Efficient Biosynthesis of Fungal Polyketides Containing the

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Efficient Biosynthesis of Fungal Polyketides Containing the Dioxabicyclo-octane Ring System Xu-Ming Mao, Zha-Jun Zhan, Matthew N. Grayson, Man-Cheng Tang, Wei Xu, YongQuan Li, Wen-Bing Yin, Hsiao-Ching Lin, Yit-Heng Chooi, K. N. Houk, and Yi Tang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b07816 • Publication Date (Web): 04 Sep 2015 Downloaded from http://pubs.acs.org on September 6, 2015

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E Efficientt Biosyntthesis off Fungal Polyketides Con ntaining the Dio oxabiccyclo-occtane Rin ng System m X Xu-Ming Mao o1,2‡, Zha-Jun n Zhan2,4‡, Maatthew N. Graayson3, Man--Cheng Tangg2, Wei Xu2, Y Yong-Quan Lii1, WenB Bing Yin2, Hssiao-Ching Lin2, Yit-Heng Chooi2, K. N. N Houk3, Yi T Tang2,3* 1

Zhejiang Univeersity, College of Life Sciences, Hangzhou 3100058, China, 2Deepartment of Chhemical and Biomolecular Engineering, Department of Chemistry and Biochemistry, University U of Caalifornia, Los Anngeles CA900955, USA, 4Zhejianng University off Technoloogy, College of Pharmaceutical P Science, Hangzzhou 310014, Ch hina 3

SSupporting Infoormation Placehholder A ABSTRACT: Aurovertins A are fungal polyketides that exhibit poteent innhibition of ATP A synthase. Aurovertins contain a 2,,6ddioxabicyclo[3.2.11]-octane ring that is proposed too be derived from ma ppolyene precursorr through regiosellective oxidationss and epoxide opeeninngs. In this stud dy, we identified only four enzym mes are required to pproduce aurovertiin E 4. The core polyketide p synthaase produces a pollyeene -pyrone 10.. Following pyron ne O-methylation n by a methyltran nsfferase, a flavin-deependent monoo oxygenase (FMO O) and an epoxide hhydrolase can iterratively transform m the terminal triiene portion of the pprecursor into th he dioxabicyclo[33.2.1]-octane scaaffold. We demoonsstrate that a tetrah hydrofuranyl polyyene 12 is the firsst stable intermed diaate in the transfo ormation, which can undergo epooxidation and an ntiB Baldwin 6-endo-tet ring opening to o yield the cyclic ether product. Our O rresults further deemonstrate the highly h concise an nd efficient ways in w which fungal bio osynthetic pathw ways can generatte complex naturral pproduct scaffolds.

Linear polykettides synthesized d by fungal pollyketide synthasses ((PKSs) can be morphed m into struucturally compleex natural produccts in a few succinct steps. Examples include the biosyynthesis of decallin ccores in lovastatiin1 and equisetin n,2 the isoindoloone moieties in cyc 3 ttochalasans, and d highly oxygen nated, multicycllic rings in merrotterpenoids.4 Thee concise biosyn nthetic pathwayss of these naturral pproducts showcaase the synchron nization between n the PKS, which cconstructs the po olyketide chain with strategicallly positioned reaacttive groups such h as double bon nds and hydroxyyl groups, and th he aassociated tailoring enzymes thatt have impressivee catalytic proweess ooptimally tuned for the polykettide precursor. Therefore, T undeersstanding the mechanistic basis of these collaaborative transfoorm mations can not only lead to the discovery of new w enzymatic toools, bbut also inspire biomimetic b strateegies for organic synthesis. An unusual polyketide-derrived structuree is the 2,,6ddioxabicyclo[3.2.1]-octane (DBO O) ring system found in the aua rrovertin family of o natural produccts (1-6, Figure 1A) isolated froom ffungal species suuch as Calcarisp porium arbuscula la.5-7 Aurovertin E ((4) represents th he structurally sim mplest member of o the family and d is tthe biosynthetic precursor to oth her derivatives. The T bicyclic eth her m moiety in 4 is fuused to a methyllated -pyrone via v a triene linkeer, aand can be diffeerentially substituted to yield various v auroverttin ccongeners. Acetyylated derivativees 2 and 3 are pootent and uncom mppetitive inhibitorrs of F1 ATPasee and have been implicated as pop

tential anticancer therrapeutics.8,9 Desppite the occurreence of the DBO m moiety in otheer microbial annd plant naturaal products (Schem me S1), such as ddecurrenside A from the goldennrod plant,10 the antiibiotic sorangicinn A from myxobbacteria11 and a m marine toxin palytoxxin from zoanthidds,12 the enzymattic basis for the fformation of this mooiety has not beenn elucidated to ddate.

Figure 11. Aurovertins annd the biosynthetiic cluster. (A) Auurovertin and related fungal natural prroducts are propoosed to derive froom a polyene polyketiide precursor. Thhe 2,6-dioxabicycllo[3.2.1]-octane rring is shown in red; ((B) The aur biosyynthetic gene cluuster in C. arbuscu cula. HRPKS: highly rreducing PKS; K KS: ketosynthasee; AT: acyltranssferase; MT: methylttransferase; FMO O: flavin-dependennt monooxygenase; TF: transcriptionnal factor; AcT: acyltransferase; aand (C) Genetic knockout of aurA in C. arbuscula folloowed by HPLC annalysis of organicc extracts.

The bbiosynthetic origgin of 4 was propposed and subseqquently verified froom labeling studiies to derive from m a polyketide ppathway.13 A polyenee-fused -pyronne derived from one unit of proppionate and eight unnits of acetate w was proposed to bbe the precursorr (Figure 2). Three eepoxidation stepps and a cascade of regioselectiive epoxideopeningg reactions weree suggested to taake place and yieeld the DBO moiety..14 Labeling studdies using 18O fuurther revealed thhat the oxygen bettween C4-C8 is derived from H2O, thereby sugggesting the likely siite of epoxide hyydrolysis.14 Synthhetic studies aim ming at confirmingg the feasibility off the biosynthetiic proposals weree performed to afforrd 4 and suggestted a possible rooute via a tetrahhydrofuranyl

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F Figure 2. A proposed pathway baseed on observed inttermediates and shunt s products.

eepoxide intermed diate.15,16 Related d fungal polyenee metabolites such 17 aas citreoviridin, asteltoxin,18 an nd asteltoxin B19 are most likeely ooxidized from sim milar pyrone-polyene precursors as 4 (Scheme S22). H Here, we demon nstrate that the combination c of a flavin-dependeent m monooxygenase (FMO) and an epoxide hydrolaase are sufficient to rregioselectively in nstall the DBO functionality f starrting from the PK KS pproduct. ng Searching through the sequencced genome20 of C. arbuscula usin hhighly-reducing (HR) PKSs as leads, l we identiffied one candidaate bbiosynthetic gene cluster (aur) on o contig 1452. This cluster coonttains an HR-PKSS gene (aurA) and tailoring gen nes that potentiallly m match the structuural features of 4. 4 The domain arcchitecture of AurrA is KS-AT-DH-M MT-KR-ACP; th he absence of an enoyreductaase ((ER) is consisten nt with the form mation of a polyeene precursor. Th he nneighboring gen nes encode an OO methyltransferase (MT, aurB B), F FMO (aurC), a predicted  hydrolase (aurD D), a protein wiith ssequence homolo ogy to bacterial aromatic a polykettide cyclase SnoaaL (aurE),21 a putatiive DNA-bindin ng protein (aurF) and an acyltran nsfferase (aurG) (F Figure 1B). A gene cluster with high h sequence hoh m mology to the auur cluster was alsso found in M. anisopliae, a anoth her pproducer of aurovertins,5 and in Aspergillus A terreuus, the producer of ccitreoviridin22 (T Table S1-S2). In n M. anisopliae, a gene encodin ng A ATP synthase waas also found witthin the cluster and is likely to coonffer self-resistancee (Figure S1). Deletion D of aurA rA in C. arbuscuula ((Figure S2) led to t complete abolishment of auroovertin productioon ((Figure 1C), wheereas the wild typ pe strain produceed compounds 1-6 1 aat high titers (~330 mg/L combin ned) (Figures S116-S27 and Tablles SS7-S9), confirmin ng the role of thee aur cluster. With the genee cluster in hand, we next aimeed to identify th he ppolyketide precuursor synthesized d by AurA. Exprression of AurA in 1 tthe yeast strain BJ5464-NpgA B (F Figure S3) fed with w propionate led tto yellow-pigmen nted cell pellets and a the isolation n of a highly conjjuggated product 7 (m/z 339 [M+H H]+, 3.1 mg/L) (F Figure 3, iv). Isollattion and NMR characterization c (Figure S28-S29 and Table S10) sshowed 7 is the hexa-ene pyron ne shown in Figgure 3. Thereforre, A AurA is a highly programmed p HR R-PKS that can i)) select propionaate aas the starter uniit; ii) synthesizee a hexa-ene chain through the rer ppeated functionss of the KR and DH domains in n the first six iterrattions; iii) selectivvely introduce th hree -methyl suubstitutions at C4, C C C6 and C16 using the S-adenssylmethionine (SAM)–dependeent M MT; and iv) shuut off KR and DH H in the last three iterations to afa

ford a 11,3,5-triketo porttion that can unddergo intramoleccular cyclization to yyield the -pyroone product 7 (Fiigure 2). To deetermine if methhylation of the C17 hydroxyl grouup of 7 takes place pprior to epoxidaation of the polyene, we inacttivated aurB which eencodes an O-M MT (Figure S4). T The C. arbusculaa mutant was unable to produce auroovertins and accuumulated 7 (Figgure 3, i and ii). No desmethyl versiions of aurovertiins could be detected in the extractss. In addition, w when AurB was coexpressed wiith AurA in yeast, w we observed emeergence of a new w product 8 thatt has the expected increase in mass (+14, m/z 3533 [M+H]+, 2.8 m mg/L) compared tto 7 (Figure 3, v). NMR charracterization connfirmed the structurre of 8 to be a m methylated form m of 7 (Figure SS30-S35 and Table SS10). Furtherm more, we inactivvated aurC in C C. arbuscula (Figuree S5), which is thhe only oxidative enzyme (FMO)) in the gene cluster and is most likeely responsible ffor the subsequeent epoxidaC strain accumulaated significant aamount of 8 tion steeps. The aurC (Figuree 3, iii). Togetheer these results cconfirmed the roole of AurB, and sugggested epoxidattions, although aare to occur at thhe distal olefins of the polyene, reqquire the pyronee to be methylatted. Indeed, when A AurA and AurC w were coexpressedd in yeast, no oxiidized products weere observed andd the strain remaained producing 7 (Figure 3, vi). D Density functionnal theory (DFT T)23 calculations on 7 and 8 showedd that the preferrred conformatioon of the polyenne region is non-plaanar (C3-C4-C55-C6 dihedral = 443°, Figure 3 andd S6) due to the proxximity of the meethyl groups attached to C4 and C6 (favored over coonformer with C C3-C4-C5-C6 dihhedral ≈ 170°by approximately 1 kcal mol-1). T This implies thatt the carbon-carrbon double bond b etween C3 and C4 is not in connjugation with thhe rest of the polyenee and pyrone O-methylation dooes not change tthe electron densityy of the trisubstittuted double bonnds to any greatt extent (see methylation Mullikeen charges, Figurre S6). Therefore, the need for m to initiaate epoxidation iis likely due to thhe substrate speccificity of the downsttream monooxyggenase (i.e. AurC C). AurC C is predicted too be a membranee-anchored monnooxygenase that hhas sequence hhomology to U UbiH, the 2-octaprenyl-6methoxxyphenol hydroxxylase in ubiquinnone biosynthesiis. This sole FMO inn the gene cluster is postulated to be involved inn the epoxidation ssteps required too oxidize 8 en rooute to 4. To invvestigate the role andd timing of AurC C, we coexpresssed AurABC in yyeast, which led to tthe synthesis off a series of com mpounds with thhe molecular weight of 402, includingg major productss 9, 10 and 11 (F Figure 4, i).


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Journal of the American Chemical Society which iis predicted to bbe an integral meembrane-bound / hydrolase bassed on 1D proteiin structure preddiction.25 Knocking out aurD in C. ar arbuscula (Figuree S8) did not eliminate the prodduction of 2, but accuumulated signifiicant amounts off new shunt prodducts including 10 and 11 (Figuree S9). Furtherm more, coexpressioon of AurD with AuurABC in yeast lled to drastic attenuation of bothh 10 and 11, while 9 became the preedominant produuct with molecullar weight of 402 (F igure 4, ii). Hennce, AurD is invvolved in the reegioselective epoxidee hydrolysis of 133 to form 9.

F Figure 3. Investigaation of the PKS AurA A function. Top: HPLC analysses oof metabolites pro oduced from funggal and yeast straains. Bottom: struucttures of 7 and 8, an nd lowest energy conformation of 8.

C Comparing to th he wild type C. arbuscula strain n, however, onlyy 9 w was shared between the two extrracts (Figure 4, iii). i Purification of tthese compounds turned out to be b challenging duue to rapid isomeerization and instab bility. For examp ple, purified 9 exxisted as a mixtuure oof two interchangeable compoun nds (Figure S7). A dominant forrm ((~75%) was obsserved in CD3Cl, C which alloweed us to assign its sstructure as the (3R, 4R, 5R, 6S) S) tetrahydrofuraanyl pyrone show wn in Figure 4 (Tablle S11, Figure S336-S41). The abssolute stereochem mistry of the furan ring in 9 is the required r configuuration to generaate tthe DBO moietyy in 4 in a subsequuent epoxide opening step (Figuure 22).24 Indeed, wheen 9 was supplem mented to the C. C arbusculaaur urA sstrain, restored production p of auurovertins can bee observed (Figuure 44, v and vi). Con nfirming 9 as a biosynthetic b intermediate also suugggests that the thrree epoxidation modifications of the precursor 12 ooccur in two separate steps (Figuure 2), in which bis-epoxidation of tthe two terminal olefins takes plaace first to yield 13; another epoxxiddation would occcur at C7-C8 after a tetrahydrofu furan formation to yyield the epoxidee 14. We purified 100 from the yeast cell c extract and solved s its structuure tto be a 6R epimeer of 9 (Table S111, Figure S42-S447). The inversioon in stereochemistrry however, places the C4-OH off 10 away from th he C C7-C8 double bond and cannot lead to the form mation of 4. As exe ppected, feeding of o 10 to the aurrA strain did not restore auroverttin pproduction (Figuure 4, vii). We reeason that in thee absence of a reggiooselective epoxid de hydrolase, botth 9 and 10 can form f from 13 viaa a sspontaneous SN1-like 1 reaction to t yield the ressonance stabilized aallylic carbocation at C6, which can c be attacked by b H2O to yield th he m mixed stereoisom mers 16 (Figure 2). Attack of th he C6-OH on C3 C rresults in formation of the diasteereomers 9 and 10. Purification of 111 was not succcessful as it rapid dly degraded un nder all attempted cconditions. Based d on its identicall UV absorbancee and m/z to 9 an nd 110, 11 should also o be a spontaneo ously cyclized prooduct. Given that onlly 9 is the on-paathway intermed diate, and previous labeling studies confirmed the C4 oxygen is derived d from H2O rrather than O2, th he formation of 9 in C. arbusculaa must be catalyzed bby an epoxide hydrolase h via the 5-exo-tet mecchanism shown in F Figure 2. The on nly possible cand didate in the gen ne cluster is AurD D,

Figure 44. The roles of A AurC and AurD inn biosynthesis off aurovertins. Traces i and ii are extraccts from yeast, booth UV and extraacted ion MS traces aare shown. Tracess iii-vii are extractts from C. arbusccula. The extracted iion chromatogram m of WT is shownn for comparison.

Surprrisingly, in the yeeast AurABCD ooverexpression sttrain, we observed noticeable amoounts of 4 (Fiigure 4, ii). Seelected ionmonitooring revealed thhat even in the AurABC expresssion strain, producttion of 4 can bee detected (Figure 4, i). This sugggested that AurC aand AurD may allso be involved iin the transformaation of 9 to 4 as weell, which requirres the formationn of the epoxidee 14 and an anti-Bal aldwin 6-endo-teet epoxide openning (Figure 2)). We confirmed the involvementt of AurC in the C7-C8 epoxidaation step, as C strain could nott restore the feedingg of 9 to the C. aarbuscula aurC producttion of auroverttins. Despite reppeated attemptss, no soluble or activve AurC could bbe obtained for bbiochemical assayy. Nonetheless, thee yeast reconstituution and chemical complementaation results confirm med the multifunnctional role of A AurC in catalyzinng the epoxidation of three double bonds in two separate steps in the biosynthesis oof 2. The low am mount of 4 in AurrABC-expressingg yeast is due to the llow amount of sspontaneously foormed 9. In the presence of AurD aand the elevated amount of 9, thhe level of 4 increeased correspondinngly. We also obbserved incompllete conversion of 8 to 4 in the yeaast host, which iis most likely duue to suboptimal expression and actitivities of fungal m membrane proteeins in the simpleer yeast. Follow wing formation of 14, the C4 hyydroxyl group is spatially set up to aattack either C7 (5-exo-tet) or C C8 (6-endo-tet), to yield the dioxabiicyclo[2,2,1]-hepptane (DBH) 155 and the DBO-ccontaining 4, respectitively. No compoounds related too 15 can be founnd in C. arbusculaa or in the yeast sstrain expressingg AurABCD, sugggesting that the antti-Baldwin epoxide opening to yyield 4 is preferrred and may therefo re also be enzyymatically conttrolled. To undderstand the epoxidee opening mechhanisms, DFT ccalculations usinng a model substratte 17 were perfoormed (Figure S110). The 6-endoo-tet product was calcculated to be theermodynamicallyy favored over thhe 5-exo and



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5-endo-tet products by 4.1 and 19.6 kcal mol-1, respectively. Epoxide opening requires either an acid catalyst at the epoxide oxygen, a base catalyst at the hydroxyl, or both.26 The transition structures (TSs) for 5-exo, 5-endo and 6-endo-tet epoxide openings were located with hydroxide as the base to simulate base-catalyzed conditions (Figure S11).26,27 In agreement with Baldwin’s rules, 5-exotet cyclization is favored by 1.6 and 22.5 kcal mol-1 over the 6-endo and 5-endo-tet pathways, respectively. In contrast, the 6-endo-tet TS is favored over SN1 C-O bond cleavage and 5-exo-tet TSs under acid-catalyzed conditions by 5.3 and 7.9 kcal mol-1, respectively (Figure S12). This change in mechanism is due to stabilization of the cationic TS by the carbon-carbon double bond in the 6-endotet TS.28 Therefore, the outcome of the spontaneous reaction is predicted to be dependent upon the reaction conditions. To simulate simultaneous general acid/base catalysis which is common in enzymatic epoxide hydrolysis,26,27,29 TSs were located with formate as the base and a formic acid to protonate the epoxide oxygen. The lowest energy 6-endo-tet TS is lower in energy by 1.3 kcal mol-1 relative to 5-exo-tet TS. This energy difference increases to 3.1 kcal mol-1 if the lowest energy 6-endo-tet TS is compared to a 5-exo-tet TS with similar catalytic residue positioning (Figure S13). A 3.1 kcal mol-1 energy difference predicts almost exclusive formation of the 6-endo product (>100:1). Hence, the computational results suggest that formation of 4 from 14 is facilitated by enzyme environment, such as that of AurD. Alternatively, the active site of AurC may also provide the general acid/base residues required for formation of 4, immediately following the epoxidation reaction. Other enzymes in the pathways are not essential for the formation of the DBO moiety of 4. Deletion of the SnoaL-like enzyme AurE reduced yields of products; while yeast reconstitution studies with AurA and AurE showed that AurE enhances the rate of pyrone formation and product release off the PKS, which is consistent with its predicted role as a cyclase.16 AurG was verified to be the Oacyltransferase that converts 4 to 2, while AurF was shown to be most likely the transcriptional activator of the aur cluster (Figure S14-S15). Our studies show the aurovertin pathway is concise, and uses only two enzymes to convert the polyene pyrone into 4. The activities of the FMO and hydrolase are well-orchestrated to sequentially oxidize and regioselectively hydrolyze the epoxides. The biosynthesis of citreoviridin and asteltoxin likely involve same enzymes to generate the complexities from pyrone polyene precursors.

ASSOCIATED CONTENT Supporting Information Experimental details, spectroscopic data, computational details, complete list of authors in reference 23; and computational data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected]

Present Addresses WBY: The Institute of Microbiology, Chinese Academy of Sciences; YHC: School of Chemistry and Biochemistry, The University of Western Australia; HCL: IBC, Academia Sinica, Taiwan

Author Contributions

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‡These authors contributed equally.

ACKNOWLEDGMENT This work was supported by the NIH 1DP1GM106413 and 1R01GM085128 to Y.T.; a fellowship of New Star Project from ZJU to XMM, the English-Speaking Union (Lindemann Trust Fellowship to M.N.G.), the NSF CHE-1361104 to K.N.H., and Natural Science Foundation of China (31470178) to WBY. Computational resources were provided by the UCLA Institute for Digital Research and Education (IDRE) and the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the NSF (OCI1053575).

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Figure 2



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Figure 3 115x98mm (300 x 300 DPI)


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Figure 4 123x88mm (300 x 300 DPI)



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TOC Figure 154x59mm (300 x 300 DPI)


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Efficient Biosynthesis of Fungal Polyketides Containing the

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