University of Groningen Genetic engineering of Penicillium ...Gateway donor vectors pDoNr P4-P1r and pDoNr P2r-P3, respec-tively, using BP clonase II enzyme mix (Invitrogen, California,

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University of Groningen

Genetic engineering of Penicillium chrysogenum for the reactivation of biosynthetic pathwayswith potential pharmaceutical valueGuzmán Chávez, Fernando

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Citation for published version (APA):Guzmán Chávez, F. (2018). Genetic engineering of Penicillium chrysogenum for the reactivation ofbiosynthetic pathways with potential pharmaceutical value. University of Groningen.

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CHAPTER 4

DEREGULATION OF SECONDARY METABOLISM IN A HISTONE DEACETYLASE MUTANT OF PENICILLIUM CHRYSOGENUM

Fernando Guzmán-Chávez 1,3,§, Oleksandr Salo1,3,§, Marta Samol1,3,§, Marco Ries4,5, Jeroen Kuipers6,

Roel A.L. Bovenberg2,7, Rob J. Vreeken4,5,$, Arnold J.M. Driessen1,3,*

1Molecular Microbiology and 2Synthetic Biology and Cell

Engineering, Groningen Biomolecular Sciences and

Biotechnology Institute, University of Groningen, Groningen,

The Netherlands3kluyver Centre for Genomics of Industrial Fermentations,

Julianalaan 67, 2628 BC Delft, The Netherlands4Division of analytical Biosciences, leiden/amsterdam Center for

Drug research, leiden, The Netherlands5Netherlands Metabolomics Centre, leiden University, leiden,

The Netherlands6Department of Cell biology, University Medical Center

Groningen, 9700 aD Groningen, The Netherlands7DSM Biotechnology Center, alexander Fleminglaan 1, 2613 aX

Delft, The Netherlands

*Correspondence: [email protected]§These authors contributed equally to this work$Current address: Discovery Sciences, Janssen r&D,

Turnhoutseweg 30, 2340 Beerse, Belgium

running Title: histone deacetylase of Penicillium chrysogenum

key words:

Penicillium chrysogenum, chrysogine, sorbicillinoids, naphtha-γ-

pyrone, cross-talk, histone deacetylase

Submitted to Microbiology Open 2017

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ABSTRACT

The Pc21g14570 gene of Penicillium chrysogenum encodes an ortho-logue of a class 2 histone deacetylase termed hdaa which may play a role in epigenetic regulation of secondary metabolism. Deletion of the hdaa gene induces a significant pleiotropic effect on the expres-sion of a set of polyketide synthase (PkS) and non-ribosomal peptide synthetase (NrPS) encoding genes. The deletion mutant exhibits a de-creased conidial pigmentation that is related to a reduced expression of the PkS gene Pc21g16000 (pks17) responsible for the production of the pigment precursor naphtha-γ-pyrone. Moreover, the hdaa de-letion caused decreased levels of the yellow pigment chrysogine that is associated with the down regulation of the NrPS encoding gene Pc21g12630 and associated biosynthetic gene cluster. In constrast, transcriptional activation of the sorbicillinoids biosynthetic gene clus-ter occurred concomitantly with the overproduction of associated compounds. a new compound was detected in the deletion strain that was observed only under conditions of sorbicillinoids production, suggesting cross-talk between biosynthetic gene clusters. our pres-ent results show that an epigenomic approach can be successfully ap-plied for the activation of secondary metabolism in industrial strains of P. chrysogenum.

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INTRODUCTION

During the last decades, the filamentous fungus Penicillium chrysoge-num has been used extensively in industry for the production of the β-lactam antibiotic penicillin (Fleming, 1929). The biosynthetic path-way and the corresponded genes involved have been well described and current production strains are generated for the high-level produc-tion of penicillins through the implementation of an intense classical strain improvement program. however, the full potential of second-ary metabolism of P. chrysogenum remained unknown till the genomic sequence became available (van den Berg et al., 2008). The genome specifies multiple genes for secondary metabolite formation includ-ing 20 polyketide synthases (PkSs), 10 non-ribosomal peptide syn-thetase (NrPSs), 2 hybrids (PkS-NrPS) and 1 dimethylallyltryptophan synthase. The function of the most of these genes remains unknown (van den Berg et al., 2008). recently, a genome based identification and analysis of the roquefortine meleagrin NrPS gene cluster was performed for P. chrysogenum (Garcia-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 sec-ondary metabolite genes under laboratory conditions is low (Brakhage and Schroeckh, 2011). Therefore, more elaborate methods other than gene inactivation are required for identification and further analysis of these so-called ‘silent’ secondary metabolite genes.

New approaches have evolved during the post-genomic era to ac-tivate gene clusters such as interference with cluster specific regu-latory genes or even of pleiotropic regulator of chromatin structure like laea. This has triggered the research on the cryptic potential of fungal secondary metabolism (Brakhage and Schroeckh, 2011). a po-tential powerful approach is the epigenetic regulation of gene expres-sion. In eukaryotic cells, DNa is compacted into a complex chromatin structure. The histone proteins h2a, h2B, h3, and h4 form the core histone octamer complex with DNa called nucleosome, the structural and functional unit of the chromatin (luger, 2003). The formation of the nucleosomes may interfere with the recognition of the bound DNa by various transcriptional elements causing gene silencing (lee et al., 1993). Thus remodeling of the chromatin by the histone modifi-cations is a trigger that influences transcription, replication, and DNa

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repair (yu et al., 2011; Zhu et al., 2011). The histone acetylation sta-tus is controlled by the balanced activity of histone acetylases (haTs) and deacetylases (hDaCs) (Brosch et al., 2008). hyperacetylation of the chromatin induced by deletion or chemical inhibition of hDaCs leads to euchromatin formation and transcriptional activation of silent chromosomal regions (Gacek and Strauss, 2012). Cladochromes and calphostin B in Cladosporium cladosporioides and nygerone a from As-pergillus niger are secondary metabolites that have recently been identified with this strategy using the hDaC inhibitor suberoylanili-dehydoxamic acid (Saha) (Fisch et al., 2009; henrikson et al., 2009; Carafa et al., 2013). an altered secondary metabolite profile was also reported for Alternaria 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+ depend-ent sirtuins (de ruijter et al., 2003). Members of both families were initially described in S. cerevisiae and subsequently identified in fila-mentous fungi and human (Taunton et al., 1996). The orthologues of the rPD3 (reduced potassium dependency) transcription factor and hDa1 of S. cerevisiae belong to the major classes 1 and 2 of the “classi-cal” hDaCs, respectively. recently, multiple effects of the inactivation of hda1 orthologues on the expression of secondary metabolite genes has been reported for a number of fungal species (Tribus et al., 2005; Shwab et al., 2007; lee et al., 2009).

here, we have demonstrated that ortholog of the class 2 histone deacetylase hda1 of S. cerevisiae (Pc21g14570) is a key regulator of the secondary metabolism in the filamentous fungus P. chrysogenum. By means of the transcriptional and metabolite profiling of the indi-vidual gene deletion mutants, the role of hdaa in production of the new metabolite, conidial pigmentation as well as the broad influence of hdaa on the expression of the SM gene clusters have been shown. Furthermore, we demonstrated that transcriptional cross-talk be-tween sorbicillinoids biosynthesis and other SM genes in this fungus is mediated by hdaa.

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MATERIAL AND METHODS

STRAINS,MEDIAANDGROWTHCONDITIONS

Penicillium chrysogenum DS68530 was provided by DSM Sinochem Pharmacuticals (Delft, The Netherlands). The strains: ΔhdaA_DS68530, Δpks17 and over expression mutant oepks17 were derived from DS68530. ΔhdaA_DS68530Res13 was derived from DS68530Res13 (Sorb407) (Salo et al., 2016; Guzmán-Chávez et al., 2017) (Table 1). liq-uid yGG medium (400 ml kCl-glucose, 100 ml 5X buffered yeast Ni-trogen Base (yNB), 10 ml fresh 10% yeast extract) was used for pre-culturing the conidia for 24 hours before inoculation into secondary metabolite production medium (SMP; (ali et al., 2013)). Solid r-agar me-dium (6 ml l-1 glycerol, 7.5 ml l-1 beet molasses, 5 g l-1 yeast extract, 18 g l-1 NaCl, 50 mg ml-1 l-1 MgSo4·7h2o, 60 mg l-1 kh2Po4, 250 mg l-1 CaSo4, 1.6 ml l-1 Nh4Fe(So4)2 (1 mg ml-1), Fe(So4)2·12h2o, 10 mg l-1 CuSo4·5h2o, and 20 g l-1 agar was used for culturing the conidia and for secondary metabolites production on plate (kovalchuk et al., 2012). all cultivations were performed at 25 °C in semi dark conditions. liquid culturing of the conidia was performed in 25 ml of yGG or SPM media in 100ml flasks shaken at 200 rpm (Guzmán-Chávez et al., 2017).

PLASMIDS CONSTRUCTION

all the plasmids in this study were constructed using the modified Gate-way cloning protocol (Invitrogen, California, USa) published previously

Table 1. Strains used in this study

Strain Genotype SourceDS68530 (AFF407) 0 Penicillin BGC, Δku70, Sorbicillinoids nonproducer DSM Sinochem

PharmaceuticalsDS68530Res13 (Sorb407) 0 Penicillin BGC, Δku70, AmdS marker free,

Sorbicillinoids producer, SorA (F146L)(Guzmán-Chávez et al., 2017)

Strains derived from DS68530∆hdaA_DS68530 AmdS marker Sorbicillinoids nonproducer This studyΔpks17 AmdS marker Sorbicillinoids nonproducer This studyoepks17 AmdS marker, pcbC::Pc21g16000 Sorbicillinoids

nonproducerThis study

Strains derived from DS68530Res13∆hdaA_DS68530Res13 AmdS marker, sorbicillinoid producer , SorA (F146L) This study

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(kovalchuk et al., 2012). 5’- and 3’- fragments for the deletion cassette were amplified with Phire hot Start II PCr Master Mix (Thermo Fisher Scientific, USa) using specific primers and cloned into corresponded Gateway donor vectors pDoNr P4-P1r and pDoNr P2r-P3, respec-tively, using BP clonase II enzyme mix (Invitrogen, California, USa). The resulted plasmids were purified from kanamycin resistant E. coli Dh5α transformants and subsequently recombined with the Gateway des-tination vector pDEST r4-r3 and pDoNr221-aMDS for in vitro re-combination using lr clonase II enzyme mix. For expression, the mod-ified pDoNr221-aMDS plasmid was used. In this construct the pcbC (isopenicillin synthase) promoter region was ligated downstream of the amdS gene. after incubation, the reaction mixture was transformed to E. coli Dh5α and the final plasmids were isolated from the ampicillin re-sistant transformants (Salo et al., 2016).

FUNGAL TRANSFORMATION

For all the transformations, 5 µg of plasmid were digested with the suitable restriction enzymes. The linearized plasmid was used to trans-form protoplasts isolated from P. chrysogenum as described previously (kovalchuk et al., 2012; Weber, kovalchuk, et al., 2012). after 6 days of growth at 25 ºC on 0.1 % acetamide selection plates, the correct transformants were screened by colony PCr using Phire Plant Direct kit (life Technologies, USa) (Guzmán-Chávez et al., 2017) and follow-ing the manufacturer’s instructions. PCr product was digested with SalI restriction enzyme (restriction sites only present in positive trans-formants). Selected colonies were purified by three rounds of selec-tion in r-agar. Correct transformants were validated by sequencing the amplified integration site from gDNa (Figure S1). all the primers used are described in Table S1.

SOUTHERN BLOT ANALYSIS

The 3’ downstream region of the hdaA gene was used as a probe and amplified by PCr with primer set listed in Table S1. The probe was labeled with digoxigenin using the highPrime kit (roche applied

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Sciences, almere, The Netherlands). gDNa (10 μg) was digested with appropriate restriction enzyme and separated on 0.8 % agarose gel. after equilibration in 20x SSC buffer (3 M sodium chloride; 0.3 M so-dium citrate) the DNa was transferred overnight onto Zeta-probe pos-itively charged nylon membrane (Biorad, Munchen, Germany). Blots were treated with anti-DIG-alkaline phosphatase antibodies and sup-plemented with CDP-star (roche applied Sciences, almere, the Neth-erlands). The fluorescence signal was measured with a lumi Imager (Figure S1) (Fujifilm laS-4000, Fujifilm Co. ltd, Tokio, Japan) (Salo et al., 2016; Guzmán-Chávez et al., 2017).

GENOMICDNAEXTRACTION

The total genomic DNa (gDNa) was isolated after 96 h of cultivation in yGG liquid medium using an adapted yeast genomic DNa isolation protocol (harju et al., 2004). The mycelium was broken in a FastPrep FP120 system (Qbiogene, Cedex, France).

TOTALRNAEXTRACTIONANDCDNASYNTHESIS

Total rNa was isolated from colonies and fungal mycelium that grown on solid r-agar medium and SMP medium for 7 and 3 days respec-tively. The Trizol (Invitrogen, California, USa) extraction method was used, with additional DNase treatment using the Turbo DNa-free kit ( ambion, Carlsbad, Ca, USa). Total rNa concentration was measured with a NanoDrop ND-1000 (ISoGEN, Utrecht, The Netherlands). For the synthesis of cDNa by iScript cDNa synthesis kit (Bio-rad, Munchen, Germany), 500 ng of rNa per reaction was used (Nijland et al., 2010).

qPCR ANALYSIS

The primers used for expression analysis of the 20 PkSs, 11NrPSs, the sorbicillinoids gene cluster of [Pc21g05050 (sorR1), Pc21g05060 (sorC), Pc21g05070 (pks12; sorB), Pc21g05080 (pks 13; sorA), Pc21g05090 (sorR2), Pc21g05100 (sorT) and Pc21g05110 (sorD)

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(Salo et al., 2015; Guzmán-Chávez et al., 2017)], the genes of puta-tive DhN- melanin cluster [Pc21g16380 (abr1), Pc21g16420 (arp1), Pc21g16430 (arp2), Pc21g16440 (ayg1), Pc22g08420 (abr2)], and the chrysogine biosynthetic gene cluster [Pc21g12570 (chyE), Pc21g12590 (chyH), Pc21g12600 (chyC), Pc21g12610 (chyM), Pc21g12620 (chyD), Pc21g12630 (nrps 9; chyA), Pc21g12640 (chyR) (Viggiano et al., 2017)], are shown in the Table S1. Primers were designed at both sides of the introns in order to be able to discriminate between the amplifi-cation on gDNa and cDNa. For expression analysis, the γ-actin gene (Pc20g11630) was used as a control for normalization (Nijland et al., 2010). a negative reverse transcriptase (rT) control was used to de-termine the gDNa contamination in the isolated total rNa. The ex-pression levels were analyzed, in duplicate, with a Miniopticon sys-tem (Bio-rad) using the Bio-rad CFX™ manager software, with which the threshold cycle (ct) values were determined automatically by re-gression. The SensiMix SyBr hi-roX kit (Bioline, australia) was used as a master mix for qPCr. The following thermocycler conditions were applied: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s. Subsequently, a melting curve was generated to determine the specificity of the qPCrs (Nijland et al., 2010; Weber, Polli, et al., 2012). The expression analysis was per-formed for two biological samples with two technical replicates. The analysis of the relative gene expression was performed through the 2-ΔΔCT method (livak and Schmittgen, 2001).

SECONDARY METABOLITE ANALYSIS

The extraction of secondary metabolites from solid r-agar medium for hPlC and MS analysis was done by the modified micro-scale extraction procedure for standardized screening of fungal metabolite production in cultures (Smedsgaard, 1997). a plug of the agar medium (5 mm in diameter) was taken for extraction from the middle of the colony ob-tained after 10 days of growth. The extraction mixture (0.5 ml) con-tained methanol-dichloromethane-ethyl acetate in a ratio of 1:2:3 (v/v). The plugs were extracted ultrasonically in 1 ml glass tubes during 60 min. The liquid extract was transferred to a fresh tube and dried under vac-uum using a SpeedVac™ vacuum concentrator (Eppendorf, hamburg,

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Germany) for 30 min. The dry pellet was re-dissolved in a 1:1 solution of methanol in water, filtered via 0.2 μm PTFE syringe filter and used for hPlC and MS analysis. Samples from liquid cultures in SMP medium were collected at 3 and 5 days, whereupon the supernatants were cen-trifuged for 5 min at 14000 rpm, previous to be filtered through 0.2 μm PTFE syringe filter (Salo et al., 2016; Guzmán-Chávez et al., 2017).

Secondary metabolites were analyzed with a Shimadzu hPlC sys-tem coupled with photodiode array detector (PDa) and it was per-formed as described previously (Salo et al., 2016).

Metabolite analysis was performed with two biological samples with two technical duplicates. reserpine was used as internal standard.

SCANNING ELECTRON MICROSCOPY

Conidia were immobilized on glass cover slips and fixed with 2 % glu-taraldehyde for 1 hour followed by washing with cacodylate buffer (ph 7.4). Samples were incubated with 1 % oso4 in 0.1 M cacodylate buffer during 1 h and washed with MQ water. The immobilized spores were dehydrated with a concentration gradient of 30, 50 and 70 % of ethanol within 30 min followed by 3 steps of final dehydration 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 incubation with pure TMS and air dried. Dried samples were coated with 2 nm Pd/au using leica EM SCD050 sputter coater and analyzed with SUPra 55 FE-SEM (Carl Zeiss, Jena Germany) at 2 kV.

OXIDATIVE STRESS ASSAY

Fungal conidia of seven day grown mycelium were re-suspended in 1 ml water contained 0.05 % of Tween-20 to prevent aggregation. The equal amount of the spores (3×103 spores per ml) in solution were ad-justed by series of dilutions and measured with Bürker-Türk counting chamber using olympus CX20™ light microscope (olympus, hamburg, Germany). a conidial suspension (100 µl) was used for inoculation to obtain approximately 300 germination events per control plate in the assay. r-agar sporulation medium with increasing concentrations

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of hydrogen peroxide from 0.5 to 3.5 mM was used in this assay. To prepare each plate the corresponding amounts of hydrogen peroxide have been mixed with 25 ml of r-agar medium before solidification to provide equal distribution of the supplement in the plate. The experi-ment has been performed twice using two sets of the hydrogen perox-ide supplemented r-agar plates as the technical replicas.

OTHER METHODS

To study the effect of sorbicillinoids on the secondary metabolism of P. chrysogenum the feeding experiment has been performed as it was reported previously (Guzmán-Chávez et al., 2017). The pre-culture of the strain DS368530 has been grown on yGG medium for 24 hours and subsequently used (3 ml) to supplement 20 ml of fresh SMP me-dium. The filtered supernatant (2 ml) obtained from the growth me-dium of the stain DS68530res13 grown in SPM for 3 days has been collected as the source of sorbicillinoids used for the feeding experi-ment. The control culture was supplemented with the supernatant de-rived from the non-sorbicillinoids producing strain DS68530.

RESULTS

DELETION OF THE hdaA GENE

The gene, Pc21g14570 of P. chrysogenum encodes an orthologue to the hda1 histone deacetylase gene of Saccharomyces cerevisiae. This gene, termed hdaA, was deleted from the chromosome in order to investi-gate its effect on development and secondary metabolite production. The complete hdaA gene was replaced by the acetoamidase (amdS) se-lection marker gene. a standard protocol was used for cloning of the corresponded phdaa deletion plasmid (kovalchuk et al., 2012) contain-ing the 3’ and 5’ flanking regions of the hdaA open reading frame. Pro-toplasts of the amdS marker-free strain DS68530 and DS68530res13 that both lack all copies of the penicillin biosynthesis cluster were used for transformation to simplify the detection of other secondary metab-olites. acetamide supplemented medium (0.1 % aMDS) was used for

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the positive selection of transformants, and the correct inactivation of the hdaA gene was validated by sequencing the locus of the inser-tion (Figure S1).

EFFECT OF THE hdaADELETIONONTHEEXPRESSIONOFSECONDARY METABOLITE GENES

To examine the effect of inactivation of hdaA on the transcription of secondary metabolite genes, the expression of all 20 PkS and 11 NrPS genes was examined using Quantitative real Time PCr anal-ysis. rNa was isolated from the mycelium of the deletion and the pa-rental strains grown on SMP medium for 3 days. The related superna-tant fractions obtained after 3 and 5 days of culture growth were used for secondary metabolite profiling (see below). The qPCr analysis of the various secondary metabolite genes was performed using primers listed in Table S1. out of the 31 analyzed secondary metabolite genes, the expression of eight genes was dramatically altered in ΔhdaA mu-tants from the different genetic backgrounds (sorbicillinoids producer and none producer strains).

an up to 500-fold increase in expression occurred for the PkS en-zymes SorB (pks12; Pc21g05070) and Sora (pks13; Pc21g05080) in the sorbicillinoids producing strain, while the transcript levels of the corre-sponding genes in the ΔhdaA mutant that is not able to produce sorb-icillinoids, was only 12-fold higher. Interestingly, the deletion of hadA showed the same positive impact in the expression levels of pks4, 7, 8, 11 and 17 (Pc16g00370, Pc16g11480, Pc21g00960, Pc21g04840, Pc21g16000, respectively), compared to the parental sorbicillinoids producer strain (Figure1a). Pks7 and pks17 expression levels were in-creased 11 and 58- fold, respectively, while the expression of pks8 was reduced 33-fold. also, the expression of two NrPS genes nrps3 and chyA (Pc13g08690 and Pc21g12630, respectively) were significantly altered (Figure 1B). The chyA (nrps9) gene that encodes for a dipep-tide synthase that belongs to the chrysogine biosynthetic gene cluster (BGC) (Viggiano et al., 2017) was 25-fold down regulated. In the ge-nome of P. chrysogenum, secondary metabolite genes are distributed over the four chromosomes. however, in particular genes that local-ize to chromosome 2 were influenced by the hdaA deletion, except

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0,016

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B)

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Pc16

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Pc21

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Figure 1. Relative expression of all secondary metabolite genes in ∆hdaA mu-tants. A) Polyketide synthases genes (PKS). B) Non-ribosomal peptide synthetase (NRPS). Genes are grouped according to the genome annotation number. Sam-ples were taken after 3 days of growth on SMP medium. Strains: DS685Res13 (black bars), ΔhdaA_DS68530Res13 (withe bars), ΔhdaA_DS68530 (grey bars). (*) Indicates lack of expression. Data are shown as fold change relative to P. chrysogenum DS68530. (ΔhdaA/DS68530). Error bars indicate the standard deviation of two biological with two technical replicates.

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for pks7 and nrps3 that localize at the opposite ends of chromosome 1. For the remainder of the secondary metabolite genes no transcrip-tional response was observed (Figure 2S).

EPIGENETIC ACTIVATION OF THE SORBICILLINOIDS BIOSYNTHESIS GENE CLUSTER

The genes belonging to the sorbicillinoids BGC (Guzmán-Chávez et al., 2017) were highly up regulated in the ΔhdaA strain (Figure 2a; ∆hdaA_DS68530Res13 strain). The sorC gene showed an increase in the expression levels of more than 100 times, while sorD and sorT were 2500-fold upregulated relative to the DS68530 strain. overexpres-sion of this BGC resulted in the high-level production of sorbicillinoids in the supernatant fraction (Figure 2B). The production of sorbicillinol [3,3*] and dihydrosorbicillinol [4,4*] — the main products of the path-way, increased up to 2.5 folds in ΔhdaA strain. however, the most sig-nificant changes were related to the downstream intermediates of the pathway. For instance, at day 5, the levels of tetra- and dihydrobisverti-nolone [8;9] were 17 and 22-fold higher with the hdaA gene deletion strain compared to the parental strain, while production of sorbicilli-noids was detected one day earlier in fermentation (data not showed). Importantly, the hdaA deletion (ΔhdaA_DS68530) also enhanced the expression of the sorbicillinoids BGC in the strain that contains a de-fect copy of the sorA gene (Figure 2a) although the effect was not as strong as in the sorbicillinoids producing strain (ΔhdaA_DS68530Res13 strain). These observations are consistent with previous findings that sorbicillinoids act as autoinducers (Guzmán- Chávez et al., 2017) and further demonstrate that hdaa silences the expression of this BCG.

Considering the significant transcriptional deregulation of the sec-ondary metabolite genes in the ΔhdaA strain and in particular the ex-pression of functionally uncharacterized PkSs and NrPSs genes, met-abolic profiling was employed to search for novel compounds. Indeed, an unknown compound was detected at elevated levels in the ΔhdaA strain that does not produce sorbicillinoids (ΔhdaA_DS68530). This compound has a m/z [M+h]+ of 369.0810 and a retention time (rT) of 6.88 min. Interestingly, the same compound was also present in the culture broth of the DS68530 strain that was supplemented with

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No Compound Name Formula Acquired RT DS68530 �HdaA_DS68530 DS68530Res13 DHdaA_DS68530Res13[M+H]+ (min) 3 5 3 5 3 5 3 5

1 Sorbici l l in C14H16O3 233,1172 30,65 0,0 0,0 0,0 0,0 0,0 0,3 0,1 0,4

2 Dihydrosorbici l l in C14H18O3 235,1327 31,90 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0

3 Sorbici l l inol C14H16O4 249,1119 20,02 0,0 0,0 0,0 0,0 1,8 3,0 1,9 7,1

4 Dihydrosorbici l l inol C14H18O4 251,1274 21,48 0,0 0,0 0,0 0,0 4,4 8,9 6,7 23,8

5 Oxosorbici l l inol C14H16O5 265,1069 19,36 0,0 0,0 0,0 0,0 0,0 0,8 0,4 1,2

2* Dihydrosorbici l l in* C14H18O3 235,1329 47,08 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0

3* Sorbici l l inol* C14H16O4 249,1119 21,05 0,0 0,0 0,0 0,0 0,8 3,6 1,9 9,3

4* Dihydrosorbici l l inol* C14H18O4 251,1274 23,33 0,0 0,0 0,0 0,0 4,4 7,0 4,7 19,6

5* Oxosorbici l l inol* C14H16O5 265,1069 28,16 0,0 0,0 0,0 0,0 0,1 0,4 0,4 1,4

6 Bisorbici l l inol C28H32O8 497,2163 28,87 0,0 0,0 0,0 0,0 0,0 0,1 0,1 0,3

7 Bisver�nolon C28H32O9 513,2112 33,23 0,0 0,0 0,0 0,0 0,0 0,1 0,1 1,0

8 Dihydrobisver�nolone C28H34O9 515,2266 33,07 0,0 0,0 0,0 0,0 0,0 0,2 0,2 3,4

9 Tetrahydrobisver�nolone C28H36O9 517,2425 33,91 0,0 0,0 0,0 0,0 0,0 0,1 0,1 2,2

10 Sorbici l l in related C12H14O3 207,1015 23,45 0,0 0,0 0,0 0,0 0,0 0,1 0,2 0,4

11 Sorbici l l in related C11H12O3 193,0858 21,56 0,0 0,0 0,0 0,0 0,1 0,8 0,3 1,5

12 Sorbici l l in related C12H17ON 192,1382 13,50 0,0 0,0 0,0 0,0 0,4 0,3 0,6 0,9

13 Sorbici l l in related C15H20O5N2 309,1440 15,42 0,0 0,0 0,0 0,0 0,0 1,3 0,7 3,5

14 Sorbici l l in related C15H20O4N2 293,1490 17,25 0,0 0,0 0,0 0,0 0,8 18,1 16,3 42,3

15 Sorbici l l in related C22H40O15N8 657,2683 33,91 0,0 0,0 0,0 0,0 0,0 1,4 0,2 5,1

16 Sorbici l l in related C11H10O5 223,0600 14,92 0,0 0,0 0,0 0,0 0,0 0,0 2,2 1,9

17 Sorbici l l in related C14H18O5 267,1224 17,13 0,0 0,0 0,0 0,0 0,2 0,3 0,2 0,6

18 Sorbici l l in related C12H32O15N8 529,2059 27,36 0,0 0,0 0,0 0,0 0,7 2,4 1,5 9,8

Max

Min

Resp

onse

ra�

o

0.1

0.3

0.5

1.0

2.0

4.0

8.0

16.0

32.0

64.0

128.0

256.0

512.0

1024.0

2048.0

4096.0

SorR1 SorC SorB SorA SorR2 SorT SorD

A)

B)

Fold

cha

nge

C)

Sorbicillin (1)2'-3'Dihydrosorbicillin (2,2*)

CH3

OH O

CH3

OH

H C3

Sorbicillinol (3,3*)2'-3'Dihydrosorbicillinol (4,4*)

H CCH3

O

O

3

OH

OHH C3

Oxosorbicillinol (5,5*)

H CCH3

O

OH

3

OH

OHH C3

Bisvertinolon (7)Bisorbicillinol (6)

CH3

OO

CH3

OHOHCH3

H C3

H C3

H C3OOOH

OH

OOH

CH3

O

H C3

CH3

OHO

O

H C3

CH3OH

CH3OH OH

Dihydrobisvertinolone(8)

Tetrahydrobisvertinolone (9)

O

O

O

O

CH3

OH OHCH3H

OHH C3

CH3 OHOH CH3

O

O

CH3

O

O

CH3

OH OHCH3H

OHH C3

CH3 OHOH CH3

CH3

Page 16

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sorbicillinoids derived from a 3 day culture of DS68530res13 strain (Figure 3). The identity of this compound is unknown.

hdaA REGULATES THE TRANSCRIPTION OF THE CHRYSOGINE BIOSYNTHETIC GENE CLUSTER

Pc21g12630 encodes an NrPS (chyA) that is involved in chrysogine production (Viggiano et al., 2017) and its expression is down regulated in the hdaA deletion strain. ChyA is part of a cluster of seven genes that in addition specifies a malonyl transferase (chyE; Pc21g12570), two asparagine synthetase (chyC, chyD [Pc21g12600 Pc21g12620]), two hypothetical proteins involved in oxidation reactions (chyH, chyM [Pc21g12590, Pc21g12610]), and a putative regulator (chyR, Pc21g12640)). The expression of the cluster was analyzed by qPCr re-vealing the down-regulation of the entire BGC in the hdaA mutants. however, down-regulation was also observed with the sorbicillinoids production DS68530res13 strain independent of the hdaA deletion. The transcriptional levels of chyA and chyD were reduced up to 25-fold, while gene expression of chyE, chyC and chyM were lowered 2.8 and 2-fold, respectively (Figure 4a). To investigate the effect of the hdaA gene deletion on the production of chrysogine, the reference strain

Figure 2. Transcription and metabolite profile analysis of the activated sorbicil-linoids biosynthetic gene cluster in the ΔhdaA mutant. A) Quantitative Real Time PCR analysis of the sorbicillinoids BGC. Strains: DS685Res13 (black bars), ΔhdaA_DS68530Res13 (white bars), ΔhdaA_DS68530 (grey bars). Samples were taken after 3 days of growth on SMP medium. Data are shown as fold change relative to P. chrysogenum DS68530 (ΔhdaA/DS68530). B) Response ratio of the sorbicillinoids concentrations in the supernatant of the indicated P. chrysogenum strains. Samples were collected after 3 and 5 days of growth in SPM medium. C) Sorbicillinoids related compounds (with known chemical structure) detected in this study. Reserpine was used as internal standard for normalization. The mass-to-charge ration (m/z) of the protonated metabolites, retention time (RT) and empirical formulas are described. (*) Indicates an iso-mer of the known sorbicillinoids. Error bars indicate the standard deviation of two biological replicates with two technical replicates.

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DS68530

DS68530_Mock

DS68530_Induced

DhdaA_DS68530

C8H9O5

C15H11O5N7

C16H17O10

A)

B)

Figure 3. A) LC-MS extracted ion chromatogram (EIC) of the extracellular me-tabolite spectrum of different strains after 5 days of growth in SPM medium. B) LC-MS spectra containing the empirical chemical formulas and calculated exact mass (<2.0 ppm). Data obtain from extracted ion chromatogram (EIC) in positive mode.

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0.02

0.03

0.06

0.13

0.25

0.50

1.00

2.00

Chy

EC

hyH

Chy

CC

hyM

Chy

DC

hyA

Chy

R

Fold change

***

***

A)M

ax

Response ra�o

Min

No

Com

poun

d N

ame

Form

ula

Acqu

ired

RT[M

+H]+

(min

)3

53

53

53

5

19Ch

ryso

gine

C 10H

10O

2N2

191.

0811

12.8

622

2.9

146.

094

.888

.510

9.6

117.

276

.811

9.9

20N

-ace

tyla

lany

lant

hran

ilam

ide

C 12H

15O

3N3

250.

1182

10.6

63.

72.

80.

70.

61.

21.

00.

71.

0

21C 1

0H10

O3N

220

7.07

6210

.05

0.8

2.1

1.4

3.7

1.4

5.5

1.6

7.4

22Ch

ryso

gine

CC 1

3H15

O5N

329

4.10

7611

.71

7.0

10.1

3.8

3.5

4.4

3.6

3.6

5.3

23Ch

ryso

gine

rela

ted

C 15H

20O

5N4

337.

1502

8.59

2.1

0.0

0.0

0.0

0.0

0.0

1.1

0.1

24Ch

ryso

gine

rela

ted

C 15H

18O

6N3

336.

1182

10.5

40.

00.

00.

00.

20.

00.

20.

00.

3

25Ch

ryso

gine

rela

ted

C 13H

12O

5N2

277.

0814

11.6

96.

79.

73.

63.

23.

73.

03.

04.

5

26Ch

ryso

gine

rela

ted

C 20H

20O

6N4

413.

1444

16.7

90.

70.

50.

30.

30.

20.

20.

20.

2

27Ch

ryso

gine

rela

ted

C 13H

13O

4N3

276.

0972

13.7

80.

40.

30.

00.

00.

20.

20.

10.

2

28Ch

ryso

gine

rela

ted

C 15H

19O

6N3

338.

1338

13.1

72.

20.

10.

60.

01.

00.

20.

70.

0

DS68

530

�Hd

aA_D

S685

30DS

6853

0Res

13�

HdaA

_DS6

8530

Res1

3

N-p

yrov

oyla

nthr

anila

mid

B)

Chr

ysog

ine

(19)

N H

N

O

OH

N-a

cety

lala

nyla

nthr

anila

mid

e (2

0)

N HN

H2

O

NH

OO

N-p

yrov

oyla

nthr

anila

mid

(21)

NHN

H2

O

OO

Chr

ysog

ine

C (1

9)

4

NHN

H2

O

NH

O OH

O

O

C) Figu

re 4

. Tra

nscr

iptio

nal a

nd m

etab

olite

pro

file

anal

ysis

of c

hrys

ogin

e bi

osyn

theti

c ge

ne c

lust

er in

Δhd

aA m

utan

t. A

) Qua

ntita

tive

Real

Ti

me

PCR

anal

ysis

of c

hrys

ogin

e ge

ne c

lust

er. S

trai

ns: D

S685

Res1

3 (b

lack

bar

s), Δ

hdaA

_DS6

8530

Res1

3 (w

ithe

bars

), Δh

daA

_DS6

8530

(g

rey

bars

). Sa

mpl

es w

ere

take

n aft

er 3

day

s of

gro

wth

on

SMP

med

ium

. Dat

a ar

e sh

own

as a

fol

d ch

ange

rel

ative

to

P. c

hrys

ogen

um

DS6

8530

(Δhd

aA /

DS6

8530

). (*

) Ind

icat

es n

on-d

etec

ted

expr

essio

n un

der t

he te

sted

str

ain.

B) R

espo

nse

ratio

on

the

conc

entr

ation

of

the

chry

sogi

ne re

late

d co

mpo

unds

in th

e cu

lture

bro

th o

f the

indi

cate

d P.

chr

ysog

enum

str

ains

. Sam

ples

wer

e co

llect

ed a

fter

3 a

nd 5

day

s of

gro

wth

in S

PM m

ediu

m. C

) Chr

ysog

ine

rela

ted

com

poun

ds (w

ith k

now

n ch

emic

al s

truc

ture

) det

ecte

d in

this

stud

y. R

eser

pine

was

use

d as

inte

rnal

sta

ndar

d fo

r nor

mal

izati

on. T

he m

ass-

to-c

harg

e ra

tion

(m/z

) of t

he p

roto

nate

d m

etab

olite

s, re

tenti

on ti

me

(RT)

and

em

piric

al

form

ulas

are

des

crib

ed. E

rror

bar

s in

dica

te th

e st

anda

rd d

evia

tion

of tw

o bi

olog

ical

repl

icat

es w

ith tw

o te

chni

cal r

eplic

ates

.

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rESUlTS

DS68530, DS68530res13 and ΔhdaA strains were grown for 3 and 5 days in SMP medium. Samples of the culture broth were filtered and analyzed by lC-MS. at day 3, chrysogine production was reduced 2-fold for the ΔhdaA mutants and DS6830res13 compared to the ref-erence DS68530 strain in line with the qPCr data (Figure 4a–B). like-wise, at day 5, also most of the chrysogine related compounds were produced at lower levels. Taken together, these results indicate that the chrysogine BGC is not only subjected to epigenetic activation, but also suppressed by the production of sorbicillinoids.

hdaAREGULATESTHEDHN-MELANINBGCINVOLVEDINPIGMENT FORMATION

Pigmentation in filamentous fungi is often attributed to the dihydroxy-naphtalene (DhN)-melanin BGC that typically consists of six genes in-cluding a PkS (Tsai et al., 1997, 1998). The DhN-melanin biosynthetic pathway was described initially for Verticillium dahliae and Wangiella dermatitidis (Bell et al., 1976; Geis et al., 1984). The pentaketide ori-gin of fungal melanins is common in other melanized fungi (langfelder et al., 2003; Wheeler et al., 2008). In A. fumigatus, the polyketide prod-uct of the PkS alb1p, the heptaketide naphthapyrone yWa1, requires the enzymatic post PkS conversion to the pentaketide 1,3,6,8-tetra-hydroxynaphthalene (T4hN) via hydrolytic polyketide shortening by ayg1p (Fujii et al., 2004). This enzymatic step is absent in C. lagenarium where the pentaketide T4hN is a direct product of PkS1 ( Fujii et al., 1999). Next, it is reduced to scytalone via the T4hN reductase arp2p, followed by dehydration to 1,3,8-tri hydroxy naphthalene (T3hN) by the scytalondehydratase arp1p. The following reduction to vermelon is arp2 dependent but the presence of other specific reductase(s) car-ing this reaction has been also proposed for Aspergilli and other fungi (Tsai et al., 1999; Wang and Breuil, 2002). The dehydration of vermelon to 1,8-dihydroxy naphthalene (DhN) is arp1p dependent. The result-ing DhN molecules are further polymerized to the structurally diverse melanins. This final enzymatic step involves the multicopper oxidase abr1p and laccase abr2p ( Jacobson, 2000; hamilton and Gomez, 2002; langfelder et al., 2003). Screening of the genome sequence of P. chrysogenum indicates the presence of the corresponded ortholog

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genes of the DhN- melanin BGC: abr1 (Pc21g16380), arp1 (Pc21g16420), arp2 (Pc21g16430), ayg1 (P21g16440), abr2 (P22g08420) and associ-ated pks17 (pcAlb1, Pc21g16000) were found partially clustered in the genome. To examine the role of hdaa in the biosynthesis of DhN-mel-anin in conidial pigmentation, the ΔhdaA mutant and DS68530 strains (no sorbicillinoids producers) were grown on solid r-agar medium for ten days, which resulted in a major decrease of the green conidial pig-mentation in the ΔhdaA mutant as compared to the reference strain. qPCr analysis of the putative DhN-melanin BGC indicated the 4-fold down-regulation of pks17 in the ΔhdaA mutant while arp1, arp2 and ayg1 were 4-fold up regulated. Expression of abr1 and abr2 was not significantly changed (Figure 5a–B). To determine if pks17 is involved in conidial pigment biosynthesis, the pks17 gene was deleted and overexpressed in order to identify the related polyketide product. a gene inactivation strain was obtained as described earlier (see materi-als and methods section) using primers listed in Table S1. The resulted Δpks17 mutant displayed an albino phenotype of the conidia while

-6

-4

-2

0

2

4

6

A

B

pks17 abr1 arp1 arp2 ayg1 abr2

pks1

7 ab

r1arp

1 arp

2 ay

g1ab

r2

Fold

cha

nge

Figure 5. A) Schematic representation of the DHN-melanin biosynthetic gene cluster. B) Quantitative Real Time PCR analysis of the DHN-melanin BGC in ∆hdaA_DS68530 mutant. Samples were taken after 7 days of growth on solid R-agar medium. The expression data is fold change (ΔhdaA/DS68530). Error bars indicate the standard deviation of two biological replicates with two technical replicates.

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grown on sporulating r-agar medium (Figure 6a). For the overexpres-sion, pks17 was placed under control of the isopenicillin N synthetase (pcbC) gene promoter and integrated into the genome. as a result, a 10–fold increase of the transcript level was obtained as compared to the reference strain. The solid medium grown mutant featured a defi-cient colouring of the conidia and intense pigmentation of the bottom surface of the colony (Figure 6a). To identify the accumulated prod-uct, extracted r-agar medium of a 7 day grown culture was analyzed

Figure 6. A) Pigmentation differences between DS68530, ΔhdaA_DS68530, Δpks17 and oepks17 strains. Top (left) and bottom (right) of the plate. The pic-ture has been taken after 14 days of growth grown on solid R-agar medium. B) Scanning electron microscopy of the condia of strain DS68530, ΔhdaA, Δpks17 (albino mutant) and the oepks17 mutant. The cell wall surface of the conidia for Δpks17 and oepks17 strains is shown. The ΔhdaA mutant displays a more pronounced relief of the conidial surface ornamentation in comparison to the reference strain DS68530. C) Percent survival of conidia grown in presence of hydrogen peroxide on R-agar medium. Germinated colonies were counted af-ter 5 days of growth. Error bars indicate the standard deviation of two biolog-ical replicates with two technical replicates.

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by the full mass range lC-ESI-MS orbitrap (Thermo Fisher Scientific, San Jose, Ca). The overproduced metabolite with the exact mass m/z [M-h]⁻ 275.06 has been detected. The elemental composition of the deprotonated molecule has been calculated as C14h11o6 using build-in Qual Browser tool of Excalibur 2.1 (Thermo Fisher Scientific, San Jose, Ca) with 0.35 ppm accuracy. The found mass, calculated el-emental formula and characteristic fragmentation pattern belongs to the known heptaketide yWa1 of A. nidulans produced by highly ho-mologous PkS wa that is involved in the conidial DhN-melanin bio-synthetic pathway (Figure 3S). In addition to analyzed strains, the sor-bicillinoids producer strains (mutant and parental), were also grown on SMP medium for 3 and 5 days. Transcriptional analysis showed an overexpression of the genes that belong to putative DhN-melanin BGC in all the tested strains. an up to 50-fold increase was observed in Pks17 overproducing strain (Figure 4S). however, it was not possi-ble to detect yWa1 in the supernatant obtained from the mycelium grown in liquid culture.

ROLE OF PKS17 IN CONIDIA FORMATION AND TOLERANCE TO OXIDATIVESTRESS

Melanins are important components of the conidial cell wall and its in-tegrity. They play an essential role in physical properties of the spores like surface interaction, hydrophobicity and virulence in pathogenic fungal species. The effect of hdaA deletion on the conidial surface in P. chrysogenum was examined using scanning electron microscopy. The conidia of the reference DS68530, ΔhdaA_DS68530, Δpks17 and over expression mutant oepks17 were isolated from colonies grown for seven days on the sporulating r-agar medium. The reference DS68530 exhibited a typical tuberous surface of the conidia, while the spores of albino mutant Δpks17 were smooth. The texture of the ΔhdaA_DS68530 spores surface was more pronounced compared to the parental strain but without dramatic changes of the conidial cell wall appearance (Figure 6B). These data suggest that Pks17 is involved in pigment formation and influences the morphology of the conidia in P. chrysogenum. The Pks17 protein was renamed alb1 according to the nomenclature of A. fumigatus.

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DISCUSSIoN

apart from the mechanical properties of the pigments (howard et al., 1991) and their role as ph buffering systems, the scavenging of reac-tive oxygen species is an important feature supporting UV and ther-mo-tolerance and pathogenicity of the conidia (Jacobson et al., 1995; kawamura et al., 1997; romero-Martinez et al., 2000). The P. chrysoge-num ΔhdaA_DS68530 mutant was grown on hydrogen peroxide sup-plemented medium for 5 days to verify the ability of the conidia to sur-vive oxidative stress conditions in the absence of pigmentation. The survival rate was decreased by 20% when the ΔhdaA strain was ex-posed to 2 mM of hydrogen peroxide in the media. Under the same conditions, the survival rate of the Δpks17 (Δalb1) was reduced more than 50%. There was no enhanced survival observed for the oepks17 overexpression mutant (Figure 6C).

DISCUSSION

recent genome sequencing and metabolite analysis studies revealed that the majority of the potential biosynthetic gene clusters (BGCs) present in genomes of filamentous fungi are silent or expressed at a low level under standard laboratory conditions. These non-expressed BGCs represent a potential untapped source of novel bioactive mole-cules. activation of the secondary metabolites production via deletion or chemical inhibition of histone deacetylases was recently reported for filamentous fungi as an effective tool for silent SM gene clusters activation and identification of new metabolites with potential phar-maceutical properties (Tribus et al., 2005; Shwab et al., 2007; Fisch et al., 2009; lee et al., 2009). here we have examined the effect of chromatin modification on expression of the secondary metabolism associated genes and products in the fungus P. chrysogenum. In this work, the P. chrysogenum hdaA gene encoding an orthologue of the class 2 histone deacetylase Hda1 of S. cerevisiae was deleted. The dele-tion mutant showed significant changes of secondary metabolite gene expression including PkS and NrPS genes with known and unknown function (Figure 1). In ΔhdaA mutants the transcriptional levels of the sorbicillinoids BGC were significantly increased. SorA and sorB genes, which encodes for the two polyketide synthases (highly reducing and non-reducing, respectively) involved in the sorbicillinoids pathway

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(Salo et al., 2016) were overexpressed in the ΔhdaA_DS38530 strain. Interestingly, overexpression of the sorbicillinoids BGC was also ob-served in the DS38530res13 strain, in which hdaA was not deleted. This has been attributed to a complex regulation mechanism that in-volves sorbicillinoids as auto inducers (Guzmán-Chávez et al., 2017). This phenomenon could potentially also involve hdaa altering the chromatin landscape (Brosch et al., 2008), since the deletion of the hdaA gene in the sorbicillinoids producer strain (Figure 2a) resulted in an additive effect (up to 500-fold) on the expression levels of both pks genes. Indeed all genes that belong to the BGC showed a similar trend likely because the chromatin state can module gene expression by improving the binding of transcriptional factors (Macheleidt et al., 2016). These observations agree with the pronounced effect of the hdaA deletion on sorbicillinoids production (Figure 2B), and the ear-lier onset of production (data not shown). In contrast, the transcript levels of the chyA (nrps9) gene of chrysogine biosynthesis was signif-icantly (25-fold) reduced in the ΔhdaA mutants but this reduction in expression was also observed in the sorbicillinoids producing strain (Figure 4). qPCr analysis indicated that the chrysogine BGC (Viggiano et al., 2017) was down regulated in the aforementioned strains with a corresponding decrease of chrysogine related metabolites in the cul-ture supernatant [19]. likely, the chrysogine BGS is subjected to epl-genetic activation by hdaa, but at the same time sorbicillinoids pro-duction reduces the expression of this gene cluster.

In A. nidulans and A. fumigatus, the homologous hdaA is a main con-tributor of histone deacetylase activity in these fungi. In A. nidulans, de-letion of the hdaA gene stimulated penicillin and sterigmatocystin pro-duction but not a telomere-distal gene cluster involved in terraquinone a biosynthesis. It was suggested, that hdaa silences the expression of subtelomeric chromosomal regions (Tribus et al., 2005). Contrary, the hdaa homolog in A. fumigatus was reported to activate gliotoxin biosynthesis and to repress several NrPSs including one gene of the siderophore BGC. a subtelomeric specificity of hdaa was not appar-ent for this fungus (lee et al., 2009). In silico comparative analysis of the P. chrysogenum genome sequence revealed four chromosomes on which all BGC are distributed (Specht et al., 2014). We performed ex-pression analysis of all 11 NrPS and 20 PkS genes in the ΔhdaA strain. The results show that the expression of eight secondary metabolite

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DISCUSSIoN

genes was significantly altered in the ΔhdaA strain including the acti-vation of a silent PkS cluster with unknown function. It is important to stress that the particular effect seems to be restricted to chromosome 2 and the chromosome 1 extremes. The chromosome 2 region con-tains a remarkably large number of BGCs, comprising 15 of the 32 PkS and NrPS encoding genes. The few remaining BCGs are distributed throughout the other chromosomes (Figure S2). The action of hdaa thus seems mostly to be restricted to the transcriptional co-regulation of a particular genomic area rich in BGCs (van den Berg et al., 2008).

The production of sorbicillinoids and the deletion of the hdaA gene, causing increased sorbicillinoids production, had similar effects on the expression of other PkS and NrPS genes (Figure 1,2,4) including the BGC that specifies chrysogine. one possible explanation is that sor-bicillinoids might act as hdaa inhibitors, since hdaA was transcribed at the same levels in the sorbicillinoids producer and non-producer strains (data not shown) while feed sorbicillinoids did not alter the tran-scription of hdaa in the DS68530 strain. Moreover, a novel compound was detected only when hdaA was deleted or when DS68530 was fed with sorbicillinoids (Figure 3). a similar phenomena occurs when Cla-dosporium cladosporioides and A. niger are exposed to suberoylanilide hydroxamic acid (Saha), a hDaC inhibitor, which induces the synthe-sis of two new compounds, cladochrome and nygerone a, respectively (rutledge and Challis, 2015). our work respresents the first example of a regulatory cross talk between BGCs in P. chrysogenum. In A. nid-ulans, overexpression of a regulator (scpR) was found to activates the expression of two cryptic NrPS genes, belonging to the same cluster, as well as the induction of genes responsible for the production of the polyketide asperfuranone (Bergmann et al., 2010; Brakhage, 2012).

The deletion of the hdaA gene also has a functional effect in P. chrysogenum, since it decreases green conidial pigmentation and an altered surface structure of the spores. The function of the 4-fold down-regulated pks17 (Pc21g16000) gene was elucidated via gene deletion and overexpression. This PkS enzyme shows a high similar-ity to the A. nidulans wa and A. fumigatus PksP proteins involved in conidial pigment biosynthesis (van den Berg et al., 2008). To iden-tify the polyketide product, pks17 was overexpressed causing the accumulation of the yellow naphtho-γ-pyrone (a polyketide pre-cursor of the conidial pigment) into the medium. This indicates that

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P. chrysogenum uses the DhN-melanin biosynthetic pathway like previously reported for Aspergillus (Jacobson, 2000; hamilton and Gomez, 2002; langfelder et al., 2003). The corresponding Δpks17 strain showed an albino phenotype confirming the primary role of this gene in the conidial pigmentation. In the closely related fungus A. fumigatus at least six genes are required for DhN-melanin bio-synthesis, which were found to be partially clustered in the genome of P. chrysogenum. qPCr analysis (Figure 5) showed the 4-fold up regulation of the arp1 (scytalondehydratase), arp2 (T4hN reductase) and ayg1 (enzyme of hydrolytic polyketide chain shortening activity) genes, while the transcript level of abr1 (multicopper oxidase) and abr2 (laccase) which products catalyze the last steps of the polymer-ization of 1,8-dihydroxynaphthalene were not significantly changed. Scavenging of reactive oxygen species by fungal melanins provides an important defence mechanism during growth under oxidative stress condition. We examined the effect of hdaA deletion on the ability of the conidia to survive high concentrations of hydrogen per-oxide. an increased sensitivity of the hdaa was noted towards hy-drogen peroxide while this effect was even more pronounced for the Δpks17 albino mutant (Figure 6C). These results suggest that the ox-idative stress response in P. chrysogenum involves hdaa and is medi-ated by the transcriptional regulation of DhN-melanin gene cluster.

In conclusion, our results demonstrate that hdaa has a broad im-pact on secondary metabolism of P. chrysogenum at the transcriptional level causing marked changes in metabolite production. Furthermore, hdaa influences conidial pigmentation and the surface structure of spores. This work provides evidence of cross-talk between gene clus-ters, which impacts secondary metabolism. The presented data sug-gest that an epigenome approach can be successfully applied for novel biosynthetic pathways discovery in P. chrysogenum.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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aUThor CoNTrIBUTIoNS

AUTHOR CONTRIBUTIONS

FGC, oS and MS designed the study, performed the experiments, wrote the manuscript and carried out the data analysis. Jk performed the elec-tron microscopy analysis. Mr performed the initial lC-MS analysis and with rJV, helped in the identification of metabolites. ralB contributed in the coordination of the project and the revision of the manuscript. aJMD conceived the study, supervised and coordinated the design, the data interpretation and corrected the manuscript.

ACKNOWLEDGEMENTS

This work was supported by the Perspective Genbiotics program subsidized by Stichting toegepaste wetenschappen (STW) and (co) financed by the Netherlands organization for Scientific research (NWo), the Netherlands Metabolomics Centre (NMC) which is a part of the Netherlands Genomics Initiative, and the Integration of Bio-synthesis and organic Synthesis (IBoS) programme residing under advanced Chemical Technologies for Sustainability (aCTS) which is subsidized by NWo. FGC was supported by Consejo Nacional de Ciencia y Tecnología (CoNaCyT, México) and Becas Complemento SEP ( México). oS and Mr were supported by STW. MS was sup-ported by NWo. The authors acknowledge DSM Sinochem Pharma-ceuticals (Delft, The Netherlands) for kindly providing the DS68530 strain. The authors wish to thank Carsten Pohl for help with the schematic representation of NrPS and PkS over the chromosomes of P. chrysogenum.

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SUPPorTING INForMaTIoN

SUPPORTING INFORMATION

Table S1. Primer list used in this study.

Primer name Gene Sequence 5’–3’ Function ReferencePKS1F Pc12g05590 GCTACAGCCCTGACGCCATGG

qPCR

PKS

s/N

RPSs

Salo et al.,2015

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 ACGTACGCTCGAGCTGGACTNRPS3R Pc14g00080 GCCGTCGCGTTGATAATTGGNRPS4F Pc16g03850 TGGTTGAAAGAGGGCAGTCTCNRPS4R Pc16g03850 CGCGAACATACACAACACCACNRPS5F Pc16g04690 CTTTCCAGAACAGTTGGCTGGTNRPS5R Pc16g04690 GCTGCATCTTACCCAGGTAATTGNRPS6F Pc16g13930 CCACCCTTGTTCAGCCGCTGAATTCCNRPS6R Pc16g13930 GGACGAGGCGAACAACATCGGACNRPS7F Pc21g01710 GCTATCTCGGTGGAGGATCTTCTGTCCNRPS7R Pc21g01710 GTGCTGCTGAGAACACGGGATTGTNRPS8F Pc21g10790 GTGAGGCAGCTTTGTTCAACACCATT

Page 34

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Primer name Gene Sequence 5’–3’ Function ReferenceNRPS8R Pc21g10790 TTCTGCAGCAGGCTGTCGGCCTGAG

qPCR

PKS

s/N

RPSs

Salo et al.,2015

NRPS9F Pc21g12630 GAGCCAACTCTGTTGTCTACGNRPS9R Pc21g12630 CAGGGCAATTTGCCTCATTCTGNRPS10F Pc21g15480 CTTGGTGGATGCAGCGAAGGNRPS10R Pc21g15480 CTGTGAGAGAGGCTCTTGAGTANRPS11F Pc22g20400 TTCGCGAACATCCGAAGAAGCNRPS11R Pc22g20400 TCGGGCGAAGACACTGTTCA 12.50F Pc21g05050 GAAGCGTGGATAGAGACCGAAGAGAAC

qPCR

of t

he e

xpre

ssed

So

rbic

illin

oids

gen

e cl

uste

r

Salo et al.,2015Guzman-Chavezet al., 2017

12.50R Pc21g05050 GGAGCCAGACTCCGGAAAGGATACTG12.60F Pc21g05060 GATAGTGAGTACAAATGCGCCTGGACC12.60R Pc21g05060 CGTTCAATACCGGAAATGGCTAGATTCG12.70F/PKS12F Pc21g05070 GTCGGAGGCAATTCGGGAAGGC12.70R/PKS12R Pc21g05070 GCAAAGTTCCACCACAATGCCGCG12.80F/PKS13F Pc21g05080 CCGAGGATCTCCGCCAGGC12.80R/PKS13R Pc21g05080 GGTTGTGCAGGTTCCAGGTGCC12.90F Pc21g05090 CGTTAACTAATGACGCCACCTGTTGC12.90R Pc21g05090 GGAAAATAGTATCCCCAGCGATTGGC12.100F Pc21g05100 CATCAGCACCGAGGTCTTCATTGTCG12.100R Pc21g05100 GCAACGCAATAGATGGTCAATGCCAG12.110F Pc21g05110 CTGCAGCACTTCAGCATGGATGAAACC12.110R Pc21g05110 TCGTTGTGAGACTTGGATGCTCGGACG12.120F Pc21g05120 CCTGCTTCTTAATCTTGCCCTGGC12.120R Pc21g05120 CCAAGCCGATGCCAAGAAGGAAGAG s570F Pc21g12570 GGCAAGGGAAATGAATCCAGGTGGC

qPCR

of t

he C

hrys

ogin

ege

ne c

lust

er

s570R Pc21g12570 GATAGATGCCGCTTGTTCGGACCs590F Pc21g12590 GGTTGTGGAGCTCTACGAGGCTGs590R Pc21g12590 CTGGCAGGGCTCGTCGGTCS600F Pc21g12600 GTAGACGCCGGTGAGACTTTGATCGS600R Pc21g12600 CAACCTAAGCGTCTAATTTTCATCGCs610F Pc21g12610 CCTGCATGCAGCTCCATACGAGC Viggiano s610R Pc21g12610 CCAACAATAGGTGGAAACAGCTCAGAC et al., 2017s620F Pc21g12620 GGAATTCGCTGGCTAACTGGTCTCGs620R Pc21g12620 GGCATGTGGTAGACGAATTGGAGCs630F/ NRPS9F Pc21g12630 GAGCCAACTCTGTTGTCTACGs630R/ NRPS9R Pc21g12630 CAGGGCAATTTGCCTCATTCTGs640F Pc21g12640 TGTCTCTCTGTGGGCTGTTCTCAGs640R Pc21g12640 CAAGAGTTCTTACGATGCGTGGCTG

abr1F Pc21g16380 GTCTACCTGAACTGGAACCTCACTTGG

qPCR

of D

HN

-mel

anin

gen

e cl

uste

r

abr1R Pc21g16380 GGTGAGTGTCAACTCTTCATCAAAGTGGarp1F Pc21g16420 TCTTCAATTCTTCAGTCTCGTAGACCTGGarp1R Pc21g16420 CCGAGCCCAACTTGGACATCAGC This studyarp2F Pc21g16430 GAGGGTTTTACTCGCTGCTTTGCTGCarp2R Pc21g16430 GATGTTAGCTCCACCGTTGCAAGGCayg1F Pc21g16440 GCTATGGCGGAGAAGTATGGCTATGACayg1R Pc21g16440 CTCTCCAACCACTTGTAAGCCACAGGabr2F Pc22g08420 GCTCAATGTAATGTCCATCCACCTCGabr2R Pc22g08420 GACCCTGAGTATCTGACAAATCTCCAGC

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SUPPorTING INForMaTIoN

Table S1—Continued. Primer list used in this study.

Primer name Gene Sequence 5’–3’Func-tion

attB4F∆HdaPF Pc21g14570 GGGGACAACTTTGTATAGAAAAGTTGCGTTTAAAGGCAGCCAAAGACTAAACTCAGTA

Clon

ing/

pHda

A/S

eque

ncin

g

attB1R∆HdaPR Pc21g14570 GGGGACTGCTTTTTTGTACAAACTTGCAAGGGAAAGCCACGGGAAGC

attB2F∆HdaPF Pc21g14570 GGGGACAGCTTTCTTGTACAAAGTGGCCTGATTCGAGCGTGAACCC

attB3R∆HdaPR Pc21g14570 GGGGACAACTTTGTATAATAAAGTTGTTTAAATGGTTGGTCACGACAGCGTT

ColonyPCR F1 Pc21g14570 CTCTTCACTGGCTTGTACATTCTGCGColonyPCR R1 Pc21g14570 ATTCACACGTGCTAGTGGACSeq∆Hda1 Pc21g14570 AGGCAGCCAAAGACTAAACTSeq∆Hda2 Pc21g14570 GGAGGCAATAACTGCAGTAGSeq∆Hda3 Pc21g14570 GACGAGTGATGGTGATAGTTCSeq∆Hda4 Pc21g14570 GAACGTTGACATCGATCACSeq∆Hda5 AMDS cassette GCGAGACAGTCAACAACATCSeq∆Hda6 AMDS cassette AGGAGCCATGGAAATACGProbeF Pc21g14570 GTACATCCATGGATGTTTCTGCTCATATTTGCProbeR Pc21g14570 CTTATATAGTTTCCCTGCTGGTGGATTGAGC attB4F∆pks17 Pc21g16000 GGGGACAACTTTGTATAGAAAAGTTGGCTGTCATTGAGTCGCT

AGGTTATCTCC

Clon

ing

/ pK

O17

attB1R∆pks17 Pc21g16000 GGGGACTGCTTTTTTGTACAAACTTGCCAGTGGCGAATTATTGGTTTCAGGCG

attB2F∆pks17 Pc21g16000 GGGGACAGCTTTCTTGTACAAAGTGGGTGCCTACTTCCAGGACATTTGTATATGGG

attB3R∆pks17 Pc21g16000 GGGGACAACTTTGTATAATAAAGTTGGATTCAACTAACATTTGTGGCAGGACGAGG

attB4Foepks17 Pc21g16000 GGGGACAACTTTGTATAGAAAAGTTGGATGACCCACGTGCATA

AGTGACAGC

Clon

ing

/ pO

E17

attB1Roepks17 Pc21g16000 GGGGACTGCTTTTTTGTACAAACTTGCCAGTGGCGAATTATTGGTTTCAGGCG

attB2Foepks17 Pc21g16000 GGGGACAGCTTTCTTGTACAAAGTGGATGGAAGGCCCCGGTCATGTATATCTC

attB3Roepks17 Pc21g16000 GGGGACAACTTTGTATAATAAAGTTGCAAACATTCCGGCGTCGTTATACCAGC

Page 36

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149SUPPorTING INForMaTIoN

23

9.46.5

4.3

2.32.0

(kb)

atg

atg

amdS

hdaA

3.1 kb

4.3 kb

DS68530

ΔhdaApgpdA

5’ FR hdaA 3’ FR hdaA

5’ FR hdaA 3’ FR hdaA

A) B)

C)

L

atg

atg

5845 bp

5692 bp

DS68530

ΔhdaA

pgpdA5’ FR hdaA 3’ FR hdaA

5’ FR hdaA 3’ FR hdaA

amdsSalI

1551 bp

*SalI is a noncu�er

hdaA

Sequenced PCR products

Southern Blot

hdaA_DS68530

hdaA_DS68530Res13

DS68530

Figure S1. A) Southern blot analysis of P. chrysogenum strains with individ-ual hdaA gene deletions. B) Scheme of the replacement of the hdaA gene in P. chrysogenum with the amdS cassette. The length of DNA fragments detected by Southern Blot is indicated for ΔhdaA and DS56530 strains. C) Scheme of PCR products sequenced from ΔhdaA and DS56530 strains. Black arrows in-dicate the used primers (Colony PCR primers). Restriction enzyme used during the screening for positive colonies is marked.

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SUPPorTING INForMaTIoN

140

120

100 80 60 40 20 0

Chr

1C

hr2

Chr

3C

hr4

Pc06

g015

40 N

RPS

-Lik

e 1(

-)

Pc16

g099

30 N

RPS

-Lik

e 6(

-)

Pc16

g114

80 P

KS 7

(-)

Pc13

g086

90 P

KS 3

(-)

Pc16

g139

30 N

RPS

6(-)

Pc13

g052

50 p

SSC

(Fer

richr

ome)

Pc13

g044

70 P

KS 2

(-)

Pc

18g0

0380

NR

PS-L

ike

7 (S

afra

myc

in)

Pc

12g1

3170

NR

PS-L

ike

3(-)

Pc

22g0

9430

NR

PS-L

ike

15(-)

Pc22

g204

00 p

SSB(

Fusa

rinin

e C

)

Pc22

g228

50 A

drD

(And

rast

in A

)Pc

22g2

3750

PKS

20(

-)

Pc

14g0

1790

NR

PS-L

ike

5 (-)

Pc

14g0

0080

NR

PS 3

(-)

Pc21

g226

50 N

RPS

-Lik

e 13

(-)Pc

21g2

2530

NR

PS-L

ike

12(-)

Pc21

g213

90 p

cbAB

(Pen

icilli

n)

Pc20

g126

70 N

RPS

-Lik

e 11

(-)

Pc20

g096

90 N

RPS

-Lik

e 10

(-)

Pc20

g025

90 N

RPS

-Lik

e 9(

-)

Pc20

g022

60 N

RPS

-Lik

e 8(

-)

Pc21

g009

60 P

KS 8

(-)

Pc21

g017

10 N

RPS

7(B

revi

anam

ide

F)

Pc21

g039

30 P

KS 9

(-) P

c21g

0399

0 PK

S 10

(-)Pc

21g0

4840

PKS

11(

-)Pc

21g0

5080

Sor

A (S

orbi

cillin

oids

)Pc

21g0

5070

Sor

B (S

orbi

cillin

oids

)

Pc21

g107

90 N

RPS

8(H

expe

ptid

e)Pc

21g1

2450

PKS

15(

-)Pc

21g1

2440

PKS

14(

-)Pc

21g1

2630

Chy

A(C

hrys

ogin

e)

Pc21

g154

80 R

oqA(

Roq

uefo

rtine

/Mel

eagr

ine)

Pc21

g160

00 P

cAlb

1(YW

A1/D

HN

-mel

anin

e)

Pc21

g151

60 P

KS 1

6(-)

Pc22

g081

70 P

cPat

K(6-

MSA

/Pat

ulin

e) P

c22g

0903

0 PK

S-Li

ke 3

(-)

Pc12

g099

80 N

RPS

-Lik

e 2(

-)

Pc12

g055

90 P

KS 1

(-)

Pc12

g055

90 P

KS-L

ike

1(-)

Pc13

g143

30 N

RPS

3(-)

Pc13

g125

70 N

RPS

-Lik

e 4(

-) P

c16g

0376

0 PK

S-Li

ke 2

(-) P

c16g

0489

0 PK

S 6(

-) P

c16g

0469

0 hc

pA (F

ungi

spor

in)

Pc1

6g03

850

pssA

(Cop

roge

n) P

c16g

0380

0 PK

S 5(

-)

Pc16

g003

70 P

cYan

A(6-

MSA

/Yan

utho

nes)

MBFi

gure

S2.

Sch

emati

c re

pres

enta

tion

and

dis-

trib

ution

of P

KS a

nd

NRP

S (-l

ike)

gen

es o

ver

the

four

chr

omos

omes

of

P. c

hrys

ogen

um. I

n sil

ico a

naly

sis w

as p

er-

form

ed th

e ge

nom

e of

P.

chry

soge

num

Wis5

4-12

55 a

nd o

f the

indu

s-tr

ial p

roge

nito

r str

ain

P. ch

ryso

genu

m P

2nia

D18

(S

pech

t et a

l., 20

14).

Blue

line

s in

dica

te

know

n co

mpo

unds

. Red

lin

es in

dica

te u

nkno

wn

com

poun

ds. A

dapt

ed

from

(Ali

et a

l., 2

013;

Sp

echt

et a

l., 2

014;

Sal

o et

al.,

201

6; S

amol

et a

l.,

2016

; Guz

mán

-Chá

vez

et a

l., 2

017)

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Figure S3. A) HPLC-MS extracted ion chromatogram (range m/z [M-H]- 275.05-275.06) of the naphtho-γ-pyrone (YWA1) produced by oepks17 strain (above) versus no production by DS68530 (below). B) HPLC-MS spectra containing ex-act mass (m/z [M-H]⁻ 275.06) and calculated elemental formula (ppm 0.33) of the deprotonated naphtho-γ-pyrone (YWA1) together with the related frag-ment acquired by in-source (ESI) fragmentation in negative mode. Schematic representation of the fragmentation is shown.

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 320m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

191.03

275.06

217.01

265.15233.05173.02 289.04205.01 246.97

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

+

B

C10H7O4 C14H12O6

YWA1

C14H11O6

[M-H]-C4H4O2

C10H6O4

[M-H]-

A

Time (min)

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Figure S4: Quantitative Real Time PCR analysis of the gene cluster of DHN- melanin biosynthesis by cells grown for 3 days in SMP medium. Strains: DS685Res13 (black bars), ΔhdaA_DS68530Res13 (withe bars), ΔhdaA_DS68530 (grey bars). Data are shown as a fold change relative to P. chrysogenum DS68530. (ΔhdaA / DS68530). (*) Indicates lack of expression. Error bars indicate the stan-dard deviation of two biological with two technical replicates.

pks17 abr1 arp1 arp2 ayg1 abr2

0.3

0.5

1.0

2.0

4.0

8.0

16.0

32.0

64.0

128.0

***

Fold

cha

nge

Page 40

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