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Nature Chemical Biology (2026)
Decarboxylative condensation drives chain elongation and translocation of polyketides and fatty acids. However, the mechanism by which nonelongating modules in trans-acyltransferase polyketide synthases (trans-AT PKSs) enable intramodular polyketide chain translocation without decarboxylation remains poorly understood. Here we elucidate a condensation-independent intramodular translocation mechanism in which KS0 within the nonelongating module operates as a transacylase, directly translocating the polyketide chain to its downstream cognate acyl carrier protein (ACP). Notably, the inherent demalonylation activity of trans-ATHtmA7 facilitates efficient ACP recycling ensuring intramodular translocation. Structural modeling and site-directed mutagenesis studies uncover a conserved KS0–ACP binding mode that underpins intramodular translocation across diverse nonelongating modules. Additionally, the strict discrimination of polyketide intermediate by the nonelongating module highlights its critical role in maintaining biosynthetic precision and efficiency. These findings provide mechanistic insights into evolutionary adaptation and sophisticated crosstalk between catalytic domains within trans-AT PKS, illuminating how metabolic flux and fidelity are maintained and opening avenues for polyketide engineering.
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All data supporting the findings of this study are available in the main paper and Supplementary Information. Protein structural models were generated using AlphaFold version 3.0.1 and the corresponding structures and molecular dynamics simulations files are provided in Supplementary Data 2 and 3. Additional materials, including plasmids and primers, are available from the corresponding authors upon reasonable request.
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This work was supported by the National Key R&D Program of China (2024YFA0917600) and the National Natural Science Foundation of China (32470033 to Y.S. and 32400042 to G.Z.).
These authors contributed equally: Zhicheng Guo, Shijuan Wu.
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education) and School of Pharmaceutical Sciences, Wuhan University, Wuhan, People’s Republic of China
Zhicheng Guo, Shijuan Wu, Minghe Luo, Yulu Dong, Guo Sun, Zixin Deng & Yuhui Sun
School of Pharmacy, Huazhong University of Science and Technology, Wuhan, People’s Republic of China
Zhicheng Guo, Gen Lu, Guifa Zhai & Yuhui Sun
Pharmacy Faculty, Hubei University of Chinese Medicine, Wuhan, People’s Republic of China
Shijuan Wu
College of Life Sciences, Wuchang University of Technology, Wuhan, People’s Republic of China
Xiaohua Wang
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Z.G. and G.Z. designed the experiments and wrote the paper. S.W. designed and constructed the mutants. X.W. synthesized the substrate mimics. G.L. performed the computational calculations. M.L., Y.D. and G.S. identified the structures of HTM. Z.D. analyzed the data and revised the paper. Y.S. and G.Z. conceptualized the overall project, analyzed the data and revised the paper.
Correspondence to Guifa Zhai or Yuhui Sun.
The authors declare no competing interests.
Nature Chemical Biology thanks the anonymous reviewers for their contribution to the peer review of this work.
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a, Schematics of intermodular translocation, intramodular translocation and intermodular retrotranslocation. b-d, In vitro reconstitution of retrotranslocation of ({{rm{KS}}}_{4}^{0}) -ATd4 with upstream holo-ACP3 (b), ({{rm{KS}}}_{3}^{0}) with upstream holo-ACP2 (c) and KS2-ATd2 with upstream holo-ACP1 (d). These assays were monitored by LC-ESI-HRMS and the results were deconvoluted. Each experiment was performed with three biological replicates, yielding similar results. Asterisk indicates that the corresponding protein is not detected.
In canonical PKS modules, ACPs typically adopt two distinct binding modes with KS domains: one for intramodular elongation with the upstream KS, and another for intermodular translocation with the downstream KS. By contrast, ACPs in non-elongating modules 3 and 4 adopt a unified binding mode compatible with their cognate KS0 and the upstream ACP. This structural adaptation facilitates the unique intramodular translocation process characteristic of these modules.
a, Functional characterization of the dehydratase domain DH3. b, Functional investigation of ({{rm{KS}}}_{4}^{0}) in mediating intermodular polyketide chain translocation. These assays were monitored by LC-ESI-HRMS and the results were deconvoluted. Each experiment was performed with three biological replicates, yielding similar results. Asterisk indicates that the corresponding protein is not detected.
Supplementary Figs. 1–75 and Methods.
Mass spectrometry data.
Structures of AlphaFold-generated proteins in PDB format.
Molecular dynamics simulation files.
Source data for Supplementary Fig. 21.
Lists of primers, strains and plasmids used in this study.
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Guo, Z., Wu, S., Wang, X. et al. Condensation-independent intramodular translocation mechanism of the trans-AT polyketide synthase assembly line. Nat Chem Biol (2026). https://doi.org/10.1038/s41589-026-02209-x
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