IDEAS home Printed from https://ideas.repec.org/a/nat/natcom/v16y2025i1d10.1038_s41467-025-62886-5.html
   My bibliography  Save this article

Matrix regulation: a plug-and-tune method for combinatorial regulation in Saccharomyces cerevisiae

Author

Listed:
  • Xiaolong Teng

    (Beijing University of Chemical Technology)

  • Zibai Wang

    (Beijing University of Chemical Technology)

  • Yueping Zhang

    (China Agricultural University)

  • Binhao Wang

    (Beijing University of Chemical Technology)

  • Guiping Gong

    (Beijing University of Chemical Technology)

  • Jinmiao Hu

    (Beijing University of Chemical Technology)

  • Yifan Zhu

    (Beijing University of Chemical Technology)

  • Baoyi Peng

    (Beijing University of Chemical Technology)

  • Junyang Wang

    (Beijing University of Chemical Technology)

  • James Chen

    (Beijing University of Chemical Technology)

  • Shuobo Shi

    (Beijing University of Chemical Technology)

  • Jens Nielsen

    (Beijing University of Chemical Technology
    Chalmers University of Technology
    BioInnovation Institute)

  • Zihe Liu

    (Beijing University of Chemical Technology)

Abstract

Transcriptional fine-tuning of long pathways is complex, even in the extensively applied cell factory Saccharomyces cerevisiae. Here, we present Matrix Regulation (MR), a CRISPR-mediated pathway fine-tuning method enabling the construction of 68 gRNA combinations and screening for the optimal expression levels across up to eight genes. We first identify multiple tRNAs with efficient gRNA processing capacities to assemble a gRNA regulatory matrix combinatorially. Then, we expand the target recognition of CRISPR regulation from NGG PAM to NG PAM by characterizing dCas9 variants. To increase the dynamic range of modulation, we test 101 candidate activation domains followed by mutagenesis and screening the best one to further enhance its activation capability in S. cerevisiae by 3-fold. The regulations generate combinatorial strain libraries for both the mevalonate pathway and the heme biosynthesis pathway and increase squalene production by 37-fold and heme by 17-fold, respectively, demonstrating the versatility of our method and its applicability in fundamental research.

Suggested Citation

  • Xiaolong Teng & Zibai Wang & Yueping Zhang & Binhao Wang & Guiping Gong & Jinmiao Hu & Yifan Zhu & Baoyi Peng & Junyang Wang & James Chen & Shuobo Shi & Jens Nielsen & Zihe Liu, 2025. "Matrix regulation: a plug-and-tune method for combinatorial regulation in Saccharomyces cerevisiae," Nature Communications, Nature, vol. 16(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-62886-5
    DOI: 10.1038/s41467-025-62886-5
    as

    Download full text from publisher

    File URL: https://www.nature.com/articles/s41467-025-62886-5
    File Function: Abstract
    Download Restriction: no
    File URL: https://libkey.io/10.1038/s41467-025-62886-5?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. Johnny H. Hu & Shannon M. Miller & Maarten H. Geurts & Weixin Tang & Liwei Chen & Ning Sun & Christina M. Zeina & Xue Gao & Holly A. Rees & Zhi Lin & David R. Liu, 2018. "Evolved Cas9 variants with broad PAM compatibility and high DNA specificity," Nature, Nature, vol. 556(7699), pages 57-63, April.
    2. 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.
    3. 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.
    4. Gita Naseri & Jessica Behrend & Lisa Rieper & Bernd Mueller-Roeber, 2019. "COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors," Nature Communications, Nature, vol. 10(1), pages 1-18, December.
    5. Chenrui Qin & Yanhui Xiang & Jie Liu & Ruilin Zhang & Ziming Liu & Tingting Li & Zhi Sun & Xiaoyi Ouyang & Yeqing Zong & Haoqian M. Zhang & Qi Ouyang & Long Qian & Chunbo Lou, 2023. "Precise programming of multigene expression stoichiometry in mammalian cells by a modular and programmable transcriptional system," Nature Communications, Nature, vol. 14(1), pages 1-10, December.
    6. Jiao Liu & Moshi Liu & Tuo Shi & Guannan Sun & Ning Gao & Xiaojia Zhao & Xuan Guo & Xiaomeng Ni & Qianqian Yuan & Jinhui Feng & Zhemin Liu & Yanmei Guo & Jiuzhou Chen & Yu Wang & Ping Zheng & Jibin Su, 2022. "CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an l-proline exporter for l-proline hyperproduction," Nature Communications, Nature, vol. 13(1), pages 1-16, December.
    7. Yueping Zhang & Juan Wang & Zibai Wang & Yiming Zhang & Shuobo Shi & Jens Nielsen & Zihe Liu, 2019. "A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae," Nature Communications, Nature, vol. 10(1), pages 1-10, December.
    8. Carlos Barajas & Hsin-Ho Huang & Jesse Gibson & Luis Sandoval & Domitilla Vecchio, 2022. "Feedforward growth rate control mitigates gene activation burden," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    9. Joy L. Nishikawa & Andras Boeszoermenyi & Luis A. Vale-Silva & Riccardo Torelli & Brunella Posteraro & Yoo-Jin Sohn & Fei Ji & Vladimir Gelev & Dominique Sanglard & Maurizio Sanguinetti & Ruslan I. Sa, 2016. "Inhibiting fungal multidrug resistance by disrupting an activator–Mediator interaction," Nature, Nature, vol. 530(7591), pages 485-489, February.
    10. Won-Hee Ryu & Forrest S. Gittleson & Julianne M. Thomsen & Jinyang Li & Mark J. Schwab & Gary W. Brudvig & André D. Taylor, 2016. "Heme biomolecule as redox mediator and oxygen shuttle for efficient charging of lithium-oxygen batteries," Nature Communications, Nature, vol. 7(1), pages 1-10, December.
    11. 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.
    12. 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. 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.
    3. 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.
    4. Ci Kong & Yin Yang & Tiancong Qi & Shuyi Zhang, 2025. "Predictive genetic circuit design for phenotype reprogramming in plants," Nature Communications, Nature, vol. 16(1), pages 1-14, December.
    5. Fang Liang & Yu Zhang & Lin Li & Yexin Yang & Ji-Feng Fei & Yanmei Liu & Wei Qin, 2022. "SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    6. 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.
    7. Roberto Di Blasi & Mara Pisani & Fabiana Tedeschi & Masue M. Marbiah & Karen Polizzi & Simone Furini & Velia Siciliano & Francesca Ceroni, 2023. "Resource-aware construct design in mammalian cells," Nature Communications, Nature, vol. 14(1), pages 1-10, December.
    8. Zhaohui Zhong & Guanqing Liu & Zhongjie Tang & Shuyue Xiang & Liang Yang & Lan Huang & Yao He & Tingting Fan & Shishi Liu & Xuelian Zheng & Tao Zhang & Yiping Qi & Jian Huang & Yong Zhang, 2023. "Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    9. Jian Wang & Yuxi Teng & Ruihua Zhang & Yifei Wu & Lei Lou & Yusong Zou & Michelle Li & Zhong-Ru Xie & Yajun Yan, 2021. "Engineering a PAM-flexible SpdCas9 variant as a universal gene repressor," Nature Communications, Nature, vol. 12(1), pages 1-10, December.
    10. 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.
    11. Marion Rosello & Malo Serafini & Luca Mignani & Dario Finazzi & Carine Giovannangeli & Marina C. Mione & Jean-Paul Concordet & Filippo Del Bene, 2022. "Disease modeling by efficient genome editing using a near PAM-less base editor in vivo," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    12. Péter István Kulcsár & András Tálas & Zoltán Ligeti & Eszter Tóth & Zsófia Rakvács & Zsuzsa Bartos & Sarah Laura Krausz & Ágnes Welker & Vanessza Laura Végi & Krisztina Huszár & Ervin Welker, 2023. "A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets," Nature Communications, Nature, vol. 14(1), pages 1-20, December.
    13. Xinyu Lu & Klaudia Ciurkot & Glen-Oliver F. Gowers & William M. Shaw & Tom Ellis, 2025. "Iterative SCRaMbLE for engineering synthetic genome modules and chromosomes," Nature Communications, Nature, vol. 16(1), pages 1-15, December.
    14. Giulia I. Corsi & Kunli Qu & Ferhat Alkan & Xiaoguang Pan & Yonglun Luo & Jan Gorodkin, 2022. "CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    15. Angelo Miskalis & Shraddha Shirguppe & Jackson Winter & Gianna Elias & Devyani Swami & Ananthan Nambiar & Michelle Stilger & Wendy S. Woods & Nicholas Gosstola & Michael Gapinske & Alejandra Zeballos , 2024. "SPLICER: a highly efficient base editing toolbox that enables in vivo therapeutic exon skipping," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    16. Zeyu Lu & Lingtian Zhang & Qing Mu & Junyang Liu & Yu Chen & Haoyuan Wang & Yanjun Zhang & Rui Su & Ruijun Wang & Zhiying Wang & Qi Lv & Zhihong Liu & Jiasen Liu & Yunhua Li & Yanhong Zhao, 2024. "Progress in Research and Prospects for Application of Precision Gene-Editing Technology Based on CRISPR–Cas9 in the Genetic Improvement of Sheep and Goats," Agriculture, MDPI, vol. 14(3), pages 1-17, March.
    17. Noor Radde & Genevieve A. Mortensen & Diya Bhat & Shireen Shah & Joseph J. Clements & Sean P. Leonard & Matthew J. McGuffie & Dennis M. Mishler & Jeffrey E. Barrick, 2024. "Measuring the burden of hundreds of BioBricks defines an evolutionary limit on constructability in synthetic biology," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    18. Dawn G. L. Thean & Hoi Yee Chu & John H. C. Fong & Becky K. C. Chan & Peng Zhou & Cynthia C. S. Kwok & Yee Man Chan & Silvia Y. L. Mak & Gigi C. G. Choi & Joshua W. K. Ho & Zongli Zheng & Alan S. L. W, 2022. "Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    19. Jian Wang & Ke Wang & Zhe Deng & Zhiyu Zhong & Guo Sun & Qing Mei & Fuling Zhou & Zixin Deng & Yuhui Sun, 2024. "Engineered cytosine base editor enabling broad-scope and high-fidelity gene editing in Streptomyces," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    20. Ju Yeon Chung & Yoon-Kyoung Hong & Eunhee Jeon & Seungju Yang & Ayoung Park & Ralph Weissleder & Yong Pil Chong & Hyun Jung Chung, 2025. "Effective treatment of systemic candidiasis by synergistic targeting of cell wall synthesis," Nature Communications, Nature, vol. 16(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_s41467-025-62886-5. 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.