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

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.