An FAD-dependent monooxygenase encoding gene (SorbC) was cloned from Penicillium chrysogenum E01-10/3 and expressed as a soluble protein in Escherichia coli. The enzyme efficiently performed the oxidative dearomatisation of sorbicillin and dihydrosorbicillin to give sorbicillinol and dihydrosorbicillinol respectively. Bioinformatic examination of the gene cluster surrounding SorbC indicated the presence of two polyketide synthase (PKS) encoding genes designated sorbA and sorbB. The gene sorbA-encodes a highly reducing iterative PKS while SorbB encodes a non-reducing iterative PKS which features a reductive release domain usually involved in the production of polyketide aldehydes. Using these observations and previously reported results from isotopic feeding experiments a new and simpler biosynthetic route to the sorbicillin class of secondary metabolites is proposed which is consistent with all reported experimental results.Al Baha University, Saudi ArabiaEP/F066104/1BB/I003355/1BMBFERASMUS programm

Abood, Amira

Al-Fahad, Ahmed

Avramovic, Marija

Butts, Craig

Cox, Russell J.

Davison, Jack

Fisch, Katja M.

Osipow, Anna

Piel, Joern

Simpson, Thomas J.

Chemical Science

PubMed

An FAD-dependent monooxygenase encoding gene (SorbC) was cloned from Penicillium chrysogenum E01-10/3 and expressed as a soluble protein in Escherichia coli. The enzyme efficiently performed the oxidative dearomatisation of sorbicillin and dihydrosorbicillin to give sorbicillinol and dihydrosorbicillinol respectively. Bioinformatic examination of the gene cluster surrounding SorbC indicated the presence of two polyketide synthase (PKS) encoding genes designated sorbA and sorbB. The gene sorbA-encodes a highly reducing iterative PKS while SorbB encodes a non-reducing iterative PKS which features a reductive release domain usually involved in the production of polyketide aldehydes. Using these observations and previously reported results from isotopic feeding experiments a new and simpler biosynthetic route to the sorbicillin class of secondary metabolites is proposed which is consistent with all reported experimental results.</p

al Fahad, Ahmed

Butts, Craig P.

Explore Bristol Research

Oxidative dearomatisation:the key step of sorbicillinoid biosynthesis

Institutionelles Repositorium der Leibniz Universität Hannover

ChemicalScienceEDGE ARTICLEOpen Access Article. Published on 20 November 2013. Downloaded on 03/12/2015 10:36:32.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View IssueaSchool of Chemistry, University of BristolE-mail: r.j.cox@bris.ac.uk; Fax: +44 (0) 117bInstitute of Microbiology, Eidgeno¨ssischeWolfgang-Pauli-Str. 10, 8093 Zu¨rich, SwitzecKekule´ Institute of Organic Chemistry and BDomagk-Str. 1, 53121 Bonn, GermanydInstitut fu¨r Organische Chemie, Leibniz U30167 Hannover, Germany. E-mail: russell.ceChemistry of Natural and Microbial Product† Electronic supplementary informatioexperimental details. See DOI: 10.1039/c3Cite this: Chem. Sci., 2014, 5, 523Received 21st October 2013Accepted 15th November 2013DOI: 10.1039/c3sc52911hwww.rsc.org/chemicalscienceThis journal is © The Royal Society of COxidative dearomatisation: the key step ofsorbicillinoid biosynthesis†Ahmed al Fahad,a Amira Abood,ae Katja M. Fisch,a Anna Osipow,a Jack Davison,aMarija Avramovic´,b Craig P. Butts,a Jo¨rn Piel,bc Thomas J. Simpsonaand Russell J. Cox*adAn FAD-dependent monooxygenase encoding gene (SorbC) was cloned from Penicillium chrysogenumE01-10/3 and expressed as a soluble protein in Escherichia coli. The enzyme eﬃciently performed theoxidative dearomatisation of sorbicillin and dihydrosorbicillin to give sorbicillinol and dihydrosorbicillinolrespectively. Bioinformatic examination of the gene cluster surrounding SorbC indicated the presence oftwo polyketide synthase (PKS) encoding genes designated sorbA and sorbB. The gene sorbA-encodes ahighly reducing iterative PKS while SorbB encodes a non-reducing iterative PKS which features areductive release domain usually involved in the production of polyketide aldehydes. Using theseobservations and previously reported results from isotopic feeding experiments a new and simplerbiosynthetic route to the sorbicillin class of secondary metabolites is proposed which is consistent withall reported experimental results.IntroductionThe sorbicillinoid natural products, such as sorbicillactones A1a and B 1b, and dimeric and trimeric species 2–4, comprise alarge class of highly complex cyclic and polycyclic compounds,oen with unusual biological activities (Fig. 1).1 They occur infungi, especially among species of Penicillium, and Trichodermabut also sporadically in other genera of ascomycetes from bothterrestrial and marine environments. The biosynthesis of thesecompounds has been proven to be of polyketide origin throughlabelling experiments using stable isotopes. For example, bis-vertinolone 2 is a b-1,6-glucan biosynthesis inhibitor andradical scavenger which has been demonstrated to be derivedfrom acetate.2 Bisorbicillinol 3 is also a radical scavenger,3 whiletrisorbicillinone C 4 is cytotoxic vs. P388 and HL60 cell lines.4 Ithas been hypothesised that sorbicillinol 5a forms a key pathwayintermediate and is a precursor to almost all of the sorbicilli-noids.2,5 Labelling studies have been used to suggest the, Cantock's Close, Bristol BS8 1TS, UK.925 1295; Tel: +44 (0) 117 928 9184Technische Hochschule (ETH) Zu¨rich,rlandiochemistry, University of Bonn, Gerhard-niversita¨t Hannover, Schneiderberg 1B,ox@oci.uni-hannover.des, National Research Centre, Dokki, Egyptn (ESI) available: Containing allsc52911hhemistry 2014biosynthetic route shown in Scheme 1, but until now littlebiochemical and no genetic information has been available.The proposed biosynthetic route is rather unusual, featuringthe formation of an unprecedented uncyclised tetracarbonyl 6by a fungal polyketide synthase (PKS),6 which is then hydrox-ylated to give 7 prior to cyclisation to form oxosorbicillinol 8.This is then reduced to the alcohol 9 which has not beenobserved. Dehydration is then proposed to give sorbicillinol 5a,which must be reduced to the observed sorbicillin 10a (Scheme1).Support for this proposed route came from the incorporationof [1,2-13C2]-acetate into sorbicillinol 5a which suggested that asymmetrical intermediate (i.e. 11, 110) was not formed.7 Thisconclusion was arrived at because free rotation of thesymmetrical aromatic moiety of 11, formed by cyclisation of 6,would scramble the position of the intact acetate units(Scheme 1) and scrambling of label was not observed. Theproposed pathway is rather unsatisfactory because of thehydroxylation prior to cyclisation; the fact that 9 has not beenobserved; and lengthy reductive chemistry required to synthe-sise sorbicillin 10a.The proposal that hydroxylation of the polyketide occursbefore cyclisation rests upon the assumption that the sorbicillinPKS forms the putative thiolester precursor 6. However, recentresults from our laboratories have shown that some fungal PKScan release their products as aldehydes, instead of thiolesters.8Furthermore we have also recently shown that oxidative dear-omatisation of PKS-products is a common early transformationduring the biosynthesis of a number of fungal secondarymetabolites9 and this led us to reassess the biosyntheticChem. Sci., 2014, 5, 523–527 | 523Scheme 1 Earlier proposed biosynthesis of sorbicillinol.2Fig. 1 Structural diversity among the sorbicillinoid family. R ¼ CH]CHCH]CHMe.Chemical Science Edge ArticleOpen Access Article. Published on 20 November 2013. Downloaded on 03/12/2015 10:36:32.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinepathway to sorbicillinol 5a. Here we describe molecular exper-iments which support a diﬀerent route to sorbicillinol 5a whichis entirely consistent with the results from the previouslydescribed isotopic labelling experiments, and with recentlyreported results in other systems.ResultsBringmann and coworkers reported the production of sorbi-cillactones A 1a and B 1b by the fungus Penicillium chrysogenumE01-10/3, as well as sorbicillin 10a itself.10 In our handsfermentation of this organism produced these compounds ingood titres, but also produced 20,30-dihydrosorbicillin 10b.5,11We hypothesised that 10a and 10b were likely to be precursorsof sorbicillactones A and B respectively. 10a and 10b might beformed by synthesis of intermediate polyketide aldehydeswhich could undergo Knoevenagel cyclisation to form 5a and 5bdirectly. Aldehydes have recently been recognised as directproducts of fungal non-reducing iterative polyketide synthases(NR-iPKS), for example during the biosynthesis of methyl-orcinaldehyde by the NR-iPKS methylorcinaldehyde synthase(MOS, encoded by aspks1), and also precursors of the azaphi-lones.12 The formation of the aldehyde is achieved by a reductiverelease domain instead of the more usual hydrolytic releasedomain.13 This class of NR-iPKS has been recognised in phylo-genetic analyses and designated non-reducing clade III(NRcIII).14 We have previously described the design of selective524 | Chem. Sci., 2014, 5, 523–527degenerate PCR primers which can recognise and amplifyfragments of NRcIII genes: for example KHKS2/KHKS3c primerswere used in the successful cloning of aspks1.8We thus applied PCR using the KHKS2/KHKS3c primers togDNA isolated from P. chrysogenum E01-10/3. Three distinct 245bp fragments of iPKS genes were amplied and sequenced, andone of these (KHKS32) was shown to be closely related to theiPKS citrinin synthase15 which belongs to NRcIII. Next, a 4800-clone fosmid library was constructed from P. chrysogenum E01-10/3 gDNA. The fosmid library was screened by PCR usingprimers based on the sequence of KHKS32. This process yieldeda number of potential fosmid clones, and the largest of thesewas subjected to random shotgun sequencing (GATC Biotech).This revealed the fosmid clone to be 48.8 kb in length. Analysisof the sequence (fgenesh16) revealed the presence of sevenputative open reading frames possibly involved in secondarymetabolism (orfs 1–7, Scheme 2) encoding: a transcriptionalregulator; an FAD-dependent monooxygenase; an NR-iPKS; ahighly-reducing iPKS (HR-iPKS); a transcription factor; an ABCtransporter; and a second FAD-dependent oxidoreductase(Scheme 2). Interestingly the sequence is almost identical to afragment of the sequenced genome of P. chrysogenum Wiscon-sin 54-1255,17 but although this strain is a penicillin G producerit is not a known producer of sorbicillinoids.18Domain analysis (see ESI†) of the NR-iPKS shows that itpossesses typical starter-unit acyl transferase (SAT), b-ketoa-cylsynthase (KS), acyl transferase (AT), product template (PT),This journal is © The Royal Society of Chemistry 2014Scheme 2 New proposed biosynthetic route to sorbicillinols.Scheme 3 Conversion of dihydrosorbicillin 10b to dihydrosorbicillinol5b and its acetylation. DAD HPLC traces (200–400 nm): (A), 0 min(before addition of NADPH); (B), 10 min; (C), 20 min; (D), 30 min. R ¼CH2CH2CH]CHCH3.Edge Article Chemical ScienceOpen Access Article. Published on 20 November 2013. Downloaded on 03/12/2015 10:36:32.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlineacyl carrier protein (ACP), C-methyl transferase (CMeT) andreductive release (Red) domains consistent with the chemistryrequired for the later steps of sorbicillin biosynthesis – andcrucially for the release of an aldehyde intermediate. Similaranalysis of the HR-iPKS indicated the presence of KS, AT,dehydratase (DH), CMeT, b-ketoacylreductase (KR), enoylre-ductase (ER) and ACP domains consistent with the early stagesof sorbicillin biosynthesis. Attempts were made to knockoutthese PKS genes in P. chrysogenum E01-10/3, but without success.In an alternative strategy to link these genes with thebiosynthesis of the sorbicillins we considered the activity of theputative tailoring genes. Orf2 encodes an FAD dependentoxidase and is similar to tropB present in the tropolonebiosynthetic cluster of Talaromyces stipitatus9 and also similar togenes which encode proteins responsible for the oxidativedearomatisation reactions during azaphilone biosynthesis.12We hypothesised that this protein might hydroxylate sorbicillin10a and form the key intermediate sorbicillinol 5a. In order toaddress this possibility we cloned orf2 from cDNA (to removeintrons) and expressed it in E. coli. This produced the expectedprotein of 48.6 kDa (50.3 KDa including his6 tag). Denaturationof the protein released the cofactor which was shown to be FADby LCMS analysis. The catalytic activity of the protein was thenexamined by incubating it with puried sorbicillin 10a (seeESI†) or dihydrosorbicillin 10b and NADPH in 50 mM phos-phate buﬀer pH 8.0. The reaction was followed by LCMS(Scheme 3) over a period of 30 min and this clearly showed theconsumption of dihydrosorbicillin 10b and the formation of anew peak which corresponded (m/z [M]H+ ¼ 251) with dihy-drosorbicillinol 5b which is chemically unstable (like itsunsaturated analogue 5a).19We converted 5b to its bis-acetate 13in situ by treating the reaction mixture with an excess of aceticanhydride and pyridine, followed by automated RP-HPLCpurication of the product and full structural elucidation by 1Dand 2D NMR (600 MHz) and HRMS.This journal is © The Royal Society of Chemistry 2014DiscussionDomain analysis of the two PKS genes suggest that they shouldwork together to form the rst intermediate in the pathway tothe sorbicillinoids, sorbicillin 10a itself. Similar sets of HR- andNR-iPKS are known to act together to form the resorcylic acidlactone20 and azaphilone21 classes of fungal polyketides both invivo and in vitro.22 We hypothesise that orf4 encodes a HR-iPKSwhich synthesises a triketide which is then passed to the orf3encoded NR-iPKS which extends it three more times andmethylates it twice. Alternatively, the orf3-encoded PKS mayproduce a tetraketide which is then chain extended and meth-ylated twice by the orf4 PKS. We thus propose to name orf4 andorf3 sorbA and sorbB respectively. The SorbA iPKS is homologouswith other HR-iPKS where the ER domain is functional, (the ERdomain is 32% identical to the ER domain of the mammalianfatty acid synthase)23 but here it appears that for the biosyn-thesis of sorbicillin 10a the ER does not operate, althoughduring the biosynthesis of dihydrosorbicillin 10b it presumablyoperates correctly during the second cycle of chainChem. Sci., 2014, 5, 523–527 | 525Chemical Science Edge ArticleOpen Access Article. Published on 20 November 2013. Downloaded on 03/12/2015 10:36:32.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinemodication. Although SorbA contains a CMeT domain, thisappears to be inactive similar to the compactin CMeT domain.24In contrast, SorbB must methylate the growing polyketide chaintwice before release and cyclisation. This is the same asobserved in MOS where methylation has been shown to beprocessive,25 however MOS methylates once during 3 extensioncycles, whereas SorbB appears to methylate during both exten-sion cycles. This is similar to the austinol NR-iPKS whichmethylates twice, but which has a hydrolytic release domainresulting in the formation of a carboxylic acid product.26Aer chain extension and methylation by SorbB, reductiverelease of 6 catalysed by the SorbB Red-domain would form analdehyde 12 which would rapidly cyclise to form sorbicillin 10a.The orf2 encoded FAD-dependent oxidase (hereaer referred toas SorbC) then hydroxylates sorbicillins 10a and 10b to formsorbicillinols 5a and 5b respectively, forming the precursors of1a and 1b, and all other subsequent sorbicillins andsorbicillinoids.ConclusionsThe proposal that SorbA and SorbB produce sorbicillin 10a,followed by oxidative dearomatisation performed by SorbC isfully consistent with the results of the previous isotopic label-ling experiments since 10a itself is not symmetrical. Theproposed pathway is simpler, more direct and supported bybiochemical evidence. The new pathway is also consistent withthe biosynthesis of a number of other fungal secondarymetabolites including the tropolones9 and azaphilones21 wherenon-reducing iPKS release simple aromatic aldehydes which areoxidatively dearomatised in early steps on pathways to morecomplex metabolites. Similar to the resorcyclic acid lactone20and azaphilone21 classes of fungal polyketides, the sorbicillinsalso require the action of two PKS – a HR-iPKS to provide thestarter unit for the NR-iPKS which catalyses the nal chainextension steps. Further work to delineate the later steps ofsorbicillactone biosynthesis are ongoing.AcknowledgementsThe following are thanked for funding: Al Baha University,Saudi Arabia (AAF); EPSRC (LCMS equipment, JD, EP/F066104/1); BBSRC (KF, BB/I003355/1); BMBF (JP, BiotecMarin); ERAS-MUS programme (AO). J. F. Imhoﬀ (IFM-GEOMAR) is thankedfor providing P. chrysogenum E01-10/3; G. Bringmann (Uni-versita¨t Wu¨rzburg) is thanked for providing a reference sampleof sorbicillin.Notes and references1 A. M. Harned and K. A. Volp, Nat. Prod. Rep., 2011, 28, 1790–1810.2 N. Abe, T. Arakawa and A. Hirota, Chem. Commun., 2002,204–205.3 N. Abe, T. Murata and A. Hirota, Biosci., Biotechnol., Biochem.,1998, 62, 2120–2126.526 | Chem. Sci., 2014, 5, 523–5274 D. Li, F. Wang, X. Xiao, Y. Fang, T. Zhu, Q. Gu and W. Zhu,Tetrahedron Lett., 2007, 48, 5235–5238.5 K. Sugaya, H. Koshino, Y. Hongo, K. Yasunaga, J.-I. Onose,K. Yoshikawa and N. Abe, Tetrahedron Lett., 2008, 49, 654–657; L. S. Trifonov, A. S. Dreiding, L. Hoesch andD. M. Rast, Helv. Chim. Acta, 1981, 64, 1843–1846.6 R. J. Cox, Org. Biomol. Chem., 2007, 5, 2010–2026.7 N. Abe, T. Arakawa, K. Yamamoto and A. Hirota, Biosci.,Biotechnol., Biochem., 2002, 66, 2090–2099.8 A. M. Bailey, R. J. Cox, K. Harley, C. M. Lazarus, T. J. Simpsonand E. Skellam, Chem. Commun., 2007, 4053–4055.9 J. Davison, A. Al Fahad, M. Cai, Z. Song, S. Y. Yehia,C. M. Lazarus, A. M. Bailey, T. J. Simpson and R. J. Cox,Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 7642–7647.10 G. Bringmann, T. A. M. Gulder, G. Lang, S. Schmitt, R. Sto¨hr,J. Wiese, K. Nagel and J. F. Imhoﬀ, Mar. Drugs, 2007, 5, 23–30; G. Bringmann, G. Lang, T. A. M. Gulder, H. Tsuruta,J. Mu¨hlbacher, K. Maksimenka, S. Steﬀens, K. Schaumann,R. Sto¨hr and J. Wiese, Tetrahedron, 2005, 61, 7252–7265.11 L. S. Trifonov, J. H. Bieri, R. Prewo, A. S. Dreiding, L. Hoeschand D. M. Rast, Tetrahedron, 1983, 39, 4243–4256.12 J. M. Winter, M. Sato, S. Sugimoto, G. Chiou, N. K. Garg,Y. Tang and K. Watanabe, J. Am. Chem. Soc., 2012, 134,17900–17903.13 L. Du and L. Lou, Nat. Prod. Rep., 2010, 27, 255–278.14 S. Kroken, N. Glass, J. Taylor, O. C. Yoder and B. G. Turgeon,Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 15670–15675.15 K. Sakai, H. Kinoshita, T. Shimizu and T. Nihira, J. Biosci.Bioeng., 2008, 106, 466–472.16 A. A. Salamov and V. V. Solovyev, Genome Res., 2000, 10, 516–522.17 M. A. van den Berg, R. Albang, K. Albermann, J. H. Badger,J. M. Daran, A. J. Driessen, C. Garcia-Estrada,N. D. Fedorova, D. M. Harris, W. H. Heijne, V. Joardar,J. A. Kiel, A. Kovalchuk, J. F. Mart´ın, W. C. Nierman,J. G. Nijland, J. T. Pronk, J. A. Roubos, I. J. van der Klei,N. N. van Peij, M. Veenhuis, H. von Do¨hren, C. Wagner,J. Wortman and R. A. Bovenberg, Nat. Biotechnol., 2008, 26,1161–1168.18 Other P. chrysogenum strains are known sorbicillinoidproducers, see: R. F. Miller and S. Huang, J. Antibiot., 1995,520–521.19 N. Abe, O. Sugimoto, K.-I. Tanji and A. Hirota, J. Am. Chem.Soc., 2000, 122, 12606–12607.20 I. Gaﬀoor and F. Trail, Appl. Environ. Microbiol., 2006, 72,1793–1799; H. Zhou, J. Zhan, K. Watanabe, X. Xie andY. Tang, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 6249–6254.21 Y.-M. Chiang, E. Szewczyk, A. D. Davidson, N. Keller,B. R. Oakley and C. C. C. Wang, J. Am. Chem. Soc., 2009,131, 2965–2970.22 Y. Xu, P. Espinosa-Artiles, V. Schubert, Y.-M. Xu, W. Zhang,M. Lin, A. L. Gunatilaka, R. Su¨ssmuth and I. Molna´r, Appl.Environ. Microbiol., 2013, 79, 2038–2047; H. Zhou, K. Qiao,Z. Gao, J. C. Vederas and Y. Tang, J. Biol. Chem., 2010, 285,41412–41421; Z. Gao, J. Wang, A. K. Norquay, K. Qiao,This journal is © The Royal Society of Chemistry 2014Edge Article Chemical ScienceOpen Access Article. Published on 20 November 2013. Downloaded on 03/12/2015 10:36:32.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineY. Tang and J. C. Vederas, J. Am. Chem. Soc., 2013, 135, 1735–1738.23 M. Leibundgut, T. Maier, S. Jenni and N. Ban, Curr. Opin.Struct. Biol., 2008, 18, 714–725.24 Y. Abe, T. Suzuki, C. Ono, K. Iwamoto, M. Hosobuchi andH. Yoshikawa, Mol. Genet. Genomics, 2002, 267, 636–646.This journal is © The Royal Society of Chemistry 201425 K. M. Fisch, E. Skellam, D. Ivison, R. J. Cox, A. M. Bailey,C. M. Lazarus and T. J. Simpson, Chem. Commun., 2010,46, 5331–5333.26 H.-C. Lo, R. Entwistle, C.-J. Guo, M. Ahuja, E. Szewczyk,J.-H. Hung, Y.-M. Chiang, B. R. Oakley and C. C. C. Wang,J. Am. Chem. Soc., 2012, 134, 4709–4720.Chem. Sci., 2014, 5, 523–527 | 527

Oxidative dearomatisation: the key step of sorbicillinoid biosynthesis

An FAD-dependent monooxygenase encoding gene (SorbC) was cloned from Penicillium chrysogenum E01-10/3 and expressed as a soluble protein in Escherichia coli. The enzyme efficiently performed the oxidative dearomatisation of sorbicillin and dihydrosorbicillin to give sorbicillinol and dihydrosorbicillinol respectively. Bioinformatic examination of the gene cluster surrounding SorbC indicated the presence of two polyketide synthase (PKS) encoding genes designated sorbA and sorbB. The gene sorbA-encodes a highly reducing iterative PKS while SorbB encodes a non-reducing iterative PKS which features a reductive release domain usually involved in the production of polyketide aldehydes. Using these observations and previously reported results from isotopic feeding experiments a new and simpler biosynthetic route to the sorbicillin class of secondary metabolites is proposed which is consistent with all reported experimental results.ISSN:2041-6520ISSN:2041-653

Al Fahad, Ahmed

Piel, Jörn

Repository for Publications and Research Data

A new biosynthetic pathway to the sorbicillinoid natural products is proposed based on the observation of oxidative dearomatisation of dihydrosorbicillin 10b.</p

Oxidative dearomatisation: the key step of sorbicillinoid biosynthesis

Abstract

Similar works

Full text

Available Versions

Explore Bristol Research

Institutionelles Repositorium der Leibniz Universität Hannover

Repository for Publications and Research Data

Crossref