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Product analysis of modified PKS-NRPS hybrids in filamentous fungi. Lasse Norup Andersen * Technical University of Denmark [email protected] 3rd of February Abstract Genome mining have led to the discovery of many PKS-NRPS hybrids, predicted to synthesize secondary metabolites. The present study examined chimeric PKS-NRPSs constructed by module swapping. Plasmids containing the chimeric PKS-NRPSs and PKS-NRPS hybrids were constructed and transformed into A. nidulans. The metabolic profile of the transformed strains was obtained with HPLC coupled with MS. To visualize the localization of PKS-NRPSs, all constructs were tagged in the C-terminus. The present study shows that it is possible to construct a functional PKS-NRPS by swapping the linker of ccsA(ACLA_078660) with the linker of sclA(A. sclerotioniger). Constructs, where the NRPS from ccsA was swapped with the NRPSs from flaA(ATEG_00325) and sclA, were not able to synthesize a functional enzyme, as no new compounds were observed in the metabolic profile of A. nidulans. The product synthetized by sclA was not determined. Data from fluorescence microscopy showed that the enzyme encoded by the ccsA hybrid and the linker-swapped ccsA chimera were present in specific compartments in the hyphae of A. nidulans, while not functional chimeric constructs were expressed in the cytosol. Furthermore the present study shows that fluorescence tagging of the PKS-NRPSs does not alter or disrupt the function of the enzyme. I. Introduction F ilamentous fungi are able to produce a variety of bioactive metabolites and en- zymes, which enable them to thrive in a competitive environment. Among the metabo- lites are compounds that are used as drugs, including: antibiotics, antibacterials, antivirals and anticancers [Lubertozzi et al, 2009]. Since many of the desirable products are naturally secreted in large amounts, fungi possess con- siderable potential as expression hosts for the production of small molecules as well as pro- teins [Hansen et al, 2011]. Aspergillus is an im- portant genus, including well-known species of economically significant molds, and are widely used for basic genetic research. Through the sequencing of an increased number of fungal genomes, new gene clusters are found and predicted to produce secondary metabolites, such as polyketides and nonribosomal pep- tides. At present time, finding the link be- tween compounds and the clusters responsible for their synthesis is still far from completed. Through the development of a genetic engi- neering "toolkit" for Aspergillus, heterologous expression is now possible. Polyketide syn- thases (PKSs) are a family of multi-domain enzymes that produce polyketides. The PKS genes are usually found in gene clusters in eukaryotes. PKSs can be classified into three groups: Type I PKSs are large, highly modular * Thanks to my supervisors Jakob Blaesbjerg Nielsen and Maria Lund Nielsen for help with all the practical work and advising 1

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Product analysis of modifiedPKS-NRPS hybrids in filamentous

fungi.

Lasse Norup Andersen∗

Technical University of [email protected]

3rd of February

Abstract

Genome mining have led to the discovery of many PKS-NRPS hybrids, predicted to synthesize secondarymetabolites. The present study examined chimeric PKS-NRPSs constructed by module swapping. Plasmidscontaining the chimeric PKS-NRPSs and PKS-NRPS hybrids were constructed and transformed intoA. nidulans. The metabolic profile of the transformed strains was obtained with HPLC coupled withMS. To visualize the localization of PKS-NRPSs, all constructs were tagged in the C-terminus. Thepresent study shows that it is possible to construct a functional PKS-NRPS by swapping the linker ofccsA(ACLA_078660) with the linker of sclA(A. sclerotioniger). Constructs, where the NRPS from ccsAwas swapped with the NRPSs from flaA(ATEG_00325) and sclA, were not able to synthesize a functionalenzyme, as no new compounds were observed in the metabolic profile of A. nidulans. The productsynthetized by sclA was not determined. Data from fluorescence microscopy showed that the enzymeencoded by the ccsA hybrid and the linker-swapped ccsA chimera were present in specific compartmentsin the hyphae of A. nidulans, while not functional chimeric constructs were expressed in the cytosol.Furthermore the present study shows that fluorescence tagging of the PKS-NRPSs does not alter or disruptthe function of the enzyme.

I. Introduction

Filamentous fungi are able to produce avariety of bioactive metabolites and en-zymes, which enable them to thrive in a

competitive environment. Among the metabo-lites are compounds that are used as drugs,including: antibiotics, antibacterials, antiviralsand anticancers [Lubertozzi et al, 2009]. Sincemany of the desirable products are naturallysecreted in large amounts, fungi possess con-siderable potential as expression hosts for theproduction of small molecules as well as pro-teins [Hansen et al, 2011]. Aspergillus is an im-portant genus, including well-known species ofeconomically significant molds, and are widely

used for basic genetic research. Through thesequencing of an increased number of fungalgenomes, new gene clusters are found andpredicted to produce secondary metabolites,such as polyketides and nonribosomal pep-tides. At present time, finding the link be-tween compounds and the clusters responsiblefor their synthesis is still far from completed.Through the development of a genetic engi-neering "toolkit" for Aspergillus, heterologousexpression is now possible. Polyketide syn-thases (PKSs) are a family of multi-domainenzymes that produce polyketides. The PKSgenes are usually found in gene clusters ineukaryotes. PKSs can be classified into threegroups: Type I PKSs are large, highly modular

∗Thanks to my supervisors Jakob Blaesbjerg Nielsen and Maria Lund Nielsen for help with all the practical work andadvising

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proteins, type II PKSs are aggregates of mono-functional proteins and type III PKSs, whichdo not use acyl carrier protein. Type I PKSsare further subdivided: Iterative PKSs, whichreuse domains in a cyclic manner and modu-lar PKSs, that contain a sequence of separatemodules and do not recycle domains. TypeI iterative PKSs are generally foung in fungi.Each type I PKS module consists of severaldomains with defined functions, separated byshort spacer regions. The modules consists ofa loading module, a elongation module anda releasing module and in general the follow-ing domains are found: Starter acyltransferase(SAT), acyltransferase (AT), acyl carrier protein(ACP), β-ketoacyl synthase (KS), β-keto reduc-tase (KR), dehydratase (DH), enoyl reductase(ER), methyltransferase (MT), sulfhydrolase(SH) and a thioesterase (TE) [Frandsen, 2010].Nonribosomal peptide synthases (NRPSs) areanother group of large multimodular enzymes.Three domains are ubiquitous in all NRPSs:An adenylation (A) domain responsible for theactivation of the amino acid, a thiolation orpeptidyl carrier protein (PCP) domain respon-sible for the propagation of the growing pep-tide chain and a condensation (C) domain re-sponsible for condensation of the amino acids.A fourth domain that is sometimes found inNRPSs is the thioesterase (TE) domain whichcatalyzes peptide release [Strieker et al, 2010].PKSs and NRPSs can be found as natural hy-brids (PKS-NRPSs) in which a single module ofNRPS is translationally fused to the C-terminusof a PKS. An example of such a hybrid ispresented in figure 1. This study focuses onthree PKS-NRPSs from the Aspergillus speciesA. clavatus, A. terreus and A. sclerotioniger. Theproduct of the PKS-NRPS gene cluster of A.clavatus and of A. terreus have previously beenidentified as cytochalasin E [Qiao et al, 2011]and isoflavipucine [Gressler et al, 2011] respec-tively. However, at present time, the compoundsynthesized by the PKS-NRPS gene cluster ofA. sclerotioniger remains to be identified.

Figure 1: The polyketide synthase nonriboso-mal peptide synthase (PKS-NRPS)hybrid encoded by ccsA from A.clavatus(ACLA_078660)[Fujii et al, 2013]

The protein sequence of the PKS-NRPSfor the mentioned Aspergillus species wasblasted to compare sequence identity and pos-itives. When comparing ccsA(ACLA_078660)to flaA(ATEG_00325) and sclA(A. sclerotioniger)the identities were 35 % and 48 % respectively,while the positives were 53 % and 65 % respec-tively. Additionally, the BLAST data shows thatthe sequence of the presumed location of thelinker displays no homology between the linkerof ccsA and the linkers of flaA and sclA. Thepresumed location of the linker is rationalizedby linker predictions. In this study, chimeraswere constructed by swapping the linker ofccsA with the linker of sclA, and the NRPSmodule of ccsA with NRPS modules from flaAand sclA(see figure 2). It is interesting to swapdomains as this method could lead to synthe-sis of novel polyketide - nonribosomal pep-tides and would further diversify the naturalreservoir of secondary metabolites. The aim ofthis study is to determine whether it is possi-ble to construct a functional chimera throughmodule swapping of the selected PKS-NRPSs.An earlier study [Fujii et al, 2013] showed that,when ccsA was expressed simultaneously withthe trans-acting enoyl reductase encoded byccsC(ACLA_078700) in A. oryzae it was possibleto produce a putative cytochalasin E precursor.Further studies have shown that production ofa putative cytochalasin E precursor is also pos-sible in A. nidulans by co-expression of ccsAand ccsC (unpublished results). This precursorcan be used as reference to assess the function-ality of the chimeric PKS-NRPSs. The strains

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seen in table 1 were constructed from the plas-mids seen in table 2. The strains include:NRPS module swaps (NID1768 and NID1769),linker and NRPS module swap (NID1770),linker swap (NID1771), full hybrids (NID1772,NID1774, NID1432) and tagging of the pro-teins encoded by PKS-NRPS hybrids/chimeraswith the red fluorescent protein, RFP(NID1775-NID1779), to see where in A. nidulans the PKS-NRPS hybrids/chimeras localize.

CT

PKS Linker NRPS

ccsA

flaA

sclA

CS1

PKS Linker NRPS

CSC

PKS Linker NRPS

CS2

PKS Linker NRPS

PKS Linker NRPS

sclA

PKS Linker NRPS

ccsA

Figure 2: PKS-NRPS Chimera and hybrid constructsconsisting of modules from A. clavatus(ccsA),A. terreus(flaA) and A. sclerotioniger(sclA)

Strain identifier Strain name GenotypeCT, ccsC prepop NID1768 argB2, pyrG89, veA1, nkuA∆, IS5::PgpdA-CT-TtrpC::AFpyrG, IS2::PgpdA-ccsC-TtrpCCS1, ccsC prepop NID1769 argB2, pyrG89, veA1, nkuA∆, IS5::PgpdA-CS1-TtrpC::AFpyrG, IS2::PgpdA-ccsC-TtrpCCS2, ccsC prepop NID1770 argB2, pyrG89, veA1, nkuA∆, IS5::PgpdA-CS2-TtrpC::AFpyrG, IS2::PgpdA-ccsC-TtrpCCSC, ccsC prepop NID1771 argB2, pyrG89, veA1, nkuA∆, IS5::PgpdA-CSC-TtrpC::AFpyrG, IS2::PgpdA-ccsC-TtrpCsclA prepop NID1772 argB2, pyrG89, veA1, nkuA∆, IS5::PgpdA-sclA-TtrpC::AFpyrGsclC prepop NID1773 argB2, pyrG89, veA1, nkuA∆, IS2::PgpdA-sclC-TtrpC::AFpyrGsclA, sclC prepop NID1774 argB2, pyrG89, veA1, nkuA∆, IS5::PgpdA-sclA-TtrpC::AFpyrG, IS2::PgpdA-sclC-TtrpCCT-RFP prepop NID1775 argB2, pyrG89, veA1, nkuA∆, IS5::PgpdA-CT-RFP-TtrpC::AFpyrGCS1-RFP prepop NID1776 argB2, pyrG89, veA1, nkuA∆, IS5::PgpdA-CS1-RFP-TtrpC::AFpyrGCSC-RFP, ccsC prepop NID1777 argB2, pyrG89, veA1, nkuA∆, IS5::PgpdA-CSC-RFP-TtrpC::AFpyrG, IS2::PgpdA-ccsC-TtrpCsclA-RFP prepop NID1778 argB2, pyrG89, veA1, nkuA∆, IS5::PgpdA-sclA-RFP-TtrpC::AFpyrGccsA-RFP, ccsC prepop NID1779 argB2, pyrG89, veA1, nkuA∆, IS5::PgpdA-ccsA-RFP-TtrpC::AFpyrG, IS2::PgpdA-ccsC-TtrpCccsC pop NID1525 argB2, pyrG89, veA1, nkuA∆, IS2::PgpdA-ccsC-TtrpCccsA, ccsC prepop NID1432 argB2, pyrG89, veA1, nkuA∆, IS1::PgpdA-ccsA-TtrpC::AFpyrG, IS2::PgpdA-ccsC-TtrpCsclC pop NID1780 argB2, pyrG89, veA1, nkuA∆, IS2::PgpdA-sclC-TtrpC

Table 1: List of strains constructed and/or used in this study.

In the attempt to identify the compoundsynthesized by the PKS-NRPS encoded by sclA,the gene was transformed into NID1(NID1772)and NID1780(NID1774) containing sclC. Thiswas done to see if the enzyme encoded by sclA,like ccsA, needed the trans-acting enoyl reduc-tase encoded by sclC to synthesize a new com-pound or the enzyme was functional without it.NID1779 and NID1777 were constructed to seeif the hybrid PKS-NRPS (ccsA) and the chimera(CSC) were functional after RFP-tagging of theprotein.

Description Derived from

pU2110-1-ccsA pU2115-1

pU2110-5-CT pU2115-5pU2110-5-CS1 pU2115-5pU2110-5-CS2 pU2115-5pU2110-5-sclA pU2115-5pU2110-5-CSC pU2115-5pU2110-2-ccsC pU2115-2pU2110-2-sclC pU2115-5pU2110-5-ccsA-RFP pU2115-5pU2110-5-CT-RFP pU2115-5pU2110-5-CS1-RFP pU2115-5pU2110-5-CSC-RFP pU2115-5pU2110-5-sclA-RFP pU2115-5

Table 2: List of the constructed plasmids used in thisstudy

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II. Materials and Methods

Strains and media.Genomic DNA from A. clavatus NRRL1, A.sclerotioniger FGS21 and A. terreus NIH2624provided by Maria Lund Nielsen was used forconstruction of the PKS-NRPS chimeras andhybrids. The A. nidulans strains NID1(argB2pyrG89 veA1 ∆nkuA)[Nielsen et al, 2008],NID1780 and NID1525(argB2 veA1 ∆nkuA)were used for transformation of the con-structed PKS-NRPS chimeras and hybrids.∆nkuA is a deletion of the ku protein essentialfor the non-homologous end-joining (NHEJ)activity, while argB2 and pyrG89 are muta-tions in argB and pyrG that allows for selectionand veA1 is a mutation that reduces sexualcrossover in A. nidulans. The A. nidulans strainsNID3(argB2 veA1 ∆nkuA) was used as a back-ground strain. All plasmids were propagatedin the chemically competent Escherichia colistrain DH5α.

• Trace element solution (1 l) 0.4 g CuSO4*5H2O; 0.04 g Na2B4O7* 10H2O; 0.8 gFeSO4* 7H2O; 0.8 g MnSO4* 2H2O; 0.8g Na2MoO4* 2H2O; and 8.0 g ZnSO4*7H2O.

• Mineral Mix (1 l) 26 g KCl; 26 g MgSO4*7H2O; and 76 g KH2PO4; and 50 ml Traceelement solution.

• Aspergillus protoplastation buffer(APB) (2 l) Final conc: 1.1 M MgSO4 and10 mM Na-phosphate buffer (Na2HPO4and NaH2PO4). pH is adjusted with 2 MNaOH to 5.8.

• Aspergillus transformation buffer(ATB) (2 l) Final conc: 1.2 M Sorbitol;50 mM CaCl2*2 H2O; 20 mM Tris; and0.6 M KCl. pH is adjusted with 2 M HClto 7.2.

• PCT (200 ml) Final conc: 50 % w/volPEG 8000; 50 mM CaCl2; 20 mM Tris;and 0.6 M KCl. pH is adjusted with 2 MHCl to 7.5.

• Minimal medium (MM) (1 l) Mineralmix (20 ml); 1 M NaNO3 (10 ml); 20 %-w/vol glucose (50 ml); supplement witharginine (arg); uridine (uri); uracile (ura)

and 5-flouroorotic acid (5-FOA) whennecessary (for solid medium add 20 gagar).

• Transformation medium (TM) (1 l) Min-eral mix (20 ml); 1 M NaNO3 (10 ml);sucrose (171.15 g); supplement with argi-nine (arg) (for solid medium add 20 gagar).

• Luria Broth (LB) medium (1 l) BactoTryptone (10g); Bacto Yeast Extract (5 g);NaCl (10 g); supplement with ampicilin(amp) (for solid medium add 20 g agar)

PCR and USER cloning.Amplification of DNA by PCR to produce DNAfragments suitable for USER cloning was per-formed using PfuX7 [Noerholm, 2010]. Reac-tion mix (50 µl): 10 µl 5X Phusion R©HF Reac-tion Buffer (New England Biolabs); 5 µl dNTP;0.5 µl PfuX7; 2 µl forward primer; 2 µl re-verse primer; 1 µl 20X diluted gDNA; 2 µlDMSO (Thermo Scientific); 29.5 µl mQ H2O.Reaction conditions: a denaturation step (98◦C, 2 min followed by 35 PCR cycles (98 ◦C,10 s; 64 ◦C (increment -0.2 ◦C/cycle), 30 sand 72 ◦C, 3 min) and finally an extensionstep (72 ◦C for 10 min). PCR products wereverified by gel electrophoresis and purifiedusing IllustraTM GFXTM PCR DNA and GelBand Purfication Kit (GE Healthcare) accord-ing to the manufacturer’s instructions. USERcloning was performed as previously described[Nour-Eldin et al, 2006] with minor modifica-tions. The PacI cassette-containing vector wasprepared for cloning by digesting with PacI at37 ◦C over night and subsequent nicking byNt.BbvCI 37◦C for 2 hours followed by heat-inactivation for 20 minutes at 65 ◦C. The di-gested vector solution (1 µl) was mixed withthe purified PCR product(s) amplified withprimers that were extended by the appropri-ate tails for USER cloning into the designatedUSER vector (pU2115-5 for the PKS-NRPS con-structs and pU2115-2 for enoyl reductase con-structs (see figure 3))). When more than onePCR product was cloned simultaneously, 3 µlof each PCR product was mixed together with1 µl of USER enzyme mix (New England Bio-Labs) and 2 µl Cutsmart buffer (New England

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BioLabs) and the total volume of the mix wasadjusted to 20 µl by adding milliQ water. Thereaction mix was incubated for 40 min at 37◦C, followed by 30 min at 25 ◦C. Next, the20-µl reaction mix was used directly to trans-form chemically competent DH5α E. coli cellson solid LB(amp) medium over night.

Figure 3: Vectors used in this study. Both vec-tors have pyrG, ampR, specific integrationsites and the strong constitutive promoter,PgpdA[Lubertozzi et al, 2009]. The chimeraswere integrated in the insertion site, IS5, whilethe enoyl reductases were integrated in IS2.

Colonies were inoculated in 5 ml LB(amp)for 16 hours. The constructed plasmidswere purified from the E. coli culture usingGenEluteTM Plasmid Miniprep Kit (SigmaAldrich) according to manufacturer’s instruc-tions. Verification of plasmids was performedby restriction analysis using two distinct re-striction enzymes.

Transformation of plasmids into A. nidu-lans.20 µl of plasmid prep was digested with SwaIfor 3 hours to liberate the gene-targeting sub-strate, which was used for transformationof NID1 with an integrated enoyl reductase(NID1525(ccsC)). For the transformation 20µl of digested plasmid was mixed with 75µl protoplast and 150 µl PCT. The mix incu-bated 10 minutes at room temperature, and250 µl ATB was added before plating on solidTM(arg). Streak-purified transformants weregrown on MM(arg). Verification of correct in-tegration was analysed through a gap-, up-,and downcheck by spore-PCR. Reaction mix(40 µl): 8 µl 5X Phire Reaction Buffer (ThermoScientific); 4 µl dNTP; 2 µl DMSO (ThermoScientific); 1 µl 50 mM MgCl2 (New EnglandBiolabs); 0.5 µl PfuX7; 2 µl forward primer; 2µl reverse primer; 22.5 µl mQ H2O. Reactionconditions: a denaturation step (98 ◦C, 30 minfollowed by 35 PCR cycles (98 ◦C, 10 s; 64 ◦C(increment -0.2 ◦C/cycle), 30 s and 72 ◦C, 3min) and finally an extension step (72 ◦C for10 min). 3 reactions were made for each check.Mycelia of transformants was transferred tothe first reaction, while the others served as adilution series.

Protoplastation of A. sclerotioniger enoylreductase.NID1773 was grown on MM(arg, ura, uri,5-FOA) plates to pop the pyrG marker. InpU2115-5 and pU2115-2 pyrG is flanked bytwo direct repeats(DR). Through DR recom-bination the pyrG marker is eliminated andthe DR recombinants can be selected in thepresence of 5-FOA, as transformants still con-taining pyrG will die, because pyrG converts5-FOA to the toxic intermediate 5-fluoro-UMP[Ling et al, 2011]. The pyrG marker can thenbe used as a selection marker for additionaltransformations[Nielsen et al, 2008]. Colonieswere transfered to MM(arg) and MM(arg, uri,ura) plates with a toothpick. Colonies that wereunable to grow on solid MM(arg) but grew onsolid MM(arg, uri, ura) were verified to havepopped pyrG by spore-PCR and restreaked onsolid MM(arg, uri, ura) plates. The conidia

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from the solid MM(arg, uri, ura) plates werecollected by adding 5 ml MM(arg, uri, ura) toeach plate and rubbing off the conidia with asterile drigalsky spatula. The conidial solutionwas transferred to a sterile 500 ml shake flaskcontaining 100 ml MM(arg, uri, ura) and incu-bated at 37 ◦C over night in a shake incubaterat 150 rpm. The fungal biomass was retainedby filtering in sterile mira cloth in funnels andwashed with sterile milliQ water to removeresidual glucose, which can inhibit protoplas-tation. The biomass was resuspended in 40ml filter-sterilized APB containing 40 mg Glu-canex/ml in two falcon tube and incubated at37 ◦C in a shake incubater at 150 rpm. An over-lay of 5 mL ATB diluted to 0.5X with sterilemilliQ water was carefully placed on top of theAPB solution and the mix was centrifuged withHeraeus Multifuge X3R Centrifuge (ThermoScientific) with the following settings. speed:3000 RCF; time: 10 minutes; no brake. In theinterphase of the two liquids, a white slurryconsisting of concentrated protoplasts was ob-served. The protoplasts were transferred to anew falcon tube and washed by adding ATBto a total volume of 40 ml in each tube. Theprotoplasts were resuspended in approximate1 ml of ATB in 1.5 ml eppendorf tubes andstored at -80 ◦C.

Brightfield and Fluorescence microscopy.All constructs were tagged with RFP. Sporesfrom the tagged strains were grown on solidMM(arg) on a microscope slide over night at 30◦C. Images were captured with a Nikon EclipseE1000 microscope and a QImaging RETIGAEXi camera.

Chemical Analysis.The Streak-purified transformants were culti-vated on solid MM(arg) as three-point inoc-ulations at 37 ◦C for 6 days. 5 plugs weretaken from the plates and transferred to a 2-mlHPLC vial. 800 µl extraction mix (ethylacetate:dichlormethane: methanol 3:2:1 + 1 % formicacid) and 40 µl chloramphenicol were addedto the plugs and the vials were placed in anultrasonication bath for 60 min. The liquid mixwas transferred to a new 2-ml clean HPLC vialand evaporated to dryness. The dried extract

was dissolved in 400 µl methanol and trans-ferred to a HPLC vial with inserts by filtration.Analysis was performed using UHPLC-DAD-TOFMS on a maXis 3G orthogonal accelera-tion quadrupole time-of-flight mass spectrom-eter (Bruker Daltonics or Agilent Technologies)equipped with an electrospray ionization (ESI)source and connected to an Ultimate 3000 UH-PLC system (Dionex). The column used wasa reverse-phase Kinetex 2.6 mm C18, 100 mm3 2.1 mm (Phenomenex), and the column tem-perature was maintained at 40 ◦C. A linearwater-acetonitrile gradient was used (both sol-vents were buffered with 20 mM formic acid)starting from 10 % (v/v) acetonitrile and in-creased to 100 % in 10 min, maintaining thisrate for 3 min before returning to the startingconditions in 0.1 min and staying there for 2.4min before the following run. A flow rate of 0.4ml/min was used. TOFMS was performed inESI+ with a data acquisition range of 10 scansper second at m/z 100 âAS 1,000. UV-visiblespectra were collected at wavelengths from 200to 700 nm. Data processing was performedusing DataAnalysis 4.0(Bruker Daltonics).

III. Results & Discussion

Identification of the cytochalasin E pre-cursor and new compounds. To determineif the constructed PKS-NRPS chimeras wereable to produce the cytochalasin E precur-sor (figure 4) or other novel products, CT,CS1, CS2, CSC, and ccsA, which all con-tain the PKS from ccsA were transformedinto the A. nidulans strain NID1525 contain-ing ccsC(NID1768-NID1771, NID1432). sclAwas transformed into both NID1780 containingsclC(NID1774) and NID1(NID1772). CT-RFP,CS1-RFP and sclA-RFP were transformed intoNID1 (NID1775,NID1776,NID1778). ccsA-RFPand CSC-RFP were transformed into NID1525(NID1779,NID1777). NID3(argB2 veA1 ∆nkuA)was used as a reference. An earlier study by[Fujii et al, 2013] showed that when ccsA wasexpressed together with the trans-acting enoylreductase encoded by ccsC in A. oryzae it waspossible to synthesize the cytochalasin E pre-

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cursor(see figure 8).

Figure 4: The proposed structure of the cytochalasin Eprecursor. The light area is still under research.

The data from LC-MS(figure 5 and 6)showed that the PKS-NRPS hybrid ccsA pro-duced the cytochalasin precursor(figure 4).The structure proposed by [Fujii et al, 2013](figure 8) does not match the observedmass of the obtained compound (figure7). Other studies analyzing PKS-NRPS hy-brids [Heneghan et al, 2011], [Xu et al, 2010]showed that precursors to the compounds syn-thesized by the PKS-NRPSs, all contained anaromatic ring, as seen in figure 4. No aro-matic ring is observed in figure 8, which whichcould indicate that the precursor found by[Fujii et al, 2013] is not the true cytochalasinE precursor, and therefore another mass is ob-served. It must be said that [Fujii et al, 2013]co-expressed ccsA and ccsC in A. oryzae, whilethis study co-expressed ccsA and ccsC in A.nidulans. This may affect the structure andmass of the detected compounds due to possi-ble modifications made by the host organism.Also, the lack of the remaining ccs gene clus-ter products may result in spontanious non-enzymatical rearragements in the precursor.The data showed that the PKS-NRPS chimeraCSC, which contained the PKS and NRPS fromccsA and the linker from sclA was able to pro-duce a compound with a mass correspondingto the cytochalasin E precursor (figure 7).

Figure 5: LC-MS data (Bruker Daltonics). The boxmarks the area of interest: the cytochalasinE precursor.

Figure 6: LC-MS data(Agilent Technologies). The boxesmarks the areas of interest: the cytochalasin Eprecursor.

This indicates that there might be a cer-tain tolerance in respect to both compositionand length of the linker as there is no homol-ogy between the linker from ccsA and sclA. Tosee if the linker from ccsA can be swappedwith linkers from flaA and other PKS-NRPS

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hybrids, constructs similar to CSC should betested. Both CS1, CS2 and CT were unableto form a functional enzyme, as no new com-pounds were observed in the data.

Figure 7: MS data illustrating the mass of the cytocha-lasin E precursor and adducts resulting fromionization.

Figure 8: The proposed structure of the cytochalasin Eprecursor by [Fujii et al, 2013].

NID1772 and NID1774(table 1) were con-structed in the attempt to identify the com-pound synthesized by the PKS-NRPS hybridencoded by sclA. sclA was expressed alone inNID1 and co-expressed with SclC in NID1780to see whether or not the hybrid needed thetrans-acting enoyl reductase encoded by sclCto synthesize the product. In this study nonew compounds were observed in the LC-MSdata for NID1772 and NID1774. To see if thegenes, sclA and sclC, are expressed in their na-tive host, qPCR could be applied. Deletionmutants, where sclA is disrupted or deletedin A. sclerotioniger, could be constructed. Bycomparing the chromatograms of the wildtypestrain and the mutant strain, peaks that might

display the mass of the product of sclA couldbe identified. If the PKS-NRPS gene clusteris silent in its native host (A. sclerotioniger),the expression of pathway-specific regulatorygenes, which are present in many secondary-metabolite gene clusters, could be the solution.An eventual activator can be over-expressed bya highly constitutive promoter, which wouldinduce the expression of all the genes in thePKS-NRPS gene cluster[Bergmann et al, 2007]. These gene products might be necessary forproduct formation and might be the reasonwhy no new compounds were observed in theLC-MS data for NID1772 and NID1774, as thePKS-NRPS and the trans-acting enoyl reduc-tase were the only genes from the scl genecluster that was expressed in A. nidulans. An-other approach is to try expressing the genecluster in other Aspergillus species(A. oryzae) ordistinct(Saccharomyces cerevisiae) heterologousorganisms. Heterologous expression in Saccha-romyces cerevisiae can be connected to in vitroexperiments to determine the product of theenzyme encoded by sclA. The enzyme encodedby sclA and the enoyl reductase encoded bysclC would have to be obtained in a pure andfunctional form, which can be difficult withlarge enzymes. With in vitro assays, many dif-ferent conditions can be tested simultaneouslyand the metabolic profile can be determinedby LC-MS[Xu et al, 2010].As seen in figure 6 the peaks are not very dis-tinctive and not well alligned. Another runshould be performed with more diluted sam-ples and better calibration of the Agilent Tech-nologies Mass Spectrometer. This would makeanalysis of the LC-MS data better, as new com-pounds, with a mass not found in the metabolicprofile of NID3, would be easier to identify.

Localization of PKS-NRPS in A. nidulans.To determine the localization of the PKS-NRPSs expressed in A. nidulans all constructs,except for CS2, were tagged with RFP in theC-terminus of the protein. The reason thatCS2 was not tagged is that CS1 and CS2 arealmost identical (linker is from ccsA(CS1) andsclA(CS2)) and were therefore expected to lo-calize in the same manner. Data from LC-MS

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showed that RFP tagging of the C-terminus ofccsA and CSC PKS-NRPSs did not disrupt thefunctionality of the enzyme, as the precursorwas still being produced in the RFP taggedconstructs(see figure 6). This observation indi-cates that the C-terminus of PKS-NRPS is notpositioned at the inside of the tertiary struc-ture of the enzyme. By looking at the imagespresented in figure 9E and F, it seems that thefunctional chimeras CSC(E) and ccsA(F) locatesin the same manner. This indicates that correctfolding or at least a functional fold of the pro-tein, will position the enzyme in compartmentsin the fungi hyphae. The image 10G representssimilar results as this chimera is from anotherstudy carried out by Maria Lund Nielsen. Thisconstruct is a chimera of the PKS from the A.nidulans PKS-NRPS hybrid involved in the syn-thesis of aspyridone, apdA, and the NRPS fromccsA. The image 10H visualizes the localiza-tion of the trans-acting enoyl reductase, ccsC,which was expected to locate together withccsA, but as the image shows ccsC is expressedin the cytosol and not in specific compartments.However, the functionality of the RFP-taggedccsC has not yet been assessed. To visualize theexact localization of ccsA and CSC additionalexperiments would have to be performed, in-cluding fluorecense tagging of the different or-ganelles in A. nidulans or fluorescense taggingof proteins with known localizations. Anotherexperiment that would have to be performedis to tag both ccsA and ccsC with two distinctfluorescent proteins, and run a chemical anal-ysis to verify that the construct is functionaland produces the desired product. This wouldvalidate or invalidate if ccsA and ccsC acts inclose proximity. The chimeras that were notcapable of producing any new compounds orthe cytochalasin E precursor(CS1 and CT) arerepresented in image 9B and C. The fluorescentprotein was still expressed in these chimeras,but located in the cytosol. One possible reasoncould be misfolding of the polypeptide chain.If the RFP tagged protein was not expressed atall the images would be similar to image 9A,as this is the background strain, NID3.

A

B

C

D

E

F

Figure 9: Brightfield and fluorescence images of: A:NID3, B: CS1-RFP, C: CT-RFP, D: sclA-RFP,E: CSC-RFP, F: ccsA-RFP.

A remarkable observation is that all con-structs with the NRPS module from ccsA(NID1777, NID1779 and the chimera consistingof the PKS from apdA and the NRPS from ccsA)all localize in specific compartments. This sug-gests that either the NRPS module determines

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the localization or something in the C-terminusaffects the localization. Another explanation isthat the tagging of the protein with RFP affectsthe localization, but does not disrupt or alterthe enzyme activity. Image 9D represents thePKS-NRPS of A. sclerotioniger expressed in A.nidulans NID1. As the chemical analysis of themetabolic profile of this hybrid needs furtheranalysis, it is not possible to draw any conclu-sions. The protein is expressed and seems to lo-calize in the same manner as the non-functionalchimeras. This could indicate that the sclA hy-brid is either not functional, due to incorrectfolding, or that the enzyme localizes differentlycompared to ccsA.

G H

Figure 10: Fluorescence images of: G: A chimera con-struct, consisting of the PKS from apdA andthe NRPS from ccsA, tagged with RFP, H:ccsC-mCitrine.

Concluding remarks Through this study ithas been verified that it is possible to swap thelinker connecting the PKS and NRPS from ccsAwith the linker from the related PKS-NTPS hy-brid sclA, without altering the function of theenzyme. Additionally the study shows thattagging of the C-terminus of the protein withthe fluorescent protein, RFP, does not alter ordisrupt the function of the PKS-NRPS.

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