university of groningen genomic wake-up call samol, martathe pc21g14570 gene of penicillium...

University of Groningen

Genomic Wake-Up CallSamol, Marta

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Publication date:2015

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Citation for published version (APA):Samol, M. (2015). Genomic Wake-Up Call: Activating Silent Biosynthetic Pathways for Novel Metabolites inPenicillium chrysogenum. University of Groningen.

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CHAPTER 4

Polyketide Synthesized PigmentRegulated by Histone Deacetylase in

Penicillium chrysogenum

Marta M. Samol1,3,†, Oleksandr Salo1,3,†,Marco I. Ries4,5, Jeroen Kuipers6, Roel A.L. Bovenberg2,7,

Rob J. Vreeken4,5, and Arnold J.M. Driessen1,3

† These authors contributed equally to this work

1Molecular Microbiology, 2Synthetic Biology and Cell Engineering, Groningen BiomolecularSciences and Biotechnology Institute, Zernike Institute for Advanced Materials,University of Groningen, Groningen, The Netherlands3Kluyver Centre for Genomics of Industrial Fermentations, Delft, The Netherlands2Division of Analytical Biosciences, Leiden Academic Centre for Drug Research,Leiden, The Netherlands5Netherlands Metabolomics Centre, Leiden University, Leiden, The Netherlands6Department of Cell biology, University Medical Center Groningen, Groningen,The Netherlands7DSM Biotechnology Center, Delft, The Netherlands

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84 4. Polyketide Synthesized Pigment Regulated by Histone Deacetylase...

Abstract

The Pc21g14570 gene of Penicillium chrysogenum encodes an ortholog of aclass 2 histone deacetylase termed HdaA which may play a role in epigenetic reg-ulation of secondary metabolism. Deletion of the hdaA gene induces a significantpleiotropic effect on the expression of a set of polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) encoding genes. The hdaA deletion mutantexhibits a decreased conidial pigmentation that links to a reduced expression ofthe PKS gene Pc21g16000 (pks17) responsible for the production of the conidialpigment precursor naphtha-γ-pyrone. Moreover, the hdaA deletion caused de-creased levels of the yellow pigment chrysogine that could be associated with thedown regulation of the NRPS encoding gene Pc21g12630 and neighboring genesthat are part of a cluster. In addition, transcriptional activation of an unknownPKS gene cluster containing Pc21g05070 (pks12) and Pc21g05080 (pks13) oc-curred. However, these genes already acquired single nucleotide substitutions atearly stages of the classical strain improvement of P. chrysogenum for β-lactamproduction that likely inactivated their activities and thus no specific metabolitescould be detected. Our present results suggest that an epigenomic approach canbe successfully applied for the activation of secondary metabolism in P. chryso-genum.

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4.1. Introduction 85

4.1 Introduction

During the last decades, the filamentous fungus Penicillium chrysogenum has beenused extensively in industry for the production of the β-lactam antibiotic penicillinFleming [1929]. The biosynthetic pathway and the corresponded genes involvedhave been well described and current production strains are geared for the highlevel production of penicillins through the implementation of an intense classicalstrain improvement program. However, the full potential of secondary metabolismof P. chrysogenum remained unknown till the genomic sequence became available(Berg et al. [2008]). The genome specifies multiple genes for secondary metaboliteformation including 20 polyketide synthases (PKSs), 10 nonribosomal polypeptidesynthases (NRPSs), 2 hybrids (PKS-NRPS) and 1 dimethylallyltryptophan synthase.The function of the most of these genes remains unknown (Berg et al. [2008]). Re-cently, a genome based identification and analysis of the roquefortine/meleagrinRoqA gene cluster was performed for P. chrysogenum. (García-Estrada et al. [2011];Veiga et al. [2012]; Ali et al. [2013]; Shang et al. [2013]). However, unlike the roque-fortine gene cluster, the expression level of the majority of the secondary metabolitegenes under laboratory conditions is low. Therefore, more elaborate methods otherthan gene inactivation are required for identification and further analysis of theseso-called ’silent’ secondary metabolite genes.

New approaches have evolved during the post-genomic era to activate gene clus-ters such as interference with cluster specific regulatory genes or even of pleiotropicregulator of chromatin structure like LeaA. This has triggered the research on thecryptic potential of fungal secondary metabolism (Brakhage and Schroeckh [2011]).A potential powerful approach is the epigenetic regulation of gene expression. Ineukaryotic cells, DNA is compacted into a complex chromatin structure. The his-tone proteins H2A, H2B, H3, and H4 form the core histone octamer complex withDNA called nucleosome, the structural and functional unit of the chromatin (Luger[2003]). The formation of the nucleosomes may interfere with the recognition of thebound DNA by various transcriptional elements causing gene silencing Lee et al.[1993]. Thus remodeling of the chromatin by the histone modifications is a trig-ger that influences transcription, replication, and DNA repair (Yu et al. [2011]; Zhuet al. [2011]). The histone acetylation status is controlled by the balanced activity ofhistone acetylases (HATs) and deacetylases (HDACs) (Brosch et al. [2008]). Hyper-acetylation of the chromatin induced by deletion or chemical inhibition of HDACsleads to euchromatin formation and transcriptional activation of silent chromosomalregions (Gacek and Strauss [2012]). Cladochromes and calphostin B in Cladosporiumcladosporioides and nygerone A from Aspergillus niger are secondary metabolites thathave recently been identified with this strategy using the HDAC inhibitor suberoy-lanilidehydoxamic acid (SAHA) (Fisch et al. [2009]; Hendrickson et al. [1999]; Carafaet al. [2012]). An altered secondary metabolite profile was also reported for Alternaria

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alternata and Penicillium expansum treated with HDAC inhibitor Trichostatin A (TSA)(Shwab et al. [2007]).

Histone deacetylases are represented by two protein families: the "classical"HDACs and the recently established group of NAD+ dependent sirtuins (Ruijteret al. [2003]). Members of both families were initially described in S. cerevisiae andsubsequently identified in filamentous fungi and human (Taunton et al. [1996]). Theorthologs of the RPD3 (reduced potassium dependency) transcription factor andHDA1 of S. cerevisiae belong to the major classes 1 and 2 of the "classical" HDACs,respectively. Recently, multiple effects of the inactivation of hda1 orthologs on theexpression of secondary metabolite genes has been reported for a number of fungalspecies (Tribus et al. [2005]; Shwab et al. [2007]; Lee et al. [2009]). In this study the or-tholog of the class 2 histone deacetylase hda1 of S. cerevisiae in the filamentous fungusP. chrysogenum (Pc21g14570) was deleted and its function in secondary metabolismwas analyzed.

4.2 Materials and Methods

4.2.1 Media and Culture Conditions.

Liquid YGG medium (400 ml KCl-glucose, 100 ml 5×buffered Yeast Nitrogen Base(YNB), 10 ml fresh 10% yeast extract was used for preculturing the conidia for 24hours before inoculation into minimal metabolite medium (MMM). Solid R-agarmedium (6 ml/L glycerol, 7.5 ml/L beet molasses, 5 g/L yeast extract, 18 g/L NaCl,50 mg/L MgSO4·7H2O, 60 mg/L KH2PO4, 250 mg/L CaSO4, 1.6 ml/L NH4Fe(SO4)2(1 mg/ml), Fe(SO4)2·12H2O, 10 mg/L CuSO4·5H2O, and 20 g/L agar was used forculturing the conidia and for secondary metabolites production. All cultivationswere performed at 25°C in semi dark conditions. Liquid culturing of the conidiawas performed in 25ml of YGG or MMM media in 100ml flasks shaken at 220 rpm.

4.2.2 Plasmids Construction.

All the plasmids in this study were constructed using the modified Gateway™ clo-ning protocol (Invitrogen) published previously (Kovalchuk et al. [2012]). 5-’ and3-fragments for the deletion cassette were amplified with Phire Hot Start II PCRMaster Mix™ (Finenzymes) using specific primers and cloned into correspondedGateway donor vectors pDONR P4-P1R and pDONR P2R-P3, respectively, using BPclonase II™ enzyme mix (Invitrogen). The resulted plasmids were purified fromkanamycin resistant E. coli Dh5α transformants and subsequently recombined withthe Gateway destination vector pDEST R4-R3 and pDONR221-AMDS for in vitrorecombination using LR clonase II™ enzyme mix. For cloning of the expressionplasmids the modified pDONR221-AMDS plasmid was used. In this construct the

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4.2. Materials and Methods 87

pcbC (isopenicillin synthase) promoter region was ligated downstream of the amdSgene. After incubation, the reaction mixture was transformed to E. coli Dh5α and thefinal plasmids have been isolated from the ampicillin resistant transformants.

4.2.3 LC/MS Sample Preparation.

The extraction of secondary metabolites from solid R-agar medium for HPLC andMS analysis was done by the modified micro-scale extraction procedure for stan-dardized screening of fungal metabolite production in cultures (Smedsgaard [1997]).A plug of the agar medium (5 mm in diameter) was taken for extraction from themiddle of the colony obtained after 10 days of growth. The extraction mixture (0.5ml) contained methanol-dichloromethane-ethyl acetate in a ratio of 1:2:3 (v/v). Theplugs were extracted ultrasonically in 1ml glass tubes during 60 min. The liquid ex-tract was transferred to a fresh tube and dried under vacuum using a SpeedVac™vacuum concentrator (Appendorf) for 30 min. The dry pellet was re-dissolved in a1:1 solution of methanol in water, filtered via 0.2 µm PTFE syringe filter and used forHPLC and MS analysis.

4.2.4 Genomic DNA Extraction.

The total genomic DNA (gDNA) was isolated after 96 h of cultivation in YGG liq-uid medium using an adapted yeast genomic DNA isolation protocol (Harju et al.[2004]). The mycelium was broken in a FastPrep FP120™ system (Qbiogene).

4.2.5 Total RNA Extraction and cDNA Synthesis.

Total RNA was isolated after 7 days of colonies growth on solid R-agar medium. TheTRIzol® (Invitrogen) extraction method was used, with additional DNAse treatmentusing the Turbo DNA-free™ kit (Ambion). Total RNA concentration was measuredwith a NanoDrop ND-1000™. For the synthesis of cDNA by iScript™ cDNA synthe-sis kit, 500 ng of RNA per reaction was used (Bio-Rad).

4.2.6 qPCR Analysis.

The primers used for expression analysis of the 20 PKSs, 11NRPSs, the gene cluster ofpks12/pks13 [Pc21g05030 (g30), Pc21g05050 (reg50), Pc21g05060 (mox60), Pc21g05070(pks12), Pc21g05080 (pks13), Pc21g05090 (reg90), Pc21g05100 (mfs100) and Pc21g05110(ox110)], the genes of putative DHN-melanin cluster [Pc21g16380 (abr1), Pc21g16420( arp1 ), Pc21g16430 (arp2), Pc21g16440 (ayg1), Pc22g08420 (abr2)], and the puta-tive chrysogine biosynthetic gene cluster [Pc21g12570, Pc21g12580, Pc21g12590,Pc21g12600, Pc21g12610, Pc21g12620, Pc21g12630, Pc21g12640], are shown in the

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Table 4.1. Primers were designed at both sides of the introns in order to be able todiscriminate between the amplification on gDNA and cDNA. For expression analy-sis, the γ-actin gene was used as a control for normalization. A negative reverse tran-scriptase (RT) control was used to determine the gDNA contamination in the isolatedtotal RNA. The expression levels were analyzed, in duplicate, with a MiniOpticon™system (Bio-Rad) using the Bio-Rad CFX™ manager software, with which the thresh-old cycle (CT) values were determined automatically by regression. The SensiMix™SYBR Hi-ROX kit (Bioline) was used as a master mix for qPCR. The following ther-mocycler conditions were applied: 95°C for 10 min, followed by 40 cycles of 95°C for15 s, 55°C for 30 s, and 72°C for 30 =s. Subsequently, a melting curve was generatedto determine the specificity of the qPCRs.

4.2.7 Southern Blot Analysis.

The 3’ downstream region of the hdaA gene was used as a probe and amplified byPCR with primer set listed in Table 4.1 . The probe was labeled with digoxigeninusing the HighPrime™ Kit (Roche, The Netherlands). gDNA (10 µg) was digestedwith appropriate restriction enzyme and separated on 0.8% agarose gel. After equi-libration in 20x SSC buffer (3M sodium chloride; 0.3M sodium citrate) the DNA wastransferred overnight onto Zeta-probe™ positively charged nylon membrane (Bio-Rad). Blots were treated with anti-DIG-alkaline phosphatase antibodies (Sigma) andsupplemented with CDP-star™ (Roche Applied Science). The fluorescence signalwas measured with a Lumi Imager™ (Roche Applied Science).

4.2.8 Secondary Metabolite Analysis.

Secondary metabolites were analyzed with a Shimadzu HPLC system coupled withphotodiode array detector (PDA). A concentration gradient of acetonitrile in water(from 5% to 37%) started after 7 min for a period of 20 min, followed by 10 min iso-cratic flow at 37% of acetonitrile in water. Finally, the column was equilibration for5 min at the initial 5% of acetonitrile in water. Solvents were buffered with KH2PO4

at 640 mg/l and H3PO4 at 340 mg/l to maintain stable pH 3 in the system. The Shim-pack XR-ODS™ C18 column (3.0 x 75 mm, 2.2 µm, Shimadzu, Japan) with flow rate0.5 ml/min at 40°C was used for analysis.

For identification of the metabolites and their quantitative analysis the HPLC-MS analysis has been performed using an Accella1250™ HPLC system coupled withthe benchtop ES-MS OrbitrapExactive™ (Thermo Fisher Scientific, San Jose, CA). Asample of 5 µL was injected into the column described above operating at 40°C andflow rate 0.3 ml/min. Linear gradient began with 90% of solvent A (100% water)and 5% of solvent C (100% Acetonitrile) starting after 5 minutes of isocratic flow.The first linear gradient reached 60% of C at 30 minutes, the second - 95% of C at

4444

4.2. Materials and Methods 89

35 minutes. The washing step for 10 minutes at 90% of solvent C was followed bythe column equilibration for 15 minutes at initial isocratic conditions. Solvent D (2%Formic acid) was continuously used at a concentration of 5% to maintain the final0.1% of formic acid in the system. The column fluent was directed to the Exactive™ES-MS Orbitrap operating at switching positive / negative modes. Voltage param-eters for positive mode: 4.2kV spray, 87.5V capillary and 120V of tube lens. Voltageparameters for negative mode: 3kV spray,-50V capillary, -150 tube lens. Capillarytemperature 325°C, sheath gas flow 60, auxiliary gas was off to maintain higher de-tection sensitivity for both positive and negative modes during the analysis. ThermoExcalibur™ processing software has been used for quantification of the secondarymetabolites. The expected masses and the retention time of chrysogine (191.08 [H]+,12.49 min.) were determined and used to set up the processing method for auto-mated peak integration and quantification. The ICIS algorithm for component peakdetection has been applied in this analysis.

4.2.9 Scanning Electron Microscopy.

Conidia were immobilized on glass cover slips and fixed with 2% glutaraldehydefor 1 hour followed by washing with cacodylate buffer (pH 7.4). Samples were incu-bated with 1% OsO4 in 0.1 M cacodylate buffer during 1 h and washed with MQwa-ter (Millipore). The immobilized spores were dehydrated with a concentration gra-dient of 30, 50 and 70% of ethanol within 30 min followed by 3 steps of final dehy-dration with 96% ethanol within 45 min. Next, the samples were incubated in 100%ethanol/tetramethylsilane (TMS) 1:1(v/v) for 10 min followed by 15 min incubationwith pure TMS and air dried. Dried samples were coated with 2 nm Pd/Au usingLeica EM SCD050 sputter coater and analyzed with SUPRA® 55 FE-SEM (Zeiss) at2 kV.

4.2.10 Stress Assay.

Fungal conidia of seven day grown mycelium were re-suspended in 1ml water con-tained 0,05% of Tween-20 to prevent aggregation. The equal amount of the spores(3x103 spores per ml) in solution were adjusted by series of dilutions and measuredwith Bürker-Türk counting chamber using Olympus CX20™ light microscope. Aconidial suspension (100 µl) was used for inoculation to obtain approximately 300germination events per control plate in the assay. R-agar sporulation medium withincreasing concentrations of hydrogen peroxide from 0.5 to 3.5 mM was used in thisassay. To prepare each plate the corresponding amounts of hydrogen peroxide havebeen mixed with 25 ml of R-agar medium before solidification to provide equal dis-tribution of the supplement in the plate.

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4.3 Results

4.3.1 Deletion of the hdaA Gene.

The gene, Pc21g14570 of P. chrysogenum is an ortholog to the hda1 histone deacety-lase gene of Saccharomyces cerevisiae. This gene, now termed hdaA, was deleted fromthe chromosome in order to investigate its effect on development and secondarymetabolite production. The complete hdaA gene was replaced by the acetoamidase(amdS) selection marker gene. A standard protocol was used for cloning of the cor-responded pHdaA deletion plasmid (Kovalchuk et al. [2012]) containing the 3’ and5’ flanking regions of the hdaA open reading frame (Figure S4.1, Figure S4.2A). Pro-toplasts of the amdS marker-free strain DS68530 that lacks all copies of the penicillinbiosynthesis cluster were used for transformation to simplify the detection of otherpossible bioactive secondary metabolites. The acetamide supplemented medium(AMDS) was used for the positive selection of the transformants, and the correctinactivation of the hdaA gene was validated by Southern blot analysis showing theexpected sizes of the products (Figure S4.1).

4.3.2 Effect of the hadA Deletion on the Expression of SecondaryMetabolite Genes.

To examine the effect of inactivation of hdaA on the transcription of secondary metabo-lite genes, the expression of the 20 PKSs and 11 NRPSs was examined using Quanti-tative Real Time PCR analysis. RNA was isolated from the mycelium of the ∆hdaAand the parental strain DS68530 grown on plates for seven days. The solid mediumfraction was also used for secondary metabolites analysis (see below). The cDNAof the various secondary metabolite genes was amplified using the primers listedin Table 4.1 . Out of the 31 analyzed secondary metabolite genes, the expression ofseven genes was dramatically altered in ∆hdaA mutant. An up to 10-fold increasein expression occurred for PKSs Pc21g05070 (pks12) and Pc21g05080 (pks13), whilethe transcript level of three NRPS genes [Pc21g01710 (nrpsA), Pc21g10790 (nrpsC),Pc21g12630 (chyA)] and other two PKSs [Pc21g03990 (pks10), Pc21g16000 (pks17)]was significantly reduced (Figure 4.1). The 6-fold down regulated chyA gene encodesa dipeptide synthase that has tentatively been assigned to chrysogine biosynthesisin P. chrysogenum (Berg et al. [2008]). Also a 20-fold decrease of the expression ofthe nrpsA gene was noted. This gene encodes a NRPS cyclopeptide synthase withunknown function. The nrpsC gene, putatively involved in hexapeptide biosynthe-sis, was 4-fold down regulated (Berg et al. [2008]). The secondary metabolite genesare distributed over the six largest genome sequencing contigs. However, only thetranscript levels of the genes of contig 21 were affected by the hdaA deletion, whileno significant respond was shown for the genes of the other contigs (c12, c13, c14,

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4.3. Results 91

pks

1 (P

c12g05590)

pks

2 (P

c13g04470)

pss

C (P

c13g05250)

pks

3 (P

c13g08690)

nrp

sB (

Pc1

3g14330)

hp

nG

(P

c14g00080)

pks

4 (P

c16g00370)

pks

5 (P

c16g03800)

pss

A (P

c16g03850)

hcp

A (P

c16g04690)

pks

6 (P

c16g04890)

pks

7 (P

c16g11

480)

hp

nG

(P

c16g13930)

pks

8 (P

c21g00960)

nrp

sA (P

c21g01710)

pks

9 (P

c21g03930)

pks

10

(P

c21g03990)

pks

11 (P

c21g04840)

pks

12

(P

c21g05070)

pks

13

(P

c21g05080)

nrp

sC (

Pc2

1g10790)

pks

14

(P

c21g12440)

pks

15

(P

c21g12450)

chyA

(

Pc2

1g12630)

pks

16

(P

c21g15160)

roqA (

Pc2

1g15480)

pks

17

(Pc2

1g16000)

pks

18

(P

c22g08170)

pss

B (P

c22g20400)

pks

19

(P

c22g22850)

pks

20

(P

c22g23750)-30

-20

-10

0

10

20

c12 c13 c14 c16 c21 c22

3,9Mb

3,8Mb

3,7Mb

5,6Mb

0,5Mb

6,3Mb

Re

lativ

e g

en

e e

xp

ressio

n(f

old

ch

an

ge

)

Figure 4.1: Quantitative Real Time PCR analysis of the expression of all secondary metabo-lite genes in ∆hdaA mutant. Genes are grouped according to genome annotationnumber. PcX’s represent the contig were SM genes are distributed. The size ofthe genomic region covered by the contig is indicated. Samples were taken after 7days of growth on solid R-agar medium. Data are shown as a fold change (∆hdaA/ DS68530)

c16 and c22) (Figure 4.1).

4.3.3 Epigenetic Activation of a Gene Cluster Containing Two Poly-ketide Synthases.

The PKS genes pks12 and pks13 that are highly up regulated in the ∆hdaA strainare located next to each other in opposite orientation and belong to a putative clus-ter of nine genes (Berg et al. [2008]). The presence of multiple PKSs/NRPSs/FASswithin one gene cluster has been reported previously and indicates a possible mu-tual role of these genes in a single biosynthetic pathway (Hendrickson et al. [1999];Bergmann et al. [2010]; Masschelein et al. [2013]). The predicted product of pks12 is

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Gene name Annotation name Protein function, BLAST

Identity , %

T.reesei G.graminicola M.purpureus

reg50 Pc21g05050 Fungal specific transcriptional factor 41 - 34

mox60 Pc21g05060 Monooxygenase FAD binding protein 57 - *a

pks12 Pc21g05070 Non-reduced polyketide synthase 64 61 43

pks13 Pc21g05080 Highly reduced polyketide synthase 65 63 -

reg90 Pc21g05090 Fungal specific transcriptional factor 50 - -

mfs100 Pc21g05100 MFS transporter, putative 73 - 39

ox110 Pc21g05110 Glucooligosaccharideoxidase, putative - - *b

Figure 4.2: Schematic representation of the activated gene cluster in P. chrysogenum∆hdaA and its highly homologous representatives among other fungal species.pks12/13, polyketide synthases; reg50/90, transcriptional factors; mox60, monooxy-genase; mfs100, transporter; ox110, oxidoreductase; gXs, hypothetical proteins. Thelinks between the functionally related genes are shown. The data were obtainedusing the AntiSMASH tool (for genome-wide identification and annotation of thesecondary metabolite biosynthesis gene clusters in bacterial and fungal genomes)and by protein BLAST analysis. aproteins with 2-oxoglutarate/Fe(II)-dependentdioxygenase activity, bsuperfamilly of serine hydrolases (FSH1).

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4.3. Results 93

g10

g20

g30

g40

reg5

0

mox

60

pks1

2

pks1

3

reg9

0

mfs10

0

ox11

0g1

20g1

300

5

10

15

20

25

Rela

tive g

ene e

xpre

ssi

on

(fold

change)

Figure 4.3: Quantitative Real Time PCR analysis of the activated PKS cluster in ∆hdaA mu-tant. The expression of the predicted gene cluster (gray bar) and, the expressionof the genes outside the cluster (white bar) are shown. Samples were taken after 7days of growth on solid R-agar medium. Data is calculated as fold change (∆hdaA/ DS68530).

a 2581 amino acid long non-reducing iterative type I polyketide synthase showingthe highest (64%) identity to an unknown PKS gene of Trichoderma reesei and 43%identity to citrinin biosynthesis gene of Monascus purpureus (Berg et al. [2008]). Theneighboring pks13 gene encodes a 2664 amino acid long highly reducing polyke-tide synthase with 65% identity to the unknown PKS of T. reesei and strong simi-larity (48% identity) to lovastatin diketide synthase LovF of A. terreus (Berg et al.[2008]) (Figure 4.2). Protein BLAST analysis indicated that the cluster consists ofthe two PKS genes (pks12, pks13), two transcription factor genes (Pc21g05050 andPc21g05090, which are termed reg50 and reg90, respectively), a monooxygenasegene(Pc21g05060, mox60), one gene for transport (Pc21g05100, mfs100), and an oxidore-ductase encoding gene (Pc21g05110, ox110) (Figure 4.2).

qPCR analysis shows the transcriptional activation of all above mentioned genesin the ∆hadA mutant (Figure 4.3). The two genes that precede the cluster, i.e.,Pc21g05030 (g30) and Pc21g05040 (g40) encode hypothetical proteins, and showedno expression changes and therefore may not be part of the gene cluster. To verify ifthe activated gene cluster has orthologs among other species, the antiSMASH appli-cation for secondary metabolite gene clusters identification and analysis was used(Medema et al. [2011]). As a result, the corresponding gene assemblies were foundin the genomes of Trichoderma reesei and Glomerella graminicola. In Trichoderma reeseiall homologs of pks12, pks13, reg50, reg90, mfs100, mox60 and ox110 share one locus.

4444


However, in the genome of Glomerella graminicola only the corresponding homologsof pks12 and pks13 were identified (Figure 4.2).

The regulatory protein Reg50 exhibits homology (34% identity) with the positiveregulator CtnR of citrinin biosynthesis in Monascus purpureus (Hajjaj et al. [1999];Shimizu et al. [2005]; Sakai et al. [2008]). The non-reducing Pks12 and the trans-porter Mfs100 are homologous to PksCT and CtnC of M. purpureus with 43 and 39%identity, respectively. The monooxygenase gene mox60 and oxidoreductase encod-ing gene ox110 of P. chrysogenum encode different families of oxidoreductases andno homologous proteins were found in M. purpureus (Figure 4.2). This data suggestthat the activated gene cluster may be responsible for the biosynthesis of citrinin orsimilar type of polyketide product.

4.3.4 HdaA Regulates the Transcription of the Chrysogine Biosyn-thetic Gene Cluster.

Pc21g12630 encodes an ChyA that likely is involved in chrysogine production andits expression is down regulated in the hdaA gene deletion strain. The gene is partof a cluster of eight genes that in addition specifies a glutamine amidotransferase(Pc21g12570), two hypothetical proteins (Pc21g12580 and Pc21g12610), two dehy-drogenases (Pc21g12590 and Pc21g12600), an amidotransferase (Pc21g12620), and aputative regulator (Pc21g12640). The corresponding gene annotations were obtainedby BLAST analysis. The size of the cluster has been predicted based on availabletranscriptome data suggesting the high expression of these particular genes in thegenome of P. chrysogenum (Berg et al. [2008]). The expression of the cluster was an-alyzed by qPCR analysis which revealed up to 4-fold down-regulation of the entiregene cluster in the hdaA mutant (Figure 4.4A, B, Figure S4.3).

To investigate the effect of the hdaA gene deletion on secondary metabolite (orchrysogine) production, the reference strain DS68530 and ∆hdaA strain were grownfor seven days on solid R-agar medium. Then, colony plugs were isolated and sub-jected to extraction (Smedsgaard [1997]). The obtained samples were filtered and an-alyzed by HPLC and MS. An 40% decrease of chrysogine production in the mediumwas observed for the ∆hdaA mutant in comparison to the reference DS68530 whichis in agreement with the qPCR data (Figure 4.4C, S4.3). An new metabolite was de-tected by HPLC in the medium with a retention time of 22 minutes (Figure 4.4C).The structure of this compound is not yet known. No other changes were observedunder the experimental conditions applied.

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4.3. Results 95

Pc21g

1256

0

Pc21g

1257

0

Pc21g

1258

0

Pc21g

1259

0

Pc21g

1261

0

Pc21g

1262

0

Pc21g

1263

0 (c

hyA)

Pc21g

1264

0

Pc21g

1265

0

Pc21g

1266

0

No

rmaly

zed

exp

ressio

n , f

old

ch

an

ge

-20

-15

-10

-5

0

5

RT, (min)

10 15 20 25

Ab

so

rban

ce a

t 350 n

m, (m

AU

)

0.0

5.0e+4

1.0e+5

1.5e+5

2.0e+5

2.5e+5

1 2 3

4

56

7 8

Pc21g

1256

0

Pc21g

1257

0

Pc21g

1258

0

Pc21g

1259

0

Pc21g

1261

0

Pc21g

1262

0

Pc21g

1263

0 (c

hyA)

Pc21g

1264

0

A

B

C

Figure 4.4: (A) Quantitative Real Time PCR analysis of the gene cluster of chrysogine biosyn-thesis in the ∆hdaA mutant. Samples were taken after 7 days of growth on solidR-agar medium. The expression data is fold change (∆hdaA / DS68530). (B)Proposed gene cluster of chrysogine biosynthesis. Pc21g12580: glutamine ami-dotransferase; Pc21g12580: hypothetical protein, two dehydrogenases Pc21g12590and Pc21g12600; Pc21g12610: hypothetical protein; Pc21g12620: amidotransferase;Pc21g12630: a NRPS and Pc21g12640, a putative regulator. (C) HPLC profile com-parison of DS68530 (black) and ∆hdaA (red) growth media. Metabolites were ex-tracted from the solid R-agar medium after 7 days of growth. Absorption at 350 nmis shown. The known metabolites are indicated as 1, Chrysogine; 2, GlandicolineA; 3, Glandicoline B; 4, Meleagrin; 5, Roquefortine D; 6, the unknown metaboliteoverproduced by hdaA mutant; 7, Roquefortine F.

4.3.5 HdaA Regulates the DHN-melanin Gene Cluster Involved inPigment Formation.

The ∆hdaA mutant strain grown for 10 days on solid R-agar medium showed a majordecrease of the green conidial pigmentation as compared to the DS68530 referencestrain (Figure 4.6A). Such pigmentation in filamentous fungi is often attributed to thedihydroxynaphtalene (DHN)-melanin biosynthesis cluster that typically consists ofsix genes including a polyketide synthase (Tsai et al. [1997]; Tsai et al. [1998]). TheDHN-melanin biosynthetic pathway was described initially for Verticillium dahliaeand Wangiella dermatitidis. (Bell et al. [1976]; Geis et al. [1984]), The pentaketide originof fungal melanins has been discovered and found to be common in other melanizedfungi (Langfelder et al. [2003]; Wheeler et al. [2008]). In A. fumigatus, the polyke-

4444


tide product of the PKS Alb1p, the heptaketide naphthapyrone YWA1, requires theenzymatic post PKS conversion to the pentaketide1,3,6,8-tetrahydroxynaphthalene(T4HN) via hydrolytic polyketide shortening activity of Ayg1p (Fujii et al. [2004]).This enzymatic step is absent in C. lagenarium where the pentaketide T4HN is a di-rect product of PKS1 (Fujii et al. [1999]). Next, is reduced to scytalone via the T4HNreductase Arp2p, followed by dehydration to 1,3,8-trihydroxynaphthalene (T3HN)by the scytalondehydratase Arp1p. The following reduction to vermelon is Arp2dependent but the presence of other specific reductase(s) caring this reaction hasbeen also proposed for Aspergilli and other fungi (Tsai et al. [1999]; Wang and Breuil[2002]). The dehydration of vermelon to 1,8-dihydroxynaphthalene (DHN) is Arp1pdependent. The resulting DHN molecules are further polymerized to the struc-turally diverse melanins. This final enzymatic step involves the multicopper oxidaseAbr1p and laccase Abr2p (Jacobson [2000]; Hamilton and Gomez [2002]; Langfelderet al. [2003]). Screening of the genome sequence indicated the presence of the cor-responded ortholog genes of the DHN-melanin biosynthetic pathway in P. chryso-genum: abr1 (Pc21g16380), arp1 (Pc21g16420), arp2 (Pc21g16430), ayg1 (21g16440),abr2 (22g08420) and associated pks17 (Pc21g16000) were found partially clustered inthe genome. qPCR analysis of the putative DHN-melanin gene cluster indicated the4-fold down-regulation of pks17 in the ∆hdaA mutant while arp1 , arp2 and ayg1were 4 fold up regulated. The expression of abr1 and abr2 involved in the last stepsof the conidial pigment biosynthesis was not significantly changed (Figure 4.5A, B).

To determine if pks17 is involved in conidial pigment biosynthesis, the pks17 genewas deleted and overexpressed in order to identify the related polyketide product. Agene inactivation strain was obtained as described earlier (see materials and methodssection) using primers listed in Table 4.1 (Figure S4.2B). The resulted ∆pks17 mutantdisplayed an albino phenotype of the conidia while grown on sporulating R-agarmedium (Figure 4.6A). For the overexpression, pks17 was placed under control ofthe isopenicillin N synthetase (pcbC) gene promoter and integrated into the genome(Figure S4.2C). As a result, a 10-fold increase of the transcript level was obtainedas compared to the reference strain. The solid medium grown mutant featured adeficient coloring of the conidia and intense pigmentation of the bottom surface ofthe colony (Figure 4.6A). To identify the accumulated product, the extracted R-agarmedium of 7 days grown culture was analyzed by LC/MS. A unique monoisotopicmass of 276.06 with predicted elemental composition C14H12O6 was determined.The found mass, calculated empirical formula and characteristic fragmentation pat-tern belongs to the known heptaketide YWA1 of A. nidulans produced by highly ho-mologous PKS wA that is involved in the conidial DHN-melanin biosynthetic path-way (Figure S4.4A, B).

4444

4.3. Results 97

pks1

7 (P

c21g

1600

0)

abr1

(Pc2

1g16

380)

arp1

(Pc2

1g16

420)

arp2

(Pc2

1g16

230)

ayg1

(Pc2

1g16

440)

abr2

(Pc2

2g08

420)

-6

-4

-2

0

2

4

6

A

B

Rela

tive g

ene e

xpre

ssio

n(f

old

change)

Figure 4.5: (A) Quantitative Real Time PCR analysis of the gene cluster of DHN-melaninbiosynthesis in ∆hdaA mutant. Samples were taken after 7 days of growth onsolid R-agar medium. The expression data is fold change (∆hdaA / DS68530). (B)Schematic representation of DHN-melanin gene cluster.

4.3.6 Role of pks17 in Conidia Formation and Resistance.

Melanins are important components of the conidial cell wall and its integrity. Theyplay an essential role in physical properties of the spores like surface interaction, hy-drophobicity and virulence in pathogenic fungal species. The effect of hdaA deletionon the conidial surface in P. chrysogenum was examined using scanning electron mi-croscopy. The conidia of the reference DS68530, ∆hdaA, ∆pks17 and over expressionmutant oepks17 were isolated from colonies grown for seven days on the sporulat-ing R-agar medium. The reference DS68530 exhibited a typical tuberous surface of

4444


Figure 4.6: (A) Pigmentation differences between DS68530, ∆hdaA, ∆pks17 and oepks17strains. Top (left) and bottom (right) of the plate. The picture has been takenafter14 days of growth grown on solid R-agar medium. (B) Scanning electron mi-croscopy of the DS68530, ∆hdaA, ∆pks17 (albino mutant) and oepks17 strains coni-dia. The effected cell wall surface of the conidia for ∆pks17and oepks17 strains isshown. The ∆hdaA mutant display more pronounced relief of the conidial surfaceornamentation in comparison to the reference strain DS68530. (C) Percent survivalof the conidia grown in presence of hydrogen peroxide in R-agar medium. Thegerminated colonies were counted after 5 days of growth.

the conidia, while the spores of albino mutant ∆pks17 were smooth. The texture ofthe ∆hdaA spores surface was more pronounced compared to the parental strain butwithout dramatic changes of the conidial cell wall appearance (Figure 4.6B). Thesedata suggest that pks17 is involved in pigment formation and influences the mor-phology of the conidia in P. chrysogenum. The pks17 protein was renamed Alb1 ac-cording to the nomenclature of the protein of A. fumigatus.

Apart from the mechanical properties of the pigments (Howard et al. [1991])and their role as pH buffering systems, the scavenging of reactive oxygen species

4444

4.4. Discussion 99

is an important feature supporting UV and thermo-tolerance and pathogenicity ofthe conidia (Jacobson et al. [1995]; Kawamura et al. [1997]; Romero-Martinez et al.[2000]). The P. chrysogenum ∆hdaA mutant was grown on hydrogen peroxide sup-plemented medium for 5 days to verify the ability of the conidia to survive oxidativestress conditions in the absence of pigmentation. The survival rate was decreasedby 20% when the ∆hdaA strain was exposed to 2 mM of hydrogen peroxide in themedia. Under the same conditions, the survival rate of the ∆pks17 (∆alb1) was re-duced more than 50%. There was no enhanced survival observed for the oepks17overexpression mutant (Figure 4.6C).

4.4 Discussion

Recent genome sequencing and metabolite analysis studies revealed that the ma-jority of the potential secondary metabolite gene clusters present in the genomes offilamentous fungi are silent or expressed at a low level under standard laboratoryconditions. These non-expressed secondary metabolite gene clusters represent a po-tential untapped source of novel bioactive molecules. Activation of the secondarymetabolites production via deletion or chemical inhibition of histone deacetylaseswas recently reported for filamentous fungi as an effective tool for silent SM geneclusters activation and identification of new metabolites with potential pharmaceu-tical properties (Tribus et al. [2005]; Shwab et al. [2007]; Henrikson et al. [2009]; Leeet al. [2009]) Here, we have examined the effect of chromatin modification on expres-sion of the secondary metabolism associated genes in the fungus P. chrysogenum. Inthis work, the P. chrysogenum hdaA gene encoding an ortholog of the class 2 histonedeacetylase Hda1 of S. cerevisiae was deleted. The deletion mutant showed decreasedgreen conidial pigmentation and an altered surface structure of the spores. qPCRanalysis of this mutant showed significant changes of secondary metabolite gene ex-pression including the transcriptional activation of a PKS gene cluster of unknownfunction.

Our results are in agreement with studies in A. nidulans and A. fumigatus, whichdemonstrate that the homologous hdaA is the main contributor of histone deacety-lase activity in these fungi. In A. nidulans, deletion of the hdaA gene stimulatedpenicillin and sterigmatocystin production but not a telomere-distal gene cluster in-volved in terraquinone A biosynthesis. It was suggested, that HdaA silences theexpression of subtelomeric chromosomal regions (Tribus et al. [2005]). In contrast,the HdaA homolog in A. fumigatus was reported as the activator of gliotoxin biosyn-thesis and the repressor of several NRPSs including one gene of the siderophorebiosynthesis gene cluster. A subtelomeric specificity of HdaA was not apparent forthis fungus (Lee et al. [2009]). The analysis of the P. chrysogenum genome sequencerevealed six large contigs on which all secondary metabolite genes (clusters) are dis-

4444


tributed. We performed the expression analysis of 11 NRPS and 20 PKS genes in the∆hdaA strain. The results show that the expression of seven secondary metabolitegenes was significantly altered in the ∆hdaA including the activation of a silent PKScluster with unknown function. It is important to stress that the particular effectseems to be restricted to only one contig (c21). This region contains a remarkablylarge number of the secondary metabolite genes / clusters. Out of 32 PKS and NRPSencoding gens, fourteen are located within the 5.6 Mb region covered by the contig(c21). The rest of the secondary metabolite genes are distributed in a small numbersthough the remaining contigs (c12, c13, c14, c16 and c22). The action of HdaA seemsthus restricted to the transcriptional co-regulation of a particular genomic area richin secondary metabolite gene clusters (Figure 4.1) (Berg et al. [2008]).

The transcript level of three NRPS genes (Pc21g01710, Pc21g10790, Pc21g12630)and two PKSs (Pc21g03990, Pc21g16000) was significantly reduced in the ∆hdaA mu-tant. By gene inactivation, we have recently established that Pc21g12630 is respon-sible for the biosynthesis of chrysogine and related intermediates (H. Ali & M. Ries,unpublished results). qPCR analysis indicated that the corresponding gene clusterof 5 genes was down regulated in the hdaA mutant followed by a 40% decrease ofchrysogine accumulation in the media.

The function of the 4-fold down-regulated pks17 (Pc21g16000) gene was eluci-dated via gene deletion and overexpression. This PKS enzyme shows high similarityto the A. nidulans wA and A. fumigatus PksP proteins involved in conidial pigmentbiosynthesis (Berg et al. [2008]). To identify the polyketide product the pks17 wasoverexpressed and these cells accumulated the yellow naphtho-γ-pyrone (a polyke-tide precursor of the conidial pigment) into the medium. This indicates that P. chryso-genum uses the DHN-melanin biosynthetic pathway like previously reported for As-pergilli (Jacobson [2000]; Hamilton and Gomez [2002]; Langfelder et al. [2003]). Thecorresponding ∆pks17 strain showed an albino phenotype confirming the primaryrole of this gene in the conidial pigmentation. In the closely related fungus A. fumiga-tus at least six genes are required for DHN-melanin biosynthesis, which were foundto be partially clustered in the genome of P. chrysogenum. qPCR analysis showedthe 4-fold up regulation of the arp1 (scytalondehydratase), arp2 (T4HN reductase)and ayg1 (enzyme of hydrolytic polyketide chain shortening activity) genes, whilethe transcript level of abr1 (multicopper oxidase) and abr2 (laccase) which productscatalyze the last steps of the polymerization of 1,8-dihydroxynaphthalene were notsignificantly changed. Scavenging of reactive oxygen species by fungal melaninsprovides an important defense mechanism during growth under oxidative stresscondition. We examined the effect of hdaA deletion on the ability of the conidiato survive high concentrations of hydrogen peroxide. The increased sensitivity ofthe HdaA mutant has been shown in comparison to the reference strain but an evenhigher toxic effect of hydrogen peroxide was observed for the ∆pks17 albino mutant(Figure 4.6C).This result suggests that the oxidative stress response involves HdaA

4444

4.4. Discussion 101

mediated transcriptional regulation of DHN-melanin gene cluster in P. chrysogenum.While the expression of most of the altered secondary metabolite genes was re-

duced in the ∆hdaA mutant the transcript level of two neighboring pks12 (Pc21g05070)and pks13 (Pc21g05080) genes was significantly (up to 10-fold) increased. Based ongenome annotation analysis, these PKS genes are located next to each other in op-posite orientation and belong to the putative gene cluster of nine genes (Berg et al.[2008]). qPCR analysis indicated that seven genes of the predicted cluster were acti-vated in ∆hdaA mutant, while there was no transcriptional response shown for thegenes located outside the predicted cluster. A similar gene cluster has been describedin a marine derived P. chrysogenum isolate and a role in the biosynthesis of Sorbicil-lacton A and B was suggested (Avramovic [2011]). The production of the yellowpigments like sorbicillinoids and bisorbicillinoids is typical for P. chrysogenum iso-lates but the genes of biosynthesis have been never assigned experimentally to thisfamily of the products. Based on the feeding experiments with 13C-labeled precur-sors the biosynthetic mechanism of sorbicillactones has been resolved. Incorporationof the core hexaketide (sorbicillin), alanine and methionine during sorbicillactonesbiosynthesis has been shown with the corresponding enzymatic conversions (Bring-mann et al. [2005]). It was proposed that two rounds of the reductive extension ofthe acetyl-CoA by HR PKS (which corresponds to pks13) deliver the triketide precur-sor that is further extended to the hexaketide sorbicillinol, passing three rounds ofnon-reductive elongation by the second NR PKS (analogous to pks12). In our study,however, the ∆hdaA strain was not able to produce any sorbicillinoids despite thetranscriptional activation of the entire gene cluster. On the other hand, in silico, weidentified this complete gene cluster also to be present in the genome of the knownsorbicillins producer Trichoderma reesei (Abe et al. [2000]).

An alternative possible role of the activated gene cluster is citrinin biosynthesis.This yellow toxin with prominent antibiotic properties against gram-positive bac-teria was initially isolated from P. citrinum (Hetherington and Raistrick [1931]) andlater from other Penicilli and Aspergilli species (Jabbar and Rahim [1962]; Timoninand Rouatt [1944]). The citrinin producing P. chrysogenum has been recently iso-lated from the leaves of the mangrove plant Porteresia coarctata (Devi et al. [2012]).Biosynthesis of this compound occurs via a pentaketide precursor and correspond-ing oxido-reductive conversions (Hajjaj et al. [1999]; Shimizu et al. [2005]) could beassociated with a specific biosynthetic gene cluster in Monascus purpureus (Sakai et al.[2008]). BLAST analysis indicated that three proteins of the activated gene cluster inthe ∆hdaA strain are homologous to the PksCT, CtnR and CtnC genes of citrininbiosynthetic cluster M. purpureus. The two putative oxidoreductases encoded byPc21g05060 (mox60) and Pc21g05110 (ox110) belong to the different families of oxi-doreductases so no homology was found with ctnA and ctnB genes in M. purpureusbut functionally they may fulfill the requirements for the biosynthesis of citrinin orcitrinin-like metabolites in P. chrysogenum.

4444


Secondary metabolite analysis performed under previously described conditionsshowed that neither sorbicillinoids nor citrinin are present in the culture media of∆hdaA strain. Those findings raised the question of the possible deficiency of theactivated genomic region. Unlike the natural isolate of P. chrysogenum, the parentalstrain DS68530 used in our study is a derivative of an intensive classical strain im-provement (CSI) program, which has had a significant impact on penicillin produc-tion by this fungus. However, the impact of these mutations on metabolism beyondpenicillin biosynthesis remains unknown. We sequenced the pks12/13 region in anearly strain P. chrysogenum NRRL1951 and identified two coding mutations that havebeen acquired by the common reference strain P. chrysogenum Wisconsin 54-1255 pre-sumably already at an early stage of the CSI. The mutations concern an isoleucineto arginine substitution in NR-PKS (Pks12) at the intra-domain region, and a leucineto phenylalanine substitution within the KS domain of the second HR-PKS (Pks13).Although, it remains unclear if the particular mutations have a functional impact onthe final polyketide biosynthesis, their further analysis may shed further light on themolecular basis of the production of the sorbicillinoids or citrinin in P. chrysogenum.

HdaA demonstrates a broad impact on secondary metabolism at the transcrip-tional level. Our present results suggest that an epigenome approach can be suc-cessfully applied for the novel biosynthetic pathways discovery in P. chrysogenum.However, the genetic background of the improved high penicillin producing strains,like Wisconsin 54-1255 and it derivatives, must be taken into account as a crucialfactor effecting secondary metabolite profiles.

4.5 Acknowledgements

This work was supported by the Perspective Genbiotics program subsidized byStichting toegepaste wetenschappen (STW) and (co)financed by the NetherlandsMetabolomics Centre (NMC) which is a part of the Netherlands Genomics Initia-tive/Netherlands Organization for Scientific Research (NWO) and the Integrationof Biosynthesis and Organic Synthesis (IBOS) programme residing under AdvancedChemical Technologies for Sustainability (ACTS) which is subsidized by NWO. OSand MR were supported by STW. MS was supported by NWO.

4444

4.6. Supplemental Data 103

4.6 Supplemental Data

L

23

9.4

6.5

4.3

2.32.0

(kb)

A

atg

atg

amdS

hdaADS68530

ΔhdaApgpdA

5’ FR hdaA

5’ FR hdaA

3.1 kb

4.3 kb

3’ FR hdaA

3’ FR hdaA

B

Figure S4.1: The scheme of hdaA gene knock out and its confirmation with southern blotanalysis. (A) Genomic DNA was digested with ScaI endonuclease, using hdaA3’ FR as a probe. The expected 3.1 kb DNA fragment of the parental strainDS68530 and 4.3 kb signal confirming that hdaA locus is replaced with amdSmarker. (B) Inactivation of the hdaA gene. The deletion cassette has been clonedusing the Gateway Three Fragment Vector Construction Kit (Invitrogen) and con-sists of the amdS selection marker under the control of the A. nidulance gpdApromoter. The 5’ and 3’ fragments from outside the hdaA open reading framewere used as flanking regions to provide the homologous recombination into thegenome.

4444


Figure S4.2: The plasmids used in this study. (A) pHdaA, (B) pKO17 and (C) pOE17. Fea-tures: amdS, an A. nidulans acetamidase gene for positive selection of fungaltransformants on the acetamide supplemented medium as a sole nitrogen source.bla, ampicillin resistance gene for the selection in E. coli; ori, pUC origin of repli-cation; attB3/4, Gateway recombination sites; PpcbC, pcbC (isopenicillin N syn-thase gene) promoter region.

4444


Table 4.1: Oligonucleotides used in this studyPrimer name Gene Sequence 5’-3’ FunctionPKS1F Pc12g05590 GCTACAGCCCTGACGCCATGG

qPCRPKSs/NRPSs

PKS1R Pc12g05590 CTGCGCAGGTCTACATCGGTACCPKS2F Pc13g04470 CCGAAGATGCCGGCGACGGPKS2R Pc13g04470 CGCTGGTCTGCGATGTGGCCPKS3F Pc13g08690 CGAGAGACCAGGATAAGGTTCTTGGCPKS3R Pc13g08690 GGTGGTCTGTCACCACTCTTCCCPKS4F Pc16g00370 CATGGTCAGCACCCTCAGTGCCPKS4R Pc16g00370 CCAGGTCAGGCGTCGTACGCPKS5F Pc16g03800 CGGGTGCTGCATAGATGTACTACGCPKS5R Pc16g03800 GCTGGCCACGGAAGACAACGCPKS6F Pc16g04890 CCTATTCGCGCCCTGATTATGGGCPKS6R Pc16g04890 CGAGATTTGTCTTCACAGAACCCACCPKS7F Pc16g11480 CACGATTTTAGCAAGTCAACCAGCGCGPKS7R Pc16g11480 CTCGCTCTCCCAGAATGTCAAGGCPKS8F Pc21g00960 GCCACACTCATCGGCACCACGPKS8R Pc21g00960 GCTCCACAGAGCAACCAACCCGPKS9F Pc21g03930 GACGTGGCCGGTGATGCCGPKS9R Pc21g03930 GCGATGTTGCGGACGAGGCCPKS10F Pc21g03990 CAGCGCCGAGTCCTACAGCCPKS10R Pc21g03990 GTGGACCTTGGAGGATGTCTTGCPKS11F Pc21g04840 CCTTGACGAATATCCGCACTCCGPKS11R Pc21g04840 CAAGCCACAGCTGATGAAGCGCPKS12F Pc21g05070 GTCGGAGGCAATTCGGGAAGGCPKS12R Pc21g05070 GCAAAGTTCCACCACAATGCCGCGPKS13F Pc21g05080 CCGAGGATCTCCGCCAGGCPKS13R Pc21g05080 GGTTGTGCAGGTTCCAGGTGCCPKS14F Pc21g12440 GCACCACCATCAGCCAAAGCATACCPKS14R Pc21g12440 CCGAGGTCCATTGGAACTATGCGCPKS15F Pc21g12450 CCAGTTGTCTGCAGCCGGCCPKS15R Pc21g12450 GCCCAGATCACCGCCGTACGPKS16F Pc21g15160 CAGCCGCGTAGTTTGCCTGGCPKS16R Pc21g15160 GCACAGTGTGCTGAGGTTACGGCPKS17F Pc21g16000 CTTGTCATCAGCAGCCCAGAGGPKS17R Pc21g16000 CAATTTGCGGTGGCTGAGACGCPKS18F Pc22g08170 GGTTGATACTCCTGGGACTGAATACAGPKS18R Pc22g08170 GCTGCTGTGGATCCATCTGCTCGPKS19F Pc22g22850 CGGTCAACCAGGGATCCAACTGCPKS19R Pc22g22850 CTGAAGCGGTCTCTGTGTGGCCPKS20F Pc22g23750 CGGTAATGTCCAGCTGGCACTCGPKS20R Pc22g23750 CTTCAGGCACTTCTGTACCGGGNRPS1F Pc13g05250 GCAGACCTGTATCCATCGCAANRPS1R Pc13g05250 GGAGGCAAGTGAAGGTGTGTTNRPS2F Pc13g14330 GCGACAGCCGCCGGAGTAACTATGGNRPS2R Pc13g14330 GAGAGACGGGGACACGCGTGATGNRPS3F Pc14g00080 ACGTACGCTCGAGCTGGACT

4444


Primer name Gene Sequence 5’-3’ FunctionNRPS3R Pc14g00080 GCCGTCGCGTTGATAATTGG

qPCRPKSs/NRPSs

NRPS4F Pc16g03850 TGGTTGAAAGAGGGCAGTCTCNRPS4R Pc16g03850 CGCGAACATACACAACACCACNRPS5F Pc16g04690 CTTTCCAGAACAGTTGGCTGGTNRPS5R Pc16g04690 GCTGCATCTTACCCAGGTAATTGNRPS6F Pc16g13930 CCACCCTTGTTCAGCCGCTGAATTCCNRPS6R Pc16g13930 GGACGAGGCGAACAACATCGGACNRPS7F Pc21g01710 GCTATCTCGGTGGAGGATCTTCTGTCCNRPS7R Pc21g01710 GTGCTGCTGAGAACACGGGATTGTNRPS8F Pc21g10790 GTGAGGCAGCTTTGTTCAACACCATTNRPS8R Pc21g10790 TTCTGCAGCAGGCTGTCGGCCTGAGNRPS9F Pc21g12630 GAGCCAACTCTGTTGTCTACGNRPS9R Pc21g12630 CAGGGCAATTTGCCTCATTCTGNRPS10F Pc21g15480 CTTGGTGGATGCAGCGAAGGNRPS10R Pc21g15480 CTGTGAGAGAGGCTCTTGAGTANRPS11F Pc22g20400 TTCGCGAACATCCGAAGAAGCNRPS11R Pc22g20400 TCGGGCGAAGACACTGTTCA

12.10F Pc21g05010 GATGCTTTCTTGAATAGCGGATCCACG

qPCR ofthe

expressedgene

cluster ofpks12/13

12.10R Pc21g05010 GAACTTTGCGTTGATCGACTATTTCCACG12.20F Pc21g05020 CTTAACATCTCCGATTCCCTACTCGG12.20R Pc21g05020 CAACCAATCCTGCGATCCATCCTCC12.30F Pc21g05030 GGATAGCCAGAGAGTCAGAGAGACC12.30R Pc21g05030 GCGGACGGCAAGACTCAAGGCGCCTCC12.40F Pc21g05040 GGAGAAAGCACGGCAATCGATAGTGG12.40R Pc21g05040 CCTATTCCTGTGCATGATTCTCGGCG12.50F Pc21g05050 GAAGCGTGGATAGAGACCGAAGAGAAC12.50R Pc21g05050 GGAGCCAGACTCCGGAAAGGATACTG12.60F Pc21g05060 GATAGTGAGTACAAATGCGCCTGGACC12.60R Pc21g05060 CGTTCAATACCGGAAATGGCTAGATTCG12.90F Pc21g05090 CGTTAACTAATGACGCCACCTGTTGC12.90R Pc21g05090 GGAAAATAGTATCCCCAGCGATTGGC12.100F Pc21g05100 CATCAGCACCGAGGTCTTCATTGTCG12.100R Pc21g05100 GCAACGCAATAGATGGTCAATGCCAG

12.110F Pc21g05110 CTGCAGCACTTCAGCATGGATGAAACC qPCR ofthe

expressedgene

cluster ofpks12/13

12.110R Pc21g05110 TCGTTGTGAGACTTGGATGCTCGGACG12.120F Pc21g05120 CCTGCTTCTTAATCTTGCCCTGGC12.120R Pc21g05120 CCAAGCCGATGCCAAGAAGGAAGAG12.130F Pc21g05130 CCTAAGTCCTTGATCAGAGCCTGG12.130R Pc21g05130 GTTGAGGTCGATACCCGTAAGTCATCC

s560F Pc21g12560 CCACGAGCCGTATGGTCAATAGAC

qPCR ofthe

Chryso-gine gene

cluster

s560R Pc21g12560 GTATGCTTGGCCGTCTGGTTGGs570F Pc21g12570 GGCAAGGGAAATGAATCCAGGTGGCs570R Pc21g12570 GATAGATGCCGCTTGTTCGGACCs580F Pc21g12580 AGGCATCGAGGAAACAACGAGAAAGCs580R Pc21g12580 CTTTTAGGTCATCCGGCAGCCAACs590F Pc21g12590 GGTTGTGGAGCTCTACGAGGCTGs590R Pc21g12590 CTGGCAGGGCTCGTCGGTCs610F Pc21g12610 CCTGCATGCAGCTCCATACGAGC

4444


Primer name Gene Sequence 5’-3’ Functions610R Pc21g12610 CCAACAATAGGTGGAAACAGCTCAGAC

qPCR ofthe

Chryso-gine gene

cluster

s620F Pc21g12620 GGAATTCGCTGGCTAACTGGTCTCGs620R Pc21g12620 GGCATGTGGTAGACGAATTGGAGCs630F Pc21g12630 GCACAGGCCAAAGTAACACGTCCs630R Pc21g12630 CCGAGGGTTTGTGGTGGATGCCs640F Pc21g12640 TGTCTCTCTGTGGGCTGTTCTCAGs640R Pc21g12640 CAAGAGTTCTTACGATGCGTGGCTGs650F Pc21g12650 CGGAGGAAGGAGGAACTCTCCGs650R Pc21g12650 CCCTAACCAGCGCATCGTTCCCs660F Pc21g12660 GGAAGGCAAGTTCAGTAAACCACGTGCs660R Pc21g12660 CTGACAAGCTTCTCGATCCTCGC

abr1F Pc21g16380 GTCTACCTGAACTGGAACCTCACTTGG

qPCR ofDHN-

melaningene

cluster

abr1R Pc21g16380 GGTGAGTGTCAACTCTTCATCAAAGTGGarp1 F Pc21g16420 TCTTCAATTCTTCAGTCTCGTAGACCTGGarp1 R Pc21g16420 CCGAGCCCAACTTGGACATCAGCarp2F Pc21g16430 GAGGGTTTTACTCGCTGCTTTGCTGCarp2R Pc21g16430 GATGTTAGCTCCACCGTTGCAAGGCayg1F Pc21g16440 GCTATGGCGGAGAAGTATGGCTATGACayg1R Pc21g16440 CTCTCCAACCACTTGTAAGCCACAGGabr2F Pc22g08420 GCTCAATGTAATGTCCATCCACCTCGabr2R Pc22g08420 GACCCTGAGTATCTGACAAATCTCCAGC

attB4F∆HdaPF Pc21g14570 GGGGACAACTTTGTATAGAAAAGTTGCGTTTAAAGGCAGCCAAAGACTAAACTCAGTA

Cloning/pHdaA

attB1R∆HdaPR Pc21g14570 GGGGACTGCTTTTTTGTACAAACTTGCAAGGGAAAGCCACGGGAAGC

attB2F∆HdaPF Pc21g14570 GGGGACAGCTTTCTTGTACAAAGTGGCCTGATTCGAGCGTGAACCC

attB3R∆HdaPR Pc21g14570 GGGGACAACTTTGTATAATAAAGTTGTTTAAATGGTTGGTCACGACAGCGTT

ProbeF Pc21g14570 GTACATCCATGGATGTTTCTGCTCATATTTGCProbeR Pc21g14570 CTTATATAGTTTCCCTGCTGGTGGATTGAGC

attB4F∆pks17 Pc21g16000 GGGGACAACTTTGTATAGAAAAGTTGGCTGTCATTGAGTCGCTAGGTTATCTCC Cloning/

pKO17attB1R∆pks17 Pc21g16000 GGGGACTGCTTTTTTGTACAAACTTGCCAGTGGCGAATTATTGGTTTCAGGCG

attB2F∆pks17 Pc21g16000 GGGGACAGCTTTCTTGTACAAAGTGGGTGCCTACTTCCAGGACATTTGTATATGGG

attB3R∆pks17 Pc21g16000 GGGGACAACTTTGTATAATAAAGTTGGATTCAACTAACATTTGTGGCAGGACGAGG

attB4Foepks17 Pc21g16000 GGGGACAACTTTGTATAGAAAAGTTGGATGACCCACGTGCATAAGTGACAGC Cloning/

pOE17attB1Roepks17 Pc21g16000 GGGGACTGCTTTTTTGTACAAACTTGCCAGTGGCGAATTATTGGTTTCAGGCG

attB2Foepks17 Pc21g16000 GGGGACAGCTTTCTTGTACAAAGTGGATGGAAGGCCCCGGTCATGTATATCTC

attB3Roepks17 Pc21g16000 GGGGACAACTTTGTATAATAAAGTTGCAAACATTCCGGCGTCGTTATACCAGC

4444


RT:12.49 AV:1 NL:2.22E7

160 180 200 220 240 260 280 300

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

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100

Re

lativ

e A

bu

nd

an

ce

191.08

192.09

173.07

182.99 213.06171.15 195.09 227.98 291.01245.19 254.09 279.16

DS68530

ΔHdaADS68530

Rel

ativ

e A

bund

ance

0

20

40

60

80

100

120

NH

O

N

O

Chrysogine

C10H10O2N2

191.08 [M+H]+

Figure S4.3: HPLC-MS spectra containing chemical formula and calculated exact mass ofthe protonated chrysogine molecule.Decrease of the chrysogine production upto 40 per cent in ∆hdaA mutant compare to the parental DS68530 strain. Dataare calculated based on the peak area ratio obtained from the extracted ion chro-matogram (EIC) of the chrysogine in positive mode (RT=12,49 min.).

4444


RT:21.86 - 24.05

22.0 22.5 23.0 23.5 24.0

Time (min)

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

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Re

lativ

e A

bu

nd

an

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22.54

21.92

22.10

22.6922.29 22.89 22.99

23.4323.11 23.9023.68

21.97

22.02

22.12

22.17

22.2522.32

22.4422.86

22.49

22.9622.6923.01 23.68 23.8223.23 23.55

NL: 3.44E6

TIC F: FTMS {1,2} - p ESI Full ms [150.00-2000.00]

NL: 1.21E6

TIC F: FTMS {1,2} - p ESI Full ms [150.00-2000.00]

A

Figure S4.4: (A) HPLC-MS total ion chromatogram (TIC) of the naphtho-γ-pyrone (YWA1)produced by oepks17 strain (above) versus no production by DS68530 (below).

4444


RT:22.57 AV:1 NL:9.99E5T:FTMS {1,2} - p ESI Full ms [150.00-2000.00]

160 180 200 220 240 260 280 300 3200

5

10

15

20

25

30

35

40

45

50

55

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Rela

tive A

bundance

191.03

275.06

217.01

265.15233.05173.02289.04205.01 246.97

193.04

161.02 189.02

277.06

230.99 297.04 314.96

OHHOCH3

O

OH

OH OH

OHO CH3

O

OH

OH OH

+

C14H12O 6 YWA1 C10H7O4

C14H11O6

[M-H]

C10H6O4

[M-H]

C4H4O2

B

Figure S4.4: (B) HPLC-MS spectra containing chemical formula and calculated exact mass ofthe deprotonated naphtho-γ-pyrone (YWA1) and related fragment acquired byin-source (ESI) fragmentation in negative mode. Schematic representation of thefragmentation mechanism is shown.

ACP KSATMT DH ACPKS ERAT DH KR

ile2210 → arg

2581 AA (Pc21g05070) 2664 AA (Pc21g05080)

leu146 → phe

Figure S4.5: The domain composition of the NR-PKS Pc21g05070 (pks12) and HR-PKSPc21g05080 (pks13). Ketosynthase, KS; Acetyltransferase, AT; Methyltransferase,MT; β-ketoreductase; Enoylreductase, ER; Dehydratase, DH; Acyl carrier pro-tein, ACP. The amino acid substitutions caused by CSI and their localization areshown with triangles.

4444

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university of groningen genomic wake-up call samol, martathe pc21g14570 gene of penicillium...

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