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Engineering site-selective incorporation of fluorine into polyketides

Nature Chemical Biology volume 18, pages 886–893 (2022)Cite this article

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Abstract

Although natural products and synthetic small molecules both serve important medicinal functions, their structures and chemical properties are relatively distinct. To expand the molecular diversity available for drug discovery, one strategy is to blend the effective attributes of synthetic and natural molecules. A key feature found in synthetic compounds that is rare in nature is the use of fluorine to tune drug behavior. We now report a method to site-selectively incorporate fluorine into complex structures to produce regioselectively fluorinated full-length polyketides. We engineered a fluorine-selective trans-acyltransferase to produce site-selectively fluorinated erythromycin precursors in vitro. We further demonstrated that these analogs could be produced in vivo in Escherichia coli on engineering of the fluorinated extender unit pool. By using engineered microbes, elaborate fluorinated compounds can be produced by fermentation, offering the potential for expanding the identification and development of bioactive fluorinated small molecules.

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Fig. 1: Merging biological and chemical strategies to expand the scope of fluorinated small molecule production.
Fig. 2: Engineering of fluoromalonyl-CoA-specific trans-AT.
Fig. 3: In vitro generation of regioselectively fluorinated 6dEB analog.
Fig. 4: Cellular production of fluorinated polyketide model compounds by engineered E. coli.
Fig. 5: Regiospecific incorporation of fluorine into complex polyketides by E. coli production host.

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Source data are provided with this paper. All data associated with this study are contained in the published article or are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank the Pfeifer laboratory at University at Buffalo, The State University of New York for pBP130 and pBP144 plasmids. We thank J. Fang for helpful discussions as well as the C. Chang, Zhang and Francis laboratories at UCB for use of equipment. This work was supported by grant nos. NIH 1 R01 GM123181-01 (M.C.Y.C.) and NIH 1 R35 GM141799 (C.K.). S.S. and O.A. acknowledge the support of a National Institutes of Health NRSA Training grant 1 T32 GM066698. H.D. acknowledges the UC Berkeley Tang Distinguished Scholars Program for a postdoctoral fellowship. Instruments in the UC Berkeley College of Chemistry NMR Facility were supported in part by grant no. NIH S10OD024998.

Author information

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    Sasilada Sirirungruang, Swetha Ramesh, Joel L. Sax & Michelle C. Y. Chang

  2. Department of Chemistry, University of California, Berkeley, CA, USA

    Omer Ad, Hongjun Dong & Michelle C. Y. Chang

  3. Department of Chemistry, Stanford University, Stanford, CA, USA

    Thomas M. Privalsky & Chaitan Khosla

  4. Joint Bioenergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA

    Edward E. K. Baidoo & Bashar Amer

  5. Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Edward E. K. Baidoo & Bashar Amer

  6. Department of Energy, Agile BioFoundry, Emeryville, CA, USA

    Edward E. K. Baidoo

  7. Department of Chemical Engineering, Stanford University, Stanford, CA, USA

    Chaitan Khosla

  8. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA

    Michelle C. Y. Chang

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  1. Sasilada Sirirungruang
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Contributions

S.S. performed in vivo polyketide production experiments, enzyme characterization experiments, physiological experiments and construction of DNAs with S.R. O.A. synthesized TKL standards and carried out DszAT library generation and screening with J.L.S. T.M.P. and S.S. jointly performed in vitro polyketide production experiments. H.D. assisted with construction of DNAs. E.E.K.B. and B.A. assisted with high-resolution fragmentation analysis of 6dEB and its analogs. M.C.Y.C. and C.K. administered the project. S.S. and M.C.Y.C. wrote the paper with contributions from all authors. All authors designed experiments and analyzed data.

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Correspondence to Michelle C. Y. Chang.

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Nature Chemical Biology thanks Constance Bailey and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 trans-AT library screening.

(A) Lysate of E. coli expressing following DszAT mutants were screened through the triketide lactone production assay with purified Mod3DEBS + TE(AT0) protein, malonate or fluoromalonate extender unit, malonate-coeznzyme A ligase MatB and NDK-SNAC: 1, F190A; 2, F190C; 3, F190D; 4, F190E; 5, F190G; 6, F190H; 7, F190I; 8, F190K; 9, F190L; 10, F190M; 11, F190N; 12, F190P; 13, F190Q; 14, F190R; 15, F190S; 16, F190T; 17, F190V; 18, F190W; 19, F190Y; 21, F190G L87V; 22, F190G L87A; 23, F190I L87V; 24, F190I L87A; 25, F190P L87A; 26, F190S L87V; 27, F190S L87A; 28, F190T L87V; 29, F190T L87A; 30, F190V L87V; 31, F190V L87A. Product formation was monitored by LC-QQQ using negative ionization mode (transition): F-TKL (173→59), H-TKL (155→97), H-TTKP (197→95), TKL (169→111). The amounts of F-TKL (red), H-TKL (black), and H-TTKP (gray) products were determined by integrating extracted ion counts for the relevant species and are shown normalized by the amount of corresponding product produced by wild-type enzyme. H-TTKP arises from two chain extensions with malonyl-CoA. Mutants that showed potentially increased selectivity towards fluoromalonyl-CoA (underlined) compared to wild-type enzyme were selected for in vitro screening. (B) Selected DszAT mutants from lysate screen (F190G, F190T, F190V, F190P L87A and F190I L87A) were overexpressed, purified and assessed through in vitro triketide lactone experiment with purified Mod3DEBS + TE(AT0) protein, MatB and NDK-SNAC for the production of H-TKL and H-TTKP from malonyl-CoA (top panels), F-TKL from fluoromalonyl-CoA (bottom left) and TKL from methylmalonyl-CoA (bottom right). The amounts of products were determined as in (A). Data are mean of two technical replicates.

Source data

Extended Data Fig. 2 Characterization of DszAT F190V.

(A) Steady-state kinetic analysis of transacylation reaction of malonyl-CoA (left), methylmalonyl-CoA (middle) and fluoromalonyl-CoA (right) by wild-type (black) and F190V (red) DszAT with 75 uM of ACPDEBSMod6 as acyl accepter. Transacylation activity was measured by α-ketoglutarate dehydrogenase coupled assay monitoring CoA release. The dose-response curves for transacylation of methylmalonyl-CoA by WT and F190V DszAT were fit with Michaelis-Menten equation. The dose-response curves for WT and F190V DszAT transacylation with malonyl- and fluoromalonyl-CoA exhibited sigmoidal behavior with evidence of inhibition at high substrate concentrations and were therefore fit with the Hill equation modified for substrate inhibition. Data shown are mean ± s.d. of three technical replicates. (B) Steady-state kinetic analysis of hydrolysis reaction of fluoromalonyl-CoA by wild-type and F190V DszAT. Hydrolytic activity was measured by DTNB assay monitoring CoA release. The dose-response curves were obtained from non-linear fitting of data to Michaelis-Menten equation. Data points are mean ± s.d. of three technical replicates.

Source data

Extended Data Fig. 3 Generation of 2-fluoro-2-desmethyl-6dEB analog through in vitro reconstitution of Mod6 AT0 DEBS.

(A) Extracted ion chromatograms showing exact mass of in vitro production of monofluorinated desmethyl 6dEB by Mod6 AT0 DEBS complemented with WT DszAT (left) or F190V DszAT (right). Reactions containing 2 μM each of LDDDEBS, Mod1DEBS, Mod2DEBS, DEBS2 and DEBS3(Mod6 AT0), 6 μM WT or F190V DszAT, 2 mM methylmalonate and when used 10 mM fluoromalonate (+Fmal, red) were analyzed by LC-QTOF. Chromatograms are representative of at least three technical replicates. (B) Fragmentation pattern of the monofluorinated desmethyl 6dEB analog produced by Mod6 AT0 DEBS complemented by WT DszAT or F190V DszAT is consistent with 2-fluoro-2-desmethyl 6dEB, which is expected when fluoromalonyl-CoA is incorporated by module 6. Fragmentation pattern shows presence of ions in A, B and D families (Supplementary Fig. 5) with mass shifts according to a substitution of -CH3 with -F group on carbon 2, 4, 6 or 8 of 6dEB. Observed loss of HF from daughter ions expected to contain fluorine substitution further supports the presence of fluorine in the molecule. Notably, the most prominent fragment in 6dEB MS/MS spectrum, the C ion, was entirely missing in the MS/MS spectrum of the monofluorinated desmethyl 6dEB analog39. This lack of detectable fragment prevents narrowing down the definite location of the fluorine substitution. However, the suppression of ion C was similarly observed when malonate was provided to the system in place of fluoromalonate (Supplementary Fig. 6) and is consistent with the reactivity of 6dEB analog with a modification near the C3 position. The lack of methyl substituent at C2 as in 2-fluoro-2-methyl 6dEB would reduce the reactivity of A ion with respect to the hydride shift from C3 to C9 necessary to form C ion. Spectra are representative of at least three technical replicates (nd, not detected). (C) Relative amounts of 2-fluoro-2-desmethyl 6dEB and 6dEB by complementation of Mod6 AT0 DEBS by WT or F190V DszAT. The amounts of products were determined by integrating extracted ion counts for the relevant species monitored by LC-QQQ (transition): 6dEB (369.2→239.1) and 2-fluoro-2-desmethyl 6dEB (373.2 → 275.1). Although the absolute amounts of the two observed products vary among replicates; F190V DszAT reactions consistently produce higher ratio of fluorinated to non-fluorinated product than WT DszAT.

Source data

Extended Data Fig. 4 Generation of 4-fluoro-4-desmethyl-6dEB analog through in vitro reconstitution of Mod5 AT0 DEBS.

(A) Extracted ion chromatograms showing exact mass of in vitro production of monofluorinated desmethyl 6dEB by Mod5 AT0 DEBS complemented with WT DszAT (left) or F190V DszAT (right). Reactions containing 2 μM each of LDDDEBS, Mod1DEBS, Mod2DEBS, DEBS2 and DEBS3(Mod5 AT0), 6 μM WT or F190V DszAT, 2 mM methylmalonate and when used 10 mM fluoromalonate (+Fmal, red) were analyzed by LC-QTOF. Chromatograms are representative of at least three technical replicates. (B) Fragmentation pattern of the monofluorinated desmethyl 6dEB analog produced by Mod5 AT0 DEBS complemented by WT DszAT or F190V DszAT is consistent with 4-fluoro-4-desmethyl 6dEB, which is expected when fluoromalonyl-CoA is incorporated by Mod5. The observed daughter ion masses are necessarily similar to those of 2-fluoro-2-desmethyl 6dEB observed in Mod6 AT0 DEBS system, due to fluorine substitution in similar positions. In short, mass shifts according to a substitution of -CH3 with -F group on carbon 2, 4, 6 or 8 of 6dEB, loss of HF from daughter ions and absence of C ion are observed. Spectra are representative of at least three technical replicates (nd, not detected). (C) Relative amounts of 4-fluoro-4-desmethyl 6dEB and 6dEB by complementation of Mod5 AT0 DEBS by WT or F190V DszAT. The amounts of products were determined by integrating extracted ion counts for the relevant species monitored by LC-QQQ (transition): 6dEB (369.2 → 239.1) and 2-fluoro-2-desmethyl 6dEB (373.2 → 275.1).

Source data

Extended Data Fig. 5 Sequence and structural analysis of E. coli FabD.

(A) Sequence comparison between FabD and DszAT. Conserved functional residues are shown in red; specificity-determining residue are shown in blue; and ACP-interacting residues are shown in green. (B) Structural comparison between Fab (blue) and DszAT (grey). The two proteins have the same overall structure (left, rmsd = 1.7 Å), active site architecture (middle) and ACP-interacting surface (right). Both proteins contain two subdomains with the active site in between. The substrate-binding pocket is largely polar. In both structures, malonate is bound by salt bridge interactions between its β-carboxy group and the side chain amine groups of R117/R111, positioning the carbonyl carbon for nucleophilic attack near the catalytic serine S84/S92, which is found in nearly identical position in both proteins. Notably, in DszAT, F190 is found positioned near the Cα of malonate that may prevent binding of substrate with bulkier substituent such as a methyl group at the α-position. In FabD, this residue is replaced with S200, which may provide more flexibility in terms of substrate binding. Interactions between AT and ACP domains has previously been shown to rely on a small number of charged amino acid residues located near the active sites of the two protein domains40,54. R278 and R279 of DszAT interact with E70 of its native protein partner, DSZS ACP1. Similarly, K180 of DszAT interacts with D45 of DSZS ACP1. Structural alignment shows that these positively charged interface residues of DszAT, R278, R279 and K180, correspond to K276, R277 and R190 of FabD, maintaining the charge at the interface for interaction with an ACP partner. Active site, specificity-determining, and interface residues are shown in sticks.

Extended Data Fig. 6 Characterization of E. coli FabD transacylation activity with the DEBS PKS.

(A) Steady-state kinetic characterization of transacylation reaction of malonyl-CoA (left), methylmalonyl-CoA (middle) and fluoromalonyl-CoA (right) catalyzed by FabD with 75 μM of ACPDEBSMod6 as acyl accepter. Activity was measured by α-ketoglutarate dehydrogenase coupled assay, monitoring coenzyme A release by NADH fluorescence. Curves were obtained from non-linear curve fitting of data to Hill equation modified for substrate inhibition model. Table contains kcat, K0.5, kcat/K0.5, Ki and n values. Data shown are mean ± s.d of three technical replicates. Error in kcat/KM is obtained by propagation from individual kinetic terms. (B) In vitro triketide lactone assay for FabD complementation of Mod3DEBS + TE(AT0). Normalized representation of the amount of F-TKL (left), H-TKL (middle) and TKL (right) products are shown for reactions containing 10 μM Mod3DEBS + TE(AT0)), 30 μM FabD or WT DszAT, 1 mM NDK-SNAC, 1 mM fluoromalonate (left), malonate (middle) or methylmalonate (right), and 1 mM coenzyme A under regenerative condition. Reactions were quenched and analyzed by LC-QQQ after 24 h. The amounts of products were determined by integrating extracted ion counts for the relevant species (transition): F-TKL (175 → 157), H-TKL (157 → 139) and TKL (171 → 153). Data are mean of two technical replicates.

Source data

Extended Data Fig. 7 Influence of extender unit availability on the in vivo selectivity of chain elongation by single-modular DEBS constructs in engineered E. coli.

Concentrated cell suspension of E. coli BAP1 expressing Mod3DEBS + TE(AT0) (left) or Mod6DEBS + TE(AT0) (right), MatB and MadLM were provided with 1 mM NDK-SNAC and 5 mM fluoromalonate, malonate and/or methylmalonate and analyzed by LC-QQQ after 24 h. The concentrations of products were determined by integrating extracted ion counts for the relevant species (transition): F-TKL (175 → 157), H-TKL (157 → 139) and TKL (171 → 153), and comparing to external standard curves generated using synthetic standards of the molecules. Data revealed that the product profile follows the corresponding precursor profile, suggesting that the outcome is mainly governed by the provided precursors. Data are mean ± s.d. of three biological replicates.

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Extended Data Fig. 8 In vivo production of desmethyl 6dEB analogs by engineered E. coli.

(A) Extracted ion chromatograms showing the production of 6dEB by E. coli expressing DEBS, Mod5 AT0 DEBS, or Mod6 AT0 DEBS. E. coli BAP1 harboring variants of pBP130 and pBP144 were grown to OD600 = 0.4–0.6 at 37 °C with shaking at 200 rpm, at which point protein expression was induced with 1 mM IPTG and 0.2% arabinose and cultures provided with 20 mM sodium propionate. Cultures were then incubated with substrates at 22 °C with shaking at 250 rpm for 1 day, after which, culture media were collected, extracted and analyzed. 6dEB product was monitored by LC-QQQ using transition 369.1→239.1. Chromatograms are representative of at least three biological replicates. (B) Extracted ion chromatograms showing the production of desmethyl-6dEB analogs by E. coli expressing DEBS, Mod5 AT0 DEBS, or Mod6 AT0 DEBS. Samples were prepared as in (A). Desmethyl-6dEB products were monitored by LC-QQQ using transition: 355.2→225.1. Chromatograms are representative of at least three biological replicates. (C) Fragmentation patterns of the desmethyl 6dEB analogs produced by E. coli harboring Mod5 AT0 DEBS (top) or Mod6 AT0 DEBS (bottom). The observed patterns are consistent with those of the desmethyl-6dEB analogs produced by in vitro reconstitution of corresponding enzyme systems. Spectra are representative of at least three biological replicates (nd, not detected).

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Extended Data Fig. 9 Growth curves of E. coli BAP1 expressing pFmal plasmids.

Various E. coli BAP1 strains were grown to OD600 ~ 0.4 at 37 °C with shaking at 200 rpm, at which point 100 μL of culture was transferred to 96-well plate and induced with 1 mM IPTG/0.2% (w/v) arabinose. The no plasmid control strain (left), strain expressing only the MadLM transporter (middle) and strain expressing the MadLM transporter and MatB (right) were supplemented with 20 mM sodium propionate, 5 mM malonate or 5 mM fluoromalonate as noted in the legend. Cell growth was monitored at OD600 using a microplate reader. Data are representative of 3 biological replicates.

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Extended Data Fig. 10 In vivo production of monofluorinated desmethyl 6dEB analogs by engineered E. coli.

(A) Fragmentation pattern and tabulated masses of daughter ions of monofluorinated desmethyl 6dEB analog produced by E. coli expressing Mod5 AT0 DEBS (top)or Mod6 AT0 DEBS (bottom). The observed patterns are consistent with those of the monofluorinated desmethyl-6dEB analogs produced by in vitro reconstitution of corresponding enzyme systems. Spectra are representative of at least three biological replicates. (B) 19F-NMR spectrum of 2-fluoro-2-desmethyl 6dEB isolated from culture media extract of E. coli expressing Mod6 AT0 DEBS, MatB and MadLM. Concentrated cell suspension was provided with 5 mM fluoromalonate and 20 mM propionate and incubated at 22 °C with shaking at 250 rpm. After 24 h incubation, culture medium was extracted with ethyl acetate, dried via rotary evaporation. 2-fluoro-2-desmethyl 6dEB was then purified from ethyl acetate extract of culture medium through HPLC and fractions were screened using LC-QQQ. Fractions showing presence of 2-fluoro-2-desmethyl 6dEB with transition 373.2 → 275.1 were combined, lyophilized and resuspended in 500 µL of 50:50 MeOH:D2O mixture for 19F-NMR analysis. Spectrum was collected on Bruker AV600 with following parameters: o1p = −200, sw = 200, d1 = 1 s, d0 -20, n = 3200. 5-fluorouracil (1 mM) was used as internal standard. Spectrum reveals a set of signals between -195 and -196 ppm displaying a doublet of doublet splitting pattern (J = 54, 12 Hz) consistent with the β-hydroxy-α-fluoro-carbonyl motif expected of 2-fluoro-2-desmethyl 6dEB. The observed chemical shift value is similar to that observed of other compounds with similar α-fluoro-β-hydroxy ester motif55. Based on vicinal coupling constant (3JF-H) and known (S)-orientation of hydroxy group on carbon 3, the observed molecule is assigned as 2-(R)-fluoro-2-desmethyl 6dEB.

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Supplementary information

Supplementary Information

Supplementary Tables 1–3, Figs. 1–7 and Notes 1 and 2.

Reporting Summary

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Supplementary Data

NMR FID Data for Extended Data Fig. 10.

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Sirirungruang, S., Ad, O., Privalsky, T.M. et al. Engineering site-selective incorporation of fluorine into polyketides. Nat Chem Biol 18, 886–893 (2022). https://doi.org/10.1038/s41589-022-01070-y

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