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A polyketoacyl-CoA thiolase-dependent pathway for the synthesis of polyketide backbones

Abstract

Polyketides found in nature originate from backbones synthesized through iterative decarboxylative Claisen condensations catalysed by polyketide synthases (PKSs). However, PKSs suffer from complicated architecture, energy inefficiencies, complex regulation, and competition with essential metabolic pathways for extender unit malonyl-CoA, all combining to limit the flux of polyketide biosynthesis. Here we show that certain thiolases, which we term polyketoacyl-CoA thiolases (PKTs), catalyse polyketide backbone formation via iterative non-decarboxylative Claisen condensations, hence offering a synthetic and efficient alternative to PKSs. We show that PKTs can synthesize polyketide backbones for representative lactone, alkylresorcinolic acid, alkylresorcinol, hydroxybenzoic acid and alkylphenol polyketide families, and elucidate the basic catalytic mechanism and structural features enabling this previously unknown activity. PKT-catalysed reactions offer a route to polyketide formation that leverages the simple architecture of thiolases to achieve higher ATP efficiencies and reduced competition with essential metabolic pathways, all of which circumvent intrinsic inefficiencies of PKSs for polyketide product synthesis.

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Fig. 1: Synthesis of polyketide backbones through polyketide synthases and polyketoacyl-CoA thiolases.
Fig. 2: Synthesis of polyketide triacetic acid lactone through the proposed PKT pathway.
Fig. 3: Formation of ORA and orcinol through the PKT-based pathway.
Fig. 4: Synthesis of polyketides 6-MSA and m-cresol.
Fig. 5: Probing the basic catalytic mechanism of PKT activity.
Fig. 6: Probing structural determinants of PKT activity.

Data availability

The authors declare that data supporting the findings of this study are available within the paper and its Supplementary Information. Supplementary Table 3 provides a list of the GenBank accession numbers of the 17 key enzymes used in this study. Data for Figs. 1–6 and Extended Data Fig. 6 are available as Source Data with this paper. All other data are available from the authors on reasonable request.

References

  1. 1.

    Hertweck, C. The biosynthetic logic of polyketide diversity. Angew. Chem. 48, 4688–4716 (2009).

    CAS  Google Scholar 

  2. 2.

    Ma, S. M. et al. Complete reconstitution of a highly reducing iterative polyketide synthase. Science 326, 589–592 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Yu, D. Y., Xu, F. C., Zeng, J. & Zhan, J. X. Type III polyketide synthases in natural product biosynthesis. IUBMB Life 64, 285–295 (2012).

    CAS  PubMed  Google Scholar 

  4. 4.

    Bond, C., Tang, Y. & Li, L. Saccharomyces cerevisiae as a tool for mining, studying and engineering fungal polyketide synthases. Fungal Genet. Biol. 89, 52–61 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Staunton, J. & Weissman, K. J. Polyketide biosynthesis: a millennium review. Nat. Prod. Rep. 18, 380–416 (2001).

    CAS  PubMed  Google Scholar 

  6. 6.

    Shen, B. Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms. Curr. Opin. Chem. Biol. 7, 285–295 (2003).

    CAS  PubMed  Google Scholar 

  7. 7.

    Huo, L. et al. Heterologous expression of bacterial natural product biosynthetic pathways. Nat. Prod. Rep. 36, 1412–1436 (2019).

    CAS  PubMed  Google Scholar 

  8. 8.

    Pfeifer, B. A. & Khosla, C. Biosynthesis of polyketides in heterologous hosts. Microbiol. Mol. Biol. Rev. 65, 106–118 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Lowry, B. et al. In vitro reconstitution and analysis of the 6-deoxyerythronolide B synthase. J. Am. Chem. Soc. 135, 16809–16812 (2013).

    CAS  PubMed  Google Scholar 

  10. 10.

    Chan, Y. A., Podevels, A. M., Kevany, B. M. & Thomas, M. G. Biosynthesis of polyketide synthase extender units. Nat. Prod. Rep. 26, 90–114 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Brownsey, R., Boone, A., Elliott, J., Kulpa, J. & Lee, W. Regulation of acetyl-CoA carboxylase. Biochem. Soc. Trans. 34, 223–227 (2006).

    CAS  PubMed  Google Scholar 

  12. 12.

    Yan, D. et al. Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria. Proc. Natl Acad. Sci. USA 115, 9835–9844 (2018).

    Google Scholar 

  13. 13.

    Keatinge-Clay, A. T. The structures of type I polyketide synthases. Nat. Prod. Rep. 29, 1050–1073 (2012).

    CAS  PubMed  Google Scholar 

  14. 14.

    Jiang, C., Kim, S. Y. & Suh, D. Y. Divergent evolution of the thiolase superfamily and chalcone synthase family. Mol. Phylogenetetics Evol. 49, 691–701 (2008).

    CAS  Google Scholar 

  15. 15.

    Austin, M. B. & Noel, A. J. P. The chalcone synthase superfamily of type III polyketide synthases. Nat. Prod. Rep. 20, 79–110 (2003).

    CAS  PubMed  Google Scholar 

  16. 16.

    Haapalainen, A. M., Merilainen, G. & Wierenga, R. K. The thiolase superfamily: condensing enzymes with diverse reaction specificities. Trends Biochem. Sci. 31, 64–71 (2006).

    CAS  PubMed  Google Scholar 

  17. 17.

    Tang, S. Y. et al. Screening for enhanced triacetic acid lactone production by recombinant Escherichia coli expressing a designed triacetic acid lactone reporter. J. Am. Chem. Soc. 135, 10099–10103 (2013).

    CAS  PubMed  Google Scholar 

  18. 18.

    Campobasso, N. et al. Staphylococcus aureus 3-hydroxy-3-methylglutaryl-CoA synthase: crystal structure and mechanism. J. Biol. Chem. 279, 44883–44888 (2004).

    CAS  PubMed  Google Scholar 

  19. 19.

    Duncombe, G. R. & Frerman, F. E. Molecular and catalytic properties of the acetoacetyl-coenzyme A thiolase of Escherichia coli. Arch. Biochem. Biophys. 176, 159–170 (1976).

    CAS  PubMed  Google Scholar 

  20. 20.

    Yang, S. Y., Yang, X. Y. H., Healylouie, G., Schulz, H. & Elzinga, M. Nucleotide-sequence of the fadA Gene—primary structure of 3-ketoacyl-coenzyme-A thiolase from Escherichia coli and the structural organization of the fadAB operon. J. Biol. Chem. 265, 10424–10429 (1990).

    CAS  PubMed  Google Scholar 

  21. 21.

    Teufel, R. et al. Bacterial phenylalanine and phenylacetate catabolic pathway revealed. Proc. Natl Acad. Sci. USA 107, 14390–14395 (2010).

    CAS  PubMed  Google Scholar 

  22. 22.

    Harwood, C. S., Nichols, N. N., Kim, M. K., Ditty, J. L. & Parales, R. E. Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate. J. Bacteriol. 176, 6479–6488 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Cheong, S., Clomburg, J. M. & Gonzalez, R. Energy- and carbon-efficient synthesis of functionalized small molecules in bacteria using non-decarboxylative Claisen condensation reactions. Nat. Biotechnol. 34, 556–561 (2016).

    CAS  PubMed  Google Scholar 

  24. 24.

    Parke, D., Garcia, M. A. & Ornston, L. N. Cloning and genetic characterization of dca genes required for β-oxidation of straight-chain dicarboxylic acids in Acinetobacter sp. strain ADP1. Appl. Environ. Microbiol. 67, 4817–4827 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Slater, S. et al. Multiple β-ketothiolases mediate poly(β-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha. J. Bacteriol. 180, 1979–1987 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Shanks, B. H. & Keeling, P. L. Bioprivileged molecules: creating value from biomass. Green. Chem. 19, 3177–3185 (2017).

    CAS  Google Scholar 

  27. 27.

    Noor, E. et al. Pathway thermodynamics highlights kinetic obstacles in central metabolism. PLoS Comput. Biol. 10, e1003483 (2014).

    PubMed  PubMed Central  Google Scholar 

  28. 28.

    Xie, D. et al. Microbial synthesis of triacetic acid lactone. Biotechnol. Bioeng. 93, 727–736 (2006).

    CAS  PubMed  Google Scholar 

  29. 29.

    Johnson, A. O. et al. Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factories. Metab. Eng. 44, 253–264 (2017).

    CAS  PubMed  Google Scholar 

  30. 30.

    Qian, S., Clomburg, J. M. & Gonzalez, R. Engineering Escherichia coli as a platform for the in vivo synthesis of prenylated aromatics. Biotechnol. Bioeng. 116, 1116–1127 (2019).

    CAS  PubMed  Google Scholar 

  31. 31.

    Collie, N. & Myers, W. V. I. I. The formation of orcinol and other condensation products from dehydracetic acid. J. Chem. Soc. Trans. 63, 122–128 (1893).

    CAS  Google Scholar 

  32. 32.

    Gagne, S. J. et al. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides. Proc. Natl Acad. Sci. USA 109, 12811–12816 (2012).

    CAS  PubMed  Google Scholar 

  33. 33.

    Tan, Z., Clomburg, J. M. & Gonzalez, R. Synthetic pathway for the production of olivetolic acid in Escherichia coli. ACS Synth. Biol. 7, 1886–1896 (2018).

    CAS  PubMed  Google Scholar 

  34. 34.

    Lim, Y. P., Go, M. K. & Yew, W. S. Exploiting the biosynthetic potential of type III polyketide synthases. Molecules 21, 806 (2016).

    PubMed Central  Google Scholar 

  35. 35.

    Zhou, W. et al. Biosynthesis of phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone in Escherichia coli from glucose. Microb. Cell Fact. 15, 149 (2016).

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Huang, L., Xue, Z. & Zhu, Q. Method for the production of resveratrol in a recombinant bacterial host cell. US patent WO2006124999A2 (2007).

  37. 37.

    Choi, O. et al. Biosynthesis of plant-specific phenylpropanoids by construction of an artificial biosynthetic pathway in Escherichia coli. J. Ind. Microbiol. Biotechnol. 38, 1657–1665 (2011).

    CAS  PubMed  Google Scholar 

  38. 38.

    Mizuuchi, Y. et al. Novel type III polyketide synthases from Aloe arborescens. FEBS J. 276, 2391–2401 (2009).

    CAS  PubMed  Google Scholar 

  39. 39.

    Fujii, I. Functional analysis of fungal polyketide biosynthesis genes. J. Antibiot. 63, 207–218 (2010).

    CAS  PubMed  Google Scholar 

  40. 40.

    Moriguchi, T., Kezuka, Y., Nonaka, T., Ebizuka, Y. & Fujii, I. Hidden function of catalytic domain in 6-methylsalicylic acid synthase for product release. J. Biol. Chem. 285, 15637–15643 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Sabatini, M. et al. Biochemical characterization of the minimal domains of an iterative eukaryotic polyketide synthase. FEBS J. 285, 4494–4511 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Crawford, J. M. et al. Structural basis for biosynthetic programming of fungal aromatic polyketide cyclization. Nature 461, 1139–1243 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Shen, B. & Hutchinson, C. R. Deciphering the mechanism for the assembly of aromatic polyketides by a bacterial polyketide synthase. Proc. Natl Acad. Sci. USA 93, 6600–6604 (1996).

    CAS  PubMed  Google Scholar 

  44. 44.

    Kim, E. J., Son, H. F., Kim, S., Ahn, J. W. & Kim, K. J. Crystal structure and biochemical characterization of β-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16. Biochem. Biophys. Res. Commun. 444, 365–369 (2014).

    CAS  PubMed  Google Scholar 

  45. 45.

    Jez, J. M. et al. Structural control of polyketide formation in plant-specific polyketide synthases. Chem. Biol. 7, 919–930 (2000).

    CAS  PubMed  Google Scholar 

  46. 46.

    Blaisse, M. R., Fu, B. & Chang, M. C. Y. Structural and biochemical studies of substrate selectivity in Ascaris suum thiolases. Biochemistry 57, 3155–3166 (2018).

    CAS  PubMed  Google Scholar 

  47. 47.

    Clomburg, J. M. et al. Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain omega-functionalized carboxylic acids. Metab. Eng. 28, 202–212 (2015).

    CAS  PubMed  Google Scholar 

  48. 48.

    Dellomonaco, C., Clomburg, J. M., Miller, E. N. & Gonzalez, R. Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355–359 (2011).

    CAS  PubMed  Google Scholar 

  49. 49.

    Kim, S., Clomburg, J. M. & Gonzalez, R. Synthesis of medium-chain length (C6-C10) fuels and chemicals via β-oxidation reversal in Escherichia coli. J. Ind. Microbiol. Biotechnol. 42, 465–475 (2015).

    CAS  PubMed  Google Scholar 

  50. 50.

    Krivoruchko, A., Zhang, Y., Siewers, V., Chen, Y. & Nielsen, J. Microbial acetyl-CoA metabolism and metabolic engineering. Metab. Eng. 28, 28–42 (2015).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank C. Pennington at Rice University for assistance with LC–MS analysis.

Author information

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Authors

Contributions

R.G. designed the research. Z.T., S.C. and J.M.C. performed the research. S.Q. performed structure simulation and alignment. Z.T., J.M.C. and R.G. wrote the paper.

Corresponding author

Correspondence to Ramon Gonzalez.

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The authors have filed a patent application (US patent application no. PCT/US2016/045037).

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Extended data

Extended Data Fig. 1 Reactions catalysed by members of the thiolase superfamily and structures of corresponding enzymes.

a, Reactions catalysed by members of the thiolase superfamily; (b) Three-dimensional structures of corresponding enzymes (monomer). The structures shown are those of Medicago sataiva chalcone synthase PKS III (1bi5), Streptomcyes albus ketosynthase domain of polyketide synthase KS/PKS I (4wky), Streptomyces coelicolor KS/PKS II (1tqy), E. coli β-ketoacyl-acyl-carrier-protein synthase KAS I (2vb8), E. coli KAS II (1kas), E. coli KAS III (1hn9), Arabidopsis thaliana KCS (modelling structure), human mitochondria thiolase I (4c2j), E. coli thiolase II (5f0v), and Bacillus subtilis HMGS (4yxq).

Extended Data Fig. 2 Purified enzymes used in this study.

The sources of enzymes are as follows. PKS III 2-PS was from Gerbera hybrid; DcaF was from Acinetobacter sp. ADP1; FadAx was from Pseudomonas putida; PcaF was from Pseudomonas putida; BktB was from Ralstonia eutropha; AtoB was from E. coli; EcFadA was from E. coli; PaaJ was from E. coli; HMGS was from Staphylococcus aureus; FadB was from E. coli; PaaH was from E. coli; The expected molecular weights of enzymes with N terminal 6 x His-tag calculated by tool from ExPASy, (https://web.expasy.org/compute_pi/) are indicated in parentheses. BktB (5#, 11#, 16#), PaaJ (8#, 14#) and EcFadA (7#, 12#) appeared more than once as we initially employed them for in vitro TAL biosynthesis tests (Fig. 2), and then we further purified and used them for the in vitro assay of ketoacyl-CoA thiolase, 3-ketoadipyl-CoA thiolase and PKT activities (see Fig. 6a). For EcFadA WT (7#, 12#) and EcFadA_BktB (21#), the expected MWs from ExPASy calculations are 41.9 kDa and 43.2 kDa, while actual sizes on SDS-PAGE appear ~40 kDa and ~41 kDa.

Extended Data Fig. 3 Primary sequence alignment of thiolases and PKS III.

a, Primary sequence alignment of PKS III 2-PS (2-pyrone synthase from Gerbera hybrid) with AtoB (from E. coli). The sequence identity is only 10.0%; (b) Primary sequence alignment of PKS III 2-PS with FadAx (from Pseudomonas putida). The sequence identity is only 8.7%; (c) Primary sequence alignment of PKS III 2-PS with BktB (from Ralstonia eutropha). The sequence identity is only 13.6%. d, Primary sequence alignment of PKS III ORS (from Rhododendron dauricum) with BktB. The sequence identity is only 9.9%.

Extended Data Fig. 4 Max-min Driving Force (MDF) for the synthesis of specified polyketides from acetyl-CoA through the proposed PKT-based pathway.

MDF calculations assume minimum and maximum metabolite concentrations of 0.000001 and 0.01 M, respectively, and a fixed NADPH/NADP + ratio of 10.

Extended Data Fig. 5 Summary of SDS-PAGE analysis of samples in this study.

a, SDS-PAGE analysis of the soluble expression of BktB and 2-PS in JC01 (DE3) ΔatoB host strain. M: marker; 1: purified BktB; 2: purified 2-PS; 3: soluble fraction of JC01 (DE3) ΔatoB expressing pCDF-bktB, the arrow indicates the soluble BktB protein; 4: soluble fraction of JC01 (DE3) ΔatoB harbouring pCDF-2-ps, the arrow indicates the soluble 2-PS protein. b, SDS-PAGE analysis of the soluble expression of BktB WT and C90S, H350A, C380S mutants. M: marker; 1: soluble fraction of BL21 (DE3) expressing BktB WT; 2: soluble fraction of BL21 (DE3) expressing BktB C90S; 3: soluble fraction of BL21 (DE3) expressing BktB H350A; 4: soluble fraction of BL21 (DE3) expressing BktB C380S. c, SDS-PAGE analysis of the soluble expression of EcFadA and BktB mutants after segment swapping. M: marker; 1: soluble fraction of BL21 (DE3) expressing EcFadA WT; 2: soluble fraction of BL21 (DE3) expressing EcFadA_BktB; 3: soluble fraction of BL21 (DE3) expressing BktB WT; 4: soluble fraction of BL21 (DE3) expressing BktB_EcFadA.

Extended Data Fig. 6 Assessment of malonyl-CoA availability through the flaviolin biosynthesis pathway.

Upper, flaviolin biosynthesis pathway: 5 molecules of malonyl-CoA are condensed by RppA to form flaviolin, which has a specific absorbance at 340 nm. Bottom, RppA was expressed with different MCS/ACC enzymes for characterization of malonyl-CoA availability in strain JC01 (DE3) ΔatoB. Engineered E. coli strains were cultured in LB-like MOPS medium + 2% (wt/v) glycerol. For strains harbouring MCS, 12 mM malonate sodium was also included. Error bars represent s.d. calculated from at least three biological replicates.

Source data

Extended Data Fig. 7 Mass spectrometry (MS) information of the samples.

a, MS information of orcinol standard and orcinol sample extracted from in vivo samples; (b) MS information of ORA standard and ORA sample extracted from in vivo samples; (c) MS of 6-MSA standard and 6-MSA sample extracted from in vitro samples. d, MS of m-cresol standard and m-cresol sample extracted from in vitro samples.

Extended Data Fig. 8 The proposed PKT-based platforms for aloesone, octaketide SEK4, norsolorinate and tetracenomycin F1 biosynthesis.

a, The proposed PKT-based platform for aloesone biosynthesis. b, The proposed PKT-based platform for SEK4 biosynthesis. c, The proposed PKT-based platform for norsolorinate biosynthesis. d, The proposed PKT-based platform for tetracenomycin F1 biosynthesis.

Extended Data Fig. 9 Catalytic mechanism of polyketoacyl-CoA thiolase (PKT) and representative PKS III 2-PS.

Cys/His/Cys is the catalytic triad of PKT activity, enabling a two-step, ping-pong mechanism for the thiolytic condensation reaction using substrates with multiple keto-groups (for example acetoacetyl-CoA). Notably, this catalytic mechanism is distinct from that of PKS III, where the core catalytic triad is Cys/His/Asn, with Asn (asparagine) playing a critical role in catalysing malonyl-CoA decarboxylation and stabilizing the condensation transition state.

Extended Data Fig. 10 Exploiting PKT to synthesize additional PK backbones and corresponding PKs (R = groups other than CH3).

Synthesis of polyketide backbones for representative lactone, alkylresorcinolic acid, and alkylresorcinol polyketide families through up to 3 rounds of iterative non-decarboxylative Claisen condensations catalyzed by PKTs.

Supplementary information

Supplementary Information

Supplementary Tables 1–4.

Reporting Summary

Source data

Source Data Fig. 1

All amino acid sequences of thiolases superfamily used in Fig. 1b.

Source Data Fig. 2

Statistical Source Data for Figs. 2b and 2d.

Source Data Fig. 3

Statistical Source Data for Figs. 3b and 3d.

Source Data Fig. 4

Statistical Source Data for Fig. 4c.

Source Data Fig. 5

Statistical Source Data for Fig. 5c.

Source Data Fig. 6

Statistical Source Data for Figs. 6a and 6d.

Source Data Extended Data Fig. 6

Statistical Source Data for Extended Fig. 6.

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Tan, Z., Clomburg, J.M., Cheong, S. et al. A polyketoacyl-CoA thiolase-dependent pathway for the synthesis of polyketide backbones. Nat Catal 3, 593–603 (2020). https://doi.org/10.1038/s41929-020-0471-8

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