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<p>Triacetic acid lactone (TAL) is a versatile platform chemical traditionally biosynthesized via decarboxylative Claisen condensation by 2-pyrone synthase. However, this route is limited by poor efficiency and dependence on malonyl-CoA. Here, we show that non-decarboxylative Claisen condensation by polyketoacyl-CoA thiolases offers a more efficient alternative. Through mining homologs of a previously reported enzyme from <i>Cupriavidus necator</i>, we identify five thiolases with TAL production activity. One candidate, BktBbr from <i>Burkholderia</i> sp. RF2-non_BP3, exhibits approximately 30-fold higher activity in vitro and supports 30-fold higher TAL titers in <i>Escherichia coli</i> compared to the original enzyme. Fed-batch fermentation achieves titers up to 2.8 g L⁻¹. Structural analysis of BktBbr co-crystallized with CoA esters guides rational engineering to further enhance performance. Our discovery of a highly active thiolase establishes an alternative enzymatic route to produce TAL efficiently, providing a scalable foundation for sustainable biomanufacturing.</p>

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A highly active Burkholderia polyketoacyl-CoA thiolase for production of triacetic acid lactone

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月9日

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  • Zilong Wang,
  • Seokjung Cheong,
  • Jose Henrique Pereira,
  • Weixi Hu,
  • Yifan Guo,
  • Andy DeGiovanni,
  • Guangxu Lan,
  • Jinho Kim,
  • Robert W. Haushalter,
  • Taek Soon Lee,
  • Paul D. Adams,
  • Jay D. Keasling

摘要

Triacetic acid lactone (TAL) is a versatile platform chemical traditionally biosynthesized via decarboxylative Claisen condensation by 2-pyrone synthase. However, this route is limited by poor efficiency and dependence on malonyl-CoA. Here, we show that non-decarboxylative Claisen condensation by polyketoacyl-CoA thiolases offers a more efficient alternative. Through mining homologs of a previously reported enzyme from Cupriavidus necator, we identify five thiolases with TAL production activity. One candidate, BktBbr from Burkholderia sp. RF2-non_BP3, exhibits approximately 30-fold higher activity in vitro and supports 30-fold higher TAL titers in Escherichia coli compared to the original enzyme. Fed-batch fermentation achieves titers up to 2.8 g L⁻¹. Structural analysis of BktBbr co-crystallized with CoA esters guides rational engineering to further enhance performance. Our discovery of a highly active thiolase establishes an alternative enzymatic route to produce TAL efficiently, providing a scalable foundation for sustainable biomanufacturing.