Two Separate Gene Clusters Encode the Biosynthetic Pathway for the Meroterpenoids Austinol and...

Two Separate Gene Clusters Encode the Biosynthetic Pathway forthe Meroterpenoids Austinol and Dehydroaustinol in AspergillusnidulansHsien-Chun Lodagger Ruth EntwistleDagger Chun-Jun Guodagger Manmeet AhujaDagger Edyta Szewczyksect

Jui-Hsiang Hung∥ Yi-Ming Chiangdaggerperp Berl R OakleyDagger and Clay C C Wangdaggerpara

daggerDepartment of Pharmacology and Pharmaceutical Sciences School of Pharmacy University of Southern California 1985 ZonalAvenue Los Angeles California 90089 United StatesDaggerDepartment of Molecular Biosciences University of Kansas 1200 Sunnyside Ave Lawrence Kansas 66045 United StatessectDepartment of Molecular Genetics Ohio State University 484 West 12th Avenue Columbus Ohio 43210 United States∥Department of Biotechnology and perpGraduate Institute of Pharmaceutical Science Chia Nan University of Pharmacy and ScienceTainan 71710 Taiwan ROCparaDepartment of Chemistry College of Letters Arts and Sciences University of Southern California Los Angeles California 90089United States

S Supporting Information

ABSTRACT Meroterpenoids are a class of fungal naturalproducts that are produced from polyketide and terpenoidprecursors An understanding of meroterpenoid biosynthesis atthe genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds The filamentous fungus Aspergillusnidulans has previously been found to produce twomeroterpenoids austinol and dehydroaustinol Using targeted deletions that we created we have determined that surprisinglytwo separate gene clusters are required for meroterpenoid biosynthesis One is a cluster of four genes including a polyketidesynthase gene ausA The second is a cluster of 10 additional genes including a prenyltransferase gene ausN located on a separatechromosome Chemical analysis of mutant extracts enabled us to isolate 35-dimethylorsellinic acid and 10 additionalmeroterpenoids that are either intermediates or shunt products from the biosynthetic pathway Six of them were identified asnovel meroterpenoids in this study Our data in aggregate allow us to propose a complete biosynthetic pathway for the Anidulans meroterpenoids

INTRODUCTIONFungal species are known to produce a variety of structurallycomplex secondary metabolites many of which have importantrelevance to human health1 Members of the genus Aspergilluscan combine polyketide and terpenoid precursors to producestructurally complex secondary metabolites called meroterpe-noids2 Examples of known meroterpenoids include terretoninand territrem produced by A terreus34 pyripyropene Aproduced by certain strains of A fumigatus56 austin producedby A ustus7 and austinol (1) and dehydroaustinol (2)produced by A nidulans (Figure 1)89 Territrem is a potentirreversible inhibitor of acetylcholinesterase (AChE) and is acandidate for drug development for treating Alzheimerrsquosdisease4 Pyripyropene is a potent selective inhibitor of acyl-CoA cholesterol acyltransferase (ACAT) an enzyme thatcatalyzes intracellular esterification of cholesterol and is beingdeveloped for treating and preventing atherosclerosis56

A series of labeling studies in the 1980s provided aframework for the biosynthetic mechanism of meroterpe-noids10minus12 Recently the biosynthetic gene cluster of pyripyr-

opene was identified in A fumigatus and verified using aheterologous fungal expression approach13 The pyripyropenegene cluster encodes one non-reducing polyketide synthase(NR-PKS) one prenyltransferase one CoA ligase twocytochrome P450s one FAD-dependent monooxygenase oneterpene cyclase and two acetyltransferasesIn this study we attempted to locate the biosynthesis genes

for austinol (1) and dehydroaustinol (2) in A nidulans bysearching the A nidulans genome for regions where a NR-PKSgene and a prenyltransferase gene are in proximity Interestinglyno prenyltransferase genes were located near NR-PKS genesThis suggested to us that the genes responsible for austinol (1)and dehydroaustinol (2) biosynthesis in A nidulans might beseparated in the genome Although secondary metabolitebiosynthesis genes are generally clustered in fungal genomes1

we recently showed that the genes that encode the prenyl

Received October 27 2011Published February 13 2012

Article

pubsacsorgJACS

copy 2012 American Chemical Society 4709 dxdoiorg101021ja209809t | J Am Chem Soc 2012 134 4709minus4720

xanthone biosynthesis pathway in A nidulans are located inthree separate regions of the genome14

We identified using a set of deletions of all non-reducingPKS genes the NR-PKS responsible for the biosynthesis of themeroterpenoids in A nidulans This NR-PKS has beenindependently identified by Nielsen et al and designatedausA15 Next we selected two candidate prenyltransferase geneslocated elsewhere in the genome for deletion and frommetabolomic analysis of mutant extracts we found that one wasinvolved in meroterpenoid synthesis To identify additionalgenes involved in the pathway we targeted the genessurrounding the NR-PKS gene and the prenyltransferase genefor deletion The gene deletion experiments allowed us to

identify 12 additional genes involved in meroterpenoidbiosynthesis which are located in two gene clusters onecontaining the NR-PKS and the other containing theprenyltransferase Large-scale cultures of the deletion strainsenabled us to isolate several stable intermediates (Figure 2)including six novel compounds allowing us to propose abiosynthesis pathway for austinol (1) and dehydroaustinol (2)biosynthesis

RESULTSIdentification of the Polyketide Synthase Responsible

for Austinol (1) and Dehydroaustinol (2) BiosynthesisWhen cultivated on solid yeast agar glucose (YAG) medium A

Figure 1 Meroterpenoids from Aspergillus species

Figure 2 Intermediates isolated from gene deletion mutants

Journal of the American Chemical Society Article

dxdoiorg101021ja209809t | J Am Chem Soc 2012 134 4709minus47204710

nidulans produces two meroterpenoids austinol (1) anddehydroaustinol (2) (Figure 1)89 The structural similarity ofthese two compounds suggests that they may have the samebiosynthetic origin For this study we used an A nidulans straincontaining the nkuAΔ and stcJΔ mutations The nkuAΔdeletion allows high homologous recombination rates andfacilitates gene targeting stcJΔ prevents production of themajor A nidulans polyketide sterigmatocystin The rationale forthe use of an stcJΔ strain is that the elimination ofsterigmatocystin frees up the common polyketide precursormalonyl-CoA and also facilitates the detection and isolation ofother metabolites Early studies using labeled precursorsshowed that austin which is very similar to austinol (1) isformed by the C-alkylation of 35-dimethylorsellinic acid anaromatic polyketide with farnesyl pyrophosphate10 Becausefungal secondary metabolite biosynthesis genes are usuallyclustered we first looked for a PKS near a prenyltransferase inthe A nidulans genome in an effort to identify themeroterpenoid gene cluster Surprisingly we found no PKSnear a prenyltransferase on any chromosome leading us toconclude that the PKS and prenyltransferase genes required foraustinol (1) and dehydroaustinol (2) biosynthesis must beseparated in the genome We consequently used a genome-wide gene targeting approach to locate the austinol (1) anddehydroaustinol (2) biosynthesis genesTo locate the PKS responsible for austinol (1) and

dehydroaustinol (2) we first determined on the basis of acomparison to other previously characterized fungal aromaticpolyketides that the aromatic structure of the predictedprecursor 35-dimethylorsellinic acid (3) (Figure 2) wouldlikely require a non-reducing PKS (NR-PKS) Previously wecreated NR-PKS knock out libraries in two different geneticbackgrounds in A nidulans to elucidate products of these NR-PKSs Using these libraries we identified the NR-PKS involvedin the biosynthesis of asperthecin9 emodin16 the prenylatedxanthones14 orsellinic acid and F-9775AB1617 Separately we

demonstrated that the NR-PKS gene AN10344 is involved inasperfuranone biosynthesis by inducing a nearby transcriptionfactor located within the gene cluster18 The products of theremaining six NR-PKS (encoded by AN05234 AN20324AN32304 AN33864 AN64484 AN70714) were unknownStrains carrying these deletions were cultivated under austinol(1)- and dehydroaustinol (2)-producing conditions andmetabolites were analyzed by liquid chromatographyminusdiodearray-mass spectrometry (LCminusDAD-MS) We found that all ofthese strains continued to produce the two meroterpenoids(data not shown) and thus that none of these NR-PKSs isinvolved in meroterpenoid productionWe reexamined the A nidulans genome using the gene

annotations of the Central Aspergillus Data Repository(CADRE) (httpwwwcadre-genomesorguk) and the Aspergil-lus genome database (httpwwwaspgdorg) and locatedAN83834 encoding a putative NR-PKS gene for which wehad not created a deletant mutant in our previous studies dueto an error in the initial annotation Expression of AN83834 inanother project revealed that it is responsible for the synthesisof 35-dimethylorsellinic acid a likely precursor of themeroterpenoids (M Ahuja et al submitted) We consequentlycreated deletion strains for AN83834 (AN83834Δ) using thegene-targeting procedures we have recently developed for Anidulans involving a nkuAΔ strain and fusion PCR1920

Diagnostic PCR using three different primer sets confirmedthat AN83834 was deleted correctly and we have used thesame technique to verify the deletions in all strains used in thisstudy (Supplementary Figures S3 and S4) The AN83834Δstrain was cultivated under austinoldehydroaustinol producingconditions and LCminusDAD-MS analysis showed that it failed toproduce the two meroterpenoids (Figure 3) These data allowus to identify AN83834 as the gene responsible for thebiosynthesis of austinol (1) and dehydroaustinol (2)AN83834 is located on chromosome V at bp 307999minus

315732 AN83834 is predicted to encode a 2476 amino acid

Figure 3 HPLC profile of extracts from (A) LO2026 (background control strain) (B) AN83834Δ PKS deletant (C) AN92594Δ prenyltransferasedeletant and (D) AN81424Δ as detected by UV at 254 nm and mass spectrometry Austinol 1 [M + H]+ mz = 459 and dehydroaustinol 2 [M +H]+ mz = 457 are detected using positive ion mode and 35-dimethylorsellinic acid 3 [M minus H]minus mz = 195 is detected using negative ion modeEIC = extracted ion chromatogram



NR-PKS that contains starter unit ACP transacylase (SAT) β-ketoacyl synthase (KS) acyltransferase (AT) product template(PT) acyl carrier protein (ACP) C-methyltransferase (C-MeT) and thiolesterase (TE) domains During the course ofthis work Nielson et al reported the independent discoverythat AN83834 is involved in austinoldehydroaustinol syn-thesis and designated it ausA15 For consistency we will alsouse this designationIdentification of the Prenyltransferase Involved in

Austinol and Dehydroaustinol Biosynthesis The pre-nylation of 35-dimethylorsellinic acid produced by the NR-PKS AusA (the protein product of the ausA gene) must becatalyzed by a prenyltransferase Since none of the geneslocated near ausA have homology to any known prenyltransfer-ase we looked to other parts of the genome to locate thenecessary prenyltransferase We performed a BLAST search ofthe A nidulans genome using the amino acid sequence of UbiAas a query to search for its homologues The UbiA enzyme isinvolved in the biosynthesis of prenylated quinones such asubiquinone in E coli21 This enzyme catalyzes the transfer of all-trans prenyl moieties to the acceptor molecule 4-hydrox-ybenzoate (4HB) This enzyme was selected because it

catalyzes CminusC bond formation between an aromatic substrateand an isoprenoid diphosphateThe top two candidate genes in the BLAST search were

AN92594 and AN81424 Deletions were created and verifiedfor each Cultivation and analysis of the deletant strain by LCminusDAD-MS showed that the AN81424 deletant (AN81424Δ)continued to synthesize austinol (1) and dehydroaustinol (2)whereas AN92594Δ failed to produce the two meroterpenoids(Figure 3) We deduced therefore that AN92594 is requiredfor austinol (1) and dehydroaustinol (2) biosynthesis and wedesignate it ausNFrom an ausNΔ strain we isolated and characterized by

NMR and mass spectrometry 35-dimethylorsellinic acid (3)the polyketide precursor of austin biosynthesis predicted fromearlier labeling studies10 We also isolated this precursor in ourAN83834 overexpression studies (M Ahuja et al submitted)indicating that AusA makes 3 which is subsequently modifiedby the product of ausN Both the genetic data and the isolationof 35-dimethylorsellinic acid (3) thus demonstrate that ausNmakes the prenyltransferase involved in biosynthesis of the twomeroterpenoids It is located on chromosome VIII whereasausA is on chromosome V

Figure 4 (A) Organization of the polyketide synthase gene cluster (cluster A) for austinoldehydroaustinol biosynthesis in A nidulans Each arrowindicates relative size and the direction of transcription of the ORFs deduced from analysis of nucleotide sequences On the basis of our genedeletion results genes in black are involved in austinoldehydroaustinol biosynthesis whereas those in white are not (B) HPLC profile of extracts ofstrains in the cluster as detected by UV absorption at 254 nm Numbering on peaks corresponds to the intermediates shown in Figure 2



Targeted Gene Deletions Reveal Additional GenesRequired for Meroterpenoid Biosynthesis Early labelingstudies showed that a series of oxidation steps are necessary forthe biosynthesis of the austinol-related molecule austin12 Sincethe genes involved in the biosynthesis of particular secondarymetabolites are usually clustered in fungi22 we wished to deletegenes surrounding ausA and ausN to identify other genesrequired for austinol and dehydroaustinol biosynthesis Aproblem with respect to the ausA region was that there was agap near ausA in the genome (ie the contiguous sequencecontaining ausA could not be connected to the adjacentcontiguous sequence apparently because sequences weremissing) To fill in this gap we amplified A nidulans genomicDNA using primers flanking the gap We sequenced theresulting fragment and found that the apparent gap was actually

due to the insertion of an incorrect sequence Once thissequence was eliminated the sequences on the two sides of theldquogaprdquo overlapped and matched perfectly and the gap was thuseliminated We found that AN83814 extended across thepreviously incorrectly annotated gap We communicated thisinformation to CADRE and the gap has now been corrected inthe CADRE and ASPGD databasesWe deleted 12 genes surrounding ausA (AN83764

AN83774 AN83784 AN83794 AN83804 AN83814AN83824 AN83844 AN83854 AN110774 AN110854and AN83874) (Figure 4A) Three putative deletants foreach gene were verified by diagnostic PCR using three differentprimer sets For each gene at least two of the three deletantsproved correct (Supplementary Figure S4) At least two strainsper gene were cultivated under austinoldehydroaustinol-

Figure 5 (A) Organization of the prenyltransferase gene cluster (cluster B) for austinoldehydroaustinol biosynthesis in A nidulans Each arrowindicates the relative size and the direction of transcription of the ORFs deduced from analysis of nucleotide sequences On the basis of our genedeletion results genes in black are involved in austinoldehydroaustinol biosynthesis whereas those in white are not (B) HPLC profile of extracts ofstrains in the cluster as detected by UV absorption at 254 nm Numbering on peaks corresponds to the intermediates shown in Figure 2



producing conditions and the culture extracts were screened byLCminusMS Deletions of AN83794 AN83814 AN83844 fullyeliminated the production of both meroterpenoids (Figure 4B)and we designate these genes ausB ausC and ausD respectivelyWe were able to detect by LCminusMS a new UV active compound4 from the ausBΔ strains An ausBΔ strain was grown at a largescale and compound 4 was isolated by flash chromatography

and preparative scale HPLC Using both 1D and 2D NMRspectroscopy we determined the structure of compound 4(NMR data shown in Supporting Information) which wenamed protoaustinoid A (4) Protoaustinoid A (4) has atetracyclic structure with a 13-diketone functional group Asimilar compound was proposed earlier as a key earlyintermediate in austin biosynthesis12 Protoaustinoid A contains

Figure 6 Proposed biosynthesis pathway of austinoldehydroaustinol The clusters in which the genes are found (Cluster A or Cluster B) areindicated



a methyl ester connected to carbon 8prime instead of the carboxylicfunction group in the previously proposed structure12

From both AN83814Δ and AN83844Δ strains we wereunable to obtain sufficient amounts of stable intermediates toelucidate the roles of the two genes in the biosynthetic pathwayWe believe we have established one border of the gene clustersurrounding the PKS using the AN83764Δ AN83774Δ andAN83784Δ strains since these strains continue to produce thetwo meroterpenoids The other border is established by theAN83854Δ AN110774Δ AN110854Δ and AN83874Δstrains since these strains also continue to produce the twomeroterpenoids Our deletion data have thus allowed us toidentify three genes surrounding the PKS gene ausA that areinvolved in the pathwayThe structural complexity of austinoldehydroaustinol led us

to believe that additional genes are necessary for theirbiosynthesis We hypothesized that they are located adjacentto the prenyltransferase gene ausN and that deletion of genes inthis region would reveal the remaining genes required formeroterpenoid biosynthesis Initially we targeted nine genessurrounding the prenyltransferase gene ausN The nine geneswere AN112054 AN92564 AN116474 AN92574AN112174 AN112064 AN116484 AN92604 andAN92614 (Figure 5A) Three putative deletants for eachgene were tested by diagnostic PCR using three differentprimer sets At least two correct deletants per gene werecultivated under meroterpenoid-producing conditions andscreened by reverse-phase LCminusMS Metabolite profiles ofAN116484 AN92604 and AN92614 deletants showed thatthey are not involved in austinoldehydroaustinol biosynthesisand they define one of the boundaries for this cluster (Figure 5and Supplementary Figure S2) Because LCminusMS analysisshowed that AN112054 (ausK) is involved in the biosynthesisof the two meroterpenoids we created additional genedeletants beyond AN112054 to look for the left boundaryWe deleted 12 additional genes AN92444 AN92454AN92464 AN92474 AN92484 AN92494 AN92504AN92514 AN92524 AN92534 AN92544 AN112144 atotal of 22 genes for this cluster including ausN We verified atleast two deletants for each gene and used them for LCminusMSanalysis The results show that AN92464 (ausE) AN92474(ausF) AN92484 (ausG) AN92494 (ausH) AN92534 (ausI)

AN112144 (ausJ) AN110254 (ausK) AN92574 (ausL) andAN112064 (ausM) are involved in the biosynthesis pathwaysince they fully eliminated the production of both meroterpe-noids except that deletion of AN92474 (ausF) permittedminor production of austinoldehydroaustinol (Figure 5B)The low production of austinoldehydroasutinol by AN92474deletion strains was confirmed by MSMS (data not shown)This suggests that AN92474 might be involved in a late step ofthe biosynthesis pathway or more likely may have a regulatoryfunction Our gene deletion data in total allow us to deducethat AN92464 and AN92594 define the borders of the genecluster surrounding ausNSeveral of our deletion strains produced sufficient amounts of

chemically stable metabolites to allow us to isolate them anddetermine their structures using 1D and 2D NMR spectroscopy(NMR data for all the intermediates are shown in SupportingInformation) Compounds 5minus13 isolated from large-scalecultures of the deletant strains are all meroterpenoids that areeither proposed intermediates or shunt products in the austinol(1) and dehydroaustinol (2) biosynthesis pathways Preausti-noid A3 (5) isoaustinone (7) austionolide (9) andaustinoneol A (11) have been previously isolated and identifiedfrom Penicillium sp and have been proposed to beintermediates in austin biosynthesis23 Our NMR analysis ofthese four compounds are in agreement with published NMRdata of these compounds The remaining chemicals thataccumulated in the deletion strains were structurally charac-terized and found to be novel materials We designate thempreaustinoid A4 (6) 11β-hydroxyisoaustinone (8) preausti-noid A5 (10) (5primeR)-isoaustinone (12) and neoaustinone (13)X-ray crystallographic analysis confirmed the absolute config-uration of (5primeR)-isoaustinone (12) (Supplementary Figure S5)

DISCUSSIONUsing a series of targeted gene deletions we have identified thegenes encoding the biosynthesis pathway for the meroterpe-noids austinol (1) and dehydroaustinol (2) in A nidulans(Figure 6) The pathway involves at least 14 genes in twoseparate gene clusters Three genes are clustered around thepolyketide synthase gene AN83834 (ausA) located onchromosome V and nine genes are clustered around theprenyltransferase gene AN92594 (ausN) located on chromo-

Table 1 Genes Required for AustinolDehydroaustinol Biosynthesis in A nidulans

ORFs homologues

gene predicted size (aa) match from BLAST search at NCBI (accession no) identitysimilarity ()

AN83794 (ausB) 745 cyclopentadecanone 12-monooxygenase [Pseudomonas sp HI-70] (BAE93346) 3754AN83814 (ausC) 638 cyclododecanone monooxygenase [Rhodococcus ruber] (AAL14233) 4356AN83834 (ausA) 2476 methylorcinaldehyde synthase [Acremonium strictum] (CAN87161) 3251AN83844 (ausD) 282 hypothetical proteinAN92464 (ausE) 298 fumonisin C-5 hydroxylase Fum3p [Gibberella moniliformis] (AAG27131) 3452AN92474 (ausF) 177 hypothetical proteinAN92484 (ausG) 529 P450 monooxygenase [Sphaceloma manihoticola] (CAP07651) 4059AN92494 (ausH) 174 hypothetical proteinAN92534 (ausI) 501 P450 monooxygenase [Sphaceloma manihoticola] (CAP07653) 3255AN112144 (ausJ) 162 hypothetical proteinAN112054 (ausK) 398 norsolorinic acid reductase [Aspergillus parasiticus] (Q00258) 4866AN92574 (ausL) 204 terpene cyclases pyr4 [Aspergillus fumigatus F37]13 3255AN112064 (ausM) 479 a PaxM [Penicillium paxilli] (AAK11530) a 4665

b FAD-dependent monooxygenase pyr5 [Aspergillus fumigatus F37]13 b 4258AN92594 (ausN) 330 a 4-hydroxybenzoate geranyltransferase [Lithospermum erythrorhizon] (BAB84122) a 3049

b prenyltransferase pyr6 [Aspergillus fumigatus F37]13 b 3046



some VIII For ease of reference we will call these two clusterscluster A and cluster B respectively Our findings illustrate thepower of combining genomics efficient gene targeting andnatural products chemistry Without combining these ap-proaches it would not have been possible to identify the genesinvolved in austinol and dehydroaustinol biosynthesis nor toobtain adequate data to propose a biosynthetic pathway forthese compoundsIn our proposed pathway the first gene ausA encodes a

polyketide synthase for 35-dimethylorsellinic acid (3) syn-thesis AusA contains the SAT KS AT PT ACP CMeT andTE domains and the lack of ER DH and KR domainsindicates that it is an NR-PKS24 This NR-PKS may utilize oneacetyl-CoA three malonyl-CoA and two SAM (S-adenosylmethionine) to produce compound 3In the next step C-alkylation of compound 3 an aromatic

polyketide by farnesyl pyrophosphate is catalyzed by anaromatic prenyltransferase AusN We were able to isolate 35-dimethylorsellinic acid (but not austinol dehydroaustinol norany other intermediate) from the ausN deletion strain whichprovides solid evidence that ausN encodes the prenyltransferasein the pathway and that the prenylation step occurs very earlyin the pathway before other intermediates are synthesizedBLAST analysis revealed that AusN has 49 protein sequencesimilarity to LePGT-1 an aromatic prenyltransferase (Table 1)The enzymatic activity of LePGT-1 was examined by Yazaki etal25 They showed that LePGT-1 which is homologous toUbiA makes 4-hydroxybenzoate an intermediate in shikoninbiosynthesis in Lithospermum erythrorhizon Analysis of thepredicted protein sequence of AusN led to the identification ofan aspartate-rich motif (88-NDLVDVDID-96) similar toLePGT-1 This NDXXDXXXD motif which binds the prenyldiphosphate via a Mg2+ ion is highly conserved among allprenyltransferases involved in lipoquinone biosynthesis26

Unlike prenyltransferase enzymes such as protein prenyltrans-ferases or fungal indole prenyltransferases which are water-soluble lipoquinone prenyltransferases are membrane-bound26

In addition AusN was predicted to have nine transmembranehelices by MEMPACK [on the PsiPred server (httpbioinfcsuclacukpsipred)] Therefore AusN could be a membrane-bound aromatic prenyltransferase that catalyzes the transfer of aprenyl moiety to an aromatic acceptor moleculeThe next steps include the epoxidation of the prenylated

polyketide intermediate possibly by the epoxidase encoded bythe gene ausM followed by cyclization catalyzed by a terpenecyclase to form the tetracyclic intermediate We were unable toisolate analyzable amounts of the proposed prenylatedpolyketide 15 and the proposed epoxidated intermediate 16presumably because these two intermediates are not sufficientlystable in our purification scheme (Figure 6)Although no intermediate could be isolated from ausM and

ausL deletant strains we can propose functions for them basedon homology BLAST searches with AusM showed that it has65 similarity to an epoxidase PaxM involved in paxillinebiosynthesis27 It also has 58 similarity to another epoxidaseencoded by pyr5 that catalyzes the epoxidation of the terpenoidmoiety of farnesyl 4-hydroxy-6-(3-pyridinyl)-2H-pyran-2-one(HPPO) in pyripyropene A biosynthesis13 Based on theepoxidation functions of its homologues we propose thatAusM catalyzes the epoxidation of the terminal alkene ofterpenoid moiety of 15 to form 16We propose that the terpene cyclization step is catalyzed by

AusL This enzyme has 55 similarity to the enzyme encoded

by pyr4 which is a terpene cyclase involved in pyripyropene Abiosynthesis in Aspergillus fumigatus13 Both AusL and Pyr4 areannotated as ldquointegral membrane proteinrdquo by the BroadInstitute Web site (httpwwwbroadinstituteorg) and theirsizes are smaller than any other terpene cyclases identified sofar This smaller size of pyr4 led Itoh et al to describe it as anovel terpene cyclase13 When we examined AusL we identifieda conserved residue (E62) in its sequence that in other terpenecyclases is proposed to be involved in the protonation of theepoxide ring that may trigger the following polyene cyclizationstep28

Next the lactone system in the ring A of 4 is formed via aBaeyerminusVilliger oxidation by AusB We were able to isolatelarge quantities of compound 4 from the ausBΔ strainproviding evidence that the lactone formation is catalyzed byAusB BLAST analysis showed that AusB has 54 amino acidsequence similarity to CpdB of Pseudomonas sp strain HI-70which was demonstrated to be a BaeyerminusVilliger monoox-ygenase (BVMO)29 Some conserved motifs are sharedbetween all the members of the BVMO family30 Analysis ofthe predicted amino acid sequence of AusB revealed that itindeed has conserved motifs found in typical BVMOsequences They are two Rossmann folds (178-GAGFGG-183and 374-GTGSTA-379) for binding FAD and NADPH aBMVO fingerprint (338-FQGHIFHTARWD-349) and twoother conserved motifs (210-GG-211 and 561-DLVVLATG-568) The underlined sequences are conserved amongmembers of this class of enzyme On the basis of homologyconserved motifs and the accumulation of compound 4 in theausB deletant we deduce that ausB encodes a BVMO thatcatalyzes the formation of ε-lactone A ring of austinoldehydroaustinol The methyl ester connected to carbon 8prime ofprotoaustinoid A (4) suggests that a methyltransferase shouldbe involved in the early stage of the pathway Since we wereunable to isolate the proposed compound 15 and 16 from ourmutant strains we cannot say whether the esterification stepoccurs before or after cyclization by AusLThe next steps involve the cleavage and rearrangement of the

tetraketide portion of the intermediates from compound 5 tocompound 9 Simpson Vederas and co-workers proposed thatthis rearrangement involves hydroxylation of the C-5prime followedby α-ketol rearrangement and reduction of the ketone withlactone formation by the attack of the hydroxyl group on thecarboxy group12 Our results show that these steps of thepathway involve at least four genes ausJ ausK ausH and ausIThe ausJ deletant accumulated compound 5 (Figure 5B)suggesting that this enzyme is responsible for the acid-catalyzedketo-rearrangement and ring contraction of the tetraketideportion to generate compound 6 The ausK deletant producedthe three compounds 6 10 and 11 We propose thatcompounds 10 and 11 are shunt products of the pathwayBLAST analysis showed that AusK has 66 amino acidsimilarity to Nor1 which converts the 1prime keto group ofnorsolorinic acid to the 1prime hydroxyl group of averantin in Aparasiticus31 We speculate that this enzyme is responsible forreducing the C-5prime keto of compound 6 to hydroxyl and thishydroxyl oxygen then attacks the carboxyl to form the γ-lactonering in the triketide portion of the molecule A similarmechanism has been proposed in previous labeling study12

1D and 2D NMR revealed that compounds 6 and 10 areconstitutional isomers Acetyl and hydroxyl groups are at C-6primein compound 6 and at C-4prime in compound 10 (please seeSupporting Information for details) The presence of two



isomers indicates to us that the keto rearrangement does notoccur in a stereoselective manner The alkyl shift can be fromC-5prime to either C-4prime or C-6prime to form compound 10 and 6respectively Compound 11 was also found in this deletant andmight be a shunt product made by oxidizing C-5prime of compound10 to carboxylic acid followed by β-keto-acid decarboxylationoxidation by other endogenous enzymes Since we did notdetect conversion of compound 6 to any compound similar to11 the enzymes may prefer only compound 10 as substraterather than compound 6 Among all the deletants compound10 was only found in the ausK deletion strain Thus theconsumption of compound 6 by AusK and the enzymesdownstream in the pathway may lead the reaction to gothrough the major route of the pathway instead of the shuntpathWhen we analyzed the metabolites of ausH deletants we

found an accumulation of compound 12 (5primeR configuration)which proved to be a stereoisomer of 7 (5primeS configuration) by1D and 2D NMR and X-ray crystallographic analysis AusH isannotated as a hypothetical enzyme on both Broad Instituteand CADRE databases BLAST analysis showed that none of itshomologues have been studied so far Our study suggests thatAusH may play a role in altering AusKrsquos stereospecificity for itsproduct When there is no AusH present AusK produces the5primeR γ-hydroxyl ester precursor of compound 12 When AusH ispresent AusK produces the 5primeS γ-hydroxyl ester precursor ofcompound 7 Therefore AusH could function as an accessoryenzyme working in tandem with AusK to make compound 7BLAST analysis revealed that AusI has 52 similarity to

SmP450-2 a P450 monooxygenase that mediates the lactoneformation of GA9 in gibberellin biosynthesis in Sphacelomamanihoticola32 The ausI deletant produces isoaustinone (7)The accumulation of this compound suggests that ausI encodesa BaeyerminusVilliger monooxygenase (BVMO) which inserts anoxygen atom between the C-4prime and vicinal carbon at C-3prime tocreate a lactone ring Another intermediate compound 8 wasisolated along with compound 7 from this gene deletant strainCompound 8 has an additional hydroxyl on C-11 to compound7 It is possible that this modification is performed by adownstream enzyme AusG encoded by AN92484 which wepropose to be a hydroxylase AusG has 59 similarity toSmP450-1 a P450 monooxygenase that mediates fouroxidation steps in gibberellin biosynthesis in Sphacelomamanihoticola32 Austinolide (9) was isolated from our ausGdeletant strain This suggests ausG encodes a hydroxylase thatcatalyzes C-11 hydroxylation to form austinol Finally we havebeen unable to locate a gene that we can show is responsible forconverting austinol to dehydroaustinol It is possible thatadditional genes involved in the pathway have yet to beidentified or the conversion is spontaneous and not enzyme-catalyzedInterestingly although the LCminusMS profile of AN112174

mutant suggests that this gene is not required formeroterpenoid biosynthesis we found that the metaboliteprofile of this mutant is different from other meroterpenoid-producing mutants and LO2026 (control strain) In LO2026the amount of austinol is greater than dehydroaustinol but theamount of austinol and dehydroaustinol are about the same inthe AN112174 mutant (Figures 3A and 5B) BLAST analysisshowed that this gene has 45 identity to Bcmfs1 fromBotryotinia fuckeliana (Supplementary Table S11) Bcmfs1 is anefflux pump which belongs to major facilitator superfamily

(MFS) This enzyme can protect B fuckeliana against naturaltoxins and fungicides by secreting them out33

Several MFS coding genes have been found in naturalproduct gene clusters and are responsible for transportingspecifically these metabolites out of producer organisms34 suchas TOXA in the HC toxin pathway of C carbonum35 TRI12 intrichothecene biosynthesis of F sporotrichioides36 and CFP inthe cercosporin pathway of C kikuchii37 Loss of TRI12 andCFP leads to the reduction in toxin production suggesting thatfeedback inhibition may turn off the pathway genes when thetoxic level is too high to be tolerated by these organisms343637

The MFS encoded by AN112174 could therefore beinvolved in the transportation of austinoldehydroaustionlpathway metabolites One admittedly speculative possibility isthat the lower level of austinol in the mutant is due to thetoxicity of austinol to its producer organism A nidulans Inaddition accumulation of austinol in the cell might allow moreaustinol to be converted to dehydroaustinol Both effects maylead to the observed difference in metabolite profile ofAN112174 deletion strains relative to the parental strainHowever whether the austinol is toxic to A nidulans is stillunknown and although it has been seen in many cases thatpathway-specific efflux is responsible for secreting pathwaymetabolites out of the host there are exceptions For exampleAflT a putative MFS transporter in the aflatoxin gene cluster inA parasiticus does not play a significant role in aflatoxinsecretion34 In addition it has been shown that some non-pathway-specific transporters in some cases can compensate forthe function of an MFS34 The role of the product ofAN112174 in austinoldehydroaustinol secretion is thus byno means certain and awaits further investigationRegarding the organization of the austinoldehydroaustinol

biosynthetic pathway genes in A nidulans one of the mostinteresting aspects of these genes is that they are located in twoseparate gene clusters In fungi the genes that encode theproteins of a particular secondary metabolite biosyntheticpathway are generally clustered together as is the case inprokaryotes138 Although secondary metabolite genes in plantsare not known to be clustered there is growing evidence that incertain systems they are indeed clustered38 There is strongevidence for horizontal transfer of a secondary metabolite genecluster from Aspergillus to Penicillium39 and it has beensuggested that clusters of secondary metabolite biosyntheticgenes might confer selective advantages to themselves withevolution favoring their propagation and survival as a unit40

There is some precedence however for split gene clusters infungi The genes for T-toxin biosynthesis in Cochliobolusheterostrophus are in two unlinked loci41 and the genes forprenylated xanthone biosynthesis in A nidulans are in threeseparate regions of the genome14 It is worth noting moreoverthat without the sequenced genome and efficient gene targetingtechniques it would not have been possible to elucidate theaustinoldehydroaustinol clusters It is possible that split orfragmented clusters are more common in fungi than has beenthought and that they are being discovered now because toolshave been developed that facilitate their discoveryWe wondered if the two austinoldehydroaustinol clusters

might be functionally specialized (eg the early steps of thepathway might require products of genes in cluster A and latesteps require products in cluster B) As Figure 6 demonstrateshowever this is not the caseIn cluster B (the prenyltransferase cluster) there is a

sequence between AN112054 and AN92564 (nucleotides



76655 to 77031 on linkage group VIII) that does not appear tobe part of a functional gene but has strong nucleotide identitywith a portion of AN83834 (in SAT domain of AN83834) (P= 67 times 10minus44) We speculate that the two clusters were oncepart of a single contiguous cluster that has been split bychromosomal rearrangement involving a partial duplication ofAN83834 and translocation of AN83834 AN83794AN83804 AN83814 AN83824 and AN83844 leaving onlya dysfunctional remnant of AN83834 behind in theprenyltransferase cluster If this speculation is correct it followsthat the translocation did not disrupt the co-regulation of thegenes of the separated gene clustersWe searched for synteny with secondary metabolite clusters

in other species of Aspergillus to determine if any of thesespecies might have an ancestral cluster syntenic with clusters Aand B We found a gene cluster in A terreus extending fromATEG100771 to ATEG100851 (httpwwwbroadinstituteorg) that exhibits substantial synteny with clusters A and B(Supplementary Figure S21) It is highly speculative butpossible that the putative meroterpenoid cluster in A terreusshares a common ancestor with the meroterpenoid clusters Aand B in A nidulans The product of the A terreus cluster iscurrently unreported although A terreus is known to producethe meroterpenoids terretonin and territrem34

Another interesting aspect of the two A nidulans clusters isthat there are a total of eight annotated genes internal to theclusters that are not required for austinol or dehydroaustinolbiosynthesis While it is possible that some of them are notactual genes and are simply missanotated sequences others areclearly real and some of them are on the basis of homologypredicted to have a role in secondary metabolism (Supple-mentary Table S11) While we can only speculate as to thefunctions of these genes it is worth noting that in A nidulansthe genes required for monodictyphenone synthesis are asubset of the prenyl xanthone biosynthetic pathway and thatone gene mdpE is required for monodictyphenone biosyn-thesis but not prenyl xanthone biosynthesis and a second genemdpD is required for prenyl xanthone biosynthesis but notmonodictyphenone biosynthesis14 This raises the highlyspeculative but intriguing possibility that fungi may usedifferential regulation of genes within secondary metabolitegene clusters to multiply the number of secondary metabolitesproduced by the clusters

CONCLUSIONPioneering work primarily using isotopic precursors in the1980s enabled Simpson Vederas and co-workers to proposebiosynthetic mechanisms for meroterpenoids involving the C-alkylation of 35-dimethylorsellinic acid12 The alkylatedintermediate is epoxylated and cyclized to form a tetracyclicintermediate which then undergoes drastic modifications toform austinol and dehydroaustinol Targeted gene deletionstudies of genes flanking the genes encoding the NR-PKS onchromosome V and the prenyltransferase on chromosome VIIIallowed the isolation of several stable intermediates in amountssufficient for full NMR characterization Many of theintermediates isolated share structural similarities to theproposed structures from the earlier labeling work and alsointermediates isolated from wild type strain of Penicillium spLaBioMi-02423 and provide direct support for the pathwayproposed by the Simpson and Vederas groupsIn summary we have shown that genes from two different

gene clusters on two different chromosomes are required to

make dehydroaustinol and austinol in A nidulans One clustercontains the PKS gene and the other cluster contains theprenyltransferase gene By creating a series of deletants andanalyzing the metabolite profiles using LCminusMS 14 genes wereidentified to be involved in austinoldehydroaustinol biosyn-thesis On the basis of the intermediates and bioinformaticsstudy we have proposed a biosynthetic pathway for austinoldehydroaustinol

MATERIALS AND METHODSStrains and Molecular Genetic Manipulations A nidulans

strains used in this study are listed in Table S1 in the SupportingInformation All of the deletant strains were generated by replacingeach gene with the Aspergillus fumigatus pyrG gene in the A nidulansstrain LO2026 The construction of fusion PCR products protoplastproduction and transformation were carried out as describedpreviously19 For the construction of the fusion PCR fragments twosim1000-bp fragments of genomic A nidulans DNA upstream anddownstream of the targeted gene were amplified by PCR Primersused in this study are listed in Table S2 in the Supporting InformationFusion PCR was set up with the two amplified flanking sequences andthe A fumigatus pyrG selectable marker cassette as the template DNAThe three fragments were fused into a single molecule and amplifiedwith two nested primers Diagnostic PCR analyses of the deletionmutants and their parent strain LO2026 were performed with theexternal primers used in the first round of PCR The difference in thesize between the gene replaced by the selective marker and the nativegene allowed the determination of correct gene replacement Inaddition the correctness of the gene replacements was furtherconfirmed using external primers along with primers located inside themarker gene Diagnostic PCR with multiple primer sets allowed us todetermine reliably if correct gene targeting had occurred Although thistechnique does not allow us to determine if additional heterologousintegrations have occurred extra insertions rarely happen (lt2) withthe strains used19 At least two transformants per gene were selectedand their metabolite profiles were examined The probability that tworandom extra insertions would occur and would give the same alteredmetabolite profile is vanishingly small

Fermentation and LCminusMS Analysis The A nidulans controlstrain LO2026 and deletion strains were cultivated at 37 degC on solidYAG (complete medium 5 g of yeast extractL 15 g of agarL and 20g of D-glucoseL supplemented with 10 mLL of a trace elementsolution and required supplements) at 1 times 107 spores per 10 cmdiameter plate (with 25 mL of medium per plate) After 3 days theagar was chopped into small pieces and the material was extractedwith 50 mL of methanol (MeOH) followed by 50 mL of 11 CH2Cl2MeOH each with 1 h of sonication The extract was evaporated invacuo to yield a residue which was suspended in H2O (25 mL) andthis suspension was then partitioned with ethyl acetate (EtOAc) twiceThe combined EtOAc layer was evaporated in vacuo redissolved in 1mL of DMSO and then diluted with 4 volumes of MeOH Then 4 μLof this dilute crude extract was injected for HPLCminusDAD-MS analysis

LCminusMS was carried out using a ThermoFinnigan LCQ advantageion trap mass spectrometer with a reverse-phase C18 column (21 mmby 100 mm with a 3 μm particle size Alltech Prevail) at a flow rate of125 μLmin The solvent gradient for HPLC was 95 acetonitrile(MeCN)H2O (solvent B) in 5 MeCNH2O (solvent A) bothcontaining 005 formic acid as follows 0 solvent B from 0 to 5min 0 to 100 solvent B from 5 to 35 min 100 solvent B from 35 to40 min 100 to 0 solvent B from 40 to 45 min and reequilibrationwith 0 solvent B from 45 to 50 min The positive mode was used forthe detection of austinoldehydroaustinol and their intermediatesNegative ion electrospray ionization (ESI) was used for the detectionof compound 3 Conditions for MS included a capillary voltage of 50kV a sheath gas flow rate at 60 arbitrary units an auxiliary gas flow rateat 10 arbitrary units and an ion transfer capillary temperature of 350degC

Isolation and Identification of Secondary Metabolites andCompound Spectral Data For structure elucidation each A



nidulans deletant was cultivated at 37 degC on sim20 YAG plates at 225 times107 spores per 15-cm plate (sim55 mL of medium per plate) Thedesired intermediates from crude extracts of each deletant strain wereisolated with flash chromatography and reverse phase HPLC (pleasesee Supporting Information for details) Melting points weredetermined with a Yanagimoto micromelting point apparatus andare uncorrected IR spectra were recorded on a GlobalWorks Cary 14Spectrophotometer Optical rotations were measured on a JASCO P-200 digital polarimeter NMR spectra were collected on a VarianMercury Plus 400 spectrometer High-resolution mass spectrometrywas performed on Agilent 6210 time-of-flight LCminusMS The X-raycrystallographic data were collected using a Bruker KAPPA APEX IIdiffractometer in the X-ray crystallography laboratory of the CaliforniaInstitute of Technology35-Dimethylorsellinic Acid Yellowish white solid mp 182 minus 185

degC IR maxZnSe 3448 1685 1208 1142 cmminus1 for UVminusvis and ESIMS

spectra see Supplementary Figure S1 1H NMR (acetone-d6 400MHz) 210 (3H s) 214 (3H s) 250 (3H s) 13C NMR (acetone-d6100 MHz) 88 (q) 124 (q) 190 (q) 1062 (s) 1089 (s) 1164 (s)1386 (s) 1590 (s) 1616 (s) 1753 (s) 1H and 13C NMR data werein good agreement with the published data42

Protoaustinoid A Yellowish gum for UVminusvis and ESIMS spectrasee Supplementary Figure S1 for 1H and 13C NMR data seeSupplementary Table S3Preaustinoid A3 Colorless solid mp 209 minus 212 degC [α]24D +2504

(CHCl3 c 09) IR maxZnSe 3371 1733 1707 1646 1374 1297 1230

1110 1004 923 cmminus1 for UVminusvis and ESIMS spectra seeSupplementary Figure S1 for 1H and 13C NMR data seeSupplementary Tables S9 and S10 1H and 13C NMR data were ingood agreement with the published data23

Preaustinoid A4 Colorless needles mp 167minus171 degC [α]24D+4695 (CHCl3 c 17) IR max

ZnSe 3383 1759 1715 1654 13761282 1239 1156 1109 984 911 cmminus1 for UVminusvis and ESIMSspectra see Supplementary Figure S1 for 1H and 13C NMR data seeSupplementary Table S4 HRESIMS [M + H]+ mz found 4572227calcd for C26H32O7 4572221Isoaustinone Colorless solid mp gt300 degC [α]24D +4626 (CHCl3

c 03) IR maxZnSe 3490 1752 1708 1375 1285 1236 1159 1114

1083 1053 981 925 cmminus1 for UVminusvis and ESIMS spectra seeSupplementary Figure S1 for 1H and 13C NMR data seeSupplementary Tables S9 and S10 1H and 13C NMR data were ingood agreement with the published data23

11β-Hydroxyisoaustinone Colorless needles mp 225 minus 229 degC[α]24D +2922 (CHCl3 c 01) IR max

ZnSe 3490 3378 1764 1750 16911312 1241 1177 1118 1086 1037 983 927 cmminus1 for UVminusvis andESIMS spectra see Supplementary Figure S1 for 1H and 13C NMRdata see Table S5 HRESIMS [M + H]+ mz found 4432069 calcdfor C25H30O7 4432064Austinolide Colorless needles mp 259 minus 263 degC [α]24D +4363

(CHCl3 c 10) IR n maxZnSe 3458 1780 1717 1375 1293 1218 1179

1117 1075 979 910 cmminus1 for UVminusvis and ESIMS spectra seeSupplementary Figure S1 for 1H and 13C NMR data seeSupplementary Table sS9 and S10 1H and 13C NMR data were ingood agreement with the published data23

Preaustinoid A5 Colorless amorphous solid mp unmeasurable[α]24D +1620 (CHCl3 c 06) IR max

ZnSe 3426 1760 1715 1374 12651235 1110 1048 973 908 cmminus1 for UVminusvis and ESIMS spectra seeSupplementary Figure S1 for 1H and 13C NMR data seeSupplementary Table S6 HRESIMS [M + H]+ mz found4572228 calcd for C26H32O7 4572221Austinoneol A Colorless needles mp 219 minus 224 degC [α]24D +3668

(CHCl3 c 09) IR maxZnSe 3403 1755 1721 1711 1661 1393 1375

1278 1223 1112 1060 974 906 cmminus1 for UVminusvis and ESIMSspectra see Supplementary Figure S1 for 1H and 13C NMR data seeSupplementary Table S9 and S10 1H and 13C NMR data were in goodagreement with the published data43

(5primeR)-Isoaustinone Colorless plates mp 234minus237 degC [α]24D+4287deg (CHCl3 c 03) IR max

ZnSe 3382 1755 1712 1375 12851201 1113 1066 1003 915 cmminus1 for UVminusvis and ESIMS spectrasee Supplementary Figure S1 for 1H and 13C NMR data see

Supplementary Table S7 HRESIMS [M + H]+ mz found 4272120calcd for C25H30O6 4272115

Neoaustinone Yellowish gum [α]24D +3571 (CHCl3 c 02) IRmax

ZnSe 3373 1760 1711 1657 1380 1312 1196 1112 1071 1017979 cmminus1 for UVminusvis and ESIMS spectra see Supplementary FigureS1 for 1H and 13C NMR data see Supplementary Table S8HRESIMS [M + H]+ mz found 4432070 calcd for C25H30O64432064

X-ray Crystallographic Analysis of (5primeR)-Isoaustinone Acolorless crystal of 12 with dimensions 121 times 021 times 019 mm3 wasselected for X-ray analysis Structure analysis was performed usingSHELXS-97 program The compound crystallized in the monoclinicspace group I2 with a = 169929(9) Aring b = 73879(4) Aring c =171390(9) Aring β = 95180(3)deg V = 21429(2) Aring3 Z = 4 ρcalc = 1322Mgm3 λ = 071073 Aring Intensity data were collected on a Brukerinstrument with an APEX II detector and crystals were mounted on aglass fiber using Paratone oil then placed on the diffractometer under anitrogen stream at 100 K A total of 30291 data points were collectedin the range 239deg le θ le 3183deg yielding 7321 unique reflectionsCrystallographic data have been deposited at the CCDC 12 UnionRoad Cambridge CB2 1EZ UK and copies can be obtained onrequest free of charge by quoting the publication citation and thedeposition number 773039

ASSOCIATED CONTENTS Supporting InformationA nidulans strains and primers used in this study detailedstructural characterization NMR spectral data purificationmethods and results of diagnostic PCR This material isavailable free of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding Authorboakleykuedu claywusceduPresent AddressDepartment of Biological and Agricultural EngineeringUniversity of California One Shields Avenue Davis California95616NotesThe authors declare no competing financial interest

ACKNOWLEDGMENTSThis work was supported by grant PO1GM084077 from theNational Institute of General Medical Sciences We thank ananonymous reviewer for pointing out the PKS-like sequencebetween AN112054 and AN92564

REFERENCES(1) Hoffmeister D Keller N P Nat Prod Rep 2007 24 393minus416(2) Geris R Simpson T J Nat Prod Rep 2009 26 1063minus1094(3) Mcintyre C R Simpson T J Chem Commun 1981 20 1043minus1044(4) Chen J W Luo Y L Hwang M J Peng F C Ling K H JBiol Chem 1999 274 34916minus34923(5) Omura S Tomoda H Kim Y K Nishida H Jpn J Antibiot1993 46 1168minus1169(6) Sunazuka T Hirose T Omura S Acc Chem Res 2008 41302minus314(7) Simpson T J Stenzel D J Bartlett A J OrsquoBrien E Holker JS E J Chem Soc Perkin Trans 1 1982 2687minus2692(8) Marquez-Fernandez O Trigos A Ramos-Balderas J LViniegra-Gonzalez G Deising H B Aguirre J Eukaryotic Cell 20076 710minus720(9) Szewczyk E Chiang Y M Oakley C E Davidson A DWang C C C Oakley B R Appl Environ Microb 2008 74 7607minus7612



(10) Mclntyre C R Simpson T J Stenzel D J Bartlett A JBrien E O Holkerb J S E J Chem Soc Chem Comm 1982 781minus782(11) Simpson T J Stenzel D J Moore R N Trimble L AVederas J C J Chem Soc Chem Comm 1984 1242minus1243(12) Ahmed S A Scott F E Stenzel D J Simpson T J MooreR N Trimble L A Arai K Vederas J C J Chem Soc PerkinTrans 1 1989 807minus816(13) Itoh T Tokunaga K Matsuda Y Fujii I Abe I EbizukaY Kushiro T Nat Chem 2010 2 858minus864(14) Sanchez J F Entwistle R Hung J H Yaegashi J Jain SChiang Y M Wang C C C Oakley B R J Am Chem Soc 2011133 4010minus4017(15) Nielsen M L Nielsen J B Rank C Klejnstrup M L HolmD K Brogaard K H Hansen B G Frisvad J C Larsen T OMortensen U H FEMS Microbiol Lett 2011 321 157minus166(16) Bok J W Chiang Y M Szewczyk E Reyes-domingez YDavidson A D Sanchez J F Lo H C Watanabe K Strauss JOakley B R Wang C C C Keller N P Nat Chem Biol 2009 5462minus464(17) Sanchez J F Chiang Y M Szewczyk E Davidson A DAhuja M Oakley C E Bok J W Keller N Oakley B R WangC C C Mol Biosyst 2010 6 587minus593(18) Chiang Y M Szewczyk E Davidson A D Keller NOakley B R Wang C C C J Am Chem Soc 2009 131 2965minus2970(19) Nayak T Szewczyk E Oakley C E Osmani A Ukil LMurray S L Hynes M J Osmani S A Oakley B R Genetics 2006172 1557minus1566(20) Chiang Y M Szewczyk E Nayak T Davidson A DSanchez J F Lo H C Ho W Y Simityan H Kuo E PraseuthA Watanabe K Oakley B R Wang C C C Chem Biol 2008 15527minus532(21) Melzer M Heide L Biochem Biophys Acta 1994 1212 93minus102(22) Brown D W Yu J H Kelkar H S Fernandes M NesbittT C Keller N P Adams T H Leonard T J Proc Natl Acad SciUSA 1996 93 1418minus1422(23) Fill T P Pereira G K Geris dos Santos R M Rodrigues-FoE Z Naturforsch 2007 62b 1035minus1044(24) Kroken S Glass N L Taylor J W Yoder O C Turgeon BG Proc Natl Acad Sci USA 2003 100 15670minus15675(25) Yazaki K Kunihisa M Fujisaki T Sato F J Biol Chem2002 277 6240minus6246(26) Heide L Curr Opin Chem Biol 2009 13 171minus179(27) Saikia S Parker E J Koulman A Scott B FEBS Lett 2006580 1625minus1630(28) Thoma R Schulz-Gasch T DrsquoArcy B Benz J Aebi JDehmlow H Hennig M Stihle M Ruf A Nature 2004 432 118minus122(29) Iwaki H Wang S Grosse S Lertvorachon J Yang JKonishi Y Hasegawa Y Lau P C K Appl Environ Microb 200672 2707minus2720(30) Fraaije M Kamerbeek N M Berkel W J H Janssen D BFEBS Lett 2002 518 43minus47(31) Cary J W Wright M Bhatnagar D Lee R Chu F S ApplEnviron Microb 1996 62 360minus366(32) Bomke C Rojas M C Gong F Hedden P Tudzynski BAppl Environ Microb 2008 74 5325minus5339(33) Hayashi K Schoonbeek H Waard M A D Appl EnvironMicrob 2002 68 4996minus5004(34) Chang P K Yu J Yu J H Fungal Genet Biol 2004 41 911minus920(35) Pitkin J W Panaccione D G Walton J D Microbiology1996 142 1557minus1565(36) Alexander N J McCormick S P Hohn T M Mol GenGenet 1999 261 977minus984(37) Callahan T M Rose M S Meade M J Ehrenshaft MUpchurch R G Mol Plant-Microbe Interact 1999 12 901minus910

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xanthone biosynthesis pathway in A nidulans are located inthree separate regions of the genome14

We identified using a set of deletions of all non-reducingPKS genes the NR-PKS responsible for the biosynthesis of themeroterpenoids in A nidulans This NR-PKS has beenindependently identified by Nielsen et al and designatedausA15 Next we selected two candidate prenyltransferase geneslocated elsewhere in the genome for deletion and frommetabolomic analysis of mutant extracts we found that one wasinvolved in meroterpenoid synthesis To identify additionalgenes involved in the pathway we targeted the genessurrounding the NR-PKS gene and the prenyltransferase genefor deletion The gene deletion experiments allowed us to

identify 12 additional genes involved in meroterpenoidbiosynthesis which are located in two gene clusters onecontaining the NR-PKS and the other containing theprenyltransferase Large-scale cultures of the deletion strainsenabled us to isolate several stable intermediates (Figure 2)including six novel compounds allowing us to propose abiosynthesis pathway for austinol (1) and dehydroaustinol (2)biosynthesis

RESULTSIdentification of the Polyketide Synthase Responsible

for Austinol (1) and Dehydroaustinol (2) BiosynthesisWhen cultivated on solid yeast agar glucose (YAG) medium A

Figure 1 Meroterpenoids from Aspergillus species

Figure 2 Intermediates isolated from gene deletion mutants






































ORFs homologues



















































(CHCl3 c 09) IR maxZnSe 3371 1733 1707 1646 1374 1297 1230




c 03) IR maxZnSe 3490 1752 1708 1375 1285 1236 1159 1114








(CHCl3 c 09) IR maxZnSe 3403 1755 1721 1711 1661 1393 1375





















































ORFs homologues



















































(CHCl3 c 09) IR maxZnSe 3371 1733 1707 1646 1374 1297 1230




c 03) IR maxZnSe 3490 1752 1708 1375 1285 1236 1159 1114








(CHCl3 c 09) IR maxZnSe 3403 1755 1721 1711 1661 1393 1375






























































(CHCl3 c 09) IR maxZnSe 3371 1733 1707 1646 1374 1297 1230




c 03) IR maxZnSe 3490 1752 1708 1375 1285 1236 1159 1114








(CHCl3 c 09) IR maxZnSe 3403 1755 1721 1711 1661 1393 1375


















Two Separate Gene Clusters Encode the Biosynthetic Pathway for the Meroterpenoids Austinol and...

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