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Insight into the role of a trans-AT polyketide synthase in the biosynthesis of lankacidin-type natural products

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Abstract

Modular polyketide synthases (type I PKSs) are biosynthetic assembly lines for synthesizing a diverse array of natural products. While most PKSs exhibit a linear module architecture, the PKS system for lankacidin-type natural products contains only five modules but carries out eight rounds of polyketide extension, challenging the collinearity rule. Here we show the distinct domain architecture of the polyketide synthase enzyme, CheC, which is central to chejuenolide biosynthesis. CheC not only dissociates from and interacts with both the preceding and succeeding PKS enzymes, creating two linear modules, but also independently assembles an unconventional module, facilitating multiple rounds of polyketide extension in the biosynthetic process. We also unveiled missing functions of certain redundant and absent domains within PKSs, fully elucidating the polyketide assembly process for lankacidin-like natural products. These findings not only reveal the biosynthetic pathway for lankacidin- and chejuenolin-type natural products but also enrich the diverse functions of PKSs, setting the stage for future rational design of PKSs.

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Fig. 1: Structures and biosynthetic gene clusters of lankacidin-type natural products.
Fig. 2: The proposed biosynthetic routes i and ii for chejuenolide.
Fig. 3: The circular biosynthetic diagram of CheC.
Fig. 4: Detection of elongated polyketide chains through in vitro enzymatic reaction.
Fig. 5: Detection of elongated polyketide chains tethered on the CheC-ACP1.
Fig. 6: LC–MS analysis of the engineered trans-AT PKS.

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Data availability

Data supporting the findings of this work are available within the paper and its Supplementary Information files. All the DNA or protein sequences were deposited in GenBank, and the accession numbers were listed in figure legends.

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Acknowledgements

This research was financially supported by National Key R&D Program of China (2018YFA0902000 to R.H.J., 2022YFC2303100 to H.M.G. and 2022YFC2804100 to H.M.G.) and National Natural Science Foundation of China (81925033 to H.M.G., 22193071 to H.M.G., 81803380 to B.Z., 22207052 to Z.P.M., 81991522 to B.Z. and 22107048 to J.S.).

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Authors and Affiliations

  1. State Key Laboratory of Pharmaceutical Biotechnology, Department of Neurology, Nan**g Drum Tower Hospital, School of Life Sciences, Chemistry and Biomedicine Innovation Center, Nan**g University, Nan**g, China

    Zhen Peng Mai, Bo Zhang, Zi Xuan Pang, **g Shi, Zi Fei Xu, Shi Ying Ma, Rui Hua Jiao, Ren **ang Tan & Hui Ming Ge

  2. State Key Laboratory of Coordination Chemistry, Jiangsu Key Laboratory of Advanced Organic Materials, School of Chemistry and Chemical Engineering, Nan**g University, Nan**g, China

    Bing-Bing Huang & Zhu-Jun Yao

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  1. Zhen Peng Mai
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Contributions

Z.P.M., B.Z., Z.X.P. and S.Y.M. carried out experiments; Z.F.X., J.S. and R.H.J. assisted in NMR and MS data measurement and analysis; Z.-J.Y. and B.-B.H. assisted in chemical synthesis; R.X.T. contributed materials and equipment; Z.P.M., B.Z. and H.M.G. wrote the paper. B.Z. and H.M.G. supervised the work. All authors discussed the results and analysed the data.

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Correspondence to Bo Zhang, Ren **ang Tan or Hui Ming Ge.

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Nature Synthesis thanks Yuhui Sun, Kira Weissman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Thomas West, in collaboration with the Nature Synthesis team.

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Extended data

Extended Data Fig. 1 Biosynthetic logic of cis-AT PKS and trans-AT PKS.

Comparison of canonical domain architectures in cis-AT PKS (a) and trans-AT PKS (b) for assembly of a fictional polyketide (dashed box). KS, ketosynthase domain; AT(Mal.), acyltransferase recognized malonyl-CoA; AT(mMal.), acyltransferase recognized methylmalonyl-CoA; ACP, acyl carrier protein domain; KR, ketoreductase domain; DH, dehydratase domain; ER, enoyl reductase; MeT, methyltransferase domain; TE, thioesterase domain; GNAT, GCN5-related AT.

Extended Data Fig. 2 LC-MS analysis of the one-pot enzymatic reconstitution of chejuenolin (IV).

In vitro enzymatic reaction of CheABCDEFG with glycine as start unit in the presence of AcCoA, ATP, MgCl2, malonyl-CoA, NADPH, SAM and FAD. The EIC analyzed used m/z 432.2381 with ± 0.005 mass tolerance. Each experiment was repeated three times, and similar results were obtained.

Extended Data Fig. 3 LC-MS analysis of the one-pot enzymatic reconstitution of 10.

The EIC analyzed used m/z 432.2392 with ± 0.005 mass tolerance. Each experiment was repeated three times, and similar results were obtained.

Extended Data Fig. 4 LC-MS detection of elongated polyketide chains through in vitro enzymatic reaction using 6b as substrate.

LC-MS analysis of the elongated polyketide chains released from CheF after KOH hydrolysis. The EIC analyzed used exact calculated masses with ± 0.005 tolerance. Each experiment was repeated three times, and similar results were obtained.

Extended Data Fig. 5 LC-MS analysis of the function of tandem ACP domains in CheC.

i) As a positive control, N-acetylglycine as a start unit reacts with CheA-D and CheF-G in the presence of malonyl-CoA, NADPH and SAM. ii) CheC was replaced by truncated CheC-KR-MeT, CheC-ACP1-ACP2 and CheC-KS-DH at the same reaction with i). iii-iv) The truncated CheC-ACP1-ACP2 was replaced by site-directed mutant CheC-ACP1-S871A and site-directed mutant CheC-ACP2-S974A at the same reaction with iii), respectively. v) As a negative control, N-acetylglycine works with the CheC(ACP1-S871A-ACP2-S974A) instead of CheC at the same reaction with i). vi-vii) The truncated CheC-ACP1-ACP2 was replaced by truncated CheC-ACP1 and truncated CheC-ACP2 at the same reaction with ii, respectively. viii) standard of 10. The EIC analyzed used m/z 432.2392 with ± 0.005 mass tolerance. Each experiment was repeated three times, and similar results were obtained.

Extended Data Fig. 6 LC-MS analysis of the putative trans-acting methylation.

i) Thiophenol derivative 7b reacts with CheDFG and CheC-MeT in the presence of cofactors, malonyl-CoA, NADPH and SAM, followed by KOH hydrolysis; ii) Based on reaction i, the expressed CheC-MeT domain and SAM was removed. The EIC analyzed used exact calculated masses with ± 0.005 tolerance. Each experiment was repeated three times, and similar results were obtained.

Extended Data Fig. 7 Fragmentation spectrum of the proposed polyketide 8a and 8c produced by enzymatic reaction.

a/b, MS/MS2 fragmentation spectrum of 8a and 8c, the key product ions were highlighted and the clear signatures of Δ14 Da were observed. Each experiment was repeated three times, and similar results were obtained.

Extended Data Fig. 8 LC-MS analysis of the function of the redundant KS-ACP domains in the final module.

a, LC-MS analysis of the function of CheF-KS2 and CheG-KS domains. i) Thiophenol derivative 7b reacts with CheD, CheF-G and CheC-MeT in the presence of Mal-CoA, SAM and NADPH to product 10. ii) CheF was replaced by CheF (KS2-C1952A) in the same reaction as i). iii) CheG was replaced by CheG (KS-C258A) in the same reaction as i). iv) CheF and CheG were both replaced by CheF (KS2-C1952A) and CheG (KS-C258A) int the same reaction as i). b, LC-MS analysis of the function of CheG-ACP1 and CheG-ACP2 domains. i) Thiophenol derivative 7b reacts with CheD, CheF-G and CheC-MeT in the presence of Mal-CoA, SAM and NADPH to product 10. ii) CheG was replaced by CheG (ACP1-S35A) in the same reaction as i). iii) CheG was replaced by CheG (ACP2-S715A) in the same reaction as i). iv) CheG weas replaced by CheG (ACP1-S35A-ACP2-S715A) into the same reaction as i). The EIC analyzed used m/z 432.2392 with ± 0.005 mass tolerance. Each experiment was repeated three times, and similar results were obtained.

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Mai, Z.P., Zhang, B., Pang, Z.X. et al. Insight into the role of a trans-AT polyketide synthase in the biosynthesis of lankacidin-type natural products. Nat. Synth (2024). https://doi.org/10.1038/s44160-024-00599-1

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