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Plug-and-play engineering of modular polyketide synthases

Nature Chemical Biology volume 21, pages 1361–1367 (2025)Cite this article

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Abstract

Modular polyketide synthases (PKSs) are multidomain, assembly line enzymes that biosynthesize complex antibiotics such as erythromycin and rapamycin. The modular characteristic of PKSs makes them an ideal platform for the custom production of designer polyketides by combinatorial biosynthesis. However, engineered hybrid PKS pathways often exhibit severe loss of enzyme activity, and a general principle for PKS reprogramming has not been established. Here we present a widely applicable strategy for designing hybrid PKSs. We reveal that two conserved motifs are robust cut sites to connect modules from different PKS pathways and demonstrate the custom production of polyketides with different starter units, extender units and variable reducing states. Furthermore, we expand the applicability of these cut sites to construct hybrid pathways involving cis-AT PKS, trans-AT PKS and even nonribosomal peptide synthetase. Collectively, our findings enable plug-and-play reprogramming of modular PKSs and facilitate the application of assembly line enzymes toward the bioproduction of designer molecules.

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Fig. 1: Hybrid PKS design and in vitro evaluation of product formation using the venemycin PKS pathway.
Fig. 2: Evaluation of different cut sites for the construction of various hybrid pathways.
Fig. 3: Plug-and-play construction of PKS hybrids using various assembly line pathways.

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Data availability

All data supporting the results in this study are available in the paper and its Supplementary Information. The polyketide synthase enzyme sequences are from the MIBiG database (https://mibig.secondarymetabolites.org/) and NCBI database based on the BLAST search result. The PKS sources used for hybrid enzyme construction are given in Supplementary Table 1. Data are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank S. Kazuo for helpful discussion in hybrid PKS construction. We thank the Instrumentation and Service Center for Molecular Sciences at Westlake University for the assistance in MS and NMR analyses and the Westlake High-Performance Computing Center for providing computational resource. This research was supported by the ‘Pioneer’ and ‘Leading Goose’ R&D Program of Zhejiang (grant no. 2023SDXHDX0007), the National Natural Science Foundation of China General Program (grant no. 22177092) and Zhejiang Provincial Key Laboratory Construction Project to L.Z.

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Authors and Affiliations

  1. Department of Chemistry, Zhejiang University, Hangzhou, China

    Zilei Huang, Shengling Xie & Shunyu Yao

  2. Zhejiang Key Laboratory of Precise Synthesis of Functional Molecules, Department of Chemistry, School of Science and Research Center for Industries of the Future, Westlake University, Hangzhou, China

    Zilei Huang, Shengling Xie, Run-Zhou Liu, Changjun Xiang, Shunyu Yao & Lihan Zhang

  3. Institute of Natural Sciences, Westlake Institute for Advanced Study, Hangzhou, China

    Lihan Zhang

  4. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Lihan Zhang

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  1. Zilei Huang
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Contributions

Z.H. performed all the biological experiments. Z.H., S.Y. and L.Z. performed the bioinformatic analyses. S.X., C.X. and R.-Z.L. performed the chemical synthesis. Z.H. and R.-Z.L. conducted the MS analyses. Z.H., S.X., S.Y. and L.Z. designed the experiments and analyzed the results. L.Z. conceived the research and wrote the paper with inputs from all coauthors.

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Correspondence to Lihan Zhang.

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Z.H., S.Y., S.X., and L.Z. are co-inventors on a PCT patent application PCT/CN2024/142956 that incorporates discoveries described in this paper. The other authors declare no competing interests.

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Nature Chemical Biology thanks Constance Bailey, Martin Grininger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phylogenetic analysis of the KS domains from modular PKSs.

The first elongating KS domains in the first module of cis-AT PKSs (KS1, except for KSQ which does not catalyze condensation) were curated from MIBiG database and analyzed together with KSs from PKS-NRPS hybrids and trans-AT PKSs. Clade a, KS1 with KSQ-AT for starter unit loading. Clade b, KS1 with KSQ-AT for substrate loading and with downstream unnatural extender units-accepting AT. Clade c, KS1 with amino or guanidino group-containing starter units. Clade d, KS1 with ATL (starter unit-loading AT) for starter unit loading. Clade e, KS1 with aromatic or aliphatic rings as starter units. Clade f, KS1 with long-chain fatty acid or aromatic substrates activated by an adenylating domain. Clade g, KS1 with ATL domain inserted into module 1 forming the ACP-KS-ATL-AT-ACP type of module. Clade h, KS1 with pyrrole substrates. Clade i, KSs (not necessarily KS1) from trans-AT PKS-NRPS hybrids with peptide substrates. Clade j, KSs (not necessarily KS1) from cis-AT PKS-NRPS hybrids with peptide substrates. Clade k, KSs (not necessarily KS1) from trans-AT PKSs. The PKSs used in this study was marked in black dots. Detailed tree is provided in Supplementary Fig. S7. Sequence information was provided in the Supplementary Dataset.

Source data

Extended Data Fig. 2 Abbreviated biosynthetic pathways and the products of the 10 PKS/NRPS pathways used in this study other than the venemycin PKS.

The enzymes not used in this study are omitted for clarity. For the FscA, RakPKS1, E837PKS1, DEBS1, RapA, KirA1 and PteA1, the starter units and the extender units are shown on the corresponding ACP. For AkaPKS3, AntC, and PhePKS5, the polyketide intermediates being translocated to the downstream enzyme are shown on the corresponding ACP. The superscript 0 indicates inactive domains.

Extended Data Fig. 3 Steady-state kinetic analysis of hybrid PKSs.

The product formation of the engineered PKS pathways was measured under variable concentration of the first PKS with constant concentration of 0.2 μM vemH and 1 mM start unit. The dose-response curves of enzyme turnover velocity (μM product /min /μM VemH) were obtained from non-linear fitting of data to Michaelis-Menten equation. Error bars represent mean ± s.d. from three parallel reactions as technical replicates.

Extended Data Fig. 4 In vitro substrate specificity assay of VemH.

50 μL reaction mixture containing 10 mM ATP, 5 mM TCEP, 10 mM MgCl2, 1 mM CoA, 1 mM N-acetylcysteamine thioester (SNAC) substrates SNAC1-SNAC6, 1.6 mM malonate, 1 μM MatB, 400 mM PBS (pH 7.5), 2 μM VemH were incubated at 30 °C overnight. a. Reaction scheme of VemH with SNAC5-6 and products 12a-b. b. HPLC analyses of VemH with SNAC5-6 monitored at UV 290 nm. c. Reaction scheme of VemH with SNAC1-4. VemH+SNAC1 produced the anticipated product 8b and the iteratively elongated 8a. VemH+SNAC2 produced the anticipated 9b and the iteratively elongated 9a. VemH+SNAC3 produced the iteratively elongated 8b. VemH+SNAC4 produced trace amount of the iteratively elongated 10a. d. LC-MS analysis of VemH with each substrate. Extracted ion chromatograms of m/z corresponding to the products were shown in red (8a), purple (8b), orange (9a), blue (9b), and green (10a), respectively.

Extended Data Fig. 5 Biosynthetic schemes of PKSs with β-processing domains.

a. Proposed product formation mechanism by Rak-Vem-4 pathway. 2a can be produced by β-processing domain (DH-ER-KR) skipping; 2c can be produced by β-processing domain (DH-ER-KR) skipping and an iterative elongation at VemH; 2d was produced by fully functional β-processing domains and an iterative elongation of VemH. b. Proposed product formation mechanism by DEBS-Vem-4 pathway. 3a was produced by KR skipping; 3c was produced by fully functional domains. Iterative elongation during in vitro reaction of modular PKS has been documented previously66.

Extended Data Fig. 6 In vitro substrate specificity assay of AkaPKS4 and PheNRPS.

a. Reaction schemes of AkaPKS4 with synthetic N-acetylcysteamine thioester (SNAC) substrates and the product structures of compounds 8b-9b. b. LC-MS analysis of AkaPKS4 with each substrate. 50 μL reaction mixture containing 10 mM ATP, 5 mM TCEP, 10 mM MgCl2, 1 mM CoA, 1 mM SNAC1-SNAC4, 1.6 mM malonate, 1 μM MatB, 400 mM PBS (pH 7.5), 2 μM AkaPKS4 were incubated at 30 °C overnight. Extracted ion chromatograms of m/z corresponding to the products were shown in purple (8b), orange (9a), blue (9b), respectively. c. Reaction schemes of PheNRPS with SNAC substrates and the product structures of compounds 11a–11d. d. LC-MS analysis of PheNRPS with each substrate. 50 μL reaction mixture containing 10 mM ATP, 5 mM TCEP, 10 mM MgCl2, 1 mM SNAC1-SNAC4, 1 mM alanine, 1 mM NADPH, 400 mM PBS (pH 7.5), 2 μM PheNRPS were incubated at 30 °C overnight. Extracted ion chromatograms of m/z corresponding to the products were shown in beige (11a), purple (11b), gray (11c), and blue (11d), respectively.

Extended Data Fig. 7 Predicted structural models of hybrid PKSs.

a. Sequence alignment of the proteins used to construct hybrid PKSs with the position of cut sites 1–5 marked. AntC does not have KS-AT and only the ACP region is aligned. b. Predicted structures of the enzymes by AlphoFold2. KS domain (yellow), KS-LD-AT linker (cyan), AT domain (green), post-AT linker (magenta), ACP (gray). The extended post-AT linker of VemG is shown in white. The structure files are available at Supplementary Data.

Extended Data Fig. 8 Phylogenetic analysis of KS-AT linkers from aminopolyol PKSs according to the upstream domain organization.

a. Phylogenetic tree of the KS-AT linkers. The tree was constructed by FastTree2.1.1 with branch support values, performed on Geneious Prime. Labels indicate sequence source and location in a PKS pathway. For example, ecoM2c indicates the KS-AT linker in the ECO-02301 pathway (eco) at the second PKS (2), third module (c) based on traditional module definition shown in panel b. eco: ECO-02301; med: mediomycin; tfb: tetrafibricin; nmd: neomediomycin; lin: linearmycin; cle: clethramycin; S.mel: Streptomyces melanospoorfaciens PKS; S.nbrc: Streptomyces. sp. NBRC 109436 PKS; S.RTd: Streptomyces. sp. RTd22 PKS; S.ven: S. venezuelae PKS S.mash: S. mashuensis PKS; K.med: Kitasatospora mediocidica PKS. Accession numbers for the PKSs are provided in Supplementary Information. b. The module duplication observed in PKS1s of aminopolyols, based on Zhang et al. 201714. Sequences with high similarity are colored with yellow (KR-containing module) and dark yellow (DH-KR-containing module) to illustrate module duplication. Types of KS-AT linkers from the PKS1s of each pathway are shown in circle, triangle, and box as explained in panel c. The clear separate clades were observed in the tree depending on the types of its upstream domains, suggesting that KS-AT linkers also comigrate with its upstream sequences during module duplication.

Source data

Extended Data Fig. 9 Phylogenetic analysis of KS-AT linkers from aminopolyol PKSs according to the types of the downstream AT.

Phylogenetic analysis of KS-AT linkers according to the types of the downstream AT. The data is identical to Extended Data Fig. 8 but differently labeled. The tree shows a clear separation of KS-AT linkers associated with downstream methylmalonyl-CoA specific AT (red star) and malonyl-CoA specific AT (blue circle), suggesting a conserved interaction between the linker and the downstream AT.

Source data

Supplementary information

Supplementary Information

Supplementary Methods for chemical synthesis, Table 1, Figs. 1–102 and references.

Reporting Summary

Supplementary Table 2

Lists of plasmids, primers, strains, proteins and reagents used in this study.

Supplementary Data 1

Source sequences of Supplementary Fig. 1 and statistical source data of Supplementary Fig. 3.

Supplementary Data 2

Structure files for Extended Data Fig. 7 in pdb format.

Source data

Source Data Fig. 1

Statistical source data of Fig. 1d.

Source Data Fig. 2

Statistical source data of Fig. 2b,d.

Source Data Fig. 3

Statistical source data of Fig. 3b,d,e.

Source Data Extended Data Fig. 1

Sequences used for tree building.

Source Data Extended Data Figs. 8 and 9

Sequences used for tree building.

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Huang, Z., Xie, S., Liu, RZ. et al. Plug-and-play engineering of modular polyketide synthases. Nat Chem Biol 21, 1361–1367 (2025). https://doi.org/10.1038/s41589-025-01878-4

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