Polyketide Bioderivatization Using the Promiscuous Acyltransferase

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Letter

Polyketide bio-derivatization using the promiscuous acyltransferase KirCII Ewa Maria Musiol-Kroll, Florian Zubeil, Thomas Schafhauser, Thomas Härtner, Andreas Kulik, John B. McArthur, Irina Koryakina, Wolfgang Wohlleben, Stephanie Grond, Gavin J. Williams, Sang Yup Lee, and Tilmann Weber ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00341 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 19, 2017

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ACS Synthetic Biology

Polyketide bio-derivatization using the promiscuous acyltransferase KirCII

Ewa M. Musiol-Kroll1,2,4,# Florian Zubeil3,#, Thomas Schafhauser4, Thomas Härtner4, Andreas Kulik4, John McArthur5,7, Irina Koryakina5,8, Wolfgang Wohlleben2,4, Stephanie Grond3, Gavin J. Williams5*, Sang Yup Lee1,6* and Tilmann Weber1,2,4* #

Equal contribution

Affiliations 1

Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet B220, 2800 Kgs. Lyngby, Denmark 2

German Centre for Infection Research (DZIF), Partner site Tübingen, IMIT, Auf der Morgenstelle 28, 72076 Tübingen, Germany 3

Eberhard Karls Universität Tübingen, Institut für Organische Chemie, Auf der Morgenstelle 18, 72076 Tübingen, Germany 4

Eberhard Karls Universität Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, Mikrobiologie/Biotechnologie, Auf der Morgenstelle 28, 72076 Tübingen, Germany 5

North Carolina State University, Department of Chemistry, Raleigh, NC 27695-8204, USA 6

Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea 7

Present address: Department of Chemistry, University of California, One Shields Avenue, Davis, USA 8

Present address: Intrexon Corporation, South San Francisco, California, USA

*

Corresponding authors: Gavin Williams: [email protected], Sang Yup Lee: [email protected] and Tilmann Weber: [email protected]

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ABSTRACT:

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During polyketide biosynthesis, acyltransferases (ATs) are the essential

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gatekeepers which provide the assembly lines with precursors and thus

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contribute greatly to structural diversity. Previously, we demonstrated that the

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discrete AT KirCII from the kirromycin antibiotic pathway accesses non-

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malonate extender units. Here, we exploit the promiscuity of KirCII to generate

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new kirromycins with allyl- and propargyl-side chains in vivo, the latter were

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utilized as educts for further modification by ‘click’ chemistry.

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KEYWORDS:

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antibiotic, kirromycin, polyketide synthase, trans-acyltransferase, engineering,

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click chemistry

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Polyketides are important secondary metabolites widely used as antibiotics,

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antifungals, and drugs for other clinical applications1, 2. Erythromycin, mupirocin,

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rapamycin, FK506, lovastatin and epothilone B are examples of antimicrobial,

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immunosuppressant, hypocholesterolemic and anticancer drugs, respectively. All

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these compounds are biosynthesized by complex multifunctional enzymes with

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modular architecture, the polyketide synthases (PKSs).

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In typical modular type I PKSs, acyltransferase domains (ATs) select and load

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malonyl-CoA derived precursors onto acyl carrier proteins (ACPs). The AT domains

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are either embedded in the PKS (cis-ATs) or encoded by distinct genes and function

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in trans as discrete enzymes (trans-ATs)3, 4. After the AT-dependent loading step,

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ketosynthases (KSs) catalyze the condensation of ACP-linked extender units.

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Optional domains, such as ketoreductases (KRs), dehydratases (DHs),

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enoylreductases (ERs) or methyltransferases (MTs) further process the polyketide

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chain. In most cases, the product is released from the assembly line by a

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thioesterase (TE) domain and modified by post-PKS tailoring enzymes.

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Notably, the AT domain determines which extender unit is incorporated into the

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growing polyketide chain5-7. Consequently, the extender unit specificity of the AT

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cumulatively dictates large portions of the final polyketide structure, and could be

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leveraged to improve the pharmacological properties of polyketide-based chemical

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entities through alteration of the precursors and/or ATs8. These features make AT

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engineering attractive for rational modification of polyketide assembly lines.

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Directed engineering approaches such as AT domain swapping, AT site-directed

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mutagenesis9 and cross-complementation of an AT-inactivated cis-PKS have been

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described for various polyketides, including erythromycin, rapamycin, tylosin and

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geldanamycin10. However, most of the previous attempts to vary AT substrate

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specificity target cis-AT PKS pathways. Furthermore, all studies on extender unit 3 ACS Paragon Plus Environment


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variation involve non-alkyne precursors with the exception of 2-propargyl-

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erythromycin11 and propargyl-premonensin12, which were produced by feeding N-

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acetylcysteamine (SNAC) pre-activated substrates to a strain harboring the

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engineered AT6DEBS and to the wild-type producing strain, respectively.

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For trans-AT type PKSs, only a few examples of assembly lines utilizing non-malonyl-

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CoA extender units have been reported. One of the best studied biosynthetic

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pathways, which involves a trans-AT type PKS and incorporates a branched unit into

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its product, is that of the antibiotic kirromycin (1) from Streptomyces collinus Tü

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36513. The unusual and complex trans- and cis-PKS/NRPS assembly line recruits

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two discrete ATs, KirCI and KirCII (Figure 1). In a previous study we demonstrated

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that KirCI provides the biosynthetic machinery with malonate14. The second AT,

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KirCII, was shown to transfer ethyl malonate onto the kirromycin ACP5 (Kirr-ACP5)

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and thus introduces the C-28 ethyl branch into the backbone of the antibiotic5. More

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recently, in vitro studies revealed that this enzyme accepts other non-malonyl-CoA

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substrates such as allylmalonyl-CoA, propargylmalonyl-CoA and to a lesser extent

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azidoethyl- and phenylmalonyl-CoA15, 16.

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Alkyne-containing molecules, such as propargyl-derived compounds provide

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functional groups for further derivatization by “click chemistry”. The term “click

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chemistry” describes rapid, selective, high-yield reactions of chemical moieties under

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mild, aqueous conditions. Copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) is

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the most commonly used click reaction, which fulfills those criteria. In recent years,

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different click chemistry reactions were developed, often resulting in new chemical

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structures and therefore, the approach become one of the most powerful tools in drug

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discovery, chemical biology, and proteomic applications17, 18.

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To the best of our knowledge, in vivo production of allyl- and/or propargyl-modified

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polyketides, derived from trans-AT pathways has so far not been described. This and 4 ACS Paragon Plus Environment

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the fact that KirCII continues to be the only characterized discrete AT with such a

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promising spectrum of substrate selectivity, encouraged us to study the flexibility of

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KirCII in vivo and to exploit its features for polyketide engineering.

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In our kirromycin bio-derivatization approach, the producer strain S. collinus

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Tü 365 was engineered to facilitate the utilization of non-natural polyketide

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precursors. The malonyl-CoA synthetase T207G/M306I MatB16, 19, which is able to

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activate non-natural extender units, was cloned into the vector pRM4.2 downstream

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of the strong ermE* promoter. The resulting plasmid pRM4.2-matB was transferred

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into S. collinus Tü 365 and an isolated S. collinus pRM4.2-matB clone was cultivated

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for feeding experiments. Separate cultures of the engineered strain were fed with

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allyl- and propargylmalonic acid and the production of new compounds was analyzed

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by HPLC-MS (Supplementary Figure 1). In both the allyl- and propargyl-derived

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extract, an ion consistent with kirromycin (1) (m/z = 795.5 [M-H]-) was present.

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Remarkably however, additional ions at m/z = 807.5 [M-H]- and m/z = 805.4 [M-H]-

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were detected in the allyl- and propargyl-derived samples, respectively

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(Supplementary Figure 1). These ions corresponded to the calculated masses of

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allyl-kirromycin (2) and propargyl-kirromycin (3) (Supplementary Table 1).

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In order to characterize the new metabolites, fermentation and feeding experiments

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were carried out in five liter scale. As only allylmalonic acid is commercially available

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and propargylmalonic acid is not, the latter had to be chemically synthesized to set

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up serial experiments with both substrates. Crude extracts obtained from these

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feeding experiments were subjected to preparative HPLC. Notwithstanding the

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modest structural differences between kirromycin (1) and its derivatives and, as a

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consequence, the challenging chromatographic purification of kirromycin derivatives,

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the compounds could be separated and highly enriched. Thereby 3.8 mg of a sample

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containing 90% allyl-kirromycin (2) and 10% kirromycin (1) and 3.7 mg of a sample 5 ACS Paragon Plus Environment


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containing 22 % propargyl-kirromycin (3) and 78 % kirromycin (1) were obtained.

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Interestingly, these derivative:kirromycin ratios are in better agreement with the KirCII

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in vitro activity data compared to the MatB in vitro activity data. The engineered MatB

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enzyme showed similar in vitro CoA-activation rates toward ethylmalonic acid

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(91±3%), propargylmalonic acid (91±5%) and allylmalonic acid (89±2%),16 but the

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loading efficiency of KirCII differed significantly depending on the substrate. For

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example, both ethylmalonyl-CoA and allylmalonyl-CoA are loaded by KirCII onto the

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cognate Kir-ACP5 with equal efficiency (~15% conversion), whereas

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propargylmalonyl-CoA is loaded at about half of this efficiency (8% conversion)16.

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Furthermore, the isolated yields of allyl- and propargyl-kirromycin indicate that in the

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in vivo system, the downstream kirromycin assembly line domains are more tolerant

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to the artificial substrate allylmalonyl-CoA than for propargyl-CoA.

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To confirm the product identity of allyl-kirromycin (2) and propargyl-kirromycin (3), the

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purified samples, as well as kirromycin (1) were analyzed by high resolution MS.

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Based on the kirromycin fragments20 previously detected and described by Edwards

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and coworkers, we proposed a fragmentation mechanism for kirromycin (1) and the

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derivatives, allyl-kirromycin (2) and propargyl-kirromycin (3) (Supplementary Table

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1, Supplementary Figure 3 and Supplementary Figure 4a-c). The anticipated

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fragments A-C were detected for all three molecules, which confirms that the allyl and

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propargyl moieties were introduced in place of the original ethyl side chain at C-28 in

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kirromycin (1) (Supplementary Table 1, Supplementary Figure 4b-c). In addition,

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1

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and for the propargyl-kirromycin sample (Supplementary Figure 5I-II and

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Supplementary Table 2). Complete NMR signal assignments of allyl-kirromycin (2)

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substantiated the presence of the allyl moiety. In the 1H spectrum of the enriched

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propargyl-kirromycin sample, a spin-system consisting of H-28 (δH 3.13, 1H, m), H-44

H, 13C, COSY, HSQC HMBC and NMR spectra were recorded for the allyl-kirromycin


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(δH 2.57, 2H, m) and H-46 (δH 2.30, 1H, t, J = 2.64 Hz) was detected in addition to

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“the collateral signals” from the residual traces of kirromycin (1). The corresponding

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HMBC correlations unambiguously confirm the identity of propargyl-kirromycin (2)

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(Figure 2).

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The fact that the propargyl-kirromycin derivative (2) possesses an alkyne moiety

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attracted our particular attention, as alkynes are highly desired chemical structures

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for click reactions (i.e. CuAAC). Therefore, propargyl-kirromycin (2) was subjected to

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CuAAC reaction using the fluorescent dye coumarin-343-azide. The click-reaction

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product was purified by chromatographic methods. A total yield of 0.81 mg was

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obtained and analyzed by HPLC-MS in negative ion mode. The analytical data

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demonstrated the presence of a molecule ion with the sum formula C66H84N7O17,

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which is in accordance with the coumarin-modified compound. MS/MS fragmentation

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revealed signals corresponding to the fragments A-C of the 1,2,3-triazole compound

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(Supplementary Table 1 and Supplementary Figure 4d). Despite the small quantity

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of the click-reaction product, NMR analyses were carried out leading to a partial

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spectra recording and signal assignments (Supplementary Figure 5III and

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Supplementary Table 2). The triazole signal (δH 7.78, 1H, s) was observed and the

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respective HMBC correlations were detected (Figure 2 and Supplementary Figure

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5III), confirming the identity of the click-reaction product coumarin-kirromycin (4).

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According to the HPLC-MS analysis this sample contained mainly coumarin-

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kirromycin (4) (96 %) and only traces of kirromycin (1) (4 %). Thus, taking advantage

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of the newly introduced propargyl-anchor, we could not only synthesize a new click

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product, but we also exploited its structural features as a strategy to separate

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coumarin-kirromycin (2) from kirromycin (1). The results support the application of the

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KirCII system for synthesizing biomolecules that are provided with functional groups,

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which can be used in highly selective click chemistry (Figure 1). This allows the 7 ACS Paragon Plus Environment


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production of further chemically modified valuable compounds as well as their

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straightforward purification. Moreover, coupling of azidofluorescent dyes can be used

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for fluorescent labeling of terminal alkynes in bioactive molecules, which is highly

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sought-after for studying drug-target interactions and rational drug design.

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The ability to generate and isolate the allyl-kirromycin (2) and coumarin-kirromycin (4)

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raised the question of whether the modifications at C-28 affect the antibiotic activity

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compared to kirromycin (1). The mode of action of kirromycin (1) is well understood.

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The antibiotic binds to the elongation factor Tu (EF-Tu)21, 22 and thereby inhibits the

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protein biosynthesis in some species of gram positive and gram negative bacteria

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(e.g. Neisseria gonorrhoeae, Haemophilus influenzae, Streptococci and some

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Enterococci strains). In order to examine and compare the efficiency of the new

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derivatives, a GFP-based in vitro translation assay23, 24 was used (Supplementary

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Figure 7). Increasing concentrations of the compounds were added to the assay

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reactions and changes in eGFP synthesis were monitored by measuring the eGFP

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fluorescence (Figure 3, Supplementary Tables 3a-b). In this system, allyl-

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kirromycin (IC50 = 1.0 µM) inhibited the eGFP protein synthesis comparably to the

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native compound kirromycin (1) (IC50 = 0.9 µM) (Figure 3). The derivative coumarin-

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kirromycin (4) also inhibited the system (IC50= 7.3 µM), albeit to a lesser extent. The

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data showed that the replacement of the ethyl moiety at C-28 of kirromycin (1) with

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an allyl moiety has no negative effect on the interactions with its target. According to

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the crystal structure of the complex of Thermus thermophiles EF-Tu with aurodox, an

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N-methyl derivative of kirromycin (1), the antibiotic mainly interacts through the

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pyridine, the tetrahydrofuran rings, and the aliphatic tail on the goldinoic acid with the

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EF-Tu residues25, 26. These structural moieties do not include the branch at C-28 of

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kirromycin (1). Thus, our results are in agreement with the binding site studies

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obtained from the crystal structure of the EF-Tu-aurodox complex25, 26 and confirm 8 ACS Paragon Plus Environment

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that the allyl-moiety is not directly involved in the interaction of the antibiotic with the

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target. In case of coumarin-kirromycin (4), where the ethyl group is conjugated to the

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relatively large coumarin moiety, the molecule is still able to inhibit protein

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biosynthesis, but with an approximately seven-fold lower inhibitory activity, indicating

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that the bulky coumarin residue affects the drug-target interaction. Nevertheless, our

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results demonstrate that kirromycin (1) is robust to variations at the C-28 atom and

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highlights that this position is suitable for further modifications.

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The modification to propargyl-kirromycin is of particular interest as it enables the

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“fusion” of diverse moieties to the kirromycin-backbone. The attachment of chemical

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groups, which would alter the physicochemical properties of the molecule, could be

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used to generate derivatives with optimized import into the cell and thereby extend

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the spectrum of antimicrobial activity for this family of antibiotic compounds.

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Furthermore, labelling of the structure with fluorescent agents would allow the

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monitoring of the antibiotic penetrating the cell and provides tools for studying protein

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biosynthesis (e.g. interactions with the elongation factor EF-Tu, which can be easily

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visualized by fluorescence detection methods).

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This work is the first example that leverages a tailored malonyl-CoA synthetase and a

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trans-AT for rational and regioselective engineering of a complex polyketide antibiotic

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in order to introduce artificial alkene and alkyne sidechains in vivo. Our results

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demonstrate that after the extender unit substitution, which is accomplished relatively

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early in the assembly process, the downstream NRPS and PKS modules accept the

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un-natural sidechains and continue the product biosynthesis. These findings uncover

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the flexibility of a mixed PKS/NRPS assembly line towards the un-natural building

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blocks and encourage the further exploration of trans-AT-based polyketide

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engineering. The MatB-KirCII/ACP5 system may have the potential to become a 9 ACS Paragon Plus Environment


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valuable tool, enabling the regiospecific incorporation of chemical handles into

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polyketides, which can serve as anchors for “click” chemistry. The combination of this

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“bio-derivatization” approach and “click” chemistry methods enables the generation of

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novel molecular probes and analogues of compounds, which would be otherwise

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difficult to access by using exclusively conventional organic chemistry.

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METHODS

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General methods and strain cultivation conditions.

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E. coli strains, the wild type and mutants of S. collinus Tü 365 were cultivated in liquid

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or on solid media as described previously5. Detailed information on strains, media

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composition, antibiotic concentrations, plasmids, oligonucleotides and PCR

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conditions can be obtained from Supporting Information.

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Chemical synthesis of 2-(Prop-2-yn-1-yl)malonic acid.

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Dimethyl propargylmalonate (0.94 ml, 6.2 mmol) and NaOH (2.4 g, 60 mmol) were

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added to a round bottom flask containing 10 ml H2O and the mixture was stirred for 6

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hours at 65°C. The solution was cooled on ice and 10 ml ice cold 12.1 N HCl was

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added. The reaction was extracted 6x with diethyl ether. The organic extractions

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were pooled, dried using MgSO4 and filtered. The solvent was removed under

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vacuum to give 2-(prop-2-yn-1-yl)malonic acid as a white solid (0.298 g, 34%). 1H-

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NMR (D2O, 300 MHz): δH 3.59 (1H, t, J = 7.2 Hz), 2.6 (3H, dd, J = 7.2, 2.7 Hz), 2.26

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(1H, t, J = 2.7 Hz).

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Cloning of the malonyl-CoA synthetase expression construct.

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To activate the substrates allylmalonic acid and propargylmalonic acid, the

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engineered malonyl-CoA synthetase (WP_011650729.1)16, 19 from Rhizobium

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leguminosarum (MatB T207G/M306I) was transferred into S. collinus Tü 365.

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Therefore, the engineered matB gene from the plasmid pET28a-matB, was cloned 10 ACS Paragon Plus Environment

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into the integrative vector pRM4.2 via the restriction sites NdeI and HindIII. The

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resulting construct pRM4.2-matB was introduced into S. collinus Tü 365 by

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intergeneric conjugation using E. coli ET12567 pUB307. Exconjugants were

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confirmed by control PCR with primers matB-test-up and matB-test-low (Supporting

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Information). The obtained mutant S. collinus pRM4.2-matB was applied to feeding

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experiments with allylmalonic acid (Fluka/Sigma-Aldrich) and the synthesized

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propargylmalonic acid.

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Feeding of allylmalonic acid or propargylmalonic acid to S. collinus Tü 365 and

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S. collinus pRM4.2-matB.

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Pre-cultures of S. collinus Tü 365 WT and S. collinus pRM4.2-matB were cultivated

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for three days in TSB medium under antibiotic selection, if required (for antibiotic

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concentration, see Supporting Information). A main culture of 90 ml SGG medium

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was inoculated with 10% of the pre-culture (for media, see Supporting Information)

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and supplemented with 50 mg of allylmalonic acid and propargylmalonic acid

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(t0 = 0 h, final concentration 0.5 mg ml-1). After five days of incubation, each 100 ml

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culture was extracted 1:1 with ethyl acetate (1 h, RT). The samples were centrifuged

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(10 min, RT, 4270 g, Sorvall RC6+ Centrifuge, Thermo Scientific) and the solvent

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fraction was concentrated to dryness under vacuum. Extracts were re-dissolved in 1

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ml methanol and analyzed by HPLC/ESI-MS (Supporting Information).

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Scale-up of feeding and extraction of kirromycin derivatives.

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Four and a half liters of SGG medium, complemented with apramycin (50 µg ml-1)

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were inoculated with 10% pre-culture of S. collinus pRM4.2-matB and fed with

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allymalonic acid or propargylmalonic acid (final concentration, 0.5 mg ml-1). The

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cultures were incubated in 1 L shaking flasks for five days at 29°C. After incubation

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each allymalonic- or propargylmalonic-fed culture was extracted 1:1 with ethyl

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acetate at RT for 1 h, using an extractor. Phase separation was achieved by 11 ACS Paragon Plus Environment


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incubation of the extract/ethyl acetate mixture over night at RT. The ethyl acetate

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phase was collected, evaporated and the residue dissolved in ethyl acetate and

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precipitated by addition of petroleum ether (40-60°C) and a subsequent

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centrifugation. The precipitant was dried in a desiccator. The samples were

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redissolved in methanol and analyzed by HPLC/ESI-MS (Supporting Information).

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Purification of allyl-kirromycin and propargyl-kirromycin.

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The allyl-kirromycin and propargyl-kirromycin sample were dissolved in DMSO:MeOH

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(5:2). Multiple chromatography runs were conducted, each with a solution of 750 µl,

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containing approximately 50 mg of the respective extract. The samples were

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separated on a Kromasil C18, 7 µm 250 x 20 mm HPLC column (Dr. Maisch,

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Ammerbuch, Germany) using MeCN:H2O (43:57) as mobile phase and a flow of 16

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ml min-1. The purity of the obtained yields was determined by NMR analysis. In case

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of allyl-kirromycin, the product of 3.8 mg contained 10% residual kirromycin and for

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propargyl-kirromycin, the pooled fractions resulted in a mixture of 78 % kirromycin

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and 22 % propargyl-kirromycin. The propargyl-kirromycin sample was directly utilized

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in CuAAC reaction without further purification.

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High Resolution Mass Spectrometry (HRMS) analysis of kirromycins.

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The HR-MS analysis of allyl- and propargyl-kirromycin was carried out on MaXis 4G

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instrument (Bruker Daltonics) coupled to a Ultimate 3000 HPLC (Thermo Fisher

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Scientific). An HPLC-method was applied as follows: 0.1% formic acid in water as

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solvent A and 0.06% formic acid in methanol as solvent B, with gradients from 10%

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to 100% B in 20 min and 100% B for 5 min, using a flow rate of 0.3 ml min-1. The

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separation was carried out on a Nucleoshell 2.7 µm 150 x 2mm column (Macherey-

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Nagel).

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The ESI source was operated at a nebulizer pressure of 2.0 bar and dry gas was set

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to 8.0 L min-1 at 200°C. MS/MS spectra were recorded in Auto MS/MS mode 12 ACS Paragon Plus Environment

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resulting in a collision energy stepping from 32.1 to 52.0 eV. Sodium formate was

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used as internal calibrant and Hexakis(2,2-difluoroethoxy)phosphazene (Apollo

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Scientific Ltd, Stockport, UK) as lock mass.

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Nuclear Magnetic Resonance (NMR) spectroscopy of kirromycins.

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The 1H, 13C, 1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC NMR spectra were

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recorded on Bruker Avance III HD spectrometers operating at a proton frequency of

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600 MHz equipped with a Prodigy BBO CryoProbe or 700 MHz equipped with a TCI

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CryoProbe, respectively (Bruker Biospin). CD3OD as solvent and an internal standard

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were used. All spectra were recorded at room temperature. Chemical shifts were

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expressed in parts per million (ppm, δ) relative to TMS.

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CuAAC reaction of propargyl-kirromycin and coumarin-343-azide and product

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purification.

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The propargyl-kirromycin sample (3.7mg) was dissolved in 0.9 ml MeOH and

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transferred to a Schlenk flask containing 3.75 ml t-BuOH and 3.75 ml H2O. The

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solution was mixed with 90 µL of 50 mM coumarin-343-azid (Lumiprobe GmbH,

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Hannover, Germany) in DMSO, 1 ml of 5 mM aqueous ascorbic acid and 1 ml of 2 M

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aqueous triethylammmonium acetate. The reaction was flushed with argon for 5 min

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and subsequently treated with 0.5 ml of 10 mM Cu(II)-TBTA, resolved in DMSO:H2O

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(55:45). The solution was stirred in a sealed flask, at room temperature for 16h.

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The reaction mixture was extracted with ethyl acetate and the solvent evaporated

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using a rotary evaporator. The remaining crude extract was dissolved in MeOH and

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applied to size exclusion chromatography using Sephadex LH-20 (75 x 2.6 cm,

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Methanol, 0.5 ml min-1). Coumarin-kirromycin-containing fractions were pooled and

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subjected to preparative HPLC using 43 % MeCN for 25 min, followed by a gradient

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to 100% in 5 minutes. To remove the residual tris-(benzyltriazolylmethyl)amine

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(TBTA) and kirromycin from the sample, the chromatographic separation was 13 ACS Paragon Plus Environment


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repeated using the same column and the following conditions: 12 ml min-1, gradient

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from 40(A):60(B) to 100(A):0(B) with MeOH(A):H2O(B) within 30 minutes. The final

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product was analyzed by LC-MS and a yield of 0.81 mg coumarin-kirromycin

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containing 4 % kirromycin was determined.

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In vitro activity testing of kirromycin and kirromycin derivatives.

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The in vitro translation assay was developed based on a combination of protocols of

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Kim et al.23, 24 and Calhoun and Swartz27. Briefly, an E. coli S12 cell extract was

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prepared and mixed with nucleotides and amino acid as substrates for transcription

305

and translation, fructose-1,6-bisphosphate for energy supply and the reporter plasmid

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pET28-egfp in wells of a 96 well microtiter plate to a final volume of 100 µL

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(Supporting Information). An assay reaction contained: 57 mM Hepes-KOH (pH

308

8.2), 90 mM potassium glutamate, 80 mM ammonium acetate, 12 mM magnesium

309

acetate, 1.67 % (w/v) PEG(8000), 5 mM DTT, 1.2 mM AMP, 0.85 mM each of CMP,

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GMP and UMP, 0.5 mM IPTG, 34 µg ml-1 L-5-formyl-THF, 2 mM each of all 20

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proteinogenic amino acids, 33 mM fructose-1,6-bisphosphate, 25% (v/v) E. coli S12

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extract and approximately 5 µg ml-1 of the reporter plasmid pET28-egfp (Supporting

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Information, all chemicals were obtained from Sigma, all given values represent the

314

final concentrations). The plasmid pET28-egfp was generated by cloning of the eGFP

315

gene from pRM4.328 with NdeI and HindIII into pET28a (Novagen) to take advantage

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of the strong inducible T7 promoter. The S-12 extract was prepared from E. coli BL21

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strain (DE3) (Invitrogen) as described by Kim et al.29, except that 1 L of 2xYTPG

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medium30 was used for cultivation.

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To test the susceptibility of the in vitro translation system, a microtiter plate was pre-

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loaded with different concentrations of kirromycin, allyl-kirromycin and coumarin-

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kirromycin prior to the addition of the assay master mix. All measurements have been

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performed in triplicate using the same preparation of the S12 extract. The assay was 14 ACS Paragon Plus Environment

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incubated for approximately 4 hours at 37°C. Thereafter, the fluorescence deriving

324

from eGFP was measured at 485/520 nm excitation/emission (POLARstar Galaxy,

325

BMG) (Supporting Information). The data evaluation and IC50 calculation was

326

performed using GraphPad Prism version 7.00 for Windows, GraphPad Software, La

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Jolla California USA, www.graphpad.com, with non-linear fit regression.

328

ASSOCIATED CONTENT

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The Supporting Information is available free of charge on ACS Publications

330

website.

331

AUTHOR INFORMATION

332

Corresponding authors

333

*E-mail: [email protected]

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335


336

Author Contributions

337

E.M.M-K., F.Z. contributed equally to this work.

338

S.G., G.J.W., W.W. and T.W. designed research. E.M.M-K., F.Z., T.S., T.H., and A.K.

339

performed research and analyzed data. J.M. and I.K. contributed reagent. E.M.M-K.,

340

T.S., S.Y.L, G.J.W. and T.W. wrote the paper.

341

Notes

342

The authors declare no competing financial interests.

343

ACKNOWLEDGMENTS

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This work was supported in part by the German Center for Infection Research

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(Deutsches Zentrum für Infektionsforschung, DZIF, TTU09.802 and TTU09.704 to



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T.W. and W.W.), the Novo Nordisk Foundation to SYL and TW (NNF15OC0016226

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to TW), and the National Institute of Health (GM104258-01 to G.J.W).

348 349

REFERENCES

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Weber, T. (2011) Supramolecular templating in kirromycin biosynthesis: the

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of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase

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Hansen, D. A., Sherman, D. H., and Williams, G. J. (2017) Inversion of extender

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unit selectivity in the erythromycin polyketide synthase by acyltransferase

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domain engineering, ACS Chem. Biol. 12, 114-123.

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Sanchez-Garcia, E., and Schulz, F. (2013) Enzyme-directed mutasynthesis: a

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combined experimental and theoretical approach to substrate recognition of a

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E. K., Wittinghofer, A., Schulz, F., and Sanchez-Garcia, E. (2014) Predicted

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(2013) The AT(2) domain of KirCI loads malonyl extender units to the ACPs of

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carrier protein interactions of an acyl-CoA promiscuous trans-acyltransferase,

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machinery revealed via engineered acyl-CoA synthetases, ACS Chem. Biol. 8,

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development and diverse chemical-biology applications, Chem. Rev. 113, 4905-

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[19] Koryakina, I., and Williams, G. J. (2011) Mutant Malonyl-CoA Synthetases with

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Altered Specificity for Polyketide Synthase Extender Unit Generation,

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[20] Edwards, D. M. F., Selva, E., Stella, S., Zerilli, L. F., and Gallo, G. G. (1992)

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protein biosynthesis that acts on elongation factor Tu, Proc. Natl. Acad. Sci.

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USA 71, 4910-4914.

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protein synthesis by kirromycin. Role of elongation factor Tu and ribosomes,

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Continuous cell-free protein synthesis using glycolytic intermediates as energy

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sources, J. Microbiol. Biotechnol. 18, 885-888.

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An economical and highly productive cell-free protein synthesis system utilizing

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fructose-1,6-bisphosphate as an energy source, J. Biotechnol. 130, 389-393.

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[25] Vogeley, L., Palm, G. J., Mesters, J. R., and Hilgenfeld, R. (2001)

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Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic

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binding. Crystal structure of the complex between EF-Tu.GDP and aurodox, J.

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Biol. Chem. 276, 17149-17155.

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[26] Olsthoorn-Tieleman, L. N., Palstra, R. J., van Wezel, G. P., Bibb, M. J., and Pleij,

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C. W. (2007) Elongation factor Tu3 (EF-Tu3) from the kirromycin producer

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Streptomyces ramocissimus is resistant to three classes of EF-Tu-specific

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inhibitors, J. Bacteriol. 189, 3581-3590.


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protein synthesis using glucose and nucleoside monophosphates, Biotechnol.

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Prog. 21, 1146-1153.

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[28] Menges, R., Muth, G., Wohlleben, W., and Stegmann, E. (2007) The ABC

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transporter Tba of Amycolatopsis balhimycina is required for efficient export of

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the glycopeptide antibiotic balhimycin, Appl. Microbiol. Biotechnol. 77, 125-134.

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[29] Kim, T. W., Keum, J. W., Oh, I. S., Choi, C. Y., Park, C. G., and Kim, D. M.

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(2006) Simple procedures for the construction of a robust and cost-effective

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cell-free protein synthesis system, J. Biotechnol. 126, 554-561.

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[30] Kim, R. G., and Choi, C. Y. (2001) Expression-independent consumption of

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substrates in cell-free expression system from Escherichia coli, J. Biotechnol.

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Figures

451

452 453

Figure 1. Kirromycin assembly line and the production of new kirromycin derivatives.

454 455 456 457 458

A: acyltransferase ACP: acyl carrier protein, KS: ketosynthase, DH: dehydrogenase, KR: ketoreductase, MT: methyltransferase, ER: enoylreductase, C: condensation domain, A: adenylation domain, PCP: peptidyl carrier protein, MatB: engineered malonyl-CoA synthetase (T207G/M306I), KirCII: discrete acyltransferase and R: functional group. Kirromycin (1), allyl-kirromycin (2), propargyl-kirromycin (3) and coumarin-kirromycin (4).


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459 460

Figure 2. New kirromycin derivatives.

461 462

(A) Chemical structure of allyl-kirromycin (2), propargyl-kirromycin (3), coumarin-kirromycin (4). (B) Key HMBC correlations of the kirromycin derivatives.

463 464 465

466 467

Figure 3. In vitro activity assay.

468

Inhibition of eGFP translation using kirromycin and kirromycin derivatives.



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The promiscuity of the acyltransferase KirCII was exploited to generate new kirromycins with allyl- and propargyl-side chains in vivo, the latter were utilized as educts for further modification by click chemistry. 39x22mm (300 x 300 DPI)

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Figure 1. Kirromycin assembly line and the production of new kirromycin derivatives. A: acyltransferase ACP: acyl carrier protein, KS: ketosynthase, DH: dehydrogenase, KR: ketoreductase, MT: methyltransferase, ER: enoylreductase, C: condensation domain, A: adenylation domain, PCP: peptidyl carrier protein, MatB: engineered malonyl-CoA synthetase (T207G/M306I), KirCII: discrete acyltransferase and R: functional group. Kirromycin (1), allyl-kirromycin (2), propargyl-kirromycin (3) and coumarinkirromycin (4).

182x180mm (300 x 300 DPI)



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Figure 2. New kirromycin derivatives. (A) Chemical structure of allyl-kirromycin (2), propargyl-kirromycin (3), coumarin-kirromycin (4). (B) Key HMBC correlations of the kirromycin derivatives.

172x150mm (300 x 300 DPI)


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Figure 3. In vitro activity assay. Inhibition of eGFP translation using kirromycin and kirromycin derivatives.

112x60mm (300 x 300 DPI)


Polyketide Bioderivatization Using the Promiscuous Acyltransferase

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