Jin Yuan, 2025. "Creation of a eukaryotic multiplexed site-specific inversion system and its application for metabolic engineering," Nature Communications, Nature, vol. 16(1), pages 1-11, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-57206-w
    DOI: 10.1038/s41467-025-57206-w
    as

    Download full text from publisher

    File URL: https://www.nature.com/articles/s41467-025-57206-w
    File Function: Abstract
    Download Restriction: no

    File URL: https://libkey.io/10.1038/s41467-025-57206-w?utm_source=ideas
    LibKey link: if access is restricted and if your library uses this service, LibKey will redirect you to where you can use your library subscription to access this item
    ---><---

    References listed on IDEAS

    as
    1. Weike Pei & Thorsten B. Feyerabend & Jens Rössler & Xi Wang & Daniel Postrach & Katrin Busch & Immanuel Rode & Kay Klapproth & Nikolaus Dietlein & Claudia Quedenau & Wei Chen & Sascha Sauer & Stephan , 2017. "Polylox barcoding reveals haematopoietic stem cell fates realized in vivo," Nature, Nature, vol. 548(7668), pages 456-460, August.
    2. Jiazhang Lian & Mohammad HamediRad & Sumeng Hu & Huimin Zhao, 2017. "Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system," Nature Communications, Nature, vol. 8(1), pages 1-9, December.
    3. Josh Abramson & Jonas Adler & Jack Dunger & Richard Evans & Tim Green & Alexander Pritzel & Olaf Ronneberger & Lindsay Willmore & Andrew J. Ballard & Joshua Bambrick & Sebastian W. Bodenstein & David , 2024. "Addendum: Accurate structure prediction of biomolecular interactions with AlphaFold 3," Nature, Nature, vol. 636(8042), pages 4-4, December.
    4. Jie Zhang & Lea G. Hansen & Olga Gudich & Konrad Viehrig & Lærke M. M. Lassen & Lars Schrübbers & Khem B. Adhikari & Paulina Rubaszka & Elena Carrasquer-Alvarez & Ling Chen & Vasil D’Ambrosio & Beata , 2022. "A microbial supply chain for production of the anti-cancer drug vinblastine," Nature, Nature, vol. 609(7926), pages 341-347, September.
    5. Juan Wang & Ze-Xiong Xie & Yuan Ma & Xiang-Rong Chen & Yao-Qing Huang & Bo He & Jia & Bing-Zhi Li & Ying-Jin Yuan, 2018. "Ring synthetic chromosome V SCRaMbLE," Nature Communications, Nature, vol. 9(1), pages 1-9, December.
    6. Josh Abramson & Jonas Adler & Jack Dunger & Richard Evans & Tim Green & Alexander Pritzel & Olaf Ronneberger & Lindsay Willmore & Andrew J. Ballard & Joshua Bambrick & Sebastian W. Bodenstein & David , 2024. "Accurate structure prediction of biomolecular interactions with AlphaFold 3," Nature, Nature, vol. 630(8016), pages 493-500, June.
    7. Yu Wang & Haijiao Cheng & Yang Liu & Ye Liu & Xiao Wen & Kun Zhang & Xiaomeng Ni & Ning Gao & Liwen Fan & Zhihui Zhang & Jiao Liu & Jiuzhou Chen & Lixian Wang & Yanmei Guo & Ping Zheng & Meng Wang & J, 2021. "In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
    8. Tackhoon Kim & Benjamin Weinberg & Wilson Wong & Timothy K. Lu, 2021. "Scalable recombinase-based gene expression cascades," Nature Communications, Nature, vol. 12(1), pages 1-9, December.
    9. Charlotte Cautereels & Jolien Smets & Peter Bircham & Dries De Ruysscher & Anna Zimmermann & Peter De Rijk & Jan Steensels & Anton Gorkovskiy & Joleen Masschelein & Kevin J. Verstrepen, 2024. "Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    Full references (including those not matched with items on IDEAS)

    Most related items

    These are the items that most often cite the same works as this one and are cited by the same works as this one.
    1. Charlotte Cautereels & Jolien Smets & Peter Bircham & Dries De Ruysscher & Anna Zimmermann & Peter De Rijk & Jan Steensels & Anton Gorkovskiy & Joleen Masschelein & Kevin J. Verstrepen, 2024. "Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    2. Reilly Pidgeon & Sacha Mitchell & Michael Shamash & Layan Suleiman & Lharbi Dridi & Corinne F. Maurice & Bastien Castagner, 2025. "Diet-derived urolithin A is produced by a dehydroxylase encoded by human gut Enterocloster species," Nature Communications, Nature, vol. 16(1), pages 1-18, December.
    3. Luc Provencher & Wilson Nartey & Peter M. Brownlee & Austin W. Atkins & Jean-Philippe Gagné & Lou Baudrier & Nicholas S. Y. Ting & Cortt G. Piett & Shujuan Fang & Dustin D. Pearson & Shaun Moore & Pie, 2025. "CHD6 has poly(ADP-ribose)- and DNA-binding domains and regulates PARP1/2-trapping inhibitor sensitivity via abasic site repair," Nature Communications, Nature, vol. 16(1), pages 1-24, December.
    4. Z. Faidon Brotzakis & Shengyu Zhang & Mhd Hussein Murtada & Michele Vendruscolo, 2025. "AlphaFold prediction of structural ensembles of disordered proteins," Nature Communications, Nature, vol. 16(1), pages 1-9, December.
    5. Wen Yang & Man Feng & Kuohai Yu & Jie Cao & Guangxian Cui & Yumei Zhang & Huiru Peng & Yingyin Yao & Zhaorong Hu & Zhongfu Ni & Feng Qin & Fuminori Takahashi & Qixin Sun & Mingming Xin, 2025. "The TaCLE24b peptide signaling cascade modulates lateral root development and drought tolerance in wheat," Nature Communications, Nature, vol. 16(1), pages 1-12, December.
    6. Bing Wang & Nelly Said & Tarek Hilal & Mark Finazzo & Markus C. Wahl & Irina Artsimovitch, 2025. "Nucleotide-induced hyper-oligomerization inactivates transcription termination factor ρ," Nature Communications, Nature, vol. 16(1), pages 1-16, December.
    7. Sophia Vincoff & Shrey Goel & Kseniia Kholina & Rishab Pulugurta & Pranay Vure & Pranam Chatterjee, 2025. "FusOn-pLM: a fusion oncoprotein-specific language model via adjusted rate masking," Nature Communications, Nature, vol. 16(1), pages 1-11, December.
    8. Yue Peng & Junze Wu & Yi Sun & Yuanxing Zhang & Qiyao Wang & Shuai Shao, 2025. "Contrastive-learning of language embedding and biological features for cross modality encoding and effector prediction," Nature Communications, Nature, vol. 16(1), pages 1-20, December.
    9. Yuanwei Gou & Dongfang Li & Minghui Zhao & Mengxin Li & Jiaojiao Zhang & Yilian Zhou & Feng Xiao & Gaofei Liu & Haote Ding & Chenfan Sun & Cuifang Ye & Chang Dong & Jucan Gao & Di Gao & Zehua Bao & Le, 2024. "Intein-mediated temperature control for complete biosynthesis of sanguinarine and its halogenated derivatives in yeast," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
    10. Itxaso Anso & Samira Zouhir & Thibault Géry Sana & Petya Violinova Krasteva, 2024. "Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
    11. Gita Naseri, 2023. "A roadmap to establish a comprehensive platform for sustainable manufacturing of natural products in yeast," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    12. Huiming Zhang & Xian Fu & Xuemei Gong & Yun Wang & Haolin Zhang & Yu Zhao & Yue Shen, 2022. "Systematic dissection of key factors governing recombination outcomes by GCE-SCRaMbLE," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    13. William M. Shaw & Lucie Studená & Kyler Roy & Piotr Hapeta & Nicholas S. McCarty & Alicia E. Graham & Tom Ellis & Rodrigo Ledesma-Amaro, 2022. "Inducible expression of large gRNA arrays for multiplexed CRISPRai applications," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    14. Paloma García Casas & Michela Rossini & Linnea Påvénius & Mezida Saeed & Nikita Arnst & Sonia Sonda & Tânia Fernandes & Irene D’Arsiè & Matteo Bruzzone & Valeria Berno & Andrea Raimondi & Maria Livia , 2024. "Simultaneous detection of membrane contact dynamics and associated Ca2+ signals by reversible chemogenetic reporters," Nature Communications, Nature, vol. 15(1), pages 1-21, December.
    15. Jidong Fei & Dongdong Zhao & Caiyi Pang & Ju Li & Siwei Li & Wentao Qiao & Juan Tan & Changhao Bi & Xueli Zhang, 2025. "Mismatch prime editing gRNA increased efficiency and reduced indels," Nature Communications, Nature, vol. 16(1), pages 1-10, December.
    16. Huiyu Cai & Zuobai Zhang & Mingkai Wang & Bozitao Zhong & Quanxiao Li & Yuxuan Zhong & Yanling Wu & Tianlei Ying & Jian Tang, 2024. "Pretrainable geometric graph neural network for antibody affinity maturation," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    17. Anete Romanauska & Edvinas Stankunas & Maya Schuldiner & Alwin Köhler, 2024. "Seipin governs phosphatidic acid homeostasis at the inner nuclear membrane," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    18. Jin Wang & Ning Xue & Wenjia Pan & Ran Tu & Shixin Li & Yue Zhang & Yufeng Mao & Ye Liu & Haijiao Cheng & Yanmei Guo & Wei Yuan & Xiaomeng Ni & Meng Wang, 2023. "Repurposing conformational changes in ANL superfamily enzymes to rapidly generate biosensors for organic and amino acids," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    19. Nathalie Béchon & Nitzan Tal & Avigail Stokar-Avihail & Alon Savidor & Meital Kupervaser & Sarah Melamed & Gil Amitai & Rotem Sorek, 2024. "Diversification of molecular pattern recognition in bacterial NLR-like proteins," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
    20. Jun Guo & Di Gao & Jiazhang Lian & Yang Qu, 2024. "De novo biosynthesis of antiarrhythmic alkaloid ajmaline," Nature Communications, Nature, vol. 15(1), pages 1-12, December.

    More about this item

    Statistics

    Access and download statistics

    Corrections

    All material on this site has been provided by the respective publishers and authors. You can help correct errors and omissions. When requesting a correction, please mention this item's handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-57206-w. See general information about how to correct material in RePEc.

    If you have authored this item and are not yet registered with RePEc, we encourage you to do it here. This allows to link your profile to this item. It also allows you to accept potential citations to this item that we are uncertain about.

    If CitEc recognized a bibliographic reference but did not link an item in RePEc to it, you can help with this form .

    If you know of missing items citing this one, you can help us creating those links by adding the relevant references in the same way as above, for each refering item. If you are a registered author of this item, you may also want to check the "citations" tab in your RePEc Author Service profile, as there may be some citations waiting for confirmation.

    For technical questions regarding this item, or to correct its authors, title, abstract, bibliographic or download information, contact: Sonal Shukla or Springer Nature Abstracting and Indexing (email available below). General contact details of provider: http://www.nature.com .

    Please note that corrections may take a couple of weeks to filter through the various RePEc services.

    IDEAS is a RePEc service. RePEc uses bibliographic data supplied by the respective publishers.