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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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.
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We thank C. Pennington at Rice University for assistance with LC–MS analysis.
The authors have filed a patent application (US patent application no. PCT/US2016/045037).
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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).
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.
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.
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.
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.
All amino acid sequences of thiolases superfamily used in Fig. 1b.
Statistical Source Data for Figs. 2b and 2d.
Statistical Source Data for Figs. 3b and 3d.
Statistical Source Data for Fig. 4c.
Statistical Source Data for Fig. 5c.
Statistical Source Data for Figs. 6a and 6d.
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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