DOE OSTI · 3013344
A highly active Burkholderia polyketoacyl-CoA thiolase for production of triacetic acid lactone
Abstract
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.
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Wang, Zilong [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); University of California, Berkeley, CA (United States)], Cheong, Seokjung [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); University of California, Berkeley, CA (United States)], Pereira, Jose Henrique [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)], Hu, Weixi [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); University of California, Berkeley, CA (United States)], Guo, Yifan [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); University of California, Berkeley, CA (United States)] (ORCID:0009000129667138), DeGiovanni, Andy [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)], Lan, Guangxu [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000204155849), Kim, Jinho [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)], Haushalter, Robert W. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)], Lee, Taek Soon [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000207642626), Adams, Paul D. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States)] (ORCID:0000000193338219), Keasling, Jay D. [Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); University of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Technical University of Denmark, Lyngby (Denmark)] (ORCID:0000000341706088). 2025-12-09. A highly active Burkholderia polyketoacyl-CoA thiolase for production of triacetic acid lactone. https://doi.org/10.1038/s41467-025-65946-y
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