Supporting Information for: New aspercryptins, lipopeptide natural products, revealed by HDAC inhibition in Aspergillus nidulans

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1 Supporting Information for: New aspercryptins, lipopeptide natural products, revealed by HDAC inhibition in Aspergillus nidulans Matthew T. Henke a, Alexandra A. Soukup b, Anthony W. Goering a, Ryan A. McClure c, Regan J. Thomson c, Nancy P. Keller b,d,e and Neil L. Kelleher a,c,f a Department of Molecular Biosciences, c Department of Chemistry and f Feinberg School of Medicine, Northwestern University, Evanston, IL 60208, USA b Department of Genetics, d Department of Bacteriology, and e Department of Medical Microbiology and Immunology University of Wisconsin, Madison, Wisconsin, 53706, USA Corresponding author: Neil L. Kelleher; (847) ; 2170 Campus Dr. Evanston, IL 60208; n-kelleher@northwestern.edu Table S1. Summary of NMR data for aspercryptins A1 and A2 Figure S1. Stereochemical determination of aspercryptins A1 and A2 Figure S2. Example of stable isotopes labeling with aspercryptin A1 Figure S3. Annotated MS/MS spectrum of aspercryptin A1 Figure S4. Annotated MS/MS spectrum of aspercryptin A2 Table S2. Summary of NMR data for aspercryptins A1 and A2 Figure S5. Quantitation of all aspercryptins in wildtype and rpda KD Figure S6. Putative structures for 13 additional aspercryptins Table S3. ORF analysis of genes in the aspercryptin biosynthetic gene cluster Figure S7. Transcript levels of aspercryptin gene cluster in rpda KD and overexpression of transcription factor atnn Figure S8. Conservation of the aspercryptin biosynthetic gene cluster across Aspergilli Figure S9. A proposal for the biosynthesis of the aspercryptins. Figure S10. Interrupted organization of fatty acid genes in the aspercryptin gene cluster Figure S11. Structures of members from other natural products that contain an α-amino fatty acid Table S4. Adenylation domain predictions for AN7884/AtnA Table S5. Strains used in this study NMR data for the aspercryptins and of synthetic fatty amino acid standards References 1. Chiang, Y. M., Ahuja, M., Oakley, C. E., Entwistle, R., Asokan, A., Zutz, C., Wang, C. C., and Oakley, B. R. (2016) Development of Genetic Dereplication Strains in Aspergillus nidulans Results in the Discovery of Aspercryptin, Angewandte Chemie 55, Rottig, M., Medema, M. H., Blin, K., Weber, T., Rausch, C., and Kohlbacher, O. (2011) NRPSpredictor2--a web server for predicting NRPS adenylation domain specificity, Nucleic acids research 39, W

2 Table S1. 1 H (600 MHz) and 13 C (150 MHz) NMR data for aspercryptin A1 and A2 in DMSOd 6. Full NMR data can be found below. position δ H (J, Hz) δ C position δ H (J, Hz) δ C serine 2-amino-octanoic acid (2aoa) (dd, 6.2, 4.3) (dd) (dd, 11, 4.7); 3.42 (dd, 10.6, 6.6) ; alanine a ; b threonine asparagine (broad) 25 8 (t) (d) (m) ; (d) (s, broad); 6.85 (s, broad) isoleucine (d) 2-amino-dodecanol (2adol) (t) (m, broad) (dd, 11,5.4); 3.2 (dd, 5.4, 11) ; ; ; b c a These values are for the alanine residue in aspercryptin A2 instead of the initial serine. b These positions are overlapping and cannot be disambiguated. c These values are based on synthetic standard of 2-amino-dodecanol. 2

3 Figure S1. Stereochemical determination of the aspercryptins following derivatization with Marfey s reagent and comparison with amino acid standards by HPLC-MS. (A) D- and L-serine standards (Selected Ion Chromatogram, or SIC: m/z ±10 ppm) confirm the serine of aspercryptin A1 is the D-enantiomer (B) D-/L-alanine standard (SIC: m/z ±10 ppm) confirms the alanine of aspercryptin A2 is actually a mixture of the D- and L-enantiomers. The epimer with L-alanine is epi-aspercryptin A2. (C) Standards of all four stereoisomers of threonine (SIC: m/z ) confirm that D-allo-threonine is present in the aspercryptins. (D) D-/L- isoleucine standard (SIC: m/z ±10 ppm) confirms the aspercryptins contain L- isoleucine. (E) Synthetic D-/L-2-amino-octanoic acid standard (SIC: m/z ±10 ppm) confirms the aspercryptins contain L-2-amino-octanoic acid. (F) D-/L-aspartate standard (SIC: m/z ±10 ppm) confirms that the aspercryptins contain L-asparagine. (G) Synthetic D-/L- 2-amino-dodecanol standard (SIC: m/z ±10 ppm) confirms the aspercryptins contain the L-enantiomer. 3

4 Figure S2. Metabolic labeling of aspercryptin A1 with three of its amino acid precursors containing stable isotopes. Shown are the mass spectra for aspercryptin A (A) with no label, (B) with 13 C 6 15 N 1 -isoleucine, (C) 13 C 4 15 N 1 -threonine, and (D) d 3 -serine. These results support the structural assignments made for the aspercryptins by NMR and MS. Note that the isotopic pattern in panel C does not correspond to the incorporation of more than one threonine: it is complicated due to the partial conversion of the labeled-threonine to isoleucine, and subsequent incorporation of both a heavy isoleucine and a heavy threonine into aspercryptin A1. The isotope pattern of panel D shows a +2 Da incorporation, indicative of an exchange of one deuterium for a proton; this is consistent with epimerization of the Ser residue in aspercryptin A1. 4

5 Figure S3. Annotation of the MS fragmentation spectrum of aspercryptin A1 (m/z ). For clarity b-ions are in blue and y-ions are in red. Unless noted otherwise, observed mass errors for all fragment ions were <1 part-per-million (ppm). Immonium ions for the isoleucine and 2- amino-octanoic acid residues are displayed on the spectrum (low m/z ions labelled with black text at the far left). Loss of ammonia is a common neutral loss from peptides containing asparagine. 5

6 Figure S4. Annotation of the MS fragmentation spectrum of aspercryptin A2 (m/z ). For clarity b-ions are in blue and y-ions are in red. The b-ions are shifted down by exactly the mass of an oxygen, while the y-ions have their same values as they do for aspercryptin A1. This localizes the oxygen to the N-terminus, and thus represents a serine-for-alanine substitution. Unless noted otherwise, observed mass errors for all fragment ions were <1 part-per-million (ppm). Immonium ions for the isoleucine and 2-amino-octanoic acid residues are displayed on the spectrum. Loss of ammonia is a common neutral loss from peptides containing asparagine. 6

7 Table S2. 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; b NMR structures described by Chiang et al. 1 ; c canonical sequence from which derivatives have been identified by shifts in characteristic fragment ions (in blue) Name m/z Formula aspercryptin A1 a,c C 37 H 71 N 7 O 9 [rpda KD ] [wildtype] RT Structure with diagnostic ions aspercryptin A2 a (and epi-form) aspercryptin A3 aspercryptin A C 37 H 71 N 7 O C 36 H 69 N 7 O C 35 H 67 N 7 O aspercryptin A5 aspercryptin A6 aspercryptin A C 36 H 69 N 7 O C 35 H 67 N 7 O C 34 H 66 N 6 O 7 aspercryptin B1 b C 47 H 79 N 7 O 12 aspercryptin B3 b C 46 H 77 N 7 O aspercryptin B2 aspercryptin B4 aspercryptin C1 aspercryptin C3 aspercryptin C2 aspercryptin C4 aspercryptin C6 aspercryptin D C 47 H 79 N 7 O C 45 H 75 N 7 O C 39 H 73 N 7 O C 38 H 71 N 7 O C 39 H 73 N 7 O C 37 H 69 N 7 O C 37 H 69 N 7 O C 40 H 75 N 7 O

8 Figure S5. Quantitation of 17 aspercryptins described here (aspercryptin A2 and epi-aspercryptin A2 are not distinguished here). Error bars are 1 standard deviation from 3 biological replicates. 8

9 Figure S6. Putative structures for the 13 aspercryptins based on analysis of MS/MS spectral differences. Heavily annotated MS/MS spectra of aspercryptins solved by NMR (blue asterisk) were used as the basis for comparison to other metabolites (see Supporting Information Figures S3 and S4). Note that stereochemical assignment has not been made for the MS-based structures; though, they are likely match the stereocenters of the experimentally determined aspercryptins. This chart is arranged hierarchically where the letter across the top describes the N-terminus, and the number along the side described the amino acid sequence. Note that epi-aspercryptin A2 is boxed to show that it does not slot into the chart as shown. 9

10 Table S3. Description of ORFs in the aspercryptin biosynthetic gene cluster from atna (An7884) to atnn (AN7872). Gene name AspGD designation protein size (aa) Description largely provided by AspGD atna AN NRPS; A 1 T-CA 2 TE-CA 3 T-CA 4 T-CA 5 T-CA 6 T-Red 6 modules; reductase domain atnb AN YCII-related domain atnc AN Predicted transmembrane transporter 9 TM helices atnd AN NAD(P)-binding; involved in oxidoreductase activity atne AN Cytochrome P450 heme binding; N-terminus (7-29) is likely transmembrane atnf AN Fatty acid synthase, subunit α (ACP-KR-KS) without PPT domain atng AN ABC-transporter; probably involved in export of mature compound atnh AN Aminotransferase predicted for branched chain amino acid metabolism; PLP binding site atni AN RTA-like protein; may bind to toxic compounds to prevent toxicity; integral membrane protein 6 TM helices atnj AN Aminotransferase predicted for branched chain amino acid metabolism; PLP binding site atnk AN Unknown atnl AN hydroxybenzoyl-CoA thioesterase. Hits to bacterial thioesterase domains atnm AN Fatty acid synthase, subunit β (AT-ER-DH-MPT) AN Unknown atnn AN Transcription factor 10

11 Figure S7. Transcript levels experimentally establish the borders of the AN7872-AN7884 gene cluster. (A) Northern blots show the up-regulation of the AN7872-AN7884 (aspercryptin) gene cluster in rpda KD when grown under repressive (-xylose) conditions, which increases global histone acetylation through lower levels of the histone deacetylase, RpdA. (B) Northern blots show the up-regulation of the gene cluster due to overexpression of the transcription factor AN7872 (alca::an7872) under inducing conditions. 11

12 Figure S8. Conservation of the aspercryptin biosynthetic gene cluster across various Aspergillus spp. Conservation between A. nidulans and A. versicolor appears to be greatest and shows the cluster boundaries quite clearly to be atna (AN7884) to atnn (AN7872). 12

13 Figure S9. A proposed biosynthesis for aspercryptin A1. Biosynthesis of the α-amino fatty acids likely occurs in 2 phases: 1) assembly of the fatty acids octanoic and dodecanoic acids by the FAS subunits AtnF and AtnM and 2) conversion to the 2-amino fatty acids through hydroxylation by AtnE, oxidation by AtnD and transamination by AtnH or AtnJ. Unusually for lipopeptide NPs, both fatty acid monomers are loaded as α-amino fatty acids through activation by dedicated adenylation domains. 13

14 Figure S10. Depiction of the chromosomal organization of the four copies of the genetic cassette encoding Fatty Acid Synthases (FASs) in the A. nidulans genome. It appears that six atn genes interrupt the FAS elements only in the case of the aspercryptin gene cluster (top); shown in green are the aminotransferases that are putatively required for conversion of the short chain fatty acids into their α-amino acids analogs for subsequent incorporation by NRPS to produce the aspercryptins. 14

15 Figure S11. Structures of known classes of lipopeptides where lipid portion is thought to be incorporated as an α-amino acid by an NRPS. All these compounds are cyclic, unlike the aspercryptins, which represent the first backward lipopeptide with an α-amino fatty acid. Table S4. Substrate predictions for the adenylation domains of AtnA (AN7884) by NRPSredictor2 2, which typically does not provide high-confidence predictions for substrates of fungal A domains. Module Stachelhaus code 1 D L G L S G M I L K 15 Nearest neighbor prediction 1 2-amino adipic acid Score Proposed substrate 50% serine/alanine 2 2 D V G F T G C V Y K cysteine 60% threonine 3 D A L F M G G V Y K leucine 60% isoleucine 4 D A M M V G V M I K 3,5-dihydroxyphenyl-glycine 50% 2-aminooctanoic acid 5 D V A F T G S I W K valine 60% asparagine 2-aminododecanoic acid 1 The nearest neighbor prediction is based on the most similar sequence to the extracted Stachelhaus code within a 6 D V L F I G G V A K cysteine 70% database of annotated A-domains. 2 In the case of aspercryptin A2 biosynthesis, alanine would be activated by the A- domain. Table S5. Strains used in this study

16 Strain Name Genotype RJMP1.1 TAAS393.2 TAAS394.2 TAAS176.3 TAAS395.1 TAAS217.1 pyrg89; pyroa4; ribob2; nkua::argb; vea1 AN7884(p)::A. parasticus pyrg::alca(p)::an7884); pyrg89; pyroa4; ribob2; nkua::argb; vea1 AN7872(p)::A. parasiticus pyrg::alca(p)::an7872); pyrg89; pyroa4; ribob2; nkua::argb; vea1 AN7884:: A. fumigatus ribob; pyrg89; pyroa4; ribob2; nkua::argb; vea1 AN7872(p)::A. parasiticus pyrg::alca(p)::an7872; AN7884:: A. fumigatus ribob; pyrg89; pyroa4; ribob2; nkua::argb; vea1 AN7872(p)::A. parasiticus pyrg::alca(p)::an7872; AN7880:: A. fumigatus ribob; pyrg89; pyroa4; ribob2; nkua::argb; vea1 Parent Strain(s) RJMP1.1 RJMP1.1 RJMP1.1 TAAS393.2 TAAS393.2 Source (Palmer et al., 2013) This study This study This study This study This study 16

17 1) 1 H-NMR of aspercryptins A1 and A2 in DMSO-d 6 NMR data for the aspercryptins A1 and A2 17

18 2) 13 C-NMR of aspercryptins A1 and A2 in DMSO-d 6 18

19 3) HSQC of aspercryptins A1 and A2 in DMSO-d 6 19

20 4) band-selective HSQC of aspercryptins A1 and A2 in DMSO-d 6 20

21 5) HMBC of aspercryptins A1 and A2 in DMSO-d 6 21

22 6) COSY of aspercryptins A1 and A2 in DMSO-d 6 22

23 7) TOCSY of aspercryptins A1 and A2 in DMSO-d 6 23

24 8) 1D-TOCSYs of aspercryptins A1 and A2 in DMSO-d 6 24

25 NMR data for fatty amino acid standards used to determine stereochemistry 1 H-NMR of synthetic 2-amino-dodecanol in DMSO-d 6 25

26 13 C-NMR of synthetic 2-amino-dodecanol in DMSO-d 6 + TFA 26

27 1 H-NMR of synthetic 2-amino-octanoic acid in DMSO-d 6 + TFA 27

28 13 C-NMR of synthetic 2-amino-octanoic acid in DMSO-d 6 + TFA 28