Rewiring of the Austinoid Biosynthetic Pathway in Filamentous Fungi

Subscriber access provided by READING UNIV

Letter

Rewiring of the austinoid biosynthetic pathway in filamentous fungi Derek J. Mattern, Vito Valiante, Fabian Horn, Lutz Petzke, and Axel A. Brakhage ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00814 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 29, 2017

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 free 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 accessible to all readers and 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.

ACS Chemical Biology 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 19

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

1

Rewiring of the austinoid biosynthetic pathway in filamentous fungi

2

Derek J. Matterna,b*, Vito Valiantec*, Fabian Hornd,e, Lutz Petzkef and Axel A. Brakhagea,b,#

3

a

4

Research and Infection Biology (HKI), Adolf-Reichwein-Str. 23, 07745 Jena, Germany

5

b

6

c

7

Natural Product Research and Infection Biology (HKI), Adolf-Reichwein-Str. 23, 07745 Jena,

8

Germany

9

d

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product

Friedrich Schiller University, 07745 Jena, Germany

Leibniz Research Group - Biobricks of Microbial Natural Product Syntheses, Leibniz Institute for

Systems Biology and Bioinformatics, Leibniz Institute for Natural Product Research and Infection

10

Biology (HKI), Adolf-Reichwein-Str. 23, 07745 Jena, Germany

11

e

12

Potsdam, Germany

13

f

14

* DJM and VV contributed equally to this work

15

#

16

[email protected]

17

Abbreviations: NP, natural product; PKS, polyketide synthase

18

Running title: Genome-based rewiring of meroterpenoids

19

Key words: Aspergillus, Penicillium, meroterpenoids, austinoids, genome mining, natural products

GFZ German Center for Geosciences, Section 5.3 Geomicrobiology, Telegrafenberg, 14471

BASF SE, 67056 Ludwigshafen, Germany

To

whom

correspondence

and

material

requests

should

be

addressed.

E-mail:

20 1 ACS Paragon Plus Environment


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

Page 2 of 19

21

Abstract

22

Filamentous fungi produce numerous high-value natural products (NPs). The biosynthetic genes for

23

NPs are normally clustered in the genome. A valuable NP class is represented by the insecticidal

24

austinoids. We previously determined their biosynthesis in the fungus Aspergillus calidoustus. After

25

further computational analysis looking into the austinoid gene clusters in two additional distantly

26

related fungi, Aspergillus nidulans and Penicillium brasilianum, a rearrangement of the genes was

27

observed which corresponded to the diverse austinoid derivatives produced by each strain. By

28

advanced targeted combinatorial engineering using polycistronic expression of selected genes, we

29

rewired the austinoid pathway in the fungus A. nidulans, which then produced certain compounds of

30

interest under industrially favored conditions. This was possible by exploiting the presence of

31

previously thought irrelevant genes. Our work shows that comparative analysis of genomes can be

32

used not only to discover new gene clusters, but unearth the hidden potential of known metabolic

33

pathways.

2 ACS Paragon Plus Environment

Page 3 of 19

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


34

Natural products (NPs) produced by microorganisms are invaluable sources for bioactive

35

compounds.1, 2 Typically these NPs are biosynthesized by genes that are clustered in the genome

36

and often possess multimodular enzymes, such as polyketide synthases (PKSs) and/or nonribosomal

37

peptide synthetases (NRPSs).3 Further chemical modifications of polyketide or nonribosomal

38

peptide products are catalyzed by tailoring enzymes that can change the backbone of these

39

molecules.1 Some polyketides can be further modified by prenyltransferase enzymes resulting in

40

products belonging to the meroterpenoid compound class.4

41

Meroterpenoids have been identified in various filamentous fungi such as anditomin from

42

Aspergillus variecolor, terretonin from Aspergillus terreus, andrastins from Penicillium

43

chrysogenum, and paraherquonin from Penicillium brasilianum.5-8 Interestingly, different

44

meroterpenoids have been shown to possess selective insecticidal activity.9 For example, among the

45

austinoids, derivatives have been found to even exert a species-specific activity towards different

46

insects.10

47

The austinoids were first isolated in 1976 from Aspergillus calidoustus (previously assigned as

48

Aspergillus ustus)10, 11, and later identified in both A. nidulans12, 13 and P. brasilianum.5 Recently, a

49

study involving the elucidation of the most complex austinoid gene cluster in A. calidoustus

50

revealed that a second PKS, a noniterative diketide synthase, is also taking part in the

51

biosynthesis.10 In the present study, we set out to obtain further insight on the genomic arrangement

52

of the austinoid gene clusters and look into the chemotypes within these producing strains.

53

Furthermore, we applied advanced synthetic biology techniques to produce various austinoid

54

derivatives in different growth stages in the model organism A. nidulans.

55

The work presented here includes an intriguing example of a gene cluster’s rearrangement among

56

three distantly related fungal species. These species not only produce different austinoid derivatives, 3 ACS Paragon Plus Environment


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

Page 4 of 19

57

but they accumulate these molecules during different growth stages. Using comparative genomics

58

and heterologous expression, we could reconstitute the entire austinoid pathway in A. nidulans

59

rewiring the complete production of these potentially valuable compounds.

60

The three known austinoid producing species, A. calidoustus, A. nidulans and P. brasilianum, have

61

had their genomes sequenced14-16 and it was previously shown that A. nidulans produces the least

62

number of austionoid derivatives with the end products austinol 1 and dehydroaustinol 817, while P.

63

brasilianum can produce until acetoxydehydroaustin 1018, and A. calidoustus produces the highest

64

number of identified derivatives (Figure 1).10 Comparative genome analysis confirmed that the

65

austinoid chemical variability was also accompanied by a genetic fluctuation between the different

66

gene clusters (Figure 2 and Table S1). In A. calidoustus, the austinoid gene cluster lies on a 66 kb

67

contiguous DNA region, while clusters from A. nidulans and P. brasilianum are split in their

68

respective genomes. As shown, the genetic rearrangement provoked certain variability among the

69

clusters. In particular, several A. calidoustus genes are absent in the other two species, namely

70

ausO, ausT, ausV and ausW (depicted in green in Figure 2). Moreover, in the clusters identified in

71

A. nidulans and P. brasilianum, the gene order of the loci is different than in A. calidoustus, with

72

some genes having different orientations (e.g. ausE and ausK in P. brasilianum). Additionally, A.

73

calidoustus is the only species encoding a noninterative diketide synthase in the cluster, ausV

74

(Figure 2 and Table 1), while the other two species show only residual pseudogenes having a DNA

75

sequence highly similar to the ausV locus (~ 80%, Table S1).

76

The high nucleotide similarity shared by the clusters of different species, coupled with the presence

77

of the ausV pseudogene in A. nidulans and P. brasilianum, suggests that A. calidoustus is the

78

original bearer of the austinoid gene cluster. Indeed, this species produces the highest variety of

79

derivatives that were likely lost in the others when the genetic rearrangements occurred. 4 ACS Paragon Plus Environment

Page 5 of 19

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


80

Another variation of the austinoid cluster was also suggested in P. brasilianum. A genetic

81

rearrangement was hypothesized between two separate P. brasilianum strains, NBRC 6234 and

82

MG11. Interestingly, NBRC 6234 strain is unable to produce austinoids, but the closely related

83

compound paraherquonin 5. Austinoids and paraherquonin share the initial biosynthetic steps, but

84

they differentiate when preaustinoid A is transformed either into preaustinoid A1 or

85

berkeleydione.5, 12 This biosynthetic step is catalyzed by the non-heme iron-dependent dioxygenase

86

AusE/PrhA.5, 19 The in vitro enzymatic characterization of the purified PrhA from P. brasilianum

87

NBRC 6234 revealed that the variation of this single enzyme led to the formation of berkeleydione,

88

an intermediate in paraherquonin biosynthesis.5

89

We decided to exploit the knowledge gained from the comparative analysis of the austinoid gene

90

cluster, with the goal of rewiring the pathway in more favorable fermentation conditions. A.

91

calidoustus can produce the most derivatives having insecticide activity, but only during static

92

culture conditions when conidiation occurs (Figure 1B). This condition is unfavorable for industrial

93

scale production as the usual methods involve large fermenters with planktonic growth. Oppositely,

94

A. nidulans containing our empty expression vector can produce austinoids during planktonic

95

growth, but only the early derivatives, which did not show any discernable activity (Figure 3A-B).

96

With the goal of creating a metabolically engineered A. nidulans strain that produces austinoid

97

derivatives in a more suitable condition, genes from A. calidoustus were expressed in A. nidulans

98

(Figures S1 and S2). The candidate genes were heterologously expressed under the control of the

99

inducible xylose promoter (xylp) from Penicillium chrysogenum. This promoter is induced by

100

xylose and repressed when glucose is the sole carbon source.20

101

The first step was to heterologously express the O-acetyltransferase gene ausP, as this gene is not

102

present in the A. nidulans cluster. As expected, the LC-MS analysis showed that the resulting 5 ACS Paragon Plus Environment


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

Page 6 of 19

103

transgenic A. nidulans strain was capable of producing both austin 2 and dehydroaustin 3 (Figure

104

3C). Surprisingly, the strain was also able to further synthesize 7-hydroxydehydroaustin 4 and

105

acetoxydehydroaustin 10 (Figure 3C). This finding showed that the homologues in the A. nidulans

106

austinol gene cluster for ausQ and ausR, which encode for a second O-acyltransferase (AN9250)

107

and a cytochrome P450 monooxygenase (AN9251) respectively, are still fully functional. Even

108

more, they are still being expressed during austinoid production, although irrelevant for the A.

109

nidulans-specific biosynthesis (Table S1). Moreover, this result demonstrated that the most active

110

compound, 3, could be produced during planktonic growth, which is a more industrially favorable

111

condition.

112

Additionally, three more A. calidoustus genes were introduced into A. nidulans by targeted

113

combinatorial engineering: the gene coding for the noniterative PKS AusV, the dioxygenase AusO

114

and the progesterone 5-beta-reductase-like protein AusT, all in different combinations with AusP.

115

Under xylose-inducing conditions, A. nidulans strains expressing ausT were able to produce 1,2-

116

dihydro-7-hydroxydehydroaustin 5 and shunt pathway derivatives including acetoxydehydroaustin

117

10 and 1,2-dihydro-acetoxydehydroaustin 11 (Figure 3D). By adding only ausV, it was possible to

118

obtain the shunt pathway product leading to 1,2-dehydro-precalidodehydroaustin 13 (Figure 3E).

119

Further gene combinations of three or more genes were generated by polycistronic expression of

120

selected genes. This method entails the use of 2A viral peptides that separate the individual genes,

121

which when transcribed are cleaved into their individual proteins. This technique is advantageous

122

because of its use of a single inducible promoter, which controls the expression of a polycistronic

123

mRNA in eukaryotes (Figure 3F-G).21 Interestingly, we could not combine all genes in the same

124

construct because the polycistronic expression of the O-acetyltransferase AusP could not restore

125

production of later derivatives (data not shown). This was likely due to the presence of the 2A tag 6 ACS Paragon Plus Environment

Page 7 of 19

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


126

on the C-terminus, which likely inhibited its activity. Therefore, further gene additions into A.

127

nidulans were transformed into a strain already expressing ausP.

128

The combination of ausT and ausV, used to transform the A. nidulans strain already expressing

129

ausP, led to the successful production of precalidodehydroaustin 6 (Figure 3F). Fascinatingly,

130

AusQ from A. nidulans could successfully transfer the AusV PKS product to the austinoid scaffold,

131

and the acetyl group as seen in acetoxydehydroaustin 10 and 1,2-dihydro-acetoxydehydroaustin 11.

132

To finish the reconstitution in A. nidulans, a three-gene combination, ausO, ausT and ausV, was

133

assembled on one plasmid and once introduced into A. nidulans, it was able to biosynthesize the end

134

product calidodehydroaustin 7 (Figure 3G). Unfortunately, but also of interest, was the fact that

135

austinoids adorned by the AusV diketide product, even if produced in A. nidulans, could not be

136

detected in a planktonic culture; hence, static cultures were required for production of compounds

137

precalidodehydroaustin 6, calidodehydroaustin 7, and 1,2-dehydro-precalidodehydroaustin 13

138

shown in Figure 3E, 3F and 3G respectively. Reasons for A. nidulans not able to produce the

139

diketide products under planktonic cultures range from the actual AusV enzyme directing the

140

biosynthesis in different growth stages or the chemical modification itself dictates production or

141

even have attachment to the membrane. However, it is important to note that the most active

142

insecticidal derivative, dehydroaustin 310,

143

conditions. This could be extremely beneficial in an industrial setting, not only because of the

144

different regulation in comparison to A. calidoustus, but also the fact that A. nidulans is a model

145

organism in filamentous fungi24 and if further genetic modifications are needed this fungus is

146

optimal.

147

This study gives intriguing insights into how gene clusters can be rearranged. This was

148

demonstrated by comparing three different austinoid gene clusters from three diverse filamentous

22, 23

, could be produced under planktonic growth



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

Page 8 of 19

149

fungi. Some genes studied here, were analyzed in a previous biosynthetic study appearing to have

150

no apparent function; thus, they were deemed irrelevant. This was the case in A. nidulans, where

151

ausR and ausQ were deleted, but no change was observed.12 After introduction of ausP from A.

152

calidoustus into A. nidulans, precursors for these enzymes were produced and the deduced products

153

requiring the activity of these redundant enzymes were detected with 7-hydroxydeyhdroaustin 4 and

154

acetoxydehydroaustin 10 (Figure 3C). Thus, we were able to take advantage of these remanent

155

genes and reconstruct the complete austinoid biosynthetic pathway in A. nidulans by heterologously

156

expressing the minimal number of remaining genes.

157

With this study, we highlighted that the knowledge of NPs cannot be merely restricted to the

158

chemical output of the individual fungus. Additional knowledge on genomic and physiological

159

aspects is also important along with available synthetic biology tools that can allow for the

160

production of different active austinoid derivatives under industrially favored conditions. Moreover,

161

dehydroaustin 3, the most active derivative, could even be synthesized by the model organism A.

162

nidulans.

163

In conclusion, our results demonstrated that the chemical plasticity of NPs might be hidden in

164

similar but not identical clusters of different microbial species. Therefore, comparative genomics

165

could be applied not only to discover new gene clusters, but also to investigate the full potential of

166

previously studied metabolic pathways.

167 168

Materials and Methods

169

Fungal strains and cultivation


Page 9 of 19

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


170

Strains used in this study are shown in Table S2, and are deposited in the Jena Microbial Resource

171

Collection (www.uni-jena.de/Pilz__Referenz_Zentrum.html). Two different media were used for

172

fungal growth, Aspergillus minimal medium (AMM)25 and complex medium (1% (w/v) glucose,

173

0.2% (w/v) peptone, 0.1% (w/v) yeast extract, 0.1% (w/v) casamino acids, 10.8 mM MgSO4, 17.4

174

mM KCl, 28.7 mM KH2PO4, 0.1% (v/v) trace elements, 0.1% (v/v) vitamin solution (stock: 0.07

175

mM p-aminobenzoic acid, 0.30 mM pyridoxine HCl, 0.008 mM biotin and 0.09 mM pantothenic

176

acid) and 0.33 µM riboflavin. For solid media, agar 1.5% (w/v) was added.

177

For austinoid production, fungal cultures were grown with complex medium or AMM and

178

inoculated with fresh spores with a concentration of 5x106 spores/mL. Static cultures were grown in

179

complex media at 30 °C for three days. Planktonic cultivations of A. nidulans strains were grown in

180

AMM at 37 °C for 24 hours at 200 rpm. For xylose inducing conditions, media were supplemented

181

with 2 % (w/v) xylose. Data shown in figure 3 were obtained with A. nidulans cultures grown in

182

static complex medium supplemented with 2 % (w/v) xylose.

183

Fungal molecular biology techniques

184

For polymerase chain reactions (PCR), 2x high fidelity Phusion master mix (Life Technologies,

185

Darmstadt, Germany) was used unless otherwise noted. A. nidulans transformations were carried

186

out according to standard protocols. A. nidulans RMS011 protoplasts were transformed as

187

previously reported.26, 27Strains generated in this study are shown in Table S1. Transformants were

188

verified for correct deletions by PCR (data not shown) and Southern blot analysis (Figures S1, and

189

Tables S3 and S4).

190

Genome mining



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

Page 10 of 19

191

The gene coding for the PKS ausA from A. calidoustus was used as probe. Blast analysis28 was

192

performed against the A. calidoustus and P. brasilianum open reading frame (ORF).15, 16 AusA

193

orthologues were identified in both species (Table S1). Putative ORFs surrounding ausA in A.

194

calidoustus were retrieved and subsequently blasted against the A. nidulans and P. brasilianum

195

ORF databases. The nucleotide identity among the different clusters was investigated using AlignX

196

from Vector NTI (Invitrogen) (Table S1).

197

Extraction and HPLC–HRMS analysis

198

Austinoids were extracted from A. calidoustus, A. nidulans and P. brasilianum cultures as

199

previously described.10 High–resolution mass spectrometry (HRMS) was conducted on a Thermo

200

Fischer Q Exactive Hybrid Quadrupole‐Orbitrap mass spectrometer with an electrospray ion source

201

and an Accela HPLC system (Thermo Fisher Scientific) equipped with a C18 column (Accucore 2.6

202

µm 150 x 2.1 mm). Mobile phase 0.2 ml min-1: water (0.1 % (v/v) formic acid): acetonitrile (0.1 %

203

(v/v) formic acid), start 95:5 then to 2:98 in 10 minutes, four minutes 2:98, 0.1 minutes 95:5, 6.9

204

95:5; 20 minutes total run.

205

Plasmid assembly

206

All plasmids used in this study are listed in table S5. Further information is available in the online

207

supplementary text.

208

Acknowledgements

209

We thank C. Schult for excellent technical assistance and help. We also thank A. Perner for HRMS

210

measurements. We are grateful to A. Herold and C. Fleck (BASF SE) for helpful discussions. This

211

work

was

supported

by

BASF

SE

(Ludwigshafen,

Germany),

the

Deutsche


Page 11 of 19

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

Forschungsgemeinschaft (DFG)-funded Graduate School of Excellence, Jena School for Microbial

213

Communication, and the Leibniz Research Cluster (LRC).

214

ASSOCIATED CONTENT

215

Supporting information

216

The Supporting Information is available free of charge via the Internet.

217

Experimental details regarding the assembly of the plasmids along with supplementary figures and

218

tables can be found in the supporting information. This includes Southern blots, nucleotide

219

comparisons and tables detailing the strains used in this study, the primer list and plasmid list.

220 221

Competing interest

222

The authors declare competing financial interests: part of this work was filed for patent under

223

EP15200500.5.

224

Figure legends

225

Figure 1. Proposed biosynthetic pathway for austinoids in the three austinoid producing strains

226

(figure modified from Valiante et al.10). (A) The extended pathway begins with austinol 1 and

227

dehydroaustinol 8, which are the final products identified in the A. nidulans austinoid pathway

228

indicated in sky blue. The pink background marks the products identified in P. brasilianum, which

229

end with acetoxydehydroaustin 10. AusP is marked in pink as it is only present in P. brasilianum

230

and A. calidoustus, while the proteins marked in green are unique to A. calidoustus. The remaining

231

genes are present and functional in the three analyzed species. (B) Extracted ion chromatogram

232

(EIC) overlays of the different austinoids identified in the reported species. Blue chromatograms 11 ACS Paragon Plus Environment


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

Page 12 of 19

233

refer to shunt products. Each peak corresponds to the following m/z: 1, m/z 459.20 [M + H]+; 2, m/z

234

501.21 [M + H]+; 3, m/z 499.19 [M + H]+; 4, m/z 515.19 [M + H]+; 5, m/z 517.20 [M + H]+; 6, m/z

235

599.24 [M + H]+; 7, m/z 615.24 [M + H]+; 8, m/z 457.18 [M + H]+; 9, m/z 501.21 [M + H]+; 10, m/z

236

557.20 [M + H]+; 11, m/z 559.21 [M + H]+; 12, m/z 575.21 [M + H]+; 13, m/z 597.23 [M + H]+.

237

Figure 2. Austinoid gene clusters considerably vary in different fungal species. Genes depicted in

238

sky blue represent common genes amongst the three species; pink portrays genes present only in A.

239

calidoustus and P. brasilianum; cluster genes identified in A. nidulans and A. calidoustus are

240

highlighted in yellow; unique genes in A. calidoustus are depicted in green; in grey are reported

241

genes that are not involved in austinoid biosynthesis, including external bordering coding

242

sequences. As reported, all austinoid biosynthetic genes in A. calidoustus are located in a single

243

cluster, while in A. nidulans and P. brasilianum they are found in two distinct clusters. The cluster

244

in A. nidulans seems to have been split into two pieces. This event likely determined the loss of

245

genes involved in the biosynthetic pathway, such as ausP and ausT. The identified cluster in P.

246

brasilianum was not only split, but some genes were also inverted compared to the A. calidoustus

247

and A. nidulans gene clusters (e.g. ausJ-ausK and ausE). A residual pseudogene similar to ausV

248

was identified in both A. nidulans and P. brasilianum.

249

Figure 3. Rewiring of the austinoid pathway in A. nidulans. (A) Austinoid gene cluster comparison

250

between A. nidulans and A. calidoustus. (B) Chromatograms of compounds extracted from A.

251

nidulans transformed with the argB+-encoding empty vector grown as a planktonic culture (left

252

panel) and static culture (right panel). (C) A. nidulans transformed with ausP led to the formation of

253

7-hydroxydehydroaustin 4 and related shunt pathways in planktonic cultures, demonstrating that the

254

A. nidulans ausQ and ausR genes were still expressed and functional. (D) Transformation of A.

255

nidulans with ausP and ausT led to the formation of 5 in planktonic cultures. (E) Expression of the 12 ACS Paragon Plus Environment

Page 13 of 19

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


256

nonreducing PKS ausV in combination with ausP promoted the formation of the shunt product 1,2-

257

dehydro-precalidodehydroaustin 13 in static cultures. (F) Expression of ausV and ausT via a

258

polycistronic mRNA in combination with ausP made the biosynthesis of the majority of the A.

259

calidoustus austinoid shunt pathways in A. nidulans possible during static growth. (G) The end

260

product calidodehydroaustin 7 identified in A. calidoustus was finally produced in A. nidulans by

261

co-expression of ausP with a polycistronic mRNA encoding ausO, ausT and ausV in static cultures.

262

Blue chromatograms refer to shunt products. The EICs were produced as in Figure 1.

263 264

References

265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292

[1] Brakhage, A. A. (2013) Regulation of fungal secondary metabolism, Nat. Rev. Microbiol. 11, 21-32. [2] Macheleidt, J., Mattern, D. J., Fischer, J., Netzker, T., Weber, J., Schroeckh, V., Valiante, V., and Brakhage, A. A. (2016) Regulation and role of fungal secondary metabolites, Annu. Rev. Genet. 50, 371-392. [3] Hertweck, C. (2009) The biosynthetic logic of polyketide diversity, Angew. Chem., Int. Ed. Engl. 48, 4688-4716. [4] Geris, R., and Simpson, T. J. (2009) Meroterpenoids produced by fungi, Nat. Prod. Rep. 26, 1063-1094. [5] Matsuda, Y., Iwabuchi, T., Fujimoto, T., Awakawa, T., Nakashima, Y., Mori, T., Zhang, H., Hayashi, F., and Abe, I. (2016) Discovery of key dioxygenases that diverged the paraherquonin and acetoxydehydroaustin pathways in Penicillium brasilianum, J. Am. Chem. Soc. 138, 12671-12677. [6] Guo, C. J., Knox, B. P., Chiang, Y. M., Lo, H. C., Sanchez, J. F., Lee, K. H., Oakley, B. R., Bruno, K. S., and Wang, C. C. (2012) Molecular genetic characterization of a cluster in A. terreus for biosynthesis of the meroterpenoid terretonin, Org Lett 14, 5684-5687. [7] Matsuda, Y., Wakimoto, T., Mori, T., Awakawa, T., and Abe, I. (2014) Complete biosynthetic pathway of anditomin: nature's sophisticated synthetic route to a complex fungal meroterpenoid, J. Am. Chem. Soc.136, 15326-15336. [8] Matsuda, Y., Awakawa, T., and Abe, I. (2013) Reconstituted biosynthesis of fungal meroterpenoid andrastin A, Tetrahedron 69, 8199-8204. [9] Xu, Y., Furutani, S., Ihara, M., Ling, Y., Yang, X., Kai, K., Hayashi, H., and Matsuda, K. (2015) Meroterpenoid chrodrimanins are selective and potent blockers of insect GABAgated chloride channels, PLoS One 10, e0122629. [10] Valiante, V., Mattern, D. J., Schueffler, A., Horn, F., Walther, G., Scherlach, K., Petzke, L., Dickhaut, J., Guthke, R., Hertweck, C., Nett, M., Thines, E., and Brakhage, A. A. (2017) Discovery of an extended austinoid biosynthetic pathway in Aspergillus calidoustus, ACS Chem. Biol. 12, 1227-1234. 13 ACS Paragon Plus Environment


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

293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338

Page 14 of 19

[11] Chexal, K. K., Spinger, J. P., Clardy, J., Cole, R. J., Kirksey, J. W., Dorner, J. W., Cutler, H. G., and Strawter, B. J. (1976) Austin, a novel polyisoprenoid mycotoxin from Aspergillus ustus, J. Am. Chem. Soc. 98, 6748-6750. [12] Lo, H. C., Entwistle, R., Guo, C. J., Ahuja, M., Szewczyk, E., Hung, J. H., Chiang, Y. M., Oakley, B. R., and Wang, C. C. (2012) Two separate gene clusters encode the biosynthetic pathway for the meroterpenoids austinol and dehydroaustinol in Aspergillus nidulans, J. Am. Chem. Soc. 134, 4709-4720. [13] Nielsen, M. L., Nielsen, J. B., Rank, C., Klejnstrup, M. L., Holm, D. K., Brogaard, K. H., Hansen, B. G., Frisvad, J. C., Larsen, T. O., and Mortensen, U. H. (2011) A genome-wide polyketide synthase deletion library uncovers novel genetic links to polyketides and meroterpenoids in Aspergillus nidulans, FEMS Microbiol. Lett. 321, 157-166. [14] Galagan, J. E., Calvo, S. E., Cuomo, C., Ma, L.-J., Wortman, J. R., Batzoglou, S., Lee, S.-I., Basturkmen, M., Spevak, C. C., Clutterbuck, J., Kapitonov, V., Jurka, J., Scazzocchio, C., Farman, M., Butler, J., Purcell, S., Harris, S., Braus, G. H., Draht, O., Busch, S., D'Enfert, C., Bouchier, C., Goldman, G. H., Bell-Pedersen, D., Griffiths-Jones, S., Doonan, J. H., Yu, J., Vienken, K., Pain, A., Freitag, M., Selker, E. U., Archer, D. B., Penalva, M. A., Oakley, B. R., Momany, M., Tanaka, T., Kumagai, T., Asai, K., Machida, M., Nierman, W. C., Denning, D. W., Caddick, M., Hynes, M., Paoletti, M., Fischer, R., Miller, B., Dyer, P., Sachs, M. S., Osmani, S. A., and Birren, B. W. (2005) Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae, Nature 438, 1105-1115. [15] Horn, F., Linde, J., Mattern, D. J., Walther, G., Guthke, R., Brakhage, A. A., and Valiante, V. (2015) Draft genome sequence of the fungus Penicillium brasilianum MG11, Genome Announc. 3, e00724-15. [16] Horn, F., Linde, J., Mattern, D. J., Walther, G., Guthke, R., Scherlach, K., Martin, K., Brakhage, A. A., Petzke, L., and Valiante, V. (2016) Draft genome sequences of fungus Aspergillus calidoustus, Genome Announc. 4, e00102-16. [17] Szewczyk, E., Chiang, Y. M., Oakley, C. E., Davidson, A. D., Wang, C. C., and Oakley, B. R. (2008) Identification and characterization of the asperthecin gene cluster of Aspergillus nidulans, Appl. Environ. Microbiol. 74, 7607-7612. [18] Hayashi, H., Mukaihara, M., Murao, S., Arai, M., Lee, A. Y., and Clardy, J. (1994) Acetoxydehydroaustin, a new bioactive compound, and related compound neoaustin from Penicillium sp. MG–11, Biosci., Biotechnol., Biochem. 58, 334-338. [19] Matsuda, Y., Awakawa, T., Wakimoto, T., and Abe, I. (2013) Spiro-ring formation is catalyzed by a multifunctional dioxygenase in austinol biosynthesis, J. Am. Chem. Soc. 135, 1096210965. [20] Zadra, I., Abt, B., Parson, W., and Haas, H. (2000) xylP promoter-based expression system and its use for antisense downregulation of the Penicillium chrysogenum nitrogen regulator NRE, Appl. Environ. Microbiol. 66, 4810-4816. [21] Unkles, S. E., Valiante, V., Mattern, D. J., and Brakhage, A. A. (2014) Synthetic biology tools for bioprospecting of natural products in eukaryotes, Chem. Biol. 21, 502-508. [22] Geris, R., Rodrigues-Fo, E., Garcia da Silva, H. H., and Garcia da Silva, I. (2008) Larvicidal effects of fungal meroterpenoids in the control of Aedes aegypti L., the main vector of dengue and yellow fever, Chem. Biodiversity 5, 341-345. [23] Kataoka, S., Furutani, S., Hirata, K., Hayashi, H., and Matsuda, K. (2011) Three austin family compounds from Penicillium brasilianum exhibit selective blocking action on cockroach nicotinic acetylcholine receptors, Neurotoxicology 32, 123-129. 14 ACS Paragon Plus Environment

Page 15 of 19

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

339 340 341 342 343 344 345 346 347 348 349 350 351


[24] Pontecorvo, G., Roper, J. A., Hemmons, L. M., Macdonald, K. D., and Bufton, A. W. (1953) The genetics of Aspergillus nidulans, Adv. Genet. 5, 141-238. [25] Brakhage, A. A., Browne, P., and Turner, G. (1992) Regulation of Aspergillus nidulans penicillin biosynthesis and penicillin biosynthesis genes acvA and ipnA by glucose, J. Bacteriol. 174, 3789-3799. [26] Riach, M. B., and Kinghorn, J. R. (1995) Genetic transformation and vector developments in filamentous fungi, In Fungal Genetics: Principles and Practice, C. Bos, ed. (London: Wiley), 209-234. [27] Litzka, O., Then Bergh, K., Van den Brulle, J., Steidl, S., and Brakhage, A. A. (1999) Transcriptional control of expression of fungal beta-lactam biosynthesis genes, Antonie Van Leeuwenhoek 75, 95-105. [28] Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., and Madden, T. L. (2009) BLAST+: architecture and applications, BMC Bioinf.10, 421.



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

graphical abstract 266x155mm (127 x 127 DPI)

ACS Paragon Plus Environment

Page 16 of 19

Page 17 of 19

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


Proposed biosynthetic pathway for austinoids in the three austinoid producing strains (figure modified from Valiante et al.10). (A) The extended pathway begins with austinol 1 and dehydroaustinol 8, which are the final products identified in the A. nidulans austinoid pathway indicated in sky blue. The pink background marks the products identified in P. brasilianum, which end with acetoxydehydroaustin 10. AusP is marked in pink as it is only present in P. brasilianum and A. calidoustus, while the proteins marked in green are unique to A. calidoustus.The remaining genes are present and functional in the three analyzed species. (B) Extracted ion chromatogram (EIC) overlays of the different austinoids identified in the reported species. Blue chromatograms refer to shunt products. Each peak corresponds to the following m/z: 1, m/z 459.20 [M + H]+; 2, m/z 501.21 [M + H]+; 3, m/z 499.19 [M + H]+; 4, m/z 515.19 [M + H]+; 5, m/z 517.20 [M + H]+; 6, m/z 599.24 [M + H]+; 7, m/z 615.24 [M + H]+; 8, m/z 457.18 [M + H]+; 9, m/z 501.21 [M + H]+; 10, m/z 557.20 [M + H]+; 11, m/z 559.21 [M + H]+; 12, m/z 575.21 [M + H]+; 13, m/z 597.23 [M + H]+. 659x557mm (64 x 64 DPI)



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

Austinoid gene clusters considerably vary in different fungal species. Genes depicted in sky blue represent common genes amongst the three species; pink portrays genes present only in A. calidoustus and P. brasilianum; cluster genes identified in A. nidulans and A. calidoustus are highlighted in yellow; unique genes in A. calidoustus are depicted in green; in grey are reported genes that are not involved in austinoid biosynthesis, including external bordering coding sequences. As reported, all austinoid biosynthetic genes in A. calidoustus are located in a single cluster, while in A. nidulans and P. brasilianum they are found in two distinct clusters. The cluster in A. nidulans seems to have been split into two pieces. This event likely determined the loss of genes involved in the biosynthetic pathway, such as ausP and ausT. The identified cluster in P. brasilianum was not only split, but some genes were also inverted compared to the A. calidoustus and A. nidulans gene clusters (e.g. ausJ-ausK and ausE). A residual pseudogene similar to ausV was identified in both A. nidulans and P. brasilianum. 257x107mm (127 x 127 DPI)


Page 18 of 19

Page 19 of 19

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


Rewiring of the austinoid pathway in A. nidulans. (A) Austinoid gene cluster comparison between A. nidulans and A. calidoustus. (B) Chromatograms of compounds extracted from A. nidulans transformed with the argB+-encoding empty vector grown as a planktonic culture (left panel) and static culture (right panel). (C) A. nidulans transformed with ausP led to the formation of 7-hydroxydehydroaustin 4 and related shunt pathways in planktonic cultures, demonstrating that the A. nidulans ausQ and ausR genes were still expressed and functional. (D) Transformation of A. nidulans with ausP and ausT led to the formation of 5 in planktonic cultures. (E) Expression of the nonreducing PKS ausV in combination with ausP promoted the formation of the shunt product 1,2-dehydro-precalidodehydroaustin 13 in static cultures. (F) Expression of ausV and ausT via a polycistronic mRNA in combination with ausP made the biosynthesis of the majority of the A. calidoustus austinoid shunt pathways in A. nidulans possible during static growth. (G) The end product calidodehydroaustin 7 identified in A. calidoustus was finally produced in A. nidulans by coexpression of ausP with a polycistronic mRNA encoding ausO, ausT and ausV in static cultures. Blue chromatograms refer to shunt products. The EICs were produced as in Figure 1.



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

280x371mm (127 x 127 DPI)


Page 20 of 19

Rewiring of the Austinoid Biosynthetic Pathway in Filamentous Fungi

Recommend Documents