Biosynthesis, Synthesis and Activities of Barnesin A, a NRPS-PKS

Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES

Letter

Biosynthesis, Synthesis and Activities of Barnesin A, a NRPSPKS Hybrid Produced by an Anaerobic Epsilonproteobacterium Maja Rischer, Luka Raguz, Huijuan Guo, Francois Keiff, Gabriele Diekert, Tobias Goris, and Christine Beemelmanns ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00445 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 15, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

2

Biosynthesis, Synthesis and Activities of Barnesin A, a NRPS-PKS Hybrid Produced by an Anaerobic Epsilonproteobacterium

3

Maja Rischer,[a] Luka Raguž,[a] Huijuan Guo,[a] Francois Keiff,[a] Gabriele Diekert,[b] Tobias Goris,[b]* and

4

Christine Beemelmanns[a]*

5

[a] Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute,

6

Beutenbergstraβe 11a, D‐07745 Jena, Germany

7

[b] Department of Applied and Ecological Microbiology, Institute of Microbiology, Friedrich Schiller

8

University, Philosophenweg 12, D‐07743 Jena, Germany

9

* corresponding authors: tobias.goris@uni‐jena.de; Christine.Beemelmanns@hki‐jena.de

1

10

11

Abstract

12

Despite the wealth of physiological knowledge and plentiful genomes available, only few natural

13

products of anaerobic bacteria have been identified until today and even less have been linked to

14

their biosynthetic gene cluster. Here, we analyzed a unique NRPS‐PKS hybrid gene cluster from an

15

anaerobic Epsilonproteobacterium (Sulfurospirillum barnesii). Phylogenetic analysis of key

16

biosynthetic genes, gene expression studies and comparative metabolomics resulted in the

17

identification of the first anoxically biosynthesized NRPS‐PKS hybrid metabolite, a lipo‐dipeptide with

18

a vinylogous side chain, named barnesin A. The absolute structure was verified by a modular total

19

synthesis and barnesin and derivatives were found to have antimicrobial activity as well as selective

20

and nanomolar inhibitory activity against pharmacological important cysteine proteases, such as

21

cathepsin B.

22

Introduction

23

Traditionally, the search for pharmaceutical important secondary metabolites was based on

24

activity screening of the most prolific bacterial lineages, actinomycetes and myxobacteria.1,2

25

However, high rediscovery rates are the major drawback of studying these well‐known

26

natural product producers. Recent bioinformatic studies on thousands of gene sequences

27

allowed to map the global abundance of biosynthetic gene clusters (BGCs) within all bacterial

28

lineages.3‐5 Most notably, genomes of rarely investigated (facultative) anaerobic bacteria,

29

previously believed to be unable to produce natural products,6,7 were found to carry genes

30

encoding for polyketide synthases (PKS) and non‐ribosomal peptide synthetases (NRPS); both

31

key to the production of complex and bioactive secondary metabolites.8,9 Despite their high

32

abundance and industrial and medicinal importance of anaerobes,10 almost none of the ACS Paragon Plus Environment

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

33

identified BGCs have yet been linked to the production of a specific metabolite. In particular

34

complex culture conditions, the tight regulation of gene expression and the genetic

35

inaccessibility have set back in depth metabolomic analysis.6,7 The few examples of natural

36

products from anaerobes include closthioamides, the first antibiotics isolated from the

37

anaerobic bacterium Clostridium cellulolyticum,11 clostrubin produced by the plant pathogen

38

Clostridium puniceum (Fig. 1),12 a new antibiotic lactocillin13 and a group of dipeptide

39

aldehydes and derivatives (e.g. phevalin) produced by a broad range of common gut

40

microbes.14 However, other anaerobic bacterial lineages, in particular free‐living species, have

41

so far been overlooked in the search of unique bioactive secondary metabolites and thus

42

represent an unexplored area of chemical space that holds considerable promise for natural

43

product discovery and potential new bioactivities.

44

45 46 47

Fig. 1. Absolute structure of the first anoxically biosynthesized NRPS‐PKS hybrid molecule barnesin A (1); and structures of the PKS‐derived clostrubin and closthioamide isolated from anaerobic Clostridium spp.

48

49

In our quest to explore so far undiscovered bacterial physiological capabilities and unique

50

biosynthetic pathways and based on our recent work on the genetic basis of organohalide

51

respiration in Epsilonproteobacteria,15‐17 we performed a detailed activity and genome‐wide

52

comparison of several members of this genus. Here, we report that Sulfurospirillum barnesii,18

53

a free‐living Epsilonproteobacterium found in anoxic sediments contaminated with arsenate

54

or selenate, harbors a unique and amongst anaerobic bacteria very rare NRPS‐PKS hybrid

55

biosynthetic gene cluster (BGC designated as brn), whereas all other Sulfurospirillum spp.

56

lacked any signs of NRPS or PKS related gene clusters (Fig. S2).19 The phylogenetic analysis of

57

key biosynthetic enzymes revealed the substrate specificity of single domains, and also

58

showed that they are evolutionary distant to from currently reported domains. Based on this

59

gene‐to‐molecule approach, we proved our structure proposal using an ‘omics’‐guided

60

isolation approach and total synthesis resulting in the identification of the first anoxically

61

biosynthesized NRPS‐PKS derived natural product, named barnesin A.

62

ACS Paragon Plus Environment

Page 2 of 14

Page 3 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


63

Results and Discussion

64

First, we performed a comparative gene cluster analysis. The brn gene cluster (approximately

65

20 kbp in size) encodes nine proteins, annotated by us as BrnA–BrnI (Fig. 2A, Table S1),

66

including a NRPS (BrnE, SMUL_748), a PKS (BrnF, SMUL_749), a PPTase (BrnI, SMUL_750), and

67

a cyclic peptide transporter (Sulba_0745‐747, BrnBCD). Sequence repeat search via RePuter

68

led to the identification of two conserved direct repeats (24 and 12 bp) flanking the brn

69

cluster,20 which are indicative for a possible horizontal gene transfer. A blastp query of the

70

complete cluster resulted in the identification of a highly similar non‐characterized gene

71

cluster within the genome of Geovibrio sp. L21‐Ace‐BES (RefSeq, Acc. No.

72

NZ_ATWF00000000.1) which belongs to the order Deferribacterales and was assembled from

73

a metagenome obtained from a hypersaline mat.21 No repeats were found up‐ and

74

downstream of the gvb hybrid gene cluster and no other evidence for horizontal gene

75

transfer could be detected within the Geovibrio genome. We then analyzed the specificity of

76

biosynthetic domains of both clusters (brn and gvb) to predict the possible core structure of

77

the encoded metabolites.22‐24 The first C‐domain (BrnE C1) was assigned as starter C‐domain

78

and suspected to catalyze a fatty acid transfer to form lipopetides.25 The second C‐domain

79

(BrnE C2) was found to promote the condensation of two L‐amino acids (Table S2, S3).

80

Subsequently, we analyzed both A‐domains; the first A domain (BrnE, A1) was predicted to

81

accept aromatic amino acids (Fig. 2A, darker shaded color code) and belongs to an own,

82

previously not identified, subgroup within this particular group of A‐domains. The second

83

A‐domain (BrnE, A2) analysis was ambiguous, but protein modeling using Swiss Model clearly

84

indicated a binding pocket fitting and stabilizing arginine or lysine residues (Fig. S3).26 The

85

trans‐AT PKS (BrnF) includes a ketosynthase (KS), ketoreductase (KR), dehydratase (DH) and

86

thioesterase (Fig. 2B, darker shaded color code BnrF KS1, Table S4).27 We also identified all AT

87

domains encoded within the genome of S. barnesii and Geovibrio sp. L21‐Ace‐BES and found

88

candidate genes (Sulba_0581 and WP_022851336.1) having the highest similarity to other

89

discrete AT domains that form a loose but distinctive clade distant from cognate ATs (Table

90

S4, S5).28 Overall, the phylogenetic BGC analysis revealed that brn and gvb encode enzymes

91

responsible for the production of a modified lipo‐dipeptide containing at least one tyrosine

92

moiety and one basic amino acid.



93

Page 4 of 14

94 95 96 97 98 99 100 101 102

Fig. 2. A) Comparative gene maps of brn (S. barnesii sp. SES‐3) and gvb (Geovibrio sp. L21‐Ace‐BES); each arrow represents a gene (ORF). Marked with an asterisk is the gene of BrnH (putative membrane protein not annotated as an ORF). Amino acid identities of the orthologues are given above the gvb genes. B) Phylogenetic analysis of adenylation domains with predicted substrate specificity. C) Phylogenetic analysis of trans‐AT associated ketosynthase domains. Predictions of the NRPS Prediction Blast Server were used for labeling predicted amino acids of the phylogenetic tree. Peptide sequences of adenylation domains were aligned using ClustalW The best distance matrix was calculated and the tree was constructed using Mega 6. Probability values >50 are shown at the nodes based on 1000 bootstraps. Darker shaded fields represent the S. barnesii and Geovibrio sp. subgroup.

103

104

To address whether the NRPS‐PKS hybrid cluster is transcribed under laboratory growth

105

conditions, we performed time‐dependent gene expression studies and found a robust level

106

of transcription (gene brnE) in the used cultivation conditions (Fig. 3 and S4). This prompted

107

us to conduct a comparative NMR‐ and UHPLC‐MS‐based metabolomic analysis of S. barnesii

108

and two closely related species (S. deleyianum and S. multivorans) lacking the NRPS‐PKS

109

cluster. Metabolites of each culture supernatant were enriched using C‐18 based solid‐phase

110

extraction and subsequent HPLC/LC‐HRMS analysis revealed one distinct UV‐absorption peak

111

(λmax 214 nm), which correlated to a unique protonated molecular ion peak at 488.2864

112

[M+H]+ not reported by AntiBase29 and SciFinder. For full structural analysis, we pursued

113

preparative scale fermentation (70 L) and MS‐ and NMR‐guided purification of culture

114

extracts led to the isolation of 1.0 mg pure compound, named barnesin A (1) (Fig. 1). The


Page 5 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


115

molecullar formula was assign ned as C25H 37O5N5 bassed on ESI‐HRMS anal ysis (m/z 488.2864 4

116

[M+H]+,, calculated d 488.2867, Δ = ‒0.69 9 ppm). Detailed analy ysis of the obtained 2D 2 NMR

117

spectra (DMSO‐d6) and HR‐MS S/MS fragm mentation id dentified the e predicted metabolite e to be a

118

eptide conssisting of aa tyrosine, a vinylogous argininee and an attached a modified lipo‐dipe

119

ey analysis verified tyyrosine to be b L‐configuured, which h was in unsaturrated fatty acid. Marfe

120

agreement with previous phylogenetic an nalysis.

121

122 123 124 125 126 127

Fig. 3 A A) brnE Gen ne expression at differeent cultivation time points using cconventional reverse transcrip ptase PCR (R RT‐control (w without reve rse transcrip ptase enzyme), gDNA (D NA of S. barrnesii), C‐ (control without DN NA or RNA template). t B B) HPLC‐base ed analysis of o culture exxtracts of S. barnesii obtained d from differrent cultivatiion time poi nts (represe entative UV ttrace at 214 nm). C) Com mparative HPLC‐baased analysiss of culture extracts from e m three diffe erent Sulfuro ospirillum sttrains (representative UV tracee at 214 nm).

128 129 130 131

Fig. 4. P Putative biossynthetic asssembly of baarnesin A (1 1) formation; abbreviatioon (ACP: acyl carrier protein; PCP: peptiidyl carrier protein; C: condensation; A: aden nylation; KSS: ketosynthase; DH: dehydratase; KR: kettoreductase;; TE: thioesteerase).

132



133 134 135 136 137 138 139 140 141

Scheme 1. Total synthesis of barnesin A and derivatives: a) HBTU, DIPEA, DCM, r.t., o.n., 78%; b) 10%aq KOH, MeOH, r.t., 3 h, quant.; c) 1. CuCO3∙Cu(OH)2, H2O, r.t., 15 min; 2. bis‐Boc‐ pyrazolocarboxamidine, DIPEA, formamide, dioxane, r.t., o.n., 3. EDTA∙2Na∙2H2O, NaHCO3, H2O then Fmoc‐OSu, acetone, r.t., o.n. 70% (three steps); d) N,O‐dimethylhydroxylamine hydrochloride, HBTU, DIPEA, DCM, r.t., o.n., 71%; e) 1. LiAlH4, THF, 0 °C, 30 min, 2. triethyl phosphonoacetate, NaH, THF, 0 °C, 45 min, 32% (two steps); f) 20% piperidine in DMF, r.t., 4 h, 86%; g) COMU, DIPEA, DMF, 0 °C, 6 h, 27%; h) TFA, DCM, 0 °C to r.t., o.n.; i) porcine liver esterase, DMSO, H2O, Tris/HCl buffer, 37 °C, 5 d, 47% (two steps).

142

143

The absolute structure of 1 matched the proposed biosynthetic assembly line as depicted in

144

Fig. 4. In short, octenoic acid, presumably originating from fatty acid biosynthesis, is coupled

145

via a specific condensation starter domain (C1) to L‐tyrosine. The resulting lipo‐peptide is

146

then coupled to arginine (A1) via the second condensation domain (C2). A switch to the trans‐

147

AT PKS module allows the elongation via decarboxylative addition of an ACP‐bound malonyl

148

extender unit and the combined action of the KR and DH domains yields the vinylogous

149

arginine moiety. Due to the high homologies between both gene clusters, brn and gvb, we

150

currently hypothesize that a highly similar compound is produced by the designated

151

Geovibrio sp. L21‐Ace‐BES. Here, it is interesting to note that the biosynthetic enzymes (Brn)

152

appear highly substrate specific as LC‐MS/MS analysis revealed barnesin A (1) to be the only

153

derivative present within the culture extracts.

154

Barnesin A belongs to the few natural products containing a vinylogous amino acids,30 and the

155

only other natural product known to contain a vinylogous arginine moiety is the pentapeptide

156

miraziridine A (Scheme 1), isolated from the marine sponge Theonella aff. Mirabilis.31 Other

157

structurally related natural products containing vinylogous amino acid are cyclotheonamides

158

derived from the marine sponge Theonella swinhoei,32 syringolin A isolated from the

159

bacterium Pseudomonas syringae,33‐35 and the related glidobactin A, produced by the ACS Paragon Plus Environment

Page 6 of 14

Page 7 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


160

bacterium Polyangium brachysporum sp.36,37 Here it is interesting to note that syringolin A

161

was identified as a virulence factor, inhibiting the 20S proteasome via a 1,4‐Michael addition

162

reaction of the proteasome to the electrophilic unsaturated amide moiety within the

163

macrolactam scaffold.35

164

To gain access to larger quantities of compound 1, to proof our structural hypothesis and to

165

perform bioactivity studies, we performed a short and modular synthesis (Scheme 1). First,

166

coupling of trans‐2‐octenoic acid 2 with L‐tyrosine methyl ester hydrochloride 3, followed by

167

saponification yielded acid 4 in 78% over two steps. Next, the vinylogous arginine was

168

synthesized using two different strategies.38,39 Then three‐fold Boc‐protected arginine 6 was

169

converted to the respective aldehyde, and the latter was subjected to a Horner‐Wadsworth‐

170

Emmons transformation. As we faced several synthetic unreliabilities with both, synthetic and

171

commercial protected arginine 6,40 we decided to use Fmoc‐protected derivative 7, which

172

allowed us to selectively deprotect the ‒position and pursue the coupling reaction with 4 to

173

give protected precursor 9. Final Boc‐deprotection using TFA and biocatalytic saponification

174

with porcine liver esterase resulted in the desired compound 1. The obtained spectral data of

175

the synthetic derivative 1 was in full agreement with analytical data obtained from the

176

natural isolate proving our structural assumption (Table S7). Moreover, change of fatty acid

177

precursor and reduction of the vinylogous double bond let to barnesin derivative 11‐13,

178

respectively.

179

We then evaluated the biological activities of barnesin A (1) and derivatives 10‐13, and

180

identified barnesin derivative 10, 11 and 13 to have moderate to good inhibitory activity

181

against Mycobacterium vaccae (10670), human‐pathogenic Staphyloccocus aureus (SG511)

182

and Pseudomonas aeruginosa (K799/61 B9) (Table S11). We also confirmed that all tested

183

compounds showed no significant antiproliferative or cytotoxic effects (Table S12). As small

184

peptides and vinylogous systems have a history in being highly potent protease inhibitors,41‐43

185

we analyzed the activity of all barnesin derivatives (1, 10‐13) and found derivatives (1, 10 and

186

11) to inhibit cysteine proteases, including Cathepsin B,44 in the nanomolar range, whereas

187

derivatives 12 and 13 were inactive (Table 1). These results suggest that barnesin derivatives

188

(1, 10‐13) act as a Michael acceptor responsible for irreversible covalent binding to active site

189

of cysteine protease.

190



Table 1. Representative protease inhibition assays of barnesin A (1) and its derivatives in comparison to reported inhibitors. Compound

Papaina

Ficina

Cathepsin Bb

Trypsinc

Barnesin A (1)

15.96 µM (± 5.8)

3.43 µM (± 0.15)

91.72 nM (± 3.29)

>2000 µM

Derivative (10)

2.89 µM (± 0.13)

3.43 µM (± 0.38)

23.99 nM (± 5.3)

>2000 µM

Derivative (11)

4.78 µM (± 2.9)

1.16 µM (± 0.07)

87.56 nM (± 1.8)

>2000 µM

Derivative (12)

>2000 µM

> 2000 µM

> 2000 µM

n.t.

Derivative (13)

>2000 µM

> 2000 µM

> 2000 µM

n.t.

Leupeptin

0.86 µM

n.r.

21.5 nM

2.2 µM

Miraziridin A38,39

n.r.

n.r.

2.05 µM

60 µM

44

Control reactions: inhibition: a) iodacetamide (2 mM): 98.8% inhibition of papain; 97.4% inhibition of ficin; b) leupeptin (1.5 mM): 99.9% inhibition of cathepsin B; c) PMF (2 mM): 100% inhibition of trypsin; n.r. = not reported in literature; n.t. = not tested.

191

192

Conclusions

193

Taken together, our experimental findings presented here reveal the first NRPS‐PKS based

194

hybrid natural product from an anoxically cultivated free‐living bacterium (S. barnesii,

195

Epsilonproteobacterium). Phylogenetic analysis of key genes of both biosynthetic clusters

196

allowed for a NMR‐ and LC‐MS‐guided isolation approach of the first PKS‐NRPS hybrid

197

metabolite (barnesin A (1)) produced from this bacterial lineage, and from anoxically

198

cultivated bacteria in general. The absolute structure was proven by a modular total

199

synthesis, which allowed accesses to non‐natural derivatives. A first pharmacological

200

evaluation of this compound class revealed nanomolar inhibitory activity against cysteine

201

proteases presumably via a 1,4‐Michael type addition mechanism. The ecological role and

202

function of barnesin, however, remains to be elucidated. As the gene cluster analysis suggest

203

a horizontal transfer between different phyla, similar compounds likely play a biological role

204

in more than one free‐living species. Furthermore, the constitutive expression of the brn

205

cluster and the production of such a reactive secondary metabolite impart a selective

206

advantage to the producing organism by acting, e.g., as a defensive metabolite against other

207

bacteria or protistan bacterivory.45

208

In general, our findings demonstrate that comparative genome analysis of so far neglected

209

natural product producers followed by a detailed phylogenetic biosynthetic pathways analysis

210

allows for a targeted and highly efficient isolation approach. Given the large number of

211

biosynthetic gene clusters of unknown function within many (facultative) anaerobes, such an


Page 8 of 14

Page 9 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


212

approach holds great potential for discovering new chemical scaffolds and the identification

213

of small‐molecule based microbial mediators in the anaerobic world.

214

Conflicts of interest

215

The authors declare a financial conflict of interest. A patent application has been filed

216

(registration number: EP 18 160 274.9: Barnesin A, derivatives and uses thereof) and is

217

currently under revision at the European patent office.

218

Acknowledgements

219

M. Rischer, L. Raguž and C. Beemelmanns are supported by the Leibniz Association, the Jena

220

School for Microbial Communication and the German Research Council (DFG, grant

221

BE4799/2‐1 EXSPHINGO and CRC ChemBioSys 1127), T. Goris by the DFG as part of the

222

research group FOR1530. The authors thank B. Schenz, P. Brand‐Schön, S. Kruse (FSU Jena)

223

and M. Küfner (HKI) for assisting in the cultivation of Sulfurospirillum barnesii. We thank A.

224

Perner, H. Heinecke, H.‐M. Dahse and C. Weigel (HKI) for their help with MS and NMR

225

measurements and activity assays, and H. Kries (HKI) for help in the modeling of the

226

adenylation binding pocket.

227

Notes and References

228

† Electronic Supplementary Informa on (ESI) available: Fermenta on procedures; isola on

229

procedures, ESI‐HRMS, 1H NMR, 13C NMR, and 2D NMR spectra as well as chemical modifications.

230

231 232 233 234 235 236

1. Blunt, J. W., Copp, B. R., Munro, M. H. G., Northcote, P. T., and Prinsep, M. R. (2010) Marine natural products, Nat. Prod. Rep. 27, 165‐237. 2. Weissman, K. J., and Müller, R. (2010) Myxobacterial secondary metabolites: bioactivities and modes‐of‐action, Nat. Prod. Rep. 27, 1276‐1295. 3. Ziemert, N., Alanjary, M., and Weber, T. (2016) The evolution of genome mining in microbes ‐ a review, Nat. Prod. Rep. 33, 988‐1005.

237

4. Wang, H., Fewer, D. P., Holm, L., Rouhiainen, L., and Sivonen, K. (2014) Atlas of

238

nonribosomal peptide and polyketide biosynthetic pathways reveals common

239

occurrence of nonmodular enzymes, Proc. Natl. Acad. Sci. USA 111, 9259‐9264.

240 241 242 243

5. Genilloud, O. (2017) Actinomycetes: still a source of novel antibiotics, Nat. Prod. Rep. 34, 1203‐1232. 6. Behnken, S., and Hertweck, C. (2012) Anaerobic bacteria as producers of antibiotics, Appl. Microbiol. Bio. 96, 61‐67.



244 245 246 247

7. Letzel, A.‐C., Pidot, S. J., and Hertweck, C. (2013) A genomic approach to the cryptic secondary metabolome of the anaerobic world, Nat. Prod. Rep. 30, 392‐428. 8. Robbins, T., Liu, Y.‐C., Cane, D. E., and Khosla, C. (2016) Structure and mechanism of assembly line polyketide synthases, Curr. Opin. Struc. Biol. 41, 10‐18.

248

9. Payne, J. A. E., Schoppet, M., Hansen, M. H., and Cryle, M. J. (2017) Diversity of nature's

249

assembly lines ‐ recent discoveries in non‐ribosomal peptide synthesis, Mol. Biosyst. 13,

250

9‐22.

251 252

10. Donaldson, G. P., Lee, S. M., and Mazmanian, S. K. (2015) Gut biogeography of the bacterial microbiota, Nat. Rev. Microbiol. 14, 20‐32.

253

11. Lincke, T., Behnken, S., Ishida, K., Roth, M., and Hertweck C. (2010) Closthioamide: an

254

unprecedented polythioamide antibiotic from the strictly anaerobic bacterium

255

Clostridium cellulolyticum, Angew. Chem. Int. Ed. 49, 2011‐2013.

256

12. Shabuer, G., Ishida, K., Pidot, S. J., Roth, M., Dahse, H.‐M., and Hertweck, C. (2015) Plant

257

pathogenic anaerobic bacteria use aromatic polyketides to access aerobic territory.

258

Science 350, 670‐674.

259

13. Donia, M. S., Cimermancic, P., Schulze, C. J., Wieland Brown, L. C., Martin, J., Mitreva,

260

M., Clardy, J., Linington, R. G., and Fischbach, M. A. (2014) A systematic analysis of

261

biosynthetic gene clusters in the human microbiome reveals a common family of

262

antibiotics, Cell 158, 1402‐1414.

263

14. Guo, C. J., Chang, F. Y., Wyche, T. P., Backus, K. M., Acker, T. M.; Funabashi, M.,

264

Taketani, M., Donia, M. S., Nayfach, S., Pollard, K. S., Craik, C. S., Cravatt, B. F., Clardy, J.,

265

Voigt, C. A., and Fischbach, M. A. (2017) Discovery of Reactive Microbiota‐Derived

266

Metabolites that Inhibit Host Proteases, Cell 168, 517‐526 e18.

267

15. Goris, T., Schubert, T., Gadkari, J., Wubet, T., Tarkka, M., Buscot, F., Adrian, L., and

268

Diekert, G. (2014) Insights into organohalide respiration and the versatile catabolism of

269

Sulfurospirillum multivorans gained from comparative genomics and physiological

270

studies, Environ. Microbiol. 16, 3562‐3580.

271

16. Goris, T., and Diekert, G. (2016) The Genus Sulfurospirillum in Organohalide‐Respiring

272

Bacteria, Eds. L. Adrian, F. E. Löffler, Springer Berlin Heidelberg, Berlin, Heidelberg, 209‐

273

234.

274

17. Goris, T., Schenz, B., Zimmermann, J., Lemos, M., Hackermüller, J., Schubert, T., and

275

Diekert, G. (2017) The complete genome of the tetrachloroethene‐respiring

276

Epsilonproteobacterium Sulfurospirillum halorespirans, J. Biotechnol. 255, 33‐36.

277

18. Stolz, J. F., Ellis, D. J., Blum, J. S., Ahmann, D., Lovley, D. R., and Oremland, R. S. (1999)

278

Note: Sulfurospirillum barnesii sp. nov. and Sulfurospirillum arsenophilum sp. nov., new


Page 10 of 14

Page 11 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


279

members of the Sulfurospirillum clade of the ε‐Proteobacteria, Int. J. Syst. Evol. Micr. 49,

280

1177‐1180.

281

19. Blin, K., Wolf, T., Chevrette, M. G., Lu, X., Schwalen, C. J., Kautsar, S. A., Suarez Duran, H.

282

G., de los Santos, E. L. C., Kim, H. U., Nave, M., Dickschat, J. S., Mitchell, D. A., Shelest, E.,

283

Breitling, R., Takano, E., Lee, S. Y., Weber, T., and Medema, M.H. (2017) antiSMASH 4.0‐

284

improvements in chemistry prediction and gene cluster boundary identification, Nucleic

285

Acids Res. 45, W36‐W41.

286

20. Kurtz, S., Choudhuri, J. V., Ohlebusch, E., Schleiermacher, C., Stoye, J., and Giegerich, R.

287

(2001) REPuter: the manifold applications of repeat analysis on a genomic scale, Nucleic

288

Acids Res. 29, 4633‐4642.

289

21. Spring, S., Brinkmann, N., Murrja, M., Spröer, C., Reitner, J., and Klenk, H.‐P. (2015) High

290

Diversity of Culturable Prokaryotes in a Lithifying Hypersaline Microbial Mat,

291

Geomicrobiol. J. 32, 332‐346.

292

22. Challis, G. L., Ravel, J., and Townsend, C.A. (2000) Predictive, structure‐based model of

293

amino acid recognition by nonribosomal peptide synthetase adenylation domains, Chem.

294

Biol. 7, 211‐224.

295 296

23. Ziemert, N., and Jensen, P. R. (2012) Chapter Eight ‐ Phylogenetic Approaches to Natural Product Structure Prediction, Methods Enzymol. 517, 161‐182.

297

24. Röttig, M., Medema, M. H., Blin, K., Weber, T., Rausch, C., and Kohlbacher, O. (2011)

298

NRPSpredictor2 — a web server for predicting NRPS adenylation domain specificity,

299

Nucleic Acids Res. 39, W362‐W367; http://nrps.informatik.uni‐tuebingen.de.

300

25. Rausch, C., Hoof, I., Weber, T., Wohlleben, W., and Huson, D.H. (2007) Phylogenetic

301

analysis of condensation domains in NRPS sheds light on their functional evolution, BMC

302

Evol. Biol. 7, 78.

303

26. Biasini, M., Bienert S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F.,

304

Cassarino, T. G., Bertoni, M., Bordoli, L., and Schwede, T. (2014) SWISS‐MODEL:

305

modelling protein tertiary and quaternary structure using evolutionary information,

306

Nucleic Acids Res. 42, W252‐W258.

307 308

27. Helfrich, E. J. N., and Piel, J. (2016) Biosynthesis of polyketides by trans‐AT polyketide synthases, Nat. Prod. Rep. 33, 231‐316.

309

28. Musiol, E. M., Hartner, T., Kulik, A., Moldenhauer, J., Piel, J., Wohlleben, W., and Weber,

310

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

311

KirCII loads ethylmalonyl‐CoA extender onto a specific ACP of the trans‐AT PKS, Chem.

312

Biol. 18, 438‐444.

313

29. Laatsch, H. AntiBase 2014: The Natural Compound Identifier. ISBN 978‐3‐527‐33841‐2



314

30. Walsh, C. T., O'Brien, R. V., and Khosla, C. (2013) Nonproteinogenic Amino Acid Building

315

Blocks for Nonribosomal Peptide and Hybrid Polyketide Scaffolds, Angew. Chem. Int. Ed.

316

52, 7098‐7124.

317

31. Nakao, Y., Fujita, M., Warabi, K., Matsunaga, S., and Fusetani, N. (2000) Miraziridine A, a

318

Novel Cysteine Protease Inhibitor from the Marine SpongeTheonella aff. mirabilis, J. Am.

319

Chem. Soc. 122, 10462‐10463.

320

32. Fusetani, N., Matsunaga, S., Matsumoto, H., and Takebayashi, Y. (1990) Bioactive marine

321

metabolites. 33. Cyclotheonamides, potent thrombin inhibitors, from a marine sponge

322

Theonella sp., J. Am. Chem. Soc. 112, 7053‐7054.

323

33. Wäspi, U., Blanc, D., Winkler, T., Rüedi, P., and Dudler, R. (1998) Syringolin, a novel

324

peptide elicitor from Pseudomonas syringae pv. syringae that induces resistance to

325

Pyricularia oryzae in rice, Mol. Plant. Microbe. In. 11, 727‐733.

326

34. Amrein, H., Makart, S., Granado, J., Shakya, R., Schneider‐Pokorny, J., and Dudler, R.

327

(2004) Functional Analysis of Genes Involved in the Synthesis of Syringolin A by

328

Pseudomonas syringae pv. syringae B301D‐R, Mol. Plant. Microbe. In. 17, 90‐97.

329

35. Clerc, J., Groll, M., Illich, D. J., Bachmann, A. S., Huber, R., Schellenberg, B., Dudler, R. and

330

Kaiser, M. (2009) Synthetic and structural studies on syringolin A and B reveal critical

331

determinants of selectivity and potency of proteasome inhibition, Proc. Natl. Acad. Sci.

332

106, 6507‐6512.

333

36. Oka, M., Nishiyama, Y., Ohta, S., Kamei, H., Konishi, M., Miyaki, T., Oki, T., and

334

Kawaguchi, H. (1988) Glidobactins A, B and C, new antitumor antibiotics. I. Production,

335

isolation, chemical properties and biological activity, J. Antibiot. 41, 1331‐1337.

336

37. Schellenberg, B., Bigler, L., and Dudler R. (2007) Identification of genes involved in the

337

biosynthesis of the cytotoxic compound glidobactin from a soil bacterium, Environ.

338

Microbiol. 9, 1640‐1650.

339

38. Konno, H., Kubo, K., Makabe, H., Toshiro, E., Hinoda, N., Nosaka, K., and Akaji, K. (2007)

340

Total synthesis of miraziridine A and identification of its major reaction site for cathepsin

341

B, Tetrahedron 63, 9502‐9513.

342 343

39. Schaschke, N. (2004) Miraziridine A: natures blueprint towards protease class‐spanning inhibitors, Bioorg. Med. Chem. Lett. 14, 855‐857.

344

40. Using commercial as well as synthesized 6 as reactant resulted in significant lower yields

345

as described in the literature regardless of the tested reaction conditions. Furthermore,

346

the analysis of commercial 6 indicated the presence of inadequate Boc‐protection.

347 348

41. Powers, J. C., Asgian, J. L., Ekici, Ö. D., and James, K. E. (2002) Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases, Chem. Rev. 102, 4639‐4750.


Page 12 of 14

Page 13 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


349

42. Konno H. (2012) Synthesis of Bioactive Natural Products as Protein Inhibitors, Biosci.

350

Biotech. Bioch. 76, 1257‐1261.

351

43. Siklos, M., BenAissa, M., and Thatcher, G. R. J. (2015) Cysteine proteases as therapeutic

352

targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors,

353

Acta Pharm. Sin. B 5, 506‐519.

354

44. Gobec, S., and Frlan, R. (2006) Inhibitors of Cathepsin B, Curr. Med. Chem. 13, 2309‐

355

2327.

356

45.

Gong, J., Qing, Y., Zou, S., Fu, R., Su, L., Zhang, X., and Zhang Q. (2016) Protist‐Bacteria

357

Associations: Gammaproteobacteria and Alphaproteobacteria Are Prevalent as

358

Digestion‐Resistant Bacteria in Ciliated Protozoa, Front. Microbiol. 7, 498.

359



TOC: graphical abstract 136x68mm (300 x 300 DPI)


Page 14 of 14

Biosynthesis, Synthesis and Activities of Barnesin A, a NRPS-PKS

Recommend Documents