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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

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ACS Chemical Biology

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Rewiring of the austinoid biosynthetic pathway in filamentous fungi

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

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a

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

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b

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c

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

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Germany

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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

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

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e

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Potsdam, Germany

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f

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* DJM and VV contributed equally to this work

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#

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[email protected]

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Abbreviations: NP, natural product; PKS, polyketide synthase

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Running title: Genome-based rewiring of meroterpenoids

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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:

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Abstract

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

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

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

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

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

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

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

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

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

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

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

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pathways.

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

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

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

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

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

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

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products belonging to the meroterpenoid compound class.4

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

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Aspergillus variecolor, terretonin from Aspergillus terreus, andrastins from Penicillium

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

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

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

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insects.10

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

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

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

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

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

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

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

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

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

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


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

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

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rewiring the complete production of these potentially valuable compounds.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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an intermediate in paraherquonin biosynthesis.5

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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condition.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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nidulans were transformed into a strain already expressing ausP.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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insecticidal derivative, dehydroaustin 310,

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

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

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

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optimal.

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

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

22, 23

, could be produced under planktonic growth



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

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

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

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

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

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

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

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expressing the minimal number of remaining genes.

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

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

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

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

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

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nidulans.

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

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

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

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previously studied metabolic pathways.

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Materials and Methods

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Fungal strains and cultivation


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

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

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

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

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

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

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

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

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

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

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

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

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

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Fungal molecular biology techniques

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

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

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

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

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

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Tables S3 and S4).

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Genome mining



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

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

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

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

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

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from Vector NTI (Invitrogen) (Table S1).

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Extraction and HPLC–HRMS analysis

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

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

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

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

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

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(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

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95:5; 20 minutes total run.

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Plasmid assembly

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

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supplementary text.

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Acknowledgements

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

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

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work

was

supported

by

BASF

SE

(Ludwigshafen,

Germany),

the

Deutsche


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

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Communication, and the Leibniz Research Cluster (LRC).

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ASSOCIATED CONTENT

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Supporting information

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

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

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

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

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Competing interest

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

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EP15200500.5.

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Figure legends

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

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

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

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

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

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

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

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


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

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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

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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

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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]+.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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References

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[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

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[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

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[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.



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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)



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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)


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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.



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Rewiring of the Austinoid Biosynthetic Pathway in ... - ACS Publications

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