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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Unexpected assembly machinery for 4(3H)-quinazolinone scaffold synthesis
Nature Communications Open Access 31 October 2022
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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.
Weissman, K. J. The structural biology of biosynthetic megaenzymes. Nat. Chem. Biol. 11, 660–670 (2015).
Cane, D. E. Programming of erythromycin biosynthesis by a modular polyketide synthase. J. Biol. Chem. 285, 27517–27523 (2010).
Robbel, L. & Marahiel, M. A. Daptomycin, a bacterial lipopeptide synthesized by a nonribosomal machinery. J. Biol. Chem. 285, 27501–27508 (2010).
Helfrich, E. J. & Piel, J. Biosynthesis of polyketides by trans-AT polyketide synthases. Nat. Prod. Rep. 33, 231–316 (2016).
Sussmuth, R. D. & Mainz, A. Nonribosomal peptide synthesis—principles and prospects. Angew. Chem. Int. Ed. Engl. 56, 3770–3821 (2017).
Magarvey, N. A., Haltli, B., He, M., Greenstein, M. & Hucul, J. A. Biosynthetic pathway for mannopeptimycins, lipoglycopeptide antibiotics active against drug-resistant gram-positive pathogens. Antimicrob. Agents Chemother. 50, 2167–2177 (2006).
Felnagle, E. A., Rondon, M. R., Berti, A. D., Crosby, H. A. & Thomas, M. G. Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin. Appl. Environ. Microbiol. 73, 4162–4170 (2007).
Thomas, M. G., Chan, Y. A. & Ozanick, S. G. Deciphering tuberactinomycin biosynthesis: isolation, sequencing, and annotation of the viomycin biosynthetic gene cluster. Antimicrob. Agents Chemother. 47, 2823–2830 (2003).
Du, L., Sanchez, C., Chen, M., Edwards, D. J. & Shen, B. The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase. Chem. Biol. 7, 623–642 (2000).
Gehring, A. M. et al. Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis. Chem. Biol. 5, 573–586 (1998).
Oh, D. C., Strangman, W. K., Kauffman, C. A., Jensen, P. R. & Fenical, W. Thalassospiramides A and B, immunosuppressive peptides from the marine bacterium Thalassospira sp. Org. Lett. 9, 1525–1528 (2007).
Ross, A. C. et al. Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria. J. Am. Chem. Soc. 135, 1155–1162 (2013).
Zhang, W. et al. Family-wide structural characterization and genomic comparisons decode the diversity-oriented biosynthesis of thalassospiramides by marine Proteobacteria. J. Biol. Chem. 291, 27228–27238 (2016).
Miyazaki, R. & van der Meer, J. R. A new large-DNA-fragment delivery system based on integrase activity from an integrative and conjugative element. Appl. Environ. Microbiol. 79, 4440–4447 (2013).
Martinez-Garcia, E., Nikel, P. I., Aparicio, T. & de Lorenzo, V. Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression. Microb. Cell Fact. 13, 159 (2014).
Zhang, J. J., Tang, X., Zhang, M., Nguyen, D. & Moore, B. S. Broad-host-range expression reveals native and host regulatory elements that influence heterologous antibiotic production in Gram-negative bacteria. MBio 8, e01291 (2017).
Labby, K. J., Watsula, S. G. & Garneau-Tsodikova, S. Interrupted adenylation domains: unique bifunctional enzymes involved in nonribosomal peptide biosynthesis. Nat. Prod. Rep. 32, 641–653 (2015).
Rausch, C., Hoof, I., Weber, T., Wohlleben, W. & Huson, D. H. Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution. BMC Evol. Biol. 7, 78 (2007).
Crosby, J. & Crump, M. P. The structural role of the carrier protein–active controller or passive carrier. Nat. Prod. Rep. 29, 1111–1137 (2012).
Calderone, C. T., Bumpus, S. B., Kelleher, N. L., Walsh, C. T. & Magarvey, N. A. A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both alpha- and beta-ketone reduction during chain growth. Proc. Natl Acad. Sci. USA 105, 12809–12814 (2008).
Whicher, J. R. et al. Structural rearrangements of a polyketide synthase module during its catalytic cycle. Nature 510, 560–564 (2014).
Drake, E. J. et al. Structures of two distinct conformations of holo-non-ribosomal peptide synthetases. Nature 529, 235–238 (2016).
Reimer, J. M., Aloise, M. N., Harrison, P. M. & Schmeing, T. M. Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase. Nature 529, 239–242 (2016).
Miller, B. R., Drake, E. J., Shi, C., Aldrich, C. C. & Gulick, A. M. Structures of a nonribosomal peptide synthetase module bound to MbtH-like proteins support a highly dynamic domain architecture. J. Biol. Chem. 291, 22559–22571 (2016).
Marahiel, M. A. A structural model for multimodular NRPS assembly lines. Nat. Prod. Rep. 33, 136–140 (2016).
Tarry, M. J., Haque, A. S., Bui, K. H. & Schmeing, T. M. X-ray crystallography and electron microscopy of cross- and multi-module nonribosomal peptide synthetase proteins reveal a flexible architecture. Structure 25, 783–793 e784 (2017).
Bozhuyuk, K. A. J. et al. De novo design and engineering of non-ribosomal peptide synthetases. Nat. Chem. 10, 275–281 (2018).
Dunn, B. J., Watts, K. R., Robbins, T., Cane, D. E. & Khosla, C. Comparative analysis of the substrate specificity of trans- versus cis-acyltransferases of assembly line polyketide synthases. Biochemistry 53, 3796–3806 (2014).
Awakawa, T. et al. Salinipyrone and pacificanone are biosynthetic by-products of the rosamicin polyketide synthase. Chembiochem 16, 1443–1447 (2015).
Moss, S. J., Martin, C. J. & Wilkinson, B. Loss of co-linearity by modular polyketide synthases: a mechanism for the evolution of chemical diversity. Nat. Prod. Rep. 21, 575–593 (2004).
Thomas, I., Martin, C. J., Wilkinson, C. J., Staunton, J. & Leadlay, P. F. Skipping in a hybrid polyketide synthase. Evidence for ACP-to-ACP chain transfer. Chem. Biol. 9, 781–787 (2002).
Wenzel, S. C., Meiser, P., Binz, T. M., Mahmud, T. & Muller, R. Nonribosomal peptide biosynthesis: point mutations and module skipping lead to chemical diversity. Angew. Chem. Int. Ed. Engl. 45, 2296–2301 (2006).
Mootz, H. D. et al. Decreasing the ring size of a cyclic nonribosomal peptide antibiotic by in-frame module deletion in the biosynthetic genes. J. Am. Chem. Soc. 124, 10980–10981 (2002).
Gao, L. et al. Module and individual domain deletions of NRPS to produce plipastatin derivatives in Bacillus subtilis. Microb. Cell Fact. 17, 84 (2018).
Yu, D., Xu, F., Zhang, S. & Zhan, J. Decoding and reprogramming fungal iterative nonribosomal peptide synthetases. Nat. Commun. 8, 15349 (2017).
Linne, U. & Marahiel, M. A. Control of directionality in nonribosomal peptide synthesis: role of the condensation domain in preventing misinitiation and timing of epimerization. Biochemistry 39, 10439–10447 (2000).
Belshaw, P. J., Walsh, C. T. & Stachelhaus, T. Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis. Science 284, 486–489 (1999).
Lu, L. et al. Mechanism of action of thalassospiramides, a new class of calpain inhibitors. Sci. Rep. 5, 8783 (2015).
Fischbach, M. A. & Clardy, J. One pathway, many products. Nat. Chem. Biol. 3, 353–355 (2007).
Firn, R. D. & Jones, C. G. Natural products—a simple model to explain chemical diversity. Nat. Prod. Rep. 20, 382–391 (2003).
Davidsen, J. M. & Townsend, C. A. In vivo characterization of nonribosomal peptide synthetases NocA and NocB in the biosynthesis of nocardicin A. Chem. Biol. 19, 297–306 (2012).
Thirlway, J. et al. Introduction of a non-natural amino acid into a nonribosomal peptide antibiotic by modification of adenylation domain specificity. Angew. Chem. Int. Ed. Engl. 51, 7181–7184 (2012).
Zhang, J. J., Yamanaka, K., Tang, X. & Moore, B. S. Direct cloning and heterologous expression of natural product biosynthetic gene clusters by transformation-associated recombination. Methods Enzymol. 621, 87–110 (2019).
Zhang, H., Fang, L., Osburne, M. S. & Pfeifer, B. A. The continuing development of E. coli as a heterologous host for complex natural product biosynthesis. Methods Mol. Biol. 1401, 121–134 (2016).
Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).
Tang, X. et al. Identification of thiotetronic acid antibiotic biosynthetic pathways by target-directed genome mining. ACS Chem. Biol. 10, 2841–2849 (2015).
Choi, K. H. & Schweizer, H. P. mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat. Protoc. 1, 153–161 (2006).
Davison, J. et al. Insights into the function of trans-acyl transferase polyketide synthases from the SAXS structure of a complete module. Chem. Sci. 5, 3081–3095 (2014).
Tanovic, A., Samel, S. A., Essen, L. O. & Marahiel, M. A. Crystal structure of the termination module of a nonribosomal peptide synthetase. Science 321, 659–663 (2008).
Dutta, S. et al. Structure of a modular polyketide synthase. Nature 510, 512–517 (2014).
Edwards, A. L., Matsui, T., Weiss, T. M. & Khosla, C. Architectures of whole-module and bimodular proteins from the 6-deoxyerythronolide B synthase. J. Mol. Biol. 426, 2229–2245 (2014).
Keatinge-Clay, A. Crystal structure of the erythromycin polyketide synthase dehydratase. J. Mol. Biol. 384, 941–953 (2008).
Akey, D. L. et al. Crystal structures of dehydratase domains from the curacin polyketide biosynthetic pathway. Structure 18, 94–105 (2010).
Al-Mestarihi, A. H. et al. Adenylation and S-methylation of cysteine by the bifunctional enzyme TioN in thiocoraline biosynthesis. J. Am. Chem. Soc. 136, 17350–17354 (2014).
Miller, B. R., Sundlov, J. A., Drake, E. J., Makin, T. A. & Gulick, A. M. Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases. Proteins 82, 2691–2702 (2014).
Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).
Sawitzke, J. A. et al. Recombineering: highly efficient in vivo genetic engineering using single-strand oligos. Methods Enzymol. 533, 157–177 (2013).
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.
The authors declare no competing interests.
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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 16, 42–49 (2020). https://doi.org/10.1038/s41589-019-0385-4
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