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<p>β-Elemene is a pharmaceutically important sesquiterpene, while its production from plants and chemical synthesis remains inefficient. To achieve the efficient in vitro synthesis of β-elemene from low-cost mevalonate, a hexa-enzyme cascade system was employed to improve biosynthetic productivity. Here we show that orderly cross-linked hexa-enzyme complexes constructed via bio-orthogonal protein pairs (Tag/Catcher, RIAD/RIDD<sub>2</sub>, and K/Q-Tag) enable spontaneous one-step co-purification and immobilization of six enzymes for β-elemene synthesis. These organized assemblies, confirmed for their spatial co-localization, achieve a β-elemene titer of 160.08 mg/L, a substantial improvement over the 27.54 mg/L produced by the corresponding free multi-enzyme system. In addition, the cross-linked complexes combine favorable operational stability with facile centrifugation-based recovery, retaining approximately 70% activity over six reuse cycles. Bio-orthogonal multi-enzyme assembly engineering to obtain complexes enables efficient in vitro biosynthesis of β-elemene, guided by nature’s metabolic synthesis. This strategy exploits the mevalonate pathway’s utility for sustainable engineered synthesis of terpenoid compounds.</p><p></p>

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Streamlined synthesis of β-elemene using a spatially organized multi-enzyme machinery

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年7月21日

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  • Mijun Li,
  • Chaoyue Nie,
  • Xiangxiang Zuo,
  • Xinxin Chen,
  • Jiayi Pan,
  • Ruida Chen,
  • Linmeng Du,
  • Jing Jiang,
  • Anming Wang

摘要

β-Elemene is a pharmaceutically important sesquiterpene, while its production from plants and chemical synthesis remains inefficient. To achieve the efficient in vitro synthesis of β-elemene from low-cost mevalonate, a hexa-enzyme cascade system was employed to improve biosynthetic productivity. Here we show that orderly cross-linked hexa-enzyme complexes constructed via bio-orthogonal protein pairs (Tag/Catcher, RIAD/RIDD2, and K/Q-Tag) enable spontaneous one-step co-purification and immobilization of six enzymes for β-elemene synthesis. These organized assemblies, confirmed for their spatial co-localization, achieve a β-elemene titer of 160.08 mg/L, a substantial improvement over the 27.54 mg/L produced by the corresponding free multi-enzyme system. In addition, the cross-linked complexes combine favorable operational stability with facile centrifugation-based recovery, retaining approximately 70% activity over six reuse cycles. Bio-orthogonal multi-enzyme assembly engineering to obtain complexes enables efficient in vitro biosynthesis of β-elemene, guided by nature’s metabolic synthesis. This strategy exploits the mevalonate pathway’s utility for sustainable engineered synthesis of terpenoid compounds.