New Aspercryptins, Lipopeptide Natural Products, Revealed by HDAC

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Letter

New aspercryptins, lipopeptide natural products, revealed by HDAC inhibition in Aspergillus nidulans Matthew T Henke, Alexandra A. Soukup, Anthony Whitney Goering, Ryan A. McClure, Regan J. Thomson, Nancy P. Keller, and Neil L. Kelleher ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00398 • Publication Date (Web): 16 Jun 2016 Downloaded from http://pubs.acs.org on June 19, 2016

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ACS Chemical Biology

New aspercryptins, lipopeptide natural products, revealed by HDAC inhibition in Aspergillus nidulans Matthew T. Henkea, Alexandra A. Soukupb, Anthony W. Goeringa, Ryan A. McClurec, Regan J. Thomsonc, Nancy P. Kellerb,d,e and Neil L. Kellehera,c,f* a

Department of Molecular Biosciences, cDepartment of Chemistry and fFeinberg School of Medicine, Northwestern University, Evanston, IL 60208, USA b Department of Genetics, dDepartment of Bacteriology, and eDepartment of Medical Microbiology and Immunology University of Wisconsin, Madison, Wisconsin, 53706, USA

ABSTRACT: Unlocking the biochemical stores of fungi is key for developing future pharmaceuticals. Through reduced expression of a critical histone deacetylase in Aspergillus nidulans, increases of up to 100-fold were observed in the levels of 15 new aspercryptins, recently described lipopeptides with two non-canonical amino acids derived from octanoic and dodecanoic acids. In addition to two NMR-verified structures, MS/MS networking helped uncover an additional 13 aspercryptins. The aspercryptins break the conventional structural orientation of lipopeptides and appear ‘backward’ when compared to known compounds of this class. We have also confirmed the 14-gene aspercryptin biosynthetic gene cluster, which encodes two fatty acid synthases and several enzymes to convert saturated octanoic and dodecanoic acid to α-amino acids. INTRODUCTION For decades the search for new natural products by screening for bioactivity has been hampered by rediscovering the same compounds.1 To avoid the rediscovery of natural products, some laboratories have decoupled discovery from screening for bioactivity by measuring accurate mass as the primary, highthroughput screen for the expression of new natural products.2 This new approach to molecular discovery has been applied to bacteria3,4 but less so in fungi, which offer an enormous biosynthetic potential.5 The genome of the mold Aspergillus nidulans contains more than 50 gene clusters annotated to be involved in the biosynthesis of natural products. Yet just over 20 of these biosynthetic gene clusters (BGCs) have associated natural products.6 Several strategies have been developed to better harness the biosynthetic repertoire of fungi.7 One such strategy involves the inhibition of histone deacetylase activity (HDACi) and has shown some promise.8-10 HDACi increases global histone acetylation levels and can increase transcription of otherwise repressed natural product BGCs. Our previous work used quantitative mass spectrometry-based analyses to compare the extracellular metabolomes of A. nidulans before and after both chemical and genetic HDACi.11 We found that HDACi both up-regulated and down-regulated the expression of many compounds. Among the up-regulated compounds were several not known to be produced by A. nidulans, such as the fellutamides.11 Continuing those efforts here, we have solved the structures of two new lipopeptides, aspercryptin A1 and A2, that are up-regulated by up to 90-fold using the HDACi-based strategy. The aspercryptins are sixamino-acid peptides containing two non-canonical α-amino acids derived from saturated C8- and C12-fatty acids, and a C-terminal alcohol and related to two previous aspercryptins published by Chiang et al. in 2016.12 Unlike the overwhelming majority of lipopeptides in the literature, the aspercryptins appear ‘backward’ and have their lipid tail at the C-terminus. We also map the BGC responsible for the aspercryptins by genetic disruption and Northern blotting. It features a NRPS backbone gene encoding a terminal reductase, atypical activation domains for unusual “fatty amino acid” substrates, and enzymes putatively involved in fatty amino acid biogenesis. The aspercryptins join a small group of lipopeptide natural products where incorporation of the lipid moiety occurs at sites other than the N-terminus.

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RESULTS AND DISCUSSION Comparative metabolomics of HDAC-deficient A. nidulans reveals the aspercryptins. In previous work, we generated a mutant of A. nidulans with constitutively lower levels of the HDAC RpdA (AN4493).11 Using differential metabolomics on this strain (rpdAKD, RAAS58.4) compared with wildtype fungus (Figure 1, left)), we detected many signals that were up-regulated in the rpdAKD mutant (Figure 1, middle panels). Dereplication of these metabolites based on their accurate masses allowed us to cull the list to only those that have not yet been characterized (Figure 1). Several dozen of the putatively new metabolites displayed a high degree of similarity in their MS/MS fragmentation spectra, suggesting structural and therefore biosynthetic relatedness (Figure 2). For the members of this compound family, all were up-regulated over a wide range from 2- to 130-fold in rpdAKD vs. wildtype (data for one compound are shown in Figure 1, third panel). The structures of two of these compounds, aspercryptin A1 (m/z 758.5386 [M+H]+, C37H71N7O9, -0.02 ppm) and aspercryptin A2 (m/z 742.5440 [M+H]+, C37H71N7O8, 0.45 ppm), (Figure 1, right panel) were determined completely by MS with stable isotope feeding and extensive NMR (Supporting Information Table S1).

Figure 1. Workflow to compare the metabolomes of wildtype Aspergillus nidulans and a strain where the HDAC RpdA is constitutively repressed (rpdAKD) leading to hyperacetylation in bulk chromatin. Knockdown of RpdA leads to many newly observed metabolites (second panel), from which new metabolites can be viewed selectively (third panel) and targeted for structure determination when they display unique mass signatures. Aspercryptins A1 and A2 are lipopeptides made by A. nidulans (fourth panel at far right). Full NMR data can be found in Supporting Information. Stable isotope incorporation experiments verify much of these structures (Supporting Information Figure S2). Annotated MS/MS spectra for both aspercryptin A1 and A2 (Supporting Information Figures S3 and S4) are included for reference.

The aspercryptins are ‘backward’ lipopeptides. The aspercryptins are linear lipopeptides built from six amino acids and appear to be the first example of peptide natural products with two lipid groups. They also seem to be the only known lipopeptides with a lipid tail at the C-terminus; thus they appear ‘backward’ to other lipopeptides. This is despite the overwhelming literature precedent for lipopeptides having an N-terminal lipid group. Aspercryptin A1 and A2 differ from each other only at the N-terminal residue - serine for aspercryptin A1 and alanine for aspercryptin A2. The most striking feature of the aspercryptins is that of the six amino acids, two are highly unusual and non-proteogenic: 2-amino-octanoic acid and 2-amino-dodecanol. Stereochemical assignment of the aspercryptins was determined for each residue by derivatization with Marfey’s reagent and comparison with standards using HPLC-MS (Supporting Information Figure S1). For aspercryptin A1 the first two residues (serine and threonine) are epimerized to D-serine and D-allothreonine while the remaining 4 residues are the L-isomers. The stereocenters of aspercryptin A2 are the same as those in aspercryptin A1; however, there is also an epimer of aspercryptin A2 such that the alanine is the L-isomer, thus we name this analogue epi-aspercryptin A2 (Supporting Information Figure S1). Several of these monomers (threonine, isoleucine and serine) were also confirmed by metabolic feeding of stable isotope analogues (Supporting Information Figure S2). Furthermore, stable-isotope labeling shows that when fed d3-serine, labeled aspercryptin A1 only shows incorporation of 2 deuterons (Supporting Information Figure S2d). This further supports the epimerization of this residue by loss of the deuteron on the alpha carbon. MS/MS networking reveals a large family of aspercryptins. We observed that the MS/MS spectra of many other metabolites up-regulated by rpdAKD had striking similarity to those of aspercryptin A1 and A2. We then turned to MS/MS networking to visualize the relatedness of these metabolites in the dataset.


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The ability for MS/MS networking to cluster biosynthetically related compounds is now established.13 When performing network analysis of the wildtype and rpdAKD extracts we saw several examples of biosynthetically related natural products clustering together as expected; for example, the emericellamides group tightly together based on MS/MS spectral similarity (Figure 2a, circled). One cluster of metabolites consistently upregulated in the rpdAKD extracts (Figure 2a, boxed) contained aspercryptins A1 and A2 (Figure 2b). Using the MS/MS fragmentation patterns of aspercryptin A1 and A2 (Supporting Information Figures S3 and S4) and the recently published aspercryptins B1 and B312 as anchor points, we detected and have proposed putative structures for an additional 13 aspercryptins (Figure 2b, yellow circles, Table 1). These new aspercryptins fall into sub-families and we were readily able to quantify their abundance increases in the rpdAKD mutant (Supporting Information Figure S5).

Figure 2. MS/MS networking to identify and characterize a large cluster of aspercryptins. (a) MS/MS networking clusters natural products into molecular families. Here the metabolites made by wildtype A. nidulans (blue) are compared to those made by the rpdAKD mutant (red). Some metabolites are expressed equally (grey). Some molecular families are seen in only one biological state. The aspercryptins are boxed and the emericellamides are circled. (b) Aspercryptin A1 and A2 (larger yellow circles) fall into an MS/MS cluster of metabolites mostly expressed by rpdAKD mutant. Comparing the MS/MS spectra of 5 NMR-elucidated aspercryptins allows for characterization of an additional 13 aspercryptins in this molecular family for a total of 18 structurally characterized aspercryptins, which includes aspercryptins B1 and B3 (yellow). Table 1. The formulae and expression ratios for the 18 aspercryptins from Figure 2, including the five aspercryptins elucidated by NMR (bold) and the 13 additional aspercryptins whose putative structures are supported by comparing MS/MS spectra. a NMR structures described in this work; bNMR structures described by Chiang et al;12 cratio is an unresolved combination of epimers m/z molecular [rpdAKD] m/z molecular [rpdAKD] Name Name [M+H]+ formula [wildtype] [M+H]+ formula [wildtype] aspercryptin A1a aspercryptin A2

758.539

a

742.544 a

C37H71N7O9

20

C37H71N7O8

90

c c

aspercryptin B1b

934.586

C47H79N7O12

60

aspercryptin B2

918.591

C47H79N7O11

130

920.571

C46H77N7O12

50

b

742.544

C37H71N7O8

90

aspercryptin A3

744.523

C36H69N7O9

2

aspercryptin B4

906.555

C45H75N7O12

120

aspercryptin A4

730.508

C35H67N7O9

2

aspercryptin C1

800.549

C39H73N7O10

20

aspercryptin A5

728.528

C36H69N7O8

20

aspercryptin C2

784.554

C39H73N7O9

60

aspercryptin A6

714.513

C35H67N7O8

20

aspercryptin C3

786.534

C38H71N7O10

3

epi-aspercryptin A2

aspercryptin B3

aspercryptin A7

671.507

C34H66N6O7

20

aspercryptin C4

772.518

C37H69N7O10

2

aspercryptin D1

814.566

C40H75N7O10

5

aspercryptin C6

756.523

C37H69N7O9

20

The putative structures for 8 of the additional 13 are shown in Figure 3. A subset was chosen for clarity in the main text (for a complete version of this chart see Supporting Information Figure S6). The variations in the structures of the aspercryptins are standard for what is seen from NRPS pathways: incorporation of alanine instead of serine, valine for isoleucine and a C10 fatty amino alcohol instead of the C12 version. Taking the structure of aspercryptin A1 as the ‘canonical’ sequence of the aspercryptins,



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we have made a hierarchical nomenclature system, where the letter represents the status of the Nterminus: A for a free amine, B for cichorine capped, C for acetyl and D for propionyl; and where the number represents ‘variants’ of the base amino acid sequence: 1 for canonical, 2 for serine to alanine, etc. Isolation and full NMR structure determination for each of the aspercryptins are beyond the scope of this work. While the de novo structure elucidation of natural products from analysis of MS and MS/MS data alone is not possible, such analyses can readily be used for the detection and putative characterization of highly similar structural analogues. Though the stereochemistry of the aspercryptins proposed by MS/MS analysis is likely the same as those that have been experimentally determined, we have refrained from assuming that this is indeed the case (Figure 3).

Figure 3. Putative structures for 8 of the 13 aspercryptins based on analysis of MS spectral differences. A complete version of this chart with the 18 structurally characterized aspercryptins is shown in the Supporting Information. Heavily annotated MS/MS spectra of aspercryptins solved by NMR (blue asterisk) were used as the basis for comparison to other metabolites (Supporting Information Figures S3 and S4). Conservatively, stereochemical assignment has not been made for the MS-based structures.

Confirmation of the aspercryptin biosynthetic gene cluster. Linking natural products to their gene clusters is key to annotating the biosynthetic potential of phylogenetically diverse fungi. The genome of A. nidulans contains a 14-gene BGC12 (AN7884 to AN7872, Figure 4a and Supporting Information Table S3) that was recently shown to produce aspercryptins B1 and B3.12 This BGC contains a six-module NRPS (AN7884) and two fatty acid synthase (FAS) subunits (AN7880 and AN7873). To experimentally link all of the aspercryptins to this BGC, we deleted the NRPS gene, AN7884 and observed no detectable levels of any of the aspercryptins (Figure 4b). We also generated an overexpression mutant of the nearby transcription factor, AN7872, which led to increased transcript levels for many of the genes predicted to be in the cluster. This mirrored the overexpression of these genes in the rpdAKD strain (Supporting Information Figure S7) and confirmed the predicted cluster boundaries. Further solidifying its role as the transcription factor for the cluster, we also observed a >2-fold increase in the levels of aspercryptin A1 upon overexpression of AN7872 (Figure 4c). Additionally deletion of the FAS gene AN7880 abolished production of the aspercryptins. Levels of the aspercryptins could be rescued partially by supplementing the media with octanoic and dodecanoic acids, which are likely the direct products of the FAS enzymes (Figure 4c).


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Figure 4. Evidence for the association of the aspercryptins to their BGC. (a) The BGC responsible for the biosynthesis of the aspercryptins contains a non-ribosomal peptide synthetase encoded by atnA (AN7884), which contains 6 adenylation domains and a terminal reductase domain; the two fatty acid synthase subunits atnF (AN7880) and atnM (AN7873); a transcription factor atnN (AN7872); two aminotransferases atnH (AN7878) and atnJ (AN7876); a cytochrome P450 atnE (AN7881); an oxidoreductase atnD (AN11028); and 3 for transport atnC (AN11031) and atnF (AN7879) and resistance atnI (AN7877). (b) Deletion of the NRPS gene atnA in the background of rpdAKD showed it is necessary for biosynthesis of the aspercryptins (only aspercryptin A1 is shown here for clarity). (c) Overexpression of the transcription factor atnN (OE atnN) led to a doubling of aspercryptin A1 levels. Subsequent deletion of the FAS gene atnF (OE atnN, ∆atnF) abolished levels of aspercryptins, which could rescued by supplementing media with the fatty acids octanoic and dodecanoic acids (OE atnN, ∆atnF + FAs).

The NRPS, the transcription factor, and the intervening genes have been named atnA (NRPS) through atnN (transcription factor). Upon exploring the phylogenetic distribution of the atn BGC in the Aspergillus genome repository (http://www.aspergillusgenome.org), we found the atn BGC present in six other Aspergilli and to be most conserved in A. versicolor (NRPS genes 87% identical, Supporting Information Figure S8). The BGC borders were confirmed by transcript mapping using Northern blots of all genes in the cluster (Supporting Information Figure S7). Proposed biosynthesis for the aspercryptins. Based on annotations of the BGC and the above deletion experiments, we propose the following for aspercryptin biosynthesis (Supporting Information Figure S9). The first step is the generation of the fatty acid precursors, octanoic and dodecanoic acids, by the FAS subunits AtnF and AtnM (AN7880 and AN7873). A. nidulans has 3 other pairs of FAS genes. One, fasA and fasB, is required for fatty acids involved in primary metabolism; deletion of either fasA or fasB is lethal.14 The other 2 pairs of FAS genes make precursor fatty acids for natural products – pkiB and pkiC for the polyketides made by the PKS pkiA (AN3386)15 and stcJ and stcK for sterigmatocystin biosynthesis.14 The fatty acid precursors are perhaps the most interesting aspect of the system and are likely transformed into the corresponding α-amino fatty acids in three steps. First they are hydroxylated by the cytochrome P450 AtnE (AN7881), then oxidized to the corresponding α-keto acids by the NAD(P)dependent oxidoreductase AtnD (AN11028), and finally converted to the α-amino fatty acids by the PLPdependent aminotransferases AtnH or AtnJ (AN7878 and AN7876). Similar pathways to convert a fatty acid or polyketide precursor to the corresponding α-amino acid have been proposed for the cyclosporins and apicidins/HC-toxin, and experimentally supported to varying degrees.16-18 The only experimental evidence to support this transformation pathway from these systems comes from the deletion of a branched chain aminotransferase which eliminates HC-toxin biosynthesis.19 A recent publication by Chiang et al. also proposes this pathway for production of the fatty amino acids, and showed that deletion



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of these genes abolished levels of aspercryptin B1 (except in the case of the aminotransferases, AtnH and AtnJ, which could reasonably compensate for each other).12 Unlike the other FAS genes in the A. nidulans genome, the aspercryptin FAS elements are ‘interrupted’ by the aminotransferase genes that we suggest are involved in generation of the α-amino fatty acids (Supporting Information Figure S10). Perhaps this chromosomal organization evolved to decrease the likelihood that the gene cassette necessary for the fatty amino acid biosynthesis will be separated by chromosomal reorganization. If they were not disrupted by the aminotransferases, the expression of the FAS genes could conceivably result in unproductive synthesis of free medium-chain fatty acids that could disrupt membrane stability. In fact, addition of dodecanoic acid above 10 µM for rescue experiments resulted in little to no growth. Once made, we propose that the α-amino fatty acids, 2-amino-octanoic and 2-amino-dodecanoic acids, are recognized, activated and covalently tethered to the NRPS AtnA by its fourth and sixth adenylation domains (Supporting Information Figure S9). For typical lipopeptides, lipid moieties are added to the Nterminus by an initial terminal condensation domain of the NRPS.20 Incorporation of the lipid group as an α-amino fatty acid by an adenylation domain has been proposed for members of the apicidin/HC-toxin family and demonstrated in the biosynthesis of cyclosporin, where the NRPS has been shown to adenylate and covalently bind to the polyketide-derived α-amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (bmt).21,22 Experiments are on-going to directly establish the activity of adenylation domains 4 and 6 of AtnA in recognizing and activating 2-amino-octanoic and 2-amino-dodecanoic acids, respectively. To date, only a handful of α-amino fatty acids have been observed in cyclic lipopeptides (Supporting Information Figure S11). Intriguingly all of these are derived from fungi, with the exception of the piperazimycins.23 Perhaps this strategy for lipid incorporation into natural products is a fungal innovation that spread into Streptomycetes. In general, using the NRPSpredictor2 (modelled from bacterial NRPS) did not confidently predict the amino acid substrates recognized by each of the six adenylation domains of AtnA (Supporting Information Table S4). While Chiang et al. propose cichorine-serine as the monomer activated by the first adenylation domain of AtnA for the biosynthesis of aspercryptin B1,12 we believe it to be serine, and that the N-terminal amine of mature aspercryptin A1 subsequently reacts during the biosynthesis of cichorine24 to form aspercryptin B1. Additionally, despite AtnA having only one epimerase domain in the threonine module, the first two amino acids of aspercryptin A1 are D-serine and D-allo-threonine. This suggests that serine is either loaded directly as D-serine, or that the epimerase domain in the threonine module epimerizes both L-serine and L-threonine. Iterative epimerase domains are not without precedent.25 Because we observed that the alanine residue of aspercryptin A2 exists as a mixture of L- and D- enantiomers, we predict that the epimerase domain acts iteratively. Chronologically, L-serine (for aspercryptin A1) or L-alanine (for aspercryptin A2) is activated by the first adenylation domain of AtnA, and while serine is fully epimerized to the D-enantiomer by the epimerase domain, alanine is only partially converted to the D-enantiomer. The complete conversion of serine and threonine and partial conversion of alanine suggests that the sidechain hydroxyl of serine and threonine may aid in governing substrate recognition by the epimerase domain. The final step in the biosynthesis of the aspercryptins is the reduction of the C-terminus to an alcohol. Terminal reductase domains generally use the energy from NAD(P)H to install a reactive aldehyde that serves as a warhead26 or as an intermediate that rearranges to yield a mature natural product.25 For the aspercryptins, the removal of a charge-bearing site from dominantly hydrophobic portion of the compound is a potential reason for the installation of this alcohol at the C-termini of aspercryptins. In fact, the putatively membrane-associated nature of the aspercryptins may be relevant to their function and is consistent with our ability to isolate these compounds mainly from the cell mass and little from the extracellular medium. Many microbes use lipopeptides as a means to adhere to or move across surfaces, and to establish biofilms.27 Because we could not assign an anti-bacterial activity, the aspercryptins may serve a similar structural/motility function for A. nidulans. We also note that atnH and atnJ are induced by ethanol (the other atn genes were not examined), which possibly reflects a role for this metabolite during hypoxic stress.28

CONCLUSION Here we uncovered over a dozen family members of the recently discovered lipopeptides, the aspercryptins, and mapped the complete gene cluster responsible for their biosynthesis. Instrumental in the discovery of the aspercryptins was the marriage of HDAC inhibition with MS-based metabolite screening. By inhibiting HDAC function, we were able to tease the production of a new family of natural


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products. By taking advantage of MS/MS networking, we could characterize the structural variation of the aspercryptins. Our successful use of HDACi in the discovery of the aspercryptins demonstrates its potential to survey fungal extracts for new chemical matter, even in species that have been so intensively studied, such as A. nidulans. Discovery of new natural product scaffolds from the microbial world at rates far higher than in past decades now represents a promising path for reinvigorating the pipeline of compounds flowing into pharmaceutical screening platforms. METHODS Fungal Transformations. The generation of the rpdAKD strain was previously described.11 All other strains in this study are described in Table S5. For construction of overexpression strains, 1 kb of AN7884 or AN7872 5’ flanking regions and 3’ coding regions were amplified and fused to A. parasiticus pyrG (amplified from pJW24) and a 401 bp fragment of the alcA promoter using double joint PCR.29,30 The resulting overexpression construct was transformed into RJMP1.1 to create strains TAAS393.2 and TAAS394.2. For deletion constructs, 1 kb of flanking regions were amplified and fused to A. fumigatus riboB using double joint PCR. The resulting knockout construct(s) were transformed into RJMP1.1 and/or TAAS393.2 to create strains TAAS176.3 TAAS395.1, and TAAS217.1. Transformants were examined for targeted replacement of the native loci by PCR and Southern blotting, and expression levels were confirmed by northern analysis. Growth and extraction of fungal strains for LC-MS/MS analysis. All strains were grown with initial inoculations of 106 spores/mL in 250 mL GMM in 1L unbaffled flasks, grown in the dark at 37˚C for 4 days at 200 rpm. For the initial screening of the rpdAKD, overexpression and deletant mutants, extractions and analysis were performed as previously described.11 For induction of overexpression strains, lactose minimal medium + 30 mM cyclopentanone was used. For rescue experiments, media was supplemented with 10 µM octanoic and doceanoic acids in acetone. However, for stable isotope incorporation experiments, 5 mL of GMM in 13 mL culture tubes were inoculated with 106 spores/mL and grown as described above, with the additional step of spiking with 1 mM sterile-filtered stableisotope amino acids after 48 hours growth. After a total of 4 days of growth, whole cultures were extracted with an equal volume of ethyl acetate with sonication and vortexing for several minutes, followed by overnight incubation at 4˚C. Organic layers were then dried down and analyzed as previously described.11 Isolation and structural determination of aspercryptins A1 and A2. For isolation of aspercryptin from rpdAKD mutant, large-scale growths (500 mL growths in 2L flask, total of 4 L) were performed as described above. Mycelium was separated from the spent media with coffee filters. The spent media was extracted with dichloromethane to generate an emulsion layer. The emulsion was dried down. The cellular material was washed with excess methanol. Methanol extracts and emulsions from DCM extraction were pooled in methanol and dried onto excess silica. Silica was washed with excess ethyl acetate and aspercryptins were eluted from silica with methanol. Methanol fraction was pHed to ~8.5 and run over SAX resin (Dowex® 1X2 chloride form), flowthrough contained aspercryptins. SAX Resin washed with MeOH (pH 5) and pooled with flowthrough. This was then fractionated over preparative RPLC to afford ~4 mg of aspercryptin A1 and A2 in a ~2:1 mixture that could not be chromatographically resolved. All NMR experiments were performed in DMSO-d6 on an Agilent 600 MHz DD2 with HCN cryoprobe, except for 13 C-NMR, which was acquired with AVANCE III 500 MHz with direct cryoprobe. Stereochemistry of amino acids was determined following a standard Marfey’s Test protocol.31 Briefly, 1 mg of a ~2:1 mixture of aspercryptins A1 and A2 was hydrolyzed in 6N HCl overnight at 110ºC. The hydrolyzed mixture was dried in vacuo, to which 100 µL 1% Marfey’s reagent (FDAA) in acetone and 20 µL 1M NaHCO3 were added. Amino acid standards were derivatized as described above; 2.5 µmoles were used of each DL-alanine, D-serine, Lserine, DL-threonine, DL-allo-threonine, DL-isoleucine, DL-aspartic acid (for asparagine), synthetic DL-2-aminooctanoic acid and synthetic DL-2-amino-dodecanol. MS/MS networking to identify aspercryptin molecular family. All MS/MS spectra were pre-processed by removing the 25% lowest intensity peaks, applying a non-linear transformation to the peak intensities by taking their square root, and normalizing peak intensities to the sum of the intensities of all remaining peaks in the spectrum. Spectra with less than 10 remaining peaks, along with those where the base peak constituted more than 75% of the total scan intensity were removed from the analysis. MS/MS spectra were compared using the cosine similarity method. When comparing two spectra, if more than six matching peaks were found, the remaining unmatched peaks were aligned by shifting their m/z by the difference in precursor mass. The resulting output is a cosine score between 0 and 1 that describes the similarity between two spectra, where 1 represents a perfect match. MS/MS spectra from the same precursor were determined by a cosine similarity of >0.7 and a precursor match within 0.01 m/z. In cases where multiple spectra from the same precursor were observed, the spectrum with the higher intensity was used and the lower intensity spectrum was discarded.



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Using the cosine comparisons, a network containing 1590 nodes and 3396 edges was constructed and visualized in Cytoscape, where each node is a representative spectrum from a unique precursor, and each edge is a cosine comparison with a value of 0.4 or greater. Synthesis of lipid amino acid monomers. DL-2-amino-octanoic acid. ±-2-bromo-octanoic acid (1 g, 4.5 mmol) was dissolved in 10 mL 1:1 H2O:acetone. NaN3 (0.5 g, 7.7 mmol) was added. The solution was vigorously stirred overnight at room temperature (reaction mostly complete after 1 hour) to yield ±-2-azido-octanoic acid. The 2azido-octanoic acid was then hydrogenated in dry THF with 20% Pd/C under 1 atm H2 with constant stirring at room temperature overnight. Mixture was filtered through celite to remove Pd/C.32 A small aliquot was purified by RP-LC to yield 36 µg of pure DL-2-amino-octanoic acid (m/z 160.1333 [M+H]+, C8H17NOH+, 0.6 ppm). White waxy solid.1H- and 13C-NMR spectra can be found in Supporting Information. DL-2-amino-dodecanol. ±-2-bromododecanoic acid (1.25 g, 4.5 mmol) was dissolved in 10 mL 1:1 H2O:acetone. NaN3 (0.5 g, 7.7 mmol) was added. The solution was vigorously stirred overnight at room temperature (reaction mostly complete after 1 hour) to yield ±-2-azido-dodecanoic acid. The 2-azido-dodecanoic acid was then reduced in dry THF with LiAlH4 (0.35 g, 9.34 mmol) with constant stirring on an ice bath for 2 hours.33 The reaction was quenched with 5% KHSO4 and extracted with ethyl acetate. A small aliquot was purified by RP-LC to yield 70 µg of DL-2-amino-dodecanol (m/z 202.2166 [M+H]+, C12H27NOH+, 0.2 ppm). Off-white waxy solid. 1H- and 13C-NMR spectra can be found in Supporting Information. Bioactivity assays. Fifteen µg of purified aspercryptins A1 and A2 (~2:1) were tested in a standard disk diffusion assay against M. luteus, E. coli, P. aeruginosa, S. epidermidis, K. pneumoniae, B. subtilis, A. nidulans and P. citrinum. ASSOCIATED CONTENT

Supporting Information. Figures are largely of structural validation of the aspercryptins, including MS/MS spectra. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]

Author Contributions MTH grew strains, analyzed extracts by LC-MS, determined the structures of aspercryptins A1 and A2 by NMR, the other 13 aspercryptins by MS/MS and prepared the manuscript. AAS generated all mutants used in this study. AWG performed MS/MS networking. RAM performed reduction reactions and Marfey’s derivatization. NPK annotated the distribution of the atn gene cluster. AAS, NLK and NPK heavily revised several versions of the manuscript.

Notes ACKNOWLEDGEMENTS We would like to thank the National Institutes of Health R01 AT009143 (N. Kelleher), T32 GM07133 and NRSA AI55397 (A. Soukup), and NIH 5PO1GM084077 (N. Keller). We also thank Y. Zhang of IMSERC at Northwestern University for NMR assistance (supported by the NIH 1S10OD012016-01 and 1S10RR019071-01A1).

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(2012) Identification and molecular genetic analysis of the cichorine gene cluster in Aspergillus nidulans MedChemComm, 3, 997-1002. (25) Evans, B. S.; Ntai, I.; Chen, Y.; Robinson, S. J.; Kelleher, N. L. (2011) Proteomics-based discovery of koranimine, a cyclic imine natural product J. Am. Chem. Soc. 133, 7316-7319. (26) Shigemori, H.; Wakuri, S.; Yazawa, K.; Nakamura, T.; Sasaki, T.; Kobayashi, J. i. (1991) Fellutamides A and B, cytotoxic peptides from a marine fish-possessing fungus Penicillium fellutanum Tetrahedron, 47, 8529-8534. (27) Raaijmakers, J. M.; De Bruijn, I.; Nybroe, O.; Ongena, M. (2010) Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics FEMS Microbiol. Rev. 34, 1037-1062. (28) Shimizu, M.; Fujii, T.; Masuo, S.; Takaya, N. (2010) Mechanism of de novo branched-chain amino acid synthesis as an alternative electron sink in hypoxic Aspergillus nidulans cells Appl. Environ. Microbiol. 76, 1507-1515. (29) Yu, J. 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MS/MS Backward HDACi + LC/MS ACS Chemical Biology Page 10 of 10 networking lipopeptides 577.339

594.369

549.319

581.309 532.292

565.431

583.49

546.308 564.282 548.288

739.51

593.334

541.266

525.271

539.287

553.302

523.292

964.715

295.103

1219.26

1 2 3 4 5 6 407.229

906.672

9921.559

424.255

294.15

566.298

1568.1

293.248

784.563

308.17

08.207

618.406 438.358

308.17

1511.05

590.334 1239.68

526.395

482.37

389.188

333.097

319.118

554.299

811.21

554.43

365.232

622.36

415.238

839.66

550.303

416.218

433.279

235.162 332.28 672.505 601.486 310.31

236.129 332.28

542.39

940.573 486.76 419.741

774.558 716.515

767.489

326.123 310.13

933.429

308.15 309.097

494.757

399.155

907.402 467.218

420.73 813.493

458.348

317.211 487.342

400.306

469.332 378.228

470.336

414.321

339.15

353.1 323.156

383.279

324.22

345.207

387.236

367.081 345.196 358.24

O

H2N 359.077

450.176

343.191

1078.26

519.139

519.139 519.139 474.311

491.34

519.139

345.1

965.298

1074.31 1073.31 832.24 1038.27 758.57 906.26 684.203 889.23 554.176 774.25 1072.31 628.195 924.27 371.102

HO

396.274

774.515

343.117 344.08

341.175

311.239

N H

329.248

951.389

444.332

923.358

364.342 350.326

408.369

363.31

395.259

314.27 520.304 337.18

315.09 258.145 351.196

422.25

317.171

285.098

796.541

420.238

475.306

442.255

867.544

950.586

442.3

459.31

905.561

441.311

904.576

332.123

894.556 924.566 768.56 853.528 918.592 852.581 920.571 934.587

800.55

265.253

1320.65 894.591

263.237 372.311

1016.59 812.586 730.508

756.523

772.519742.544

1000.6 714.511

455.134 440.14

633.198 442.159

457.15441.141

586.374

479.098 454.12

452.11

453.119

470.145 451.103 469.113

389.123

463.103

333.242

345.078

N H

441.264

634.2

OH H N 344.08

599.411

881.374

459.274 291.232 281.21

273.097

509.125 359.093

441.3

O

421.162

833.39 765.327

O

610.541

740.524

596.525

526.411

575.504

291.195 214.18

O 635.47

634.541

599.504

636.556

302.102

N H O

NH2

aspercryptin A2 314.24 277.158 589.447 606.471 607.463 278.148

502.41

518.406

577.519

612.557

718.537

742.539

717.53

308.14

216.196

H N

423.18 422.17

N H

293.211

309.243 295.226 296.258 625.468 312.253 313.15 294.243

476.338

697.265 747.317 815.379

441.191

O

654.182 319.081

495.11

347.258

459.311

360.181

O

615.373

598.409

454.37

439.132

451.101 449.087 544.363

424.18

574.338

313.169 394.295714.494

491.233

850.406

343.154

1211.83

282.279

281.249

285.242 257.211 243.196

998.486

754.583

362.27

H2N

1032.59

314.206

492.24

736.572

1163.77 598.42

297.28

1618.12 1587.08

484.233

782.569 782.57

639.44

585.398 613.429 599.413627.443 1180.79 655.475 582.387 568.371 622.419 612.399 640.46 641.46 625.428 1264.89 683.507 638.449 608.402 624.435 636.433 594.386 1247.86 613.428 669.491 610.381 612.397 639.444 596.405 612.43 1191.8 627.444610.418 641.46 1804.22 1131.68 655.475 1205.81 1236.85 1208.82 568.41 1222.84 626.411 1219.83 630.353 644.369

295.263 299.223

828.575

801.55

783.521 1060.62 1600.09 1557.05 933.57 1395.95 906.555 1254.68 1545.05

932.57 962.5811034.6

460.34

494.348 425.31

758.539

826.565 786.533 948.602 770.538 771.54 1000.6 814.566 842.561

330.202

427.321

782.554

786.57

769.519 798.57

466.28

420.238

415.208

784.585 782.57 784.585

742.535

810.57 812.55 772.519

906.331

443.345

1485.98

743.5

697.486 699.502

773.51

799.57

438.249

586.241

366.264 348.253

494.24

493.289

831.396

357.197 358.201

OH

494.3

474.212913.364 498.212

756.6

355.13

NH2

738.63 458.2251386.57 594.546 457.196 1512.19

576.535

584.38

640.429

758.57

782.57 784.585 760.585 770.569 786.601 784.585 522.356 756.551 830.554 772.585 768.554 680.45810.601 772.585 760.562 744.554 784.585 762.538 650.439 786.601 758.571 785.579 786.602 664.455 788.617 747.002 784.584 746.588774.558 747.603 676.455 757.584 674.439 761.59 759.569 523.267 782.569 786.601 783.57814.559 743.49 758.569 784.583 758.57 787.59 781.555 814.559 758.569

333.3

331.224 332.08 264.093 393.119 248.18 349.092 341.087

446.181

851.55

757.508 741.512612.47

776.52

347.315 336.313 319.283

394.352

752.515 488.358 515.359 664.463

397.197 556.431 371.111 300.29

574.385 372.12

301.14

644.46

792.52877.55 744.51 731.492 671.509 774.51788.52 773.502 745.507 1490.01 791.42 755.491 727.497 775.38 742.51 756.52

798.577

356.28 357.083 375.089 358.2

N H O

417.082 416.17

443.22

442.273 441.173

458.298

610.43

656.322

358.2

O

317.247

O

806.568

626.423 582.398 486.329 598.393

496.34 520.34

284.128

386.14

H N

297.221 375.251 333.242298.225

808.58

807.365

O

aspercryptin A1

850.25

519.139

519.139 519.139 519.139

267.103

249.117

989.581

297.198 315.218 316.235

O

444.21

343.19 343.191 378.228

536.166 519.139

519.371

519.139

308.128

406.17

295.198

824.574

866.62 838.589 896.631 808.579

361.177

407.209

408.317

852.605 794.563

N H

337.18

312.163

348.225 347.212 365.232 362.095

796.542

OH H N

380.207

311.195

382.259364.249

361.092

378.264 344.23

371.11

370.169

369.127

339.198

374.23 363.141

329.198

360.079

325.19

323.2

383.221327.22

341.18

342.21 358.2

387.108

385.091

395.24

377.202

380.199

519.139 929.23

386.1

327.199359.209 394.259 412.269

341.112

445.12

362.24

345.218

387.217

372.218

342.18

324.211

409.303

421.164

403.224

611.49 544.233 404.186 455.363

686.52 769.671 338.342 610.413 558.249 405.17 473.374

346.21 359.166

347.126

363.171

408.305 817.395 816.38 390.18

432.165

1096.79 599.437 467.358 762.558 1140.82 776.573 1008.74 732.547 432.28 388.254 616.457 658.51 660.49 673.53 468.39 600.469 541.395 584.473628.5572.437 440.35 778.553 498.4 603.45 453.343 528.411 556.441 542.427 748.542 704.516 514.395 820.599 558.421 630.479 614.484 702.537 476.307 497.369 734.527 690.5 646.473 674.505 672.527540.447 470.369 586.453 484.385 602.448 526.432 454.374 380.337 300.2 496.421570.487 792.568 482.405 512.418 344.228 396.332 426.342 382.299 722.505 634.452 590.427 810.563 766.531 438.379 547.4 546.4 854.584 678.479 502.374 452.394 708.489 532.385

795.484

553.392 810.272

658.474

883.685

840.16

394.222

348.225

477.26

746.562 790.589

247.097 261.077

383.149

333.672

347.221

855.653

311.16

377.197

292.071

258.28

814.37777.342 795.35 813.36 778.35 389.173

1052.77 964.714

317.066

332.671 318.656

349.2

327.15 378.227

344.23

406.246 361.237

404.242 423.257 360.23 422.254 405.234

355.191

717.364376.212 328.09

309.279 251.047 421.271 335.219 463.282 286.31 259.065 266.067 239.129

759.41

369.216 843.395 450.285 773.426 403.25 389.26

390.228 408.238

ACS Paragon Plus Environment

331.227 333.24

596.308

276.16

815.38 796.36 797.369

500.231

511.384

389.2 374.202 798.442 391.271 436.269 745.395

882.507

777.343 545.286

555.41

373.171

831.395

781.416

409.245 392.192

770.527594.42

985.555

702.351

915.38

391.226

477.358

406.222

459.202 917.396 476.228 849.406 934.422

377.098

445.331

463.342

428.181

459.201

333.24

459.488 418.113

585.399

231.065

817.395

427.217

314.109 330.17

5.168

493.281

440.23

372.14 374.149

646.415 778.581

295.188

1071.57

332.068

664.371

827.509 1228.28 1627.05

313.147

536.283

1207.69

453.32497.346

541.373

638.449

494.28

568.284

662.389

496.3 588.24 657.455 676.405 531.366 499.27550.629 615.445 497.287 557.4 483.308 494.566 639.496 675.466 522.597 633.397 587.413 689.481 647.413 543.387 497.267 284.294 353.102 519.33 631.439613.429 452.29 492.345 578.353 589.371 327.078 603.387 339.05 499.361 209.081 335.127 262.144 455.334 453.261 451.318 291.159 270.279 453.348 475.303 435.18 321.112 439.332 435.251 231.102 368.425 467.313 317.209 457.33 332.331 302.048 455.207 437.196 255.066 333.11 207.07 242.248 285.04 328.079 404.28 321.242 337.107 257.08 403.151 256.263 261.11 203.107 339.193 323.128 267.065 259.083 364.082 392.328 351.086217.105 243.088 405.17 263.128 258.279 647.262 230.248 349.143 297.279 228.232 449.303 291.05 307.227 258.076487.139 269.19 230.118 363.122 379.118 260.068 275.055 227.07238.109 481.33 283.058 806.539

682.473 726.5

564.282 575.298

604.35

660.431 648.374

934.667

1811.04 333.211

334.21

223.097

1227.78

332.28

654.305

532.277 942.465

350.233 311.09

866.708

1227.28

51.108

1207.69

600.34636.34

941.79 940.781

367.15

822.682

621.35

620.346

552.273 598.397

424.249 926.766 425.31

424.253

570.422

542.39

456.369

408.189

407.194

619.39

600.411

394.2 878.723

475.4

6

1017.55

618.365

767.529

578.437 520.395

509.276

550.303

1044.62

407.229

409.31

549.416

328.285 311.26

297.242

507.297

634.368

618.375

361

644.4

604.551

450.379

631.551 582.493 890.641 802.588 648.577 726.5 433.353 740.01 521.405 538.432 792.52 626.448 758.565 477.379 609.457 660.445 454.301 630.406 494.405626.484 670.51 538.396 714.537 524.38 616.412 748.498 666.472 744.511480.353 700.482582.399 498.33 534.4 406.353 552.411 754.531 670.472 828.603 650.484 772.542 716.48 798.558 520.421 656.459 586.38 542.354 704.471674.593 547.421 710.506 740.548 640.464684.49 554.369 452.322 578.428 714.5830.62 741.56 591.447 612.432 568.406 508.385730.474 622.451 679.499 503.394 698.541 496.348 774.5 723.525 654.479 653.38 626.448 874.647 540.375 742.568 642.421696.529 700.521698.505 655.348 635.473 672.453 652.5 608.473 728.516 652.499 564.469 512.344 600.396 596.437 686.47 644.422 686.448 628.427 612.468 484.311 584.401 674.485656.495 508.421640.5 584.401556.369 610.451 642.442 623.473 528.338 598.453 598.417 688.448 512.343 468.317 711.526816.604728.552 568.442 540.374 468.317 614.411 426.322 716.479 552.447491.39 570.386 690.479 596.477 526.359554.39 630.46 684.564 730.494 535.421 602.428 496.35 579.448 646.453 804.547 860.631 482.333 514.374 447.368 650.483 540.39 702.464 734.505 603.43 606.459 510.363 584.416 694.51 470.348 658.438 464.395 778.532 822.558 866.584 628.442 658.438 558.4 672.468 496.363 738.624 826.588 562.431 668.495 800.573 466.338 712.521 624.467 844.599 580.442 756.549 492.39 713.52

525.27

1049.54

542.312

OH

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