Pass-back chain extension expands multimodular assembly line biosynthesis

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Abstract

Modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymatic assembly lines are large and dynamic protein machines that generally effect a linear sequence of catalytic cycles. Here, we report the heterologous reconstitution and comprehensive characterization of two hybrid NRPS–PKS assembly lines that defy many standard rules of assembly line biosynthesis to generate a large combinatorial library of cyclic lipodepsipeptide protease inhibitors called thalassospiramides. We generate a series of precise domain-inactivating mutations in thalassospiramide assembly lines, and present evidence for an unprecedented biosynthetic model that invokes intermodule substrate activation and tailoring, module skipping and pass-back chain extension, whereby the ability to pass the growing chain back to a preceding module is flexible and substrate driven. Expanding bidirectional intermodule domain interactions could represent a viable mechanism for generating chemical diversity without increasing the size of biosynthetic assembly lines and challenges our understanding of the potential elasticity of multimodular megaenzymes.

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Fig. 1: Heterologous reconstitution of thalassospiramide biosynthetic gene clusters in a P. putida host.
Fig. 2: Ttc and Ttm assembly lines and structures of associated cyclic lipodepsipeptide products.
Fig. 3: Selective inactivation of assembly line enzymatic domains alters product formation.
Fig. 4: Model for thalassospiramide biosynthesis by Ttm C2 inactivation mutant.
Fig. 5: Model for thalassospiramide A biosynthesis by Ttc.

Data availability

The ttc and ttm biosynthetic gene cluster sequences are available in the MIBiG database (accession BGC0001050 and BGC0001876). Plasmids pCAP-BAC (#120229), pJZ001 (#120230) and pJZ002 (#120231) are available at Addgene.

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Acknowledgements

We thank J.R. van der Meer (University of Lausanne) for providing plasmid pRMR6K-Gm, V. de Lorenzo (National Center for Biotechnology-CSIC) for providing strain P. putida EM383, and D.L. Court (National Cancer Institute, NIH) for providing strain E. coli HME68. We are grateful to A. Edlund (J. Craig Venter Institute), P.Y. Qian (Hong Kong University of Science and Technology), H. Xia (Shanghai Institutes for Biological Sciences, CAS) and W. Fenical and P.R. Jensen (Scripps Institution of Oceanography, UCSD) for facilitating access to equipment, chemical standards and bacterial strains. We also thank Y. Kudo, P.A. Jordan, J.R. Chekan, L.T. Hoang and S. Carreto for helpful discussion and technical assistance. The research reported has been supported by National Institutes of Health grants F31-AI129299 to J.J.Z. and R01-GM085770 to B.S.M.

Author information

J.J.Z. and B.S.M. designed the study. J.J.Z., X.T. and A.C.R. performed cloning, gene deletion and other molecular biology experiments. J.J.Z. and X.T. performed heterologous expression and chemical extraction experiments. J.J.Z. and T.H. performed mass spectrometry experiments and analyzed mass spectrometry data. J.J.Z. and B.S.M. analyzed all data and wrote the manuscript with input from all authors.

Correspondence to Bradley S. Moore.

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Zhang, J.J., Tang, X., Huan, T. et al. Pass-back chain extension expands multimodular assembly line biosynthesis. Nat Chem Biol (2019) doi:10.1038/s41589-019-0385-4

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