Biosynthesis of Diverse Antimicrobial and Antiproliferative Acyloins in

Articles Cite This: ACS Chem. Biol. 2019, 14, 1490−1497

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Biosynthesis of Diverse Antimicrobial and Antiproliferative Acyloins in Anaerobic Bacteria Sebastian Schieferdecker,†,# Gulimila Shabuer,†,# Anne-Catrin Letzel,† Barbara Urbansky,† Mie Ishida-Ito,† Keishi Ishida,† Michael Cyrulies,‡ Hans-Martin Dahse,§ Sacha Pidot,∥ and Christian Hertweck*,†,⊥

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†

Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Beutenbergstr. 11a, 07745 Jena, Germany ‡ BioPilot Plant, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Beutenbergstr. 11a, 07745 Jena, Germany § Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Beutenbergstr. 11a, 07745 Jena, Germany ∥ Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, 792 Elizabeth Street, Victoria 3010, Australia ⊥ Faculty of Biological Sciences, Friedrich Schiller University Jena, 07743 Jena, Germany S Supporting Information *

ABSTRACT: Metabolic profiling and genome mining revealed that anaerobic bacteria have the potential to produce acyloin natural products. In addition to sattazolin A and B, three new sattazolin congeners and a novel acyloin named clostrocyloin were isolated from three strains of Clostridium beijerinckii, a bacterium used for industrial solvent production. Bioactivity profiling showed that the sattazolin derivatives possess antimicrobial activities against mycobacteria and pseudomonads with only low cytotoxicity. Clostrocyloin was found to be mainly active against fungi. The thiamine diphosphate (ThDP)dependent sattazolin-producing synthase was identified in silico and characterized both in vivo and in in vitro enzyme assays. A related acyloin synthase from the clostrocyloin producer was shown to be responsible for the production of the acyloin core of clostrocyloin. The biotransformation experiments provided first insights into the substrate scope of the clostrocyloin synthase and revealed biosynthetic intermediates.

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hile anaerobic bacteria play an important role in biotechnology (e.g., solvents and biofuels), they have been regarded as devoid of secondary metabolism and have thus been neglected as a source of antimicrobials.1 Mining the increasing number of sequenced genomes of anaerobic bacteria, however, revealed a giant, thus far overlooked, biosynthetic potential. Biosynthetic gene clusters for various natural product classes, such as polyketides, nonribosomal peptides, hybrids thereof, and ribosomal peptides (RiPPs) were identified.2 The discoveries of closthioamides,3 clostrubins,4,5 and barnesin6 indicated that anaerobes are indeed a valuable source of novel antibiotics.7 Here, we report the discovery of a family of antimicrobial acyloins from three strains of the anaerobic Gram-positive bacterium Clostridium beijerinckii, which is used industrially for solvent production.8,9 We also report the functional characterization of synthases involved in acyloin biosynthesis in these clostridia and show that orphan genes coding for related enzymes are found in various genomes of other anaerobic bacteria. © 2019 American Chemical Society

RESULTS AND DISCUSSION

Genome mining of C. beijerinckii (NCIMB 8052) suggested that this species has great potential for secondary metabolite production.2,10 LC-MS-based metabolic screening of three C. beijerinckii strains (NCIMB 8052, HKI805, and HKI806) led to the discovery of several nitrogen-containing compounds (1−6). To elucidate their structures, all three C. beijerinckii strains were cultured on a larger scale (20 L). The ethyl acetate extracts of the fermentation broths were subjected to open column chromatography and preparative HPLC, yielding sufficient amounts of all metabolites for their full structure elucidation. On the basis of their NMR, MS, and optical rotation data, compounds 1 and 2 were identified as (+)-sattazolin A and B Received: March 22, 2019 Accepted: June 5, 2019 Published: June 5, 2019 1490

DOI: 10.1021/acschembio.9b00228 ACS Chem. Biol. 2019, 14, 1490−1497

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ACS Chemical Biology

Figure 1. (A) Observed COSY (bold lines) and HMBC (arrows) NMR correlations of compounds 1−6. (B) Chromatographic determination of absolute configuration of compound 6 by comparison of its R-(+)-MTPA diastereomer of the cleaved alcohol (chromatogram 3) with synthetic references (R-(+)-MTPA ester of (S)- and racemic 2-hydroxy-5-methylhexan-3-one shown in chromatograms 1 and 2). (C) General scheme of acyloin condensation and chemical structures of acyloin derivatives 1−6. (D) Antimicrobial activity of compounds 1−6 determined by agar diffusion assay. (E) GI50 values of compounds 1−6 against HUVEC, K-562, and HeLa cell lines. Values with a GI50 of 50 μg mL−1 represent no detectable activity up to a concentration of 50 μg mL−1.

isobutyl side chain similar to compound 1. In addition, an olefinic bond could be detected that is linked to the indole ring, according to an HMBC correlation (H-7 to C-15). HMBC correlations of H-7 and H-3 indicated that this olefinic moiety is also connected to the isobutyl side chain with the

(see Tables S4 and S5 and Figures S1 and S2), which have been reported as antiviral metabolites from Bacillus spp.11,12 For compound 3, a sum formula of C15H17NO2 was deduced from its exact mass spectrum (m/z 244.1329 [M + H]+). NMR spectra of 3 revealed the presence of an indole ring and an 1491


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Figure 2. (A) Characterized and orphan acyloin synthase gene clusters from diverse anaerobic bacteria. Genes (A) coding for carboxymuconolactone decarboxylase (CMD); genes (B) coding for acetolactate synthase (ALS). CbeiHKI805, C. beijerinckii HKI805; CbeiHKI806, C. beijerinckii HKI806; CbeiNCIMB8052, C. beijerinckii NCIMB8052; CPUHKI742, C. puniceum HKI742; CbeiDSM6423, C. beijerinckii DSM6423, CbeiATCC35702, C. beijerinckii ATCC35702; CchrDSM23318, Clostridium chromiireducens DSM 23318; CdioNJP7, Clostridium diolis NJP7; CspMF28, Clostridium sp. MF28; CspLS8, Clostridium sp. LS8. (B) Maximum likelihood phylogenetic tree, substrates, and structures for acyloin synthases and related ThDP-dependent enzymes. The scale bar indicates amino acid substitutions per site. The detailed tree data are shown in Supporting Information Figure S10 and Table S10. Abbreviations: ACS, acyloin synthase; ALS, acetolactate synthase; BAL, benzaldehyde lyase; BFD, benzoylformate decarboxylase; CeaS, N2-(2-carboxyethyl)arginine synthase; GCL, glyoxylate carboligase; IolD, 3D-(3,5/ 4)-trihydroxycyclohexane-1,2-dione hydrolase; IpdC, indole-3-pyruvate decarboxylase; MenD, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene1-carboxylate synthase; OcaDC, oxalyl-CoA decarboxylase; PDC, pyruvate decarboxylase; PO, pyruvate oxidase; PpdC, phenylpyruvate decarboxylase; PPDC, phosphonopyruvate decarboxylase; Pyr, pyrimidine binding domain; PP, pyrophosphate binding domain; TH3, transhydrogenase dIII domain.

ketone (C-5; Figure 1A). Thus, compound 3 represents dehydrosattazolin (Table S6 and Figure S3). Since the double bond geometry of 3 could not been established by selective NOE experiments, a computational approach was chosen to identify the more likely configuration. Energy minimization using the MM2 force field indicated a lower energy for the Zisomer of 3 (18.25 kcal/mol) than for the E-isomer (23.73 kcal/mol). As both isomers are interchangeable by keto−enol tautomerization, the double bond of compound 3 is likely Zconfigured. The sum formula of compound 4 (C22H24N2O3) was deduced from HR-MS data (m/z 365.1861 [M + H]+). The NMR spectrum of 4 shares signals with the partial structure of sattazolin A. Next to the indole ring, however, we identified a second aromatic ring system (anthranilic acid), which is connected to the sattazolin backbone by an ester bond (Table S7 and Figure S4). Compound 5 possesses a sum formula of C15H19NO3. NMR spectra indicate that 5 has an isobutyl side chain like compounds 1−4. Furthermore, we identified a 7hydroxyindole moiety that is linked to a hydroxyethylene

carbon at C-2, similar to compound 1 and 4. Thus, 5 represents hydroxysattazolin (Table S8 and Figure S5). Structurally similar indole-containing acyloin natural products have been reported from taxonomically distant bacteria such as Xenorhabdus bovienii and a nematode symbiont Xenorhabdus spp.13,14 Compound 6, named clostrocyloin, was isolated as a white to yellowish solid from strain HKI805 and was not observed in the extracts of the other investigated strains of C. beijerinckii. The compound has a molecular formula of C14H19NO3, as determined by mass spectrometry (m/z 250.1437 [M + H]+, calculated for 250.1438 [M + H]+). The analysis of the 1H and 13 C NMR data (Table S9 and Figure S6) revealed the presence of a ketone (δC 213.7), an oxygen-bearing methine (δH 4.20), a disubstituted phenyl ring (δH 7.92, 7.32, 6.61, 6.54), and three methyl groups (δH 1.40, 0.90, 0.85). One partial structure was identified as an isobutyl moiety based on 1H−1H-COSY correlations (C1−C4), which could be connected through the ketone to the methine-bearing partial structure by a HMBC correlation (Figure 1A). The methine could further be 1492


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ACS Chemical Biology connected to the third methyl group by a 1H−1H-COSY experiment. On the basis of 13C chemical shifts and the calculated sum formula, the aromatic ring was identified as anthranyl moiety acid that is connected to the aliphatic partial structure by an ester bond. To elucidate the absolute configuration of 6, the alcohol partial structure was synthesized both as a racemate and the Senantiomer. Therefore, lactic acid methyl ester was TBSprotected and subsequently converted to its Weinreb amide. After reaction with isobutylmagnesium bromide, the alcohol was deprotected with TBAF to yield 2-hydroxy-5-methylhexan3-one (Figure S7). To analyze the absolute configuration of compound 6, the ester bond was cleaved with 2 M HCl. Afterward, the diastereomers 7 and 8 of the cleaved alcohol as well as the prepared synthetic standards were prepared with Mosher’s acid chloride (MTPA-Cl) and chromatographed on a RP18-HPLC column. The coelution of the prepared Senantiomer MPTA ester with the MPTA ester of cleaved 6 revealed that the stereogenic center of clostrocyloin has an S configuration (Figure 1B). While the structure of clostrocyloin is new, its alcohol partial structure is a volatile produced by a variety of microorganisms including Stachylidium sp., Pteridiospora spinosispora, Corynebacterium glutamicum, and Zygosaccharomyces bisporus.15,16 The structures of 1−6 suggest a common biosynthetic origin, as they seem to result from the fusion of various α-ketoacids (Figure 1C). Such acyloin compounds are widespread in nature and have been isolated from various sources including bacteria,12,17,18 fungi,19 and plants.20 These compounds possess a wide variety of bioactivity including antibacterial,17 antiviral,12 antifouling,21 and neuroactive22 properties. To test the antimicrobial properties of compounds 1 to 6, we subjected them to various agar diffusion assays incubated with test strains (Figure 1D and Table S1). All tested compounds showed moderate, selective antifungal activity against Penicillium notatum. Compound 2 also inhibited the growth of Candida albicans, and compound 6 was active against Sporobolomyces salmonicolor. Furthermore, all compounds except for compound 4 showed antibacterial activity against Mycobacterium vaccae. Compounds 2, 3, and 6 were active against Bacillus subtilis, and compounds 1, 2, 3, and 5 inhibited the growth of Staphylococcus aureus. None of the tested compounds were active against Escherichia coli or Pseudomonas aeruginosa. To assay the molecules for antiproliferative and cytotoxic properties, all compounds were tested against HUVEC, K-562, and HeLa cell lines (Figure 1E and Table S2). Except for compound 4, which showed moderate activity in the cell-based assays, none of the tested compounds showed any remarkable antiproliferative or cytotoxic effects. The biosynthetic pathways for acyloin-containing natural products such as xenocylins, sattabacin, and scytonemin typically involve the thiamine diphosphate (ThDP)-dependent condensation of two α-keto-acids.18,23,24 Such carboligation reactions have been the focus of intense research aimed at harnessing their biocatalytic potential for synthetic applications.25−29 The ability to form (−)-sattazolin was recently described for the protein Thzk0150 from the thermophilic bacterium Thermosporothrix hazakensis.23 This enzyme is also responsible for the production of (−)-sattabacin, which is the only acyloin compound found in culture extracts of T. hazakensis. To identify possible enzyme candidates responsible for the ThDP-dependent acyloin condensation of compounds

1 and 6, the genomes of the C. beijerinckii strains HKI805 and HKI806 were sequenced. Two recently characterized acyloincondensing enzymes, Thzk0150923 and XclA4c,18 were used as query sequences in a BLASTP search against the predicted proteomes of all three C. beijerinckii strains. In the course of these in silico analyses, we noted that putative acyloin synthase encoding genes seemed to be widely distributed among Clostridium species. An initial genome-mining approach identified genes for homologous enzymes in 10 different clostridial genomes (Figure 2A). Notably, the acyloin synthase genes were always paired with genes tentatively coding for carboxymuconolactone decarboxylase, which likely promotes the decarboxylation step of the acyloin condensation.30 Using the amino acid sequences of the tentative acyloin synthases from the C. beijerinckii strains and related ThDPdependent enzymes from aerobic organisms, we constructed a phylogenic tree (maximum likelihood algorithm; Figure 2B). While the ancestral protein of these enzymes is still unknown, structural comparisons and phylogenetic analyses indicated that an ancestor of sulfopyruvate decarboxylase (SPDC) and phosphonopyruvate decarboxylase (PPDC) shares the same evolutionary origin.31,32 The maximum likelihood tree of ThDP-dependent enzymes is divided into different clades (Figure S10). Cbei2730 and CbeiHKI805_0381 are present in the same clade with known acyloin synthases Thzk0150, NzsH,33 XclA, and ScyA.34 These ThDP-dependent enzymes possess, besides PPDC, a TH3 (transhydrogenase dIII) domain containing a FAD-binding motif. Notably, the FAD in PO (pyruvate oxidase) only plays a catalytic role as an electron transfer and is related to phosphate-linked acyl transfer in radical mediated reactions.32,35 The FAD in ALS (acetolactate synthase) and GCL (glyoxylate carboligase) is essential for the structural integrity, but does not have any catalytic function.36 In other non-FAD-dependent enzyme families including acyloin synthases, a function of the TH3 domain has not yet been reported, but it is suggested that the TH3 domain is required for homotetramer assembly.32 Furthermore, the substrate specificity of the non-FADdependent enzyme group is more diverse than FAD-utilizing enzymes. To functionally characterize the putative acyloin synthases from C. beijerinckii, we first cloned the Cbei2730 gene for heterologous expression in E. coli. By LC-MS analysis, the production of 1 and 2 was detectable in the feeding experiments of E. coli BL21 (DE3) pET28-Cbei2730 (Figure 3A). The most abundant products were the dehydrogenated compounds, 1 and 2, and compound 3, which was also isolated from C. beijerinckii strains. Furthermore, we purified soluble, His6-tagged Cbei2730 from E. coli and subjected it to an in vitro enzyme assay with thiamine pyrophosphate, MgCl2, indole-3-pyruvic acid, and calcium 4-methyl-2-oxovalerate. The crude mixture was extracted with ethyl acetate, and product formation was monitored by LC-MS. Through comparing with authentic standards, we found that sattazolins 1 and 2 were formed. In addition, we were able to detect a peak with the exact mass of the proposed intermediate (10) that still bears the carboxylate residue (Figure 3B). Furthermore, homocoupling products 9, 11, and 12 of indole-3-pyruvic acid were detected in the assay. In contrast, no homo coupling products of 4-methyl-2-oxopentanoic acid could be detected by HR-MS. Although the decarboxylated, final acyloin products were formed in the enzyme assay using the sattazolin synthase alone, it cannot be excluded that these arise from a spontaneous 1493


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decarboxylation of the 1,3-dicarbonyl intermediate. It is conceivable that a putative carboxymuconolactone decarboxylase encoded in the vicinity of the acyloin synthase genes catalyzes the decarboxylation. As the structure of compound 6 differs substantially from acyloins 1−5, we first investigated by stable isotope labeling whether an acyloin condensation was involved in its biosynthesis. Therefore, we cultured C. beijerinckii HKI805 in the presence of 2-13C-labeled leucine, the presumed precursor for 4-methyl-2-oxopentanoic acid. High-resolution LC-MS showed the incorporation of the 13C label in the backbone of 6 (Figures 4A, S61, and S62). The putative acyloin synthase CbeiHKI805_0381, which is phylogenetically related to Cbei2730, appeared to be a candidate for the production of compound 6 in C. beijerinckii HKI805. To test this, the His6tagged enzyme was heterologously produced in E. coli, and the purified enzyme was used in an in vitro assay with pyruvate and 4-methyl-2-oxovalerate as substrates. After extraction of the assay mixture with dichloromethane, the extract was subjected to LC-MS measurements, which confirmed the production of α-hydroxy acid 13 (Figures 4B and S63). Initially, the decarboxylated acyloin 29, the clostrocyloin precursor lacking the anthranilate residue, could not be detected by the applied ESI-HR-MS method, likely due to ionization issues. Thus, we attempted to verify its presence by the formation of its 2,4dinitrophenylhydrazone 14. Indeed, we could detect a peak with the expected m/z value. The identity of the compound was unequivocally confirmed by LC-MS and coelution experiments using an authentic synthetic reference (Figures 4C and S64). Again, a homocoupling of the educts was not detected. The production of two structurally diverse acyloin compounds by CbeiHKI805_0381 encouraged us to further test the substrate scope of this enzyme. Therefore, we performed an acyl acceptance assay using CbeiHKI805_0381 with either pyruvic acid or 4-methyl-2-oxopentanoic acid and one of the following compounds: 3,3-dimethyl-2-oxobutanoic acid, 3,3,3-trifluoro-2-oxopropanoic acid, 2-oxo-2-phenylacetic acid, 3-methyl-2-oxobutanoic acid, 3-methyl-2-oxopentanoic acid, and 2-oxobutanoic acid. After workup of all reaction mixtures with ethyl acetate, the extracts were analyzed by highresolution LC-MS. This assay showed that CbeiHKI805_0381 does not accept 4-methyl-2-oxopentanoic acid as a substrate. Pyruvic acid, however, can be replaced by 3,3-dimethyl-2oxobutanoic acid, 3-methyl-2-oxobutanoic acid, and 3-methyl2-oxopentanoic acid (Figures 4B, S65, S66, and S67). The observed substrate range is comparable to the sattabacin synthase Thzk0150, which tolerates a range of acyl donors.23 On the basis of these findings, we propose the following biosynthetic model for clostrocyloin (6): Pyruvic acid is initially attacked by thiamine pyrophosphate to form a tertiary alcohol. After decarboxylation, the enol derivative 22 acts as a nucleophile for the keto group of 4-methyl-2-oxopentanoic acid (24), which is biosynthesized from leucine by a transaminase, to form a diol intermediate. After elimination of thiamine pyrophosphate, the α-hydroxy acid 27 is formed. A second decarboxylation step would lead to the formation of an ene-diol intermediate which tautomerizes to the acyloin 29. This step is possibly catalyzed by the putative carboxymuconolactone decarboxylase, which is encoded by a gene in close proximity to the acyloin synthase gene. An enzymatic decarboxylation step similar to acetolactate decarboxylases would explain the high enantioselectivity of the acyloin

Figure 3. (A) Heterologous production of sattazolin A (1) and B (2). Reversed-phase HPLC profiles of the ethyl acetate extracts from (a) E. coli BL21 (DE3) pET28a, (b) E. coli BL21 (DE3) pET28Cbei2730, (c) C. beijerinckii NCIMB 8052, (d) authentic standard of 3. Asterisk indicates unidentified unstable compound. (B) The reversed-phase HPLC profiles of the ethyl acetate extracts from (a) heat-inactivated recombinant His6-Cbei2730, (b) recombinant His6Cbei2730, (c) recombinant His6-Cbei2730 without 4-methyl-2oxovaleric acid, (d) recombinant His6-Cbei2730 without iodole-3pyruvic acid, (e) C. beijerinckii NCIMB 8052, (f) authentic reference of 3. 1494


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Figure 4. Scope and proposed mechanism of acyloin condensation. (A) Stable-isotope labeling of compound 6 with 2-13C-leucine. (B) Enzyme assay for the in vitro production of compound 13 by CbeiHKI805_0381 and production of unnatural acyloins 2-hydroxy-2-isobutyl-4,4-imethyl-3oxopentanoic acid (15), 2-hydroxy-2-isobutyl-4-methyl-3-oxohexanoic acid (16), and 2-hydroxy-2-isobutyl-4-methyl-3-oxopentanoic acid (17). (C) Detection of acyloin 29 as its dinitrophenylhydrazine derivative 14. (D) Proposed biosynthetic model for compounds 1−6.

formation.37 Finally, esterification with anthranilic acid would lead to 6 (Figure 4D). In summary, we isolated seven derivatives of sattazolin and the new compound clostrocyloin from three different strains of the anaerobic bacterium Clostridium beijerinckii. Initial bioactivity profiling indicated that the compounds had several antimicrobial properties. We demonstrated that all compounds derive from a sattazolin synthase, which was characterized in vitro. The rather broad substrate specificity might be utilizable for otherwise difficult-to-prepare enantiopure acyloin com-

pounds. Finally, this study underlines the fact that anaerobic bacteria are an untapped source for novel bioactive natural products.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.9b00228. 1495


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(10) Bachmann, B. O., Van Lanen, S. G., and Baltz, R. H. (2014) Microbial genome mining for accelerated natural products discovery: is a renaissance in the making? J. Ind. Microbiol. Biotechnol. 41, 175− 184. (11) Snyder, K. M., Doty, T. S., Heins, S. P., DeSouchet, A. L., and Miller, K. A. (2013) Asymmetric total synthesis of (+)-sattazolin. Tetrahedron Lett. 54, 192−194. (12) Lampis, G., Deidda, D., Maullu, C., Madeddu, M. A., Pompei, R., Monache, F. D., and Satta, G. (1995) Sattabacins and Sattazolins: New Biologically Active Compounds with Antiviral Properties Extracted from a Bacillus sp. J. Antibiot. 48, 967−972. (13) Li, J., Chen, G., Webster, J. M., and Czyzewska, E. (1995) Antimicrobial Metabolites from a Bacterial Symbiont. J. Nat. Prod. 58, 1081−1086. (14) Paul, V. J., Frautschy, S., Fenical, W., and Nealson, K. H. (1981) Antibiotics in microbial ecology. J. Chem. Ecol. 7, 589−597. (15) Barra, L., Barac, P., König, G. M., Crüsemann, M., and Dickschat, J. S. (2017) Volatiles from the fungal microbiome of the marine sponge Callyspongia cf. f lammea. Org. Biomol. Chem. 15, 7411−7421. (16) Citron, C. A., Barra, L., Wink, J., and Dickschat, J. S. (2015) Volatiles from nineteen recently genome sequenced actinomycetes. Org. Biomol. Chem. 13, 2673−2683. (17) Huang, S.-X., Powell, E., Rajski, S. R., Zhao, L.-X., Jiang, C.-L., Duan, Y., Xu, W., and Shen, B. (2010) Discovery and Total Synthesis of a New Estrogen Receptor Heterodimerizing Actinopolymorphol A from Actinopolymorpha rutilus. Org. Lett. 12, 3525−3527. (18) Proschak, A., Zhou, Q., Schöner, T., Thanwisai, A., Kresovic, D., Dowling, A., ffrench-Constant, R., Proschak, E., and Bode, H. B. (2014) Biosynthesis of the Insecticidal Xenocyloins in Xenorhabdus bovienii. ChemBioChem 15, 369−372. (19) Uchida, R., Shiomi, K., Inokoshi, J., Masuma, R., Kawakubo, T., Tanaka, H., Wai, Y. I., and Omura, S. (1996) Kurasoins A and B, New Protein Farnesyltransferase Inhibitors Produced by Paecilomyces sp. FO-3684. J. Antibiot. 49, 932−934. (20) Damianakos, H., Sotiroudis, G., and Chinou, I. (2013) Pyrrolizidine Alkaloids from Onosma erecta. J. Nat. Prod. 76, 1829−1835. (21) Culioli, G., Ortalo-Magné, A., Valls, R., Hellio, C., Clare, A. S., and Piovetti, L. (2008) Antifouling Activity of Meroditerpenoids from the Marine Brown Alga Halidrys siliquosa. J. Nat. Prod. 71, 1121− 1126. (22) Lin, Z., Marett, L., Hughen, R. W., Flores, M., Forteza, I., Ammon, M. A., Concepcion, G. P., Espino, S., Olivera, B. M., Rosenberg, G., Haygood, M. G., Light, A. R., and Schmidt, E. W. (2013) Neuroactive diol and acyloin metabolites from cone snailassociated bacteria. Bioorg. Med. Chem. Lett. 23, 4867−4869. (23) Park, J. S., Kagaya, N., Hashimoto, J., Izumikawa, M., Yabe, S., Shin-ya, K., Nishiyama, M., and Kuzuyama, T. (2014) Identification and Biosynthesis of New Acyloins from the Thermophilic Bacterium Thermosporothrix hazakensis SK20−1T. ChemBioChem 15, 527−532. (24) Balskus, E. P., and Walsh, C. T. (2008) Investigating the Initial Steps in the Biosynthesis of Cyanobacterial Sunscreen Scytonemin. J. Am. Chem. Soc. 130, 15260−15261. (25) Müller, M., Sprenger, G. A., and Pohl, M. (2013) CC bond formation using ThDP-dependent lyases. Curr. Opin. Chem. Biol. 17, 261−270. (26) Hampel, S., Steitz, J. P., Baierl, A., Lehwald, P., Wiesli, L., Richter, M., Fries, A., Pohl, M., Schneider, G., Dobritzsch, D., and Müller, M. (2018) Structural and mutagenesis studies of the thiaminedependent, ketone-accepting YerE from Pseudomonas protegens. ChemBioChem 19, 2283−2292. (27) Bernacchia, G., Bortolini, O., De Bastiani, M., Lerin, L. A., Loschonsky, S., Massi, A., Müller, M., and Giovannini, P. P. (2015) Enzymatic chemoselective aldehyde-ketone cross-couplings through the polarity reversal of methylacetoin. Angew. Chem., Int. Ed. 54, 7171−7175. (28) Loschonsky, S., Wacker, T., Waltzer, S., Giovannini, P. P., McLeish, M. J., Andrade, S. L., and Müller, M. (2014) Extended

All experimental procedures, compound isolation, characterization, synthetic protocols, enzyme assays, and spectra of new compounds (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Christian Hertweck: 0000-0002-0367-337X Author Contributions #

Both authors have contributed equally to this work

Author Contributions

S.S., A.-C.L., and K.I. isolated and characterized compounds. S.S. conducted chemical synthesis and computational analysis. M.C. conducted fermentations. H.-M.D. performed bioassays. S.P. generated and analyzed genome data. S.S., G.S., B.U., M.I.I., and K.I. performed genetic and biochemical experiments and biotransformations and analyzed data. C.H., S.S., G.S., and K.I. designed and guided all aspects of this work. All authors contributed to manuscript preparation. C.H. edited the final version. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank A. Perner for LC HR-MS measurements, H. Heinecke for NMR measurements, T. Kindel for MALDITOF-MS measurements, and C. Weigel for the determination of antimicrobial activity. Financial support by the BMBF (InfectControl 2020), and the DFG (Leibniz award to C.H.) is gratefully acknowledged.

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REFERENCES

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