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<p>Genetically encoded fluorescent biosensors (GEFBs) are invaluable tools for spatiotemporal metabolite monitoring in cellular metabolism, yet their development for many key metabolites is hampered by a lack of specific biorecognition elements. Here, we report a versatile strategy to engineer metabolite-responsive GEFBs by leveraging the allosteric properties of regulatory domains from allosteric enzymes. Using regulatory domains from chorismate mutase, 2-acetolactate synthase, and <span>d</span>-citramalate synthase as biorecognition elements, we construct three biosensors for specific <span>l</span>-phenylalanine, <span>l</span>-valine, and <span>l</span>-isoleucine detection. We further demonstrate that multi-ligand-binding regulatory domains can be exploited to derive diverse specific biosensors, and apply this strategy to develop two <i>S</i>-adenosyl-<span>l</span>-methionine biosensors and an <i>S</i>-methyl-5’-thioadenosine biosensor. We also showcase the utility of these biosensors for real-time, in situ tracking of target metabolites in living cells, as well as bioprocess monitoring and clinical diagnostics. Overall, this study establishes a flexible strategy that provides insights to construct GEFBs targeting other metabolites.</p>

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A versatile method for designing biosensors via regulatory domains of allosteric enzymes

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月20日

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  • Zhaoqi Kang,
  • Rong Xu,
  • Ping Han,
  • Hui Zhang,
  • Shuang Hou,
  • Dan Xiao,
  • Yan Zhang,
  • Yidong Liu,
  • Leilei Guo,
  • Jie Wu,
  • Weikang Sun,
  • Xiaoxu Tan,
  • Xianzhi Xu,
  • Kaiyu Gao,
  • Chuanjuan Lü,
  • Cuiqing Ma,
  • Rong Qiang,
  • Ping Xu,
  • Chao Gao

摘要

Genetically encoded fluorescent biosensors (GEFBs) are invaluable tools for spatiotemporal metabolite monitoring in cellular metabolism, yet their development for many key metabolites is hampered by a lack of specific biorecognition elements. Here, we report a versatile strategy to engineer metabolite-responsive GEFBs by leveraging the allosteric properties of regulatory domains from allosteric enzymes. Using regulatory domains from chorismate mutase, 2-acetolactate synthase, and d-citramalate synthase as biorecognition elements, we construct three biosensors for specific l-phenylalanine, l-valine, and l-isoleucine detection. We further demonstrate that multi-ligand-binding regulatory domains can be exploited to derive diverse specific biosensors, and apply this strategy to develop two S-adenosyl-l-methionine biosensors and an S-methyl-5’-thioadenosine biosensor. We also showcase the utility of these biosensors for real-time, in situ tracking of target metabolites in living cells, as well as bioprocess monitoring and clinical diagnostics. Overall, this study establishes a flexible strategy that provides insights to construct GEFBs targeting other metabolites.