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Genome-scale CRISPRi screen identifies pcnB repression conferring improved physiology for overproduction of free fatty acids in Escherichia coli

Author

Listed:
  • Lixia Fang

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Xueyan Hao

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Jie Fan

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Xiaolei Liu

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Yaru Chen

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Lian Wang

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Xiaoying Huang

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Hao Song

    (Tianjin University
    Tianjin University
    Tianjin University)

  • Yingxiu Cao

    (Tianjin University
    Tianjin University
    Tianjin University)

Abstract

Microbial physiology plays a pivotal role in construction of superior microbial cell factories for efficient biosynthesis of desired products. Here we identify that pcnB repression confers improved physiology for overproduction of free fatty acids (FFAs) in Escherichia coli through genome-scale CRISPRi modulation combining fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS). The repression of pcnB can enhance the stability and abundance of the transcripts of genes involved in the proton-consuming system, thereby supporting global improvements in membrane properties, redox state, and energy level. Based on pcnB repression, further repression of acrD increases FFAs biosynthesis by enhancing FFAs efflux. The engineered strain pcnBi-acrDi-fadR+ achieves 35.1 g L−1 FFAs production in fed-batch fermentation, which is the maximum titer reported to date in E. coli. This study highlights the significance of uncovering hidden genetic determinants that confer improved microbial physiology for enhancing the biosynthesis of desired products.

Suggested Citation

  • Lixia Fang & Xueyan Hao & Jie Fan & Xiaolei Liu & Yaru Chen & Lian Wang & Xiaoying Huang & Hao Song & Yingxiu Cao, 2025. "Genome-scale CRISPRi screen identifies pcnB repression conferring improved physiology for overproduction of free fatty acids in Escherichia coli," Nature Communications, Nature, vol. 16(1), pages 1-16, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-58368-3
    DOI: 10.1038/s41467-025-58368-3
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    References listed on IDEAS

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    1. Tianmin Wang & Changge Guan & Jiahui Guo & Bing Liu & Yinan Wu & Zhen Xie & Chong Zhang & Xin-Hui Xing, 2018. "Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance," Nature Communications, Nature, vol. 9(1), pages 1-15, December.
    2. Jiazhang Lian & Carl Schultz & Mingfeng Cao & Mohammad HamediRad & Huimin Zhao, 2019. "Multi-functional genome-wide CRISPR system for high throughput genotype–phenotype mapping," Nature Communications, Nature, vol. 10(1), pages 1-10, December.
    3. Lun Yao & Kiyan Shabestary & Sara M. Björk & Johannes Asplund-Samuelsson & Haakan N. Joensson & Michael Jahn & Elton P. Hudson, 2020. "Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes," Nature Communications, Nature, vol. 11(1), pages 1-13, December.
    4. Hui Liu & Pei Zhou & Mengya Qi & Liang Guo & Cong Gao & Guipeng Hu & Wei Song & Jing Wu & Xiulai Chen & Jian Chen & Wei Chen & Liming Liu, 2022. "Enhancing biofuels production by engineering the actin cytoskeleton in Saccharomyces cerevisiae," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
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