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Journal of Proteomics
journal homepage: www.elsevier.com/locate/jprot
Catabolism of phenylacetic acid in Penicillium rubens. Proteome-wideanalysis in response to the benzylpenicillin side chain precursor
Mohammad-Saeid Jamia,b, Juan-Francisco Martínc,⁎, Carlos Barreiroa,Rebeca Domínguez-Santosa,c, María-Fernanda Vasco-Cárdenasa,c, María Pascuala,Carlos García-Estradaa,d,⁎⁎
a INBIOTEC, Instituto de Biotecnología de León, Avda. Real n°. 1, Parque Científico de León, 24006 León, Spainb Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iranc Área de Microbiología, Departamento de Biología Molecular, Universidad de León, Campus de Vegazana s/n, 24071 León, Spaind Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain
A R T I C L E I N F O
Keywords:Phenylacetic acidBenzylpenicillin2-hydroxyphenylacetatePhenylacetate hydroxylaseProteomicsPenicillium rubens
A B S T R A C T
Biosynthesis of benzylpenicillin in filamentous fungi (e.g. Penicillium chrysogenum - renamed as Penicillium ru-bens- and Aspergillus nidulans) depends on the addition of CoA-activated forms of phenylacetic acid to iso-penicillin N. Phenylacetic acid is also detoxified by means of the homogentisate pathway, which begins with thehydroxylation of phenylacetic acid to 2-hydroxyphenylacetate in a reaction catalysed by the pahA-encodedphenylacetate hydroxylase. This catabolic step has been tested in three different penicillin-producing strains ofP. rubens (P. notatum, P. chrysogenum NRRL 1951 and P. chrysogenum Wisconsin 54–1255) in the presence ofsucrose and lactose as non-repressing carbon sources. P. chrysogenumWisconsin 54–1255 was able to accumulate2-hydroxyphenylacetate at late culture times. Analysis of the P. rubens genome showed the presence of severalPahA homologs, but only Pc16g01770 was transcribed under penicillin production conditions. Gene knock-downexperiments indicated that the protein encoded by Pc16g01770 seems to have residual activity in phenylaceticacid degradation, this catabolic activity having no effect on benzylpenicillin biosynthesis. Proteome-wide ana-lysis of the Wisconsin 54–1255 strain in response to phenylacetic acid revealed that this molecule has a positiveeffect on some proteins directly related to the benzylpenicillin biosynthetic pathway, the synthesis of amino acidprecursors and other important metabolic processes.Significance: The adaptive response of Penicillium rubens to benzylpenicillin production conditions remains to befully elucidated. This article provides important information about the molecular mechanisms interconnectedwith phenylacetate (benzylpenicillin side chain precursor) utilization and penicillin biosynthesis, and willcontribute to the understanding of the complex physiology and adaptation mechanisms triggered by P. rubens (P.chrysogenum Wisconsin 54–1255) under benzylpenicillin production conditions.
1. Introduction
Since Fleming's fortuitous discovery of penicillin ninety years ago,constant efforts have been made by the scientific community from in-dustry and academia in order to improve penicillin titers. This has beenachieved mainly by industrial strain improvement programs, whereselected strains have been subjected during the last decades to severalrounds of radioactive and chemical mutagenesis, thus reaching producttiters and productivities three orders of magnitude higher than thoseprovided by the ancestor strains [1]. The fungal strain producing the
antimicrobial agent penicillin was initially identified by Fleming andcolleagues as Penicillium rubrum, which was later re-identified as Peni-cillium notatum and finally placed in synonymy with Penicillium chry-sogenum. However, this nomenclature has been recently reconsidered,leading to the conclusion that Fleming's original strain, the full genomesequenced strain P. chrysogenum Wisconsin 54–1255 and its ancestorstrain P. chrysogenum NRRL 1951 (wild-type), are in fact Penicilliumrubens [2]. Although all these strains are now classified as P. rubens, forthe sake of clarity the old names P. notatum, P. chrysogenum Wisconsin54–1255 and NRRL-1951 are also used in this article, since hundreds of
https://doi.org/10.1016/j.jprot.2018.08.006Received 14 May 2018; Received in revised form 17 July 2018; Accepted 4 August 2018
⁎ Corresponding author.⁎⁎ Corresponding author at: INBIOTEC, Instituto de Biotecnología de León, Avda. Real n°. 1, Parque Científico de León, 24006 León, Spain.E-mail addresses: [email protected] (J.-F. Martín), [email protected], [email protected] (C. García-Estrada).
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Available online 06 August 20181874-3919/ © 2018 Published by Elsevier B.V.
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references using the old names have been published.Much of the efforts made by the scientific community have focused
on the biochemical and genetic characterization of the penicillin bio-synthetic pathway [3] (Fig. 1), which is compartmentalized betweencytosol and peroxisomes (for reviews see [4, 5]). It starts with the non-ribosomal condensation of L-α-aminoadipic acid, L-cysteine and L-va-line by means of the nonribosomal peptide synthetase L-δ(α-aminoa-dipyl)-L-cysteinyl-D-valine (ACV) synthetase (ACVS), which is a verylarge multifunctional protein (MW 426 kDa). This protein is encoded bythe single structural 11-kbp pcbAB gene. Next, in a reaction catalysed bythe isopenicillin N (IPN) synthase or cyclase (encoded by the pcbCgene), the ACV undergoes the oxidative ring closure of the tripeptide.This leads to the formation of the bicyclic structure (penam nucleus) ofIPN in the cytosol. In the last step of the penicillin biosyntheticpathway, the α-aminoadipyl side chain of IPN is replaced inside per-oxisomes by a hydrophobic side chain activated as thioester with CoA.
In the case of benzylpenicillin, the side chain precursor is phenylaceticacid, which is activated in the form of phenylacetyl CoA. Replacementis catalysed by the penDE-encoded acyl-CoA: IPN acyltransferase (IAT),which is synthesized as a 40-kDa precursor protein (proIAT) that un-dergoes self-processing between residues Gly102 and Cys103. There-fore, the active protein is a heterodimer comprising two subunits: α(11 kDa, corresponding to the N-terminal fragment) and β (29 kDa,corresponding to the C-terminal region). Activation of the side chainprecursor is achieved by means of aryl CoA-ligases. At least three aryl-CoA ligases, encoded by the phl, phlB (aclA) and phlC genes, respec-tively, have been reported to activate phenylacetic acid [6–9]. How-ever, direct contribution to penicillin biosynthesis has only been de-scribed in the case of the phenylacetyl CoA ligase encoded by the phlgene [6].
Research has also been focused on the characterization of themodifications introduced by industrial strain improvement programs.
Fig. 1. Metabolic routes of phenylacetic acid in P. rubens: penicillin biosynthetic pathway (upper chart) and homogentisate pathway (lower chart).
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Amplification of the penicillin gene cluster is well documented in manyof the improved penicillin producers, which contain several copies ofthis cluster (e.g. the AS-P-78 strain contains 5 or 6 copies) [10]. Mi-crobodies (peroxisomes), the organelles where activation of the sidechain and its incorporation to the IPN molecule occur, are moreabundant in high-producer strains [11, 12]. Genome and transcriptomeanalyses have also revealed that transcription of genes involved in thebiosynthesis of the penicillin amino acid precursors, as well as of thosegenes encoding microbody proteins, was higher in the high-producerstrain DS17690 [12]. More recently, proteomics studies concluded thatthe increase in penicillin production along the industrial strain im-provement program was a consequence of complex metabolic re-organizations, and suggested that energetic burden, redox metabolismor the supply of precursors are crucial for the biosynthesis of this an-tibiotic [13].
Besides this background knowledge, it is well known that thehomogentisate pathway for the catabolism of phenylacetic acid (theside chain precursor in the biosynthesis of benzylpenicillin) to fumarateand acetoacetate (Fig. 1) is diminished in Wisconsin 54–1255, andpresumably, in derived strains as well [14, 15]. Phenylacetic acid is aweak acid that is toxic to cells depending on its concentration andculture pH. This compound can be metabolized in P. rubens (P. chry-sogenum) and Aspergillus nidulans (another filamentous fungus with theability to biosynthesize benzylpenicillin) through at least two routes;incorporation to the benzylpenicillin molecule or catabolism via thehomogentisate pathway, which is also used to catabolize phenylalanineand tyrosine [16–21]. The first step of the phenylacetic acid catabolicpathway is a 2-hydroxylation by a microsomal cytochrome P450monooxygenase (phenylacetate hydroxylase) encoded in P. rubens bythe pahA gene (Pc21g14280) (Fig. 1). Little is known about the globalresponses of this microorganism to the addition of the side-chain pre-cursor, such as specific effects on key enzymes of primary and sec-ondary metabolism. Harris and co-workers [22] dissected the effects ofphenylacetic acid on chemostat cultures using a microarray-basedanalysis. These authors found that the homogentisate pathway wasstrongly transcriptionally upregulated in those cultures supplementedwith the side chain precursor, as well as those genes involved in ni-trogen and sulphur metabolism. This study provided an initial globaloverview about the effect of phenylacetic acid on fungal physiology,although full exploitation of P. rubens (P. chrysogenum) requires theintegration of knowledge from other “omics”, such as proteomics.
In this work we provide information about the catabolism of phe-nylacetic acid in three different strains of P. rubens (P. notatum, P.chrysogenum NRRL 1951 and P. chrysogenum Wisconsin 54–1255),characterize the function of a putative phenylacetate hydroxylasehomolog in phenylacetate degradation and penicillin biosynthesis, andanalyse global modifications of the P. chrysogenum intracellular andextracellular proteomes to the addition of the benzylpenicillin sidechain precursor.
2. Materials and methods
2.1. Strains and growth conditions
Three strains of P. rubens (Fleming's original isolate P. notatum; thewild-type strain P. chrysogenum NRRL 1951; and the reference strain forthe genome and proteome projects, P. chrysogenum Wisconsin 54–1255[12, 13, 23]), were used in this work. They were grown on solid Powermedium [24] for seven days at 28 °C. Conidia from one Petri dish werecollected and inoculated into a 500-mL flask containing 100mL of de-fined inoculation medium (DIM) with 40 g/L glucose [24]. After 24 h ofincubation at 25 °C and at 250 rpm, a 10% of inoculum was added to a500-mL flask containing 100mL of defined production medium(MDFP), which was prepared by adding 1 g/L potassium phenylacetate,30 g/L lactose and 10 g/L sucrose to the DIM medium without glucose,and incubated under the same conditions for different times.
For proteomics experiments, P. rubens (P. chrysogenum Wisconsin54–1255) was grown as indicated above in defined medium with 1 g/Lpotassium phenylacetate. Control cultures lacked the side-chain pre-cursor potassium phenylacetate. Cultures were incubated at 25 °C and250 rpm and samples (mycelia and culture medium) were collectedafter 60 h for intracellular and extracellular proteome analysis.
For expression analysis experiments, conidia were inoculated incomplex inoculum medium CIM [25] without phenylacetate. After in-cubation at 25 °C for 20 h in an orbital shaker (250 rpm), aliquots (5%)were inoculated in CP complex penicillin production medium [25] with4 g/L potassium phenylacetate and incubated under the same condi-tions for 48 h and 60 h.
For transformation experiments, conidia were inoculated into MPPYmedium (40 g/L glucose, 3 g/L NaNO3, 2 g/L yeast extract, 0.5 g/L KCl,0.5 g/L MgSO4·7H2O, 0.01 g/L FeSO4·7H2O, pH=6.0) and grown for24 h at 25 °C and 250 rpm.
2.2. Plasmid constructions for gene silencing
Plasmid pJL43-RNAi [26], which confers phleomycin resistance,was previously digested with NcoI and used as backbone structure forthe constructions aimed to generate knock-down transformants in thePc16g01770 gene. Oligonucleotides 1770F (5′-GATCCCATGGCCATGATCCAGC-3′) and 1770R (5′-GATAGCCATGGCCGCCCGATC-3′), whichwere designed to bear NcoI restriction sites (in italics), were used toamplify a 473-bp exon fragment from Pc16g01770. The amplicon wasdigested with NcoI and cloned into pJL43-RNAi, thus yielding plasmidpJL43-RNAi-1770.
2.3. Transformation of P. rubens (P. chrysogenum Wisconsin 54–1255)protoplasts, extraction of genomic DNA and Southern blotting
Protoplasts were obtained and transformed as previously described[27]. Then, transformed protoplasts were grown in Czapek minimalmedium (30 g/L sucrose, 2 g/L NaNO3, 0.5 gLl K2HPO4, 0.5 g/LMgSO4.7H2O, 0.01 g/L FeSO4) and further selected in Czapek minimalmedium containing 30 μg/mL phleomycin.
DNA isolation and Southern blotting hybridization were carried outas previously described [28].
2.4. RNA extraction and semiquantitative RT-PCR assays
Cultures of P. rubens (P. chrysogenum Wisconsin 54–1255) weregrown in complex medium as indicated above during 48 h and 60 h.Total RNA was extracted using “RNeasy Mini Kit” columns (Qiagen),following the manufacturer's instructions. Total RNA was treated with“RQ1 RNase-Free DNase” (Promega Corporation) and quantified usinga NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific).
RT-PCR was conducted with 200 ng of total RNA using the“SuperScript One-Step RT-PCR with Platinum Taq” system (InvitrogenCorporation) and applying 40 amplification cycles as recommended bythe manufacturer. For the amplification of a 473-bp fragment fromPc16g01770, primers 1770F and 1770R (see above) were used. For theamplification of a 432-bp fragment from Pc22g02230, primers 2230F (5′-GGATGCTAAGGCCTATGAAGG-3′) and 2230R (5′-GAAGATCCAATGGTAAAGCCCTG-3′) were used. For the amplification of a 457-bp frag-ment from the actA-encoding β-actin gene, primers actAF (5′-CTGGCCGTGATCTGACCGACTAC-3′) and actAR (5′-GGGGGAGCGATGATCTTGACCT-3′) were used. The absence of contaminating DNA in the RNAsamples was confirmed by PCR.
For some experiments, densitometry analyses using the “Gel-ProAnalyser” software (Media Cybernetics) were performed in order toquantify the signals provided by the RT-PCR assays. The transcript le-vels were normalized by comparing the intensity of each mRNA signalto the β-actin mRNA signal. Expression levels were considered sig-nificantly different according to the standard deviation and when the p-
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value provided by the Student's t-test was p < 0.01.
2.5. HPLC analysis
Extraction, analysis and quantitation of benzylpenicillin were car-ried out by HPLC using an Agilent 1100 HPLC system with an analytical4.6× 250mm (5 μm) RPC18 Lichrospher® 100 column as previouslydescribed [29].
Phenylacetate and 2-hydroxyphenylacetate were extracted, ana-lysed and quantified as follows. Culture supernatants (0.8 mL) weremixed with cold HPLC-grade methanol (1:1 ratio) and left overnight at4 °C for protein precipitation. Then, samples were centrifuged at13,000 rpm for 10min at 4 °C and analysed by HPLC, which was carriedout using an Agilent 1100 HPLC system with an analytical4.6× 150mm (3 μm) Mediterranea Sea18 Teknokroma® column with aflow rate of 1mL/min. Detector wavelength was set to 217 nm (forpotassium phenylacetate) or 270 nm (for 2-hydroxyphenylacetate).Samples (10 μL) were injected in the HPLC using 1% trifluoroacetic acidas solvent A and acetonitrile as solvent B. The elution gradient was asfollows: 10%B→ 55%B linear over 15min, 55%B→ 100%B linear over1min, isocratic elution for 4min, 100%B→ 10%B for 0.5 min, isocraticelution for 5.5 min. Under these conditions, the retention time for po-tassium phenylacetate was 11.05 ± 0.15min, whereas for 2-hydro-xyphenylacetate, the retention time was 8.45 ± 0.15min. The detec-tion limit was 15 μg/mL.
2.6. Protein extraction
Proteins from either the mycelia (intracellular) or the culture su-pernatants (extracellular) were obtained as previously described [13,23]. The final pellet was solubilized in sample buffer: 8M urea, 2% (w/v) CHAPS, 0.5% (v/v) IPG Buffer (GE Healthcare), 20mM DTT, 0.002%bromophenol blue. The insoluble fraction was discarded by cen-trifugation at 16,000 x g for 5min. The supernatant was collected andprotein concentration was determined according to the Bradfordmethod, which showed a high reproducibility for this protein extractionprotocol.
2.7. 2-DE gel electrophoresis
A solution containing 350 μg of soluble intracellular proteins or450 μg of soluble extracellular proteins in the sample buffer (seeabove), was loaded onto 18-cm IPG strips (GE Healthcare), with non-linear pH 3–10 gradient (for intracellular proteins) or non-linear pH 4–7gradient (for extracellular proteins). Focusing of proteins and equili-bration of the focused IPG strips were achieved as previously described,as well as the second dimension, which was run by SDS-PAGE in 12.5%polyacrylamide in an Ettan Dalt Six apparatus (GE Healthcare) [13, 23].Gels were dyed with Colloidal Coomassie (CC) following the “BlueSilver” staining method [30], which provides high reproducibility, asindicated before [13, 23].
2.8. Analysis of differential protein expression
Scanned 2D gels were analysed using an ImageScanner II (GEHealthcare) calibrated with a grayscale marker (Eastman Kodak Co.).Labscan 5.00 (v1.0.8) software (GE Healthcare) and theImageMasterTM 2D Platinum v5.0 software (GE Healthcare) were usedfor image acquisition and analysis as previously described [13, 23].Three biological replicates were used for each condition. After auto-mated spot detection, spots were checked manually to eliminate anypossible artefacts, such as streaks or background noise. Spot normal-ization, as an internal calibration to make the data independent fromexperimental variations among gels, was made using relative volumes(volume of each spot divided by the total volume of all the spots in thegel) to quantify and compare the gel spots. Differentially expressed
proteins between two strains were considered when the ratio of therelative volume average for one specific spot (present in the threebiological replicates) was higher than 1.5 or lower than−1.5 and the p-value was 20, were collected and represented as a list of monoisotopicmolecular weights using the 4000 Series Explorer v3.5.3 software(Applied Biosystems). Well known contaminant ions (trypsin- andkeratin-derived peptides) were excluded for later MS/MS analysis.Hence, the six most intensive precursors from each MS spectra with a S/N>20 were selected for MS/MS analyses with CID (atmospheric gaswas used) in 2-kV ion reflector mode and precursor mass windowsof± 7Da. Default calibration was optimized for the MS/MS spectra.
Mascot Generic Files combining MS and MS/MS spectra were au-tomatically created for protein identification by means of a non-re-dundant protein database using a local license of Mascot v 2.2 fromMatrix Science through the Protein Global Server (GPS) v 3.6 (AppliedBiosystems). The search parameters for peptide mass fingerprints andtandem MS spectra obtained were set as follows: (i) UniprotAscomycota (date 2017.07.03; 6,766,808 sequences, 3,023,177,811residues); (ii) fixed and variable modifications were considered (Cys asS carbamidomethyl derivative and Met as oxidized methionine); (iii)one missed cleavage site was allowed; (iv) precursor tolerance was100 ppm and MS/MS fragment tolerance was 0.3 Da; (v) peptidecharge: 1+; and (vi) the algorithm was set to use trypsin as the enzyme.Protein candidates produced by this combined peptide mass finger-printing (PMF)/tandem MS search were considered valid when theglobal Mascot score was>83 with a significance level of p < 0.05.Additional criteria for confident identification were that the proteinmatch should have at least 15% sequence coverage; for lower cov-erages, only those proteins with at least two peptides identified wereconsidered valid.
3. Results
3.1. Catabolism of phenylacetic acid in three different P. rubens strains
The behaviour of three different penicillin-producing strains of P.rubens (P. notatum, P. chrysogenum NRRL 1951 and P. chrysogenumWisconsin 54–1255) was tested in the presence of phenylacetic acid.For this purpose, they were grown for 72 h in DP medium containing1 g/L potassium phenylacetate (7 mM). All strains were able to grow inthe presence of potassium phenylacetate and showed a similar growthpattern and biomass values with no significant differences along theculture time (Fig. 2A). Antibiotic production was analysed in the threestrains. As expected, P. chrysogenum Wisconsin 54–1255 produced thehighest benzylpenicillin titers (3,6 ± 1,2mg/g dry weight at 72 h),which were 300-fold and 2000-fold higher than those provided by P.chrysogenum NRRL 1951 and P. notatum, respectively (data not shown).Phenylacetate consumption was similar in the three strains until 24 h.After this time point, P. chrysogenum Wisconsin 54–1255 showed alower consumption rate than the wild-type parental strain P. chryso-genum NRRL 1951 and P. notatum, which were the only strains able tofully deplete the side chain precursor after 72 h of growth (Fig. 2B).
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This suggests that late steps in the catabolism of phenylacetic acidmight be limiting in this strain. As a control, a parallel abiotic experi-ment was run with DP medium supplemented with 1 g/L potassiumphenylacetate (7 mM) and subjected to similar conditions. Phenylace-tate levels remained constant along the culture time, thus excluding aphenomenon of non-enzymatic degradation of this compound in theabsence of P. rubens (data not shown). To test the phenylacetate cata-bolic activity of the three P. rubens strains, secretion of 2-hydro-xyphenylacetate was assessed (Fig. 2C). Similar amounts of this meta-bolite were found in the culture media of these strains at early timepoints. P. notatum produced higher amounts of 2-hydroxyphenylacetateat 48 h, and a gradual decrease in the amount of this compound was
observed from this time-point. P. chrysogenum NRRL 1951 showed aslightly different pattern, with a full depletion of this metabolite at 72 h.Interestingly, in P. chrysogenum Wisconsin 54–1255, 2-hydro-xyphenylacetate levels in the culture medium increased from 36 h ofgrowth until the end of the culture time (Fig. 2C).
3.2. Characterization of phenylacetate hydroxylase homologs in P. rubens(P. chrysogenum Wisconsin 54–1255)
With the aim of shedding light into the degradation of phenylaceticacid by the improved strain P. chrysogenum Wisconsin 54–1255, asearch for pahA-encoded phenylacetate hydroxylase (Pc21g14280)
Fig. 2. Catabolism of phenylacetic acid in three strains of P rubens (P. notatum, P chrysogenum NRRL 1951 and P. chrysogenum Wis 54–1255). A) Dry weight (mg/mL)obtained from samples taken at different time points. B) Consumption of potassium phenylacetate (μg/mL) along the culture time. C) Secretion of 2-hydro-xyphenylacetate (2-OH phenylacetate) (μg/mL) along the culture time. Data correspond to three biological replicates performed in triplicate.
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homologs was carried out in the P. rubens (P. chryosogenum Wisconsin54–1255) genome [12]. Three proteins were found with>50% simi-larity with the pahA-encoded protein: Pc22g02230 (65% similarity,46% identity), Pc16g01770 (62% similarity, 44% identity) andPc21g22560 (60% similarity, 42% identity). According to the sequence,all proteins belonged to the Cytochrome P450 superfamily. In addition,Pc21g22560 also contained GAL4-like (Zn2Cys6 binuclear cluster DNA-binding) and fungal transcription factor regulatory middle homologyregion domains, which suggested a different role from phenylacetatecatabolism.
Therefore, we focused our research on Pc22g02230 andPc16g01770. Expression of these genes was analysed in cultures of P.rubens (P. chrysogenum Wisconsin 54–1255), which was grown for 48 hand 60 h in complex medium in the presence of 4 g/L potassium phe-nylacetate. RT-PCR experiments (Fig. 3) showed that Pc16g01770 wasexpressed along the culture time, unlike Pc22g02230, whose tran-scription was not detected even after 50 amplification cycles.
According to expression data, Pc22g02230 was discarded for furtheranalysis and the role of Pc16g01770 in phenylacetate degradation wasassessed by means of gene silencing experiments. For this purpose, P.rubens (P. chrysogenum Wisconsin 54–1255) was transformed withplasmid pJL43-RNAi-1770. Integration of the silencing cassette in dif-ferent transformants was confirmed by Southern blotting (Fig. 4A) afterthe digestion of genomic DNA with SphI and HindIII, and hybridisationto the DIG-labelled exon fragment (the same DNA fragment that wasincluded in the silencing cassette). All transformants and the parentalstrain showed the 9.4-kbp hybridisation band containing the internalPc16g01770 gene. In addition, transformants 1, 5, 15, 38, 52 and 55showed the 1.8-kbp band that included the silencing cassette. At-tenuation of expression in these transformants was confirmed by RT-PCR (Fig. 4B). All transformants, except 52 and 55, showed significant(p < 0.05) reduced expression (ranging from 42% in transformant 15to 17% in transformant 38) of the Pc16g01770 gene, and were phe-notypically characterized.
Cultures of P. chrysogenum Wisconsin 54–1255 and knock-downtransformants 1, 5, 15 and 38 were conducted in defined medium in thepresence of 1 g/L potassium phenylacetate. Samples were collected at24 h, 48 h and 72 h and the presence of non-consumed phenylaceticacid, 2-hydroxyphenylacetic acid and benzylpenicillin in the culturesupernatants was analysed by HPLC. Transformants 1, 5 and 15 showeda slight (up to 20%) significant (p < 0.05) increase in the phenylaceticacid levels at 72 h regarding the values provided by the parentalWisconsin 54–1255 strain (Fig. 5A). Also, the presence of 2-hydro-xyphenylacetic acid was assessed in those transformants. In general,they showed significant (p < 0.05) slightly reduced levels (up to 25%)
of this compound at 48 h and 72 h (transformant 5 did not provide asignificant decrease at 72 h), in comparison with the values provided bythe parental strain (Fig. 5B). This behaviour may be due to a reduceddegradation of the benzylpenicillin side chain precursor in the knock-down transformants. Benzylpenicillin specific production remained si-milar between transformants and the parental Wisconsin 54–1255strains (Fig. 5C). These results suggest that Pc16g01770 may have aresidual activity in phenylacetic acid degradation in the Wisconsin54–1255 strain, this catabolic activity having no effect on benzylpeni-cillin biosynthesis (see Discussion).
3.3. Effect of phenylacetic acid on the intracellular and extracellularproteomes of P. rubens (P. chrysogenum Wisconsin 54–1255)
In order to get more insight into the metabolic processes modifiedby the presence of the benzylpenicillin side chain precursor, a pro-teome-wide analysis was carried out in P. rubens (P. chrysogenumWisconsin 54–1255).
For this purpose, cultures were conducted with this fungal strain inthe presence and absence of 1 g/L phenylacetic acid. Samples includedboth mycelia (for intracellular proteome analysis) and culture super-natants (for extracellular proteome analysis) and were taken at 60 h ofgrowth. Protein fractions were analysed by 2-DE and tandem MSspectrometry.
3.3.1. Intracellular proteomeThe 2-DE gels with the intracellular protein fractions obtained from
both conditions were compared to each other (Fig. 6). A total of 22spots (D1-D22 including 23 proteins) resulted overrepresented, whereas53 spots (C1-C53 including 56 proteins) were underrepresented afterphenylacetic acid addition (Supplementary Tables S1 and S2). Func-tions were inferred for these proteins (Tables 1 and 2) and the mainfindings are summarized below.
Only one protein from the homogentisate pathway was foundoverrepresented after the addition of phenylacetic acid (Table 1). SpotD6 (4.5-fold overrepresented) contains the fumaryl acetoacetase(Pc12g09030), which is involved in the last step of the catabolicpathway of phenylacetic acid (see Discussion).
Interestingly, several proteins related to penicillin biosynthesis werealso found overrepresented after the addition of the benzylpenicillinside chain precursor. The first one is IAT (Pc21g21370), one of thepenicillin biosynthetic enzymes, which is included in spot D11 (5.8-foldoverrepresented). Other important proteins are present in Spot D4 (2.4-fold overrepresented in the presence of phenylacetic acid and includinga hypothetical cystathionine beta synthase) Spot D23 (only detectedwith phenylacetic acid and including a probable ketol-acid re-ductoisomerase ilv-2) and Spot D22 (only detected after supplementa-tion with phenylacetic acid and containing a probable thioredoxinperoxidase (Pc22g04430)). Another interesting protein that resultedoverrepresented after phenylacetic acid addition is S-adenosylmethio-nine synthase, which is included in spot D14 (2.9-fold overrepresented)(See Discussion).
Several proteins that are underrepresented due to the presence ofphenylacetic acid (Table 2) belong to the glycolysis and tricarboxylicacid cycle. Examples are provided by spots C7 (Pc18g01220, probablefructose-bisphosphate aldolase), C12 (Pc18g06000, probable pyruvatekinase), C27 and C28 (both including Pc20g01610, a probable mi-tochondrial malate dehydrogenase), C40 (Pc22g02000, a probablemitochondrial aconitate hydratase), and C45 and C46 (both includingPc12g06870, a probable alpha subunit of succinyl coenzyme A syn-thase). In addition, three spots related to the metabolism of acetyl-CoAwere also found underrepresented under these conditions: spot C9(Pc21g20480, probable ATC citrate lyase), spot C11 (Pc12g03130, aprobable acetyl-CoA hydrolase) and spot C44 (Pc22g11710, probablealpha subunit E1 of the pyruvate dehydrogenase complex). Related tothis finding is the fact that spot C2, which includes a probable N-
Fig. 3. Expression of putative pahA homologs in P. rubens (P. chrysogenum Wis54–1255). Ethidium bromide-stained agarose gel showing the RT-PCR band(473-bp) of Pc16g01770 that has been amplified from RNA samples taken at48 h and 60 h. The absence of contaminating DNA in the RNA samples wasconfirmed by PCR (C-). Note the absence of RT-PCR bands for Pc22g02230when RNA is used and the amplification of a 432-bp PCR band from this genewhen genomic DNA is used as template (gDNA). Amplification of RT-PCRfragments (457 bp) from the actA-encoding β-actin gene was used as a referenceof transcription.
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acetylglucosamine-6-phosphate deacetylase (Pc22g10010), is 5.28-foldunderrepresented in the presence of phenylacetic acid. This enzymedeacetylates amino sugars to yield glucosamine-6-phosphate andacetate.
Another interesting group of proteins that resulted under-represented after the addition of the benzylpenicillin side chain pre-cursor were related to protein folding, modification or degradation.This is the case of spots C5 (Pc22g11240, probable heat shock protein70 hsp70), C8 (Pc22g19990, probable endonuclease SceI 75 kDa sub-unit Ens1 with putative hsp70 activity), C21 and C41 (both includingPc22g10220, a probable dnaK-type molecular chaperone), C29(Pc21g16970, vacuolar serine proteinase AAG44693 or allergen Pen n18), C33 (probable proteasome component PRE6), C36 (Pc22g13950,probable vacuolar aminopeptidase Ysci) and C49 (Pc20g09400, prob-able dipeptidyl-peptidase V).
A subset of proteins related to oxidative stress response was alsounderrepresented in the presence of phenylacetic acid. Examples areprovided by spots C3 (Pc12g14620, probable flavohemoglobin Fhp),C16 and C17 (both including Pc16g13280, a probable glutathione re-ductase), C30 (Pc16g09250, probable cytochrome-b5 reductase) andC48 (Pc18g00790, probable glutathione S-transferase).
3.3.2. Extracellular proteomeThe 2-DE gels including the extracellular protein fractions obtained
in the presence and absence of phenylacetic acid were also compared toeach other (Fig. 7). A total of 45 spots, named P1-P45 and including 49proteins (36 different proteins), resulted overrepresented, whereas 14spots, named S1-S14 and including 16 proteins (12 different proteins),were underrepresented after phenylacetic acid addition (Supplemen-tary Tables S3 and S4). Secretion of those proteins due to the presenceof classical signal peptides or through a non-classical secretory me-chanism was predicted as indicated in our previous work [23]. A totalof 38 different proteins out of the 48 proteins found differentially re-presented in the secretome were predicted to contain either classical ornon-classical signal sequences (Tables 3 and 4) (see Discussion).Functions were inferred for these proteins and the main findings aresummarized below.
The most important extracellular protein overrepresented after theaddition of phenylacetic acid is included in spot P16. This spot is onlydetected under these conditions and contains the glutamate dehy-drogenase (encoded by the gdhA gene), which lacks classical or non-classical signal sequences for secretion (see Discussion).
Some proteins from the glycolysis, tricarboxylic acid cycle and
Fig. 4. Gene silencing of Pc16g01770. A) Southern blot ana-lysis of different transformants (1, 5, 15, 38, 52 and 55) andthe parental P. rubens (P. chrysogenum Wisconsin 54–1255strain) (Wis). The 473-bp exon fragment (indicated as a blackbox inside the corresponding gene) included in the silencingcassette, was used as probe. All transformants show the 1855-bp hybridisation band corresponding to the silencing cassette.Note the presence of the 9423-bp genomic band containing theendogenous Pc16g01770 gene. Additional hybridisation bandsare likely due to partial digestion of genomic DNA. B) Relativeexpression (quantified by RT-PCR) of Pc16g01770 in differenttransformants compared to the Wisconsin 54–1255 strain(Wis; reference value set to 100). Values correspond to themean plus standard deviation of three independent experi-ments. Statistical significance by ANOVA test is representedabove error bars as “*” (0.01≤ P < 0.05); “**”(0.001≤ P < 0.01); “***” (P < 0.001).
M.-S. Jami et al. Journal of Proteomics 187 (2018) 243–259
249
pentose phosphate pathways are also overrepresented under theseconditions, including a probable mitochondrial aconitate hydrataseAco1 (Pc22g02000, spots P4, P5 and P9), an enolase (Pc14g01740,spots P18 and P43), and a probable transaldolase Tal1 (Pc21g16950,spot P26). Interestingly, none of these proteins are predicted to be se-creted (Table 3) (see Discussion).
All proteins underrepresented in the presence of the benzylpenicillinside chain precursor are predicted to be secreted (Table 4). Most ofthem are involved in plant cell wall and plant tissues degradation. Thisis the case of spots S3 and S11 (both including Pc22g20290, a probablepolygalacturonase pgaI), S5 (Pc22g24890, probable pectate lyase plyA,and Pc20g07020, endo-1,4-beta-xylanase A precursor XylP), S11(Pc22g20290, probable polygalacturonase pgaI), S12 and S13 (both
including Pc20g07030, a probable 1,4-beta-Δ-arabinoxylan arabino-furanohydrolase axhA).
The presence of phenylacetic acid downregulates the synthesis of aprobable cephalosporin esterase (Pc12g13400). This wide substratespectrum esterase forms deacetylcephalosporin C and acetate usingcephalosporin C as substrate and is included in spot S6 (5.2-fold un-derrepresented under these conditions).
4. Discussion
One of the most important milestones in the history of penicillins isthe finding that addition of specific side chain precursors (e.g. pheny-lacetic acid) to culture media, directed the biosynthetic process mainlytowards benzylpenicillin (penicillin G) [32], which is the main bio-synthetic penicillin produced under industrial conditions. Distinct P.rubens strains behave in a different way regarding detoxification ofphenylacetic acid. Unlike P. notatum, P. chrysogenum is unable to growon phenylacetic acid as sole carbon source, although it can efficientlyoxidize it, hence suggesting a block in the catabolic pathway to fuma-rate and acetoacetate [14, 15]. These authors reported that modifica-tions (L181F and A394V) in the phenylacetate hydroxylase (the firstenzyme of the catabolic pathway of phenylacetic acid) during strainimprovement programs gave rise to loss-of-functions mutations, thusleading to reduced degradation of phenylacetic acid and to penicillinoverproduction in P. chrysogenum [14, 15]. Our results (Fig. 2) indicatethat in the presence of sucrose and lactose as carbon sources, P. chry-sogenum is able not only to incorporate phenylacetic acid to the ben-zylpenicillin biosynthetic pathway, but also to convert it to 2-hydro-xyphenylacetic acid by the phenylacetate hydroxylase via thehomogentisate pathway. In addition, P. chrysogenum Wisconsin54–1255 showed an increase in 2-hydroxyphenylacetate levels alongthe culture time in comparison with P. notatum and P. chrysogenumNRRL 1951, which accumulate lower amounts of this compound likelydue to a faster metabolization to 2, 5-dihydroxyphenylacetate at latetime points. This phenomenon is similar to that reported in P. chryso-genum overproducing strains, where significant amounts of 2-hydro-xyphenylacetic acid are detected in the fermentation broth [15].Therefore, both detoxification mechanisms seem to coexist in differentspecies of P. rubens, but with different efficiencies. While catabolicdetoxification is more efficient in P. notatum and P. chrysogenum NRRL1951 than in P. chrysogenum Wisconsin 54–1255, detoxification bymeans of penicillin formation is more efficient in P. chrysogenum Wis-consin 54–1255 than in the other two strains. The fact that P. chryso-genum Wisconsin 54–1255 shows catabolic detoxification suggests apartial blockage of phenylacetate hydroxylase activity in improvedstrains of P. chrysogenum, or the presence of at least another proteinwith phenylacetate hydroxylase activity in this microorganism. Thisquestion can be elucidated by comparison to the information publishedfor A. nidulans, another penicillin-producing fungus.
A. nidulans, known to degrade efficiently phenylacetic acid by dif-ferent enzymes, is also able to utilize phenylacetate as a carbon sourcevia homogentisate, phenylacetic acid being converted to 2-hydro-xyphenylacetate by means of a 2-hydroxylation reaction catalysed by acytochrome P450 monooxygenase, which is encoded by the phacA gene[18]. In addition, the existence of another cytochrome P450 mono-oxygenase (encoded by the phacB) with 3-hydroxyphenylacetate 6-hy-droxylase and 3,4-dihydroxyphenylacetate 6-hydroxylase activities thatforms 2,5-dihydroxyphenylacetate (homogentisate) and can also con-vert phenylacetic acid into 2-hydroxyphenylacetate, has been reportedin this microorganism [20]. Interestingly, we found three proteins(Pc22g02230, Pc16g01770 and Pc21g22560) from the cytochromeP450 superfamily that show>50% similarity with the pahA-encodedprotein (Pc21g14280) in the P. rubens (P. chrysogenum Wisconsin54–1255) genome. Unlike Pc22g02230, Pc16g01770 was expressedunder the conditions tested (Fig. 3). This result is consistent with pre-vious transcriptomics data, which reported high transcription rate of
Fig. 5. Relative percentage (%) of A) potassium phenylacetate, B) 2-hydro-xyphenylacetate (2-OH phenylacetate) specific production and B) benzylpeni-cillin specific production assessed in samples obtained at 24 h, 48 h and 72 hfrom cultures of P. rubens (P. chrysogenum Wisconsin 54–1255) (W) and dif-ferent Pc16g01770 knock-down transformants (1, 5, 15 and 38). Results arerepresented as the mean ± standard deviation from three independent ex-periments carried out in triplicate. Values were normalized to those provided bythe Wisconsin 54–1255 strain (W) at each time-point, which were set to 100%.Statistical significance by ANOVA test is represented above error bars as “*”(0.01≤ P < 0.05).
M.-S. Jami et al. Journal of Proteomics 187 (2018) 243–259
250
Pc16g01770 and transcriptional induction in the presence of phenyla-cetate. However, Pc21g22560 and Pc22g02230 exhibited null expres-sion levels and no transcriptional induction under the same conditionsin chemostat cultivations [12]. The protein encoded by Pc16g01770shows 90% similarity and 82% identity with the A. nidulans phacB-en-coded cytochrome P450 monooxygenase, suggesting a possible role ofthis protein in the formation of 2,5-dihydroxyphenylacetate (homo-gentisate) and in phenylacetate hydroxylation. The latter was tested bygene knock-down experiments, where most of the transformants si-lenced in the expression of Pc16g01770 showed a slight decrease in 2-hydroxyphenylacetate levels (Fig. 5). These results point to the pre-sence of at least one additional enzyme, encoded by Pc16g01770, withresidual phenylacetate hydroxylase activity in P. chrysogenum Wis-consin 54–1255. However, additional experiments (e.g. heterologousexpression, biochemical characterization and analysis of substratespecificity) are still required to confirm the role played by the proteinencoded by Pc16g01770 in the homogentisate pathway.
Interestingly, Pc16g01770 and the pahA-encoded phenylacetatehydroxylase lack a canonical PTS1 signal, which is a target sequencepresent at the C terminus that allows import of proteins into peroxi-somes [33, 34]. Therefore, phenylacetate degradation via the homo-gentisate pathway by the protein encoded by Pc16g01770 and thepahA-encoded phenylacetate hydroxylase likely takes place during itsway to the peroxisomal matrix. The transport of phenylacetic acid fromthe culture medium to the cytoplasm and then to peroxisomes has beena matter of discrepancy. Either active transport [35, 36] or passivediffusion [37], have been suggested as mechanisms for internalizationof the benzylpenicillin side-chain precursor. More recently, a process of
two consecutive steps (facilitated diffusion in the plasma membraneand active transport in the peroxisomal membrane) has been suggestedafter the characterization of a MFS transporter (PaaT) that participatesin the translocation of phenylacetic acid from the cytosol to the per-oxisomal lumen across the peroxisomal membrane of P. rubens [38].Once phenylacetic acid is present within peroxisomes, it is activated byaryl-CoA ligases and incorporated into the penicillin biosyntheticpathway. The percentage of phenylacetic acid being incorporated intoeither of these two pathways (i.e. catabolism via homogentisate andbenzylpenicillin biosynthesis) seems to be highly dependent on thestrain, as previously suggested [14, 15].
Phenylacetic acid has a clear direct involvement in benzylpenicillinbiosynthesis due to its participation as side chain precursor. In an at-tempt to characterize other roles that this molecule can play regardingpenicillin production, we decided to carry out a global comparativeproteomics analysis in P. rubens (P. chrysogenum Wisconsin 54–1255)with and without phenylacetic acid addition. Unexpectedly, only oneprotein (fumaryl acetoacetase; Pc12g09030) involved in the catabolismof the side chain precursor was induced by phenylacetate. Previoustranscriptomics analysis reported that all genes of the homogentisatepathway for phenylacetate catabolism were strongly upregulated in thepresence of phenylacetic acid [22]. These authors used a high-produ-cing P. chrysogenum strain grown under glucose-limited chemostatconditions, which may be one of the main reasons for the upregulationof the whole catabolic pathway.
Among the intracellular proteins whose synthesis was induced inthe presence of this side chain precursors, there are several enzymesrelated to β-lactam biosynthesis. The most important one is IAT
Fig. 6. Effect of phenylacetic acid (PAA) in the intracellular proteome of P. rubens (P. chrysogenum Wisconsin 54–1255). Intracellular proteins obtained from myceliaof P. rubens (P. chrysogenum Wisconsin 54–1255) grown for 60 h in DP medium with and without 1 g/L potassium phenylacetate, were separated by 2-DE using 18-cmwide-range IPG strips (pH 3–10 NL) and 12.5% SDS-PAGE gels, which were stained with CC following the “Blue Silver” staining method. Those spots overrepresentedwithout PAA (underrepresented with PAA) are designated as “C”, whereas the letter “D” was used to designate those spots overrepresented in the presence of PAA.The spots differentially represented in each condition are numbered and correspond to those proteins listed in Tables 1 (“D” spots) and 2 (“C” spots) and Supple-mentary Tables S1 (“D” spots) and S2 (“C” spots).
M.-S. Jami et al. Journal of Proteomics 187 (2018) 243–259
251
Table1
Intracellularproteins
overrepresen
tedat
60hin
thepresen
ceof
phen
ylacetic
acid.F
oldincrease
andp-va
lueareindicated.
Proteins
that
areon
lyde
tected
afterthead
dition
ofph
enylacetic
acid
arede
notedas
N/A
.
Spot
ORF
Accession
No
Simila
rity
Fold
chan
geP-Value
Func
tion
D1
Pc22
g102
20gi|211
5920
47strong
simila
rity
todn
aK-typ
emolecular
chap
eron
eSsb2
-Sa
ccha
romyces
cerevisiae
4.7
3.20
E-03
Proteinfate.P
rotein
folding,
mod
ification
andde
stination
D2
Pc18
g053
20gi|211
5870
92strong
simila
rity
toIM
Pde
hydrog
enaseIM
H3-Can
dida
albicans
3.1
1.64
E-04
GTP
biosyn
thesis
D3
Pc12
g160
40gi|211
5830
21strong
simila
rity
toph
osph
oglycerate
mutasepg
m-Ba
cillu
ssubtilis
3.1
1.73
E-03
Glyco
lysis
D4
Pc13
g053
20gi|211
5835
86strong
simila
rity
tohy
pothetical
cystathion
ebe
ta-syn
thasecysB
-Dictyosteliu
mdiscoideum
2.4
4.13
E-05
AminoAcidMetab
olism
D5
Pc16
g047
30gi|211
5854
47ph
osph
oglycerate
kina
sepg
kA-Penicillium
chrysogenu
m20
.27.70
E-06
Glyco
lysis
D6
Pc12
g090
30gi|211
5823
50strong
simila
rity
tofumarylacetoa
cetase
-Hom
osapiens
4.5
2.48
E-03
Hom
ogen
tisate
pathway
D7
Pc12
g008
30gi|211
5816
03strong
simila
rity
tosorbitol
utilization
proteinsou2
-Can
dida
albicans
2.7
1.83
E-05
Carbo
hydrateMetab
olism.(Related
toshort-ch
ainalco
holde
hydrog
enases)
D8
Pc22
g199
90gi|211
5929
18strong
simila
rity
toen
donu
clease
SceI
75kD
asubu
nitEn
s1-Sa
ccha
romyces
cerevisiae
N/A
N/A
Proteinfate.P
rotein
folding,
mod
ification
andde
stination.
(Putativefunc
tion
ashsp7
0)D9
Pc22
g102
20gi|211
5920
47strong
simila
rity
todn
aK-typ
emolecular
chap
eron
eSsb2
-Sa
ccha
romyces
cerevisiae
34.76
E-03
Proteinfate.P
rotein
folding,
mod
ification
andde
stination
D10
Pc21
g168
70gi|211
5903
98strong
simila
rity
tohy
pothetical
proteinsm
ik_170
56-Sa
ccha
romyces
mikatae
N/A
N/A
Unk
nown
D11
Pc21
g213
70gi|211
5908
23acyl-coe
nzym
eA:isop
enicillin
Nacyltran
sferase(acyltransferase)AAT/
PenD
E-Penicillium
chrysogenu
m5.8
2.80
E-05
Penicillinbiosyn
thesis
D12
Pc13
g088
10gi|211
5839
26strong
simila
rity
toelon
gation
factor
1betaEF
-1-Oryctolagus
cuniculus
5.8
1.19
E-05
Tran
slation
Pc20
g132
70gi|211
5885
42strong
simila
rity
tona
scen
tpo
lype
ptide-associated
complex
alph
ach
ainalph
a-NAC-Mus
musculus
5.8
1.19
E-05
Proteinfate.P
rotein
folding,
mod
ification
andde
stination
D13
Pc22
g057
90gi|211
5916
28strong
simila
rity
totran
scriptionactiva
torAdr1-Sa
ccha
romyces
cerevisiae
2.6
2.95
E-03
Tran
scription
D14
Pc16
g043
80gi|211
5854
13strong
simila
rity
toS-ad
enosylmethion
inesynthe
tase
eth-1-Neurosporacrassa
2.9
2.93
E-02
Methion
inecycle
D15
Pc22
g066
90gi|211
5917
14weaksimila
rity
tove
rsicolorin
redu
ctaseve
rA-Aspergillu
snidu
lans
4.8
6.22
E-05
Red
oxmetab
olism
D16
Pc21
g192
70gi|211
5906
29strong
simila
rity
tova
losin-co
ntaining
proteinlik
eAAA-ATP
aseCdc
48-Sa
ccha
romyces
cerevisiae
N/A
N/A
Proteinfate.P
rotein
folding,
mod
ification
andde
stination
D17
Pc22
g126
70gi|211
5922
27strong
simila
rity
tohy
pothetical
proteinco
ntig_1_67_scaff
old_4.tfa_44
0wg-A
spergillu
snidu
lans
N/A
N/A
Unk
nown
D18
Pc22
g056
90gi|211
5916
18strong
simila
rity
tohy
pothetical
proteinco
ntig12
.tfa_17
30cg
-Aspergillu
sfumigatus
N/A
N/A
Unk
nown
D19
Pc12
g112
70gi|211
5825
64strong
simila
rity
tohy
pothetical
proteinco
ntig14
92_0.tfa_18
60cg
-Aspergillu
sfumigatus
N/A
N/A
Unk
nown
D20
Pc22
g056
90gi|211
5916
18strong
simila
rity
tohy
pothetical
proteinco
ntig12
.tfa_17
30cg
-Aspergillu
sfumigatus
N/A
N/A
Unk
nown
D21
Pc21
g123
10gi|211
5899
73hy
pothetical
protein[Penicillium
chrysogenu
m]
2.4
5.31
E-03
Unk
nown
D22
Pc22
g044
30gi|211
5914
98strong
simila
rity
tothioredo
xinpe
roxida
selik
eproteinAn0
6g01
660-Aspergillu
sniger
N/A
N/A
Oxida
tive
stress
respon
se
M.-S. Jami et al. Journal of Proteomics 187 (2018) 243–259
252
Table2
Intracellularproteins
unde
rrep
resented
at60
hin
thepresen
ceof
phen
ylacetic
acid.F
oldde
crease
andp-va
lueareindicated.
Proteins
that
areno
tde
tected
afterthead
dition
ofph
enylacetic
acid
arede
notedas
N/A
.
Spot
ORF
Accession
No
Simila
rity
Fold
chan
geP-Value
Func
tion
C1
Pc22
g228
10gi|211
5931
92strong
simila
rity
tosulphy
dryl
oxidaseSo
xfrom
patent
EP56
5172
-A1-
Aspergillu
sniger
−3.3
1.1E
-02
Oxida
tion
ofsulfhy
dryl
compo
unds
C2
Pc22
g100
10gi|211
5920
27strong
simila
rity
toN-acetylgluco
samine-6-ph
osph
atede
acetylase
CaN
AG2-Can
dida
albicans
−5.3
2.9E
-05
Aminosug
arsmetab
olism
C3
Pc22
g101
40gi|211
5920
39strong
simila
rity
tocytosolic
acetyl-CoA
C-acetyltransferase
Erg1
0-
Saccha
romyces
cerevisiae
−4.8
8.3E
-04
Mev
alon
atemetab
olism
(isopren
oids
biosyn
thesis)
Pc12
g146
20gi|211
5828
85strong
simila
rity
toflav
ohem
oglobinFh
p-Alcaligenes
eutrophu
s−
4.8
8.3E
-04
Oxida
tive
stress
respon
seC4
Pc20
g155
80gi|211
5887
65strong
simila
rity
toNADPH
-dep
ende
ntalde
hyde
redu
ctase-
Sporobolom
yces
salm
onicolor
−2.2
2.1E
-03
Red
uction
ofava
rietyof
alde
hyde
san
dcarbon
yls.
Detox
ification
ofalde
hyde
inhibitors
C5
Pc22
g112
40gi|211
5920
88strong
simila
rity
tohe
atshoc
kprotein70
hsp7
0-A
jello
myces
capsulatus
−4.7
1.6E
-02
Proteinfate.Proteinfolding,
mod
ification
andde
stination
C6
Pc12
g120
40gi|211
5826
39strong
simila
rity
totran
slationelon
gation
factor
eEF-2-C
ricetulusg
riseus
−4.7
3.8E
-03
Tran
slation
C7
Pc18
g012
20gi|211
5867
00strong
simila
rity
tofruc
tose-bisph
osph
atealdo
lase
Fba1
-Saccharom
yces
cerevisiae
−4.6
2.1E
-05
Glyco
lysis
C8
Pc22
g199
90gi|211
5929
18strong
simila
rity
toen
donu
clease
SceI
75kD
asubu
nitEn
s1-
Saccha
romyces
cerevisiae
−7.6
1.5E
-03
Proteinfate.Proteinfolding,
mod
ification
andde
stination.
(Putativefunc
tion
ashsp7
0)C9
Pc21
g204
80gi|211
5907
46strong
simila
rity
toATP
citratelyaseACL1
-Sorda
riamacrospora
N/A
N/A
Acetyl-C
oAmetab
olism.C
arbo
hydratean
dlip
idsmetab
olism
C10
Pc16
g061
30gi|211
5855
72strong
simila
rity
toalph
a-gluc
ansyntha
semok
1p-Schizosaccharom
yces
pombe
N/A
N/A
Cellwallan
dmorph
ogen
esis
C11
Pc12
g031
30gi|211
5818
18strong
simila
rity
toacetyl-CoA
hydrolaseAch
1-Sa
ccha
romyces
cerevisiae
N/A
N/A
Acetyl-C
oAmetab
olism.A
cetate
utilization
C12
Pc18
g060
00gi|211
5871
60strong
simila
rity
topy
ruva
tekina
sepk
iA-Aspergillu
sniger
N/A
N/A
Glyco
lysis
C13
Pc18
g009
80gi|211
5866
77strong
simila
rity
tohy
pothetical
trun
klateralc
ellspe
cificge
neHrTLC
1-
Halocyn
thia
roretzi
N/A
N/A
Red
oxmetab
olism
C14
Pc21
g023
60gi|211
5890
11strong
simila
rity
toGU4nu
cleic-bind
ingprotein1Arc1-S
accharom
yces
cerevisiae
N/A
N/A
Proteinwithbind
ingfunc
tion
orco
factor
requ
irem
ent.Bind
sto
tRNA
and
func
tion
sas
aco
factor
forthemethion
yl-tRNAsynthe
tase
(MetRS)
andglutam
yl-
tRNA
synthe
tase
(GluRS)
C15
Pc12
g033
70gi|211
5818
41strong
simila
rity
tomitoc
hond
rial
F1-ATP
asealph
a-subu
nitAtp1-
Saccha
romyces
cerevisiae
N/A
N/A
Proteintran
sportinside
mitoc
hond
ria
C16
Pc16
g132
80gi|211
5862
50strong
simila
rity
toglutathion
eredu
ctaseGlr1-S
accharom
yces
cerevisiae
N/A
N/A
Oxida
tive
stress
respon
seC17
Pc16
g132
80gi|211
5862
50strong
simila
rity
toglutathion
eredu
ctaseGlr1-S
accharom
yces
cerevisiae
N/A
N/A
Oxida
tive
stress
respon
seC18
Pc13
g134
70gi|211
5843
81strong
simila
rity
totubu
linbe
tach
ainbe
ta-tub
ulin
likeprotein
An0
8g03
190-Aspergillu
sniger
N/A
N/A
Cellularstructure
C19
Pc16
g117
90gi|211
5861
03strong
simila
rity
tofruc
tosylam
ineox
ygen
oxidored
uctase
-Aspergillu
sfumigatus
N/A
N/A
Oxida
tive
deglycationof
Amad
orip
rodu
cts(glycatedlow
molecular
weigh
tamino
acids)
toyieldam
inoacids,
gluc
oson
ean
dH2O
2C20
Pc16
g074
70gi|211
5856
95strong
simila
rity
toglycinede
carbox
ylasesubu
nitTGcv1-
Saccha
romyces
cerevisiae
N/A
N/A
Aminoa
cidmetab
olism.Catab
olism
ofglycineto
5,10
-methy
lene
-THF
C21
Pc22
g102
20gi|211
5920
47strong
simila
rity
todn
aK-typ
emolecular
chap
eron
eSsb2
-Sa
ccha
romyces
cerevisiae
N/A
N/A
Proteinfate.Proteinfolding,
mod
ification
andde
stination
C22
Pc20
g029
10gi|211
5875
80strong
simila
rity
toaspa
rtate-semialdeh
ydede
hydrog
enaseHom
2-
Saccha
romyces
cerevisiae
N/A
N/A
Aminoa
cidmetab
olism
C23
Pc22
g080
80gi|211
5918
41strong
simila
rity
toactininteractingproteinlik
eproteinAn0
2g14
620-
Aspergillu
sniger
N/A
N/A
Cellularstructure
C24
Pc20
g048
10gi|211
5877
57strong
simila
rity
toestrog
enreceptor-binding
cyclop
hilin
cypD
-Bo
sprim
igeniustaurus
N/A
N/A
Proteinwithbind
ingfunc
tion
orco
factor
requ
irem
ent
C25
Pc18
g022
90gi|211
5868
06strong
simila
rity
to2-nitrop
ropa
nediox
ygen
aseprecursornc
d-2-
Neurosporacrassa
N/A
N/A
Oxida
tion
ofnitroa
lkan
esinto
theirco
rrespo
ndingcarbon
ylco
mpo
unds
and
nitrite
C26
Pc13
g107
70gi|211
5841
17strong
simila
rity
tocA
MP-de
pend
entproteinkina
seregu
latory
subu
nit
pkaR
-Aspergillu
sniger
N/A
N/A
Proteinfate.Proteinph
osph
orylation
C27
Pc20
g016
10gi|211
5874
55strong
simila
rity
tomitoc
hond
rial
malatede
hydrog
enaseMdh
1-
Saccha
romyces
cerevisiae
N/A
N/A
Citricacid
cycle
(con
tinuedon
next
page)
M.-S. Jami et al. Journal of Proteomics 187 (2018) 243–259
253
Table2(con
tinued)
Spot
ORF
Accession
No
Simila
rity
Fold
chan
geP-Value
Func
tion
C28
Pc20
g016
10gi|211
5874
55strong
simila
rity
tomitoc
hond
rial
malatede
hydrog
enaseMdh
1-
Saccha
romyces
cerevisiae
N/A
N/A
Citricacid
cycle
Pc14
g020
10gi|211
5848
16strong
simila
rity
tohy
pothetical
protein
contig_1_98_scaff
old_6.tfa_10
90cg
-Aspergillu
snidu
lans
N/A
N/A
Unk
nown
C29
Pc21
g169
70gi|255
9558
89va
cuolar
serine
proteina
seAAG44
693-Penicillium
chrysogenu
m.A
llergen
Penn18
[Penicillium
chrysogenu
m]
N/A
N/A
Proteinfate.Proteinfolding,
mod
ification
andde
stination
C30
Pc16
g092
50gi|211
5858
66strong
simila
rity
tocytoch
rome-b5
redu
ctaseMcr1-Sa
ccha
romyces
cerevisiae
N/A
N/A
Oxida
tive
stress
respon
se
C31
Pc12
g014
20gi|211
5816
59strong
simila
rity
toribo
flav
inbiosyn
thesisproteinRib7-S
accharom
yces
cerevisiae
N/A
N/A
Cofactorbiosyn
thesis
C32
Pc12
g165
40gi|211
5830
71strong
simila
rity
tocytosolic
aspa
rtate–tRNA
ligaseDps1-
Saccha
romyces
cerevisiae
N/A
N/A
aminoa
cyl-trnabiosyn
thesis
C33
proteasomeco
mpo
nent
PRE6
[Aspergillu
sterreusNIH
2624
]gi|115
4373
66proteasomeco
mpo
nent
PRE6
[Aspergillu
sterreusNIH
2624
]N/A
N/A
Proteinfate.Proteinfolding,
mod
ification
andde
stination
C34
Pc20
g118
50gi|211
5884
06strong
simila
rity
toelon
gation
factor
1-ga
mma1Te
f3-Sa
ccha
romyces
cerevisiae
N/A
N/A
Tran
slation
C35
Pc20
g032
90gi|211
5876
16strong
simila
rity
tohy
pothetical
proteinco
ntig14
95_2.tfa_64
0cg-
Aspergillu
sfumigatus
N/A
N/A
Unk
nown
C36
Pc22
g139
50gi|211
5923
36strong
simila
rity
tova
cuolar
aminop
eptida
seYsci-Saccharom
yces
cerevisiae
N/A
N/A
Proteinfate.Proteinfolding,
mod
ification
andde
stination
C37
Pc21
g031
40gi|211
5890
89strong
simila
rity
tocellcycleregu
latorp2
1proteinwos2p
-Schizosaccha
romyces
pombe
N/A
N/A
Cellcyclean
dDNA
proc
essing
C38
Pc21
g031
40gi|211
5890
89strong
simila
rity
tocellcycleregu
latorp2
1proteinwos2p
-Schizosaccha
romyces
pombe
N/A
N/A
Cellcyclean
dDNA
proc
essing
C39
Pc06
g007
10gi|211
5812
97strong
simila
rity
to15
0kD
aox
ygen
regu
latedproteinORP1
50-Rattus
norvegicus
N/A
N/A
Unk
nown
C40
Pc22
g020
00gi|211
5912
61strong
simila
rity
tomitoc
hond
rial
acon
itatehy
drataseAco
1-
Saccha
romyces
cerevisiae
−1.2
1.3E
-03
Citricacid
cycle
C41
Pc22
g102
20gi|211
5920
47strong
simila
rity
todn
aK-typ
emolecular
chap
eron
eSsb2
-Sa
ccha
romyces
cerevisiae
−1.7
9.6E
-02
Proteinfate.Proteinfolding,
mod
ification
andde
stination
C42
Pc18
g009
80gi|211
5866
77strong
simila
rity
tohy
pothetical
trun
klateralc
ellspe
cificge
neHrTLC
1-
Halocyn
thia
roretzi
−2.5
1.8E
-03
Red
oxmetab
olism
C43
Pc18
g009
80gi|211
5866
77strong
simila
rity
tohy
pothetical
trun
klateralc
ellspe
cificge
neHrTLC
1-
Halocyn
thia
roretzi
−2.0
1.0E
-03
Red
oxmetab
olism
C44
Pc22
g117
10gi|211
5921
34strong
simila
rity
toalph
asubu
nitE1
ofthepy
ruva
tede
hydrog
enase
complex
Pda1
-Sa
ccha
romyces
cerevisiae
−2.9
7.9E
-03
Biosyn
thesis
ofacetyl
CoA
from
pyruva
te
C45
Pc12
g068
70gi|211
5821
44strong
simila
rity
tosuccinyl
coen
zymeA
syntha
sealph
asubu
nit
SYRTS
A-Rattusno
rvegicus
−1.8
2.2E
-03
Citricacid
cycle
C46
Pc12
g068
70gi|211
5821
44strong
simila
rity
tosuccinyl
coen
zymeA
syntha
sealph
asubu
nit
SYRTS
A-Rattusno
rvegicus
N/A
N/A
Citricacid
cycle
Pc22
g179
50gi|211
5927
21strong
simila
rity
tohy
pothetical
proteinco
ntig40
.tfa_68
0wg-A
spergillu
sfumigatus
N/A
N/A
Unk
nown
C47
Pc22
g012
60gi|211
5911
87strong
simila
rity
tosm
allG-protein
Gsp1-Can
dida
albicans
−1.6
1.6E
-02
Cellularco
mmun
ication/
Sign
altran
sduc
tion
mecha
nism
C48
Pc18
g007
90gi|211
5866
58strong
simila
rity
toglutathion
eS-tran
sferaselik
eproteinAn0
2g06
560-
Aspergillu
sniger
−2.9
6.2E
-03
Oxida
tive
stress
respon
se
C49
Pc20
g094
00gi|211
5881
71strong
simila
rity
todipe
ptidyl-pep
tida
seVDPP
V-Aspergillu
sfumigatus
−7.8
2.0E
-03
Proteinfate.Proteinfolding,
mod
ification
andde
stination
C50
Pc22
g252
20gi|211
5934
26strong
simila
rity
to1,4-be
nzoq
uino
neredu
ctaseqr.-
Phan
erocha
ete
chrysosporium
−1.9
1.1E
-03
Red
uction
ofmetho
xylated,
lignin-de
rive
dqu
inon
es
C51
Pc22
g095
80gi|211
5919
86strong
simila
rity
toacid
phosph
ataseap
hA-Aspergillu
sficuum
−2.3
6.2E
-05
Phosph
atemetab
olism
C52
Pc20
g058
30gi|211
5878
47strong
simila
rity
toen
oylred
uctase
ofthelova
statin
biosyn
thesislovC
-Aspergillu
sterreus
−4.0
3.0E
-04
Red
uctase
activity.F
atty
acid
biosyn
thesis
C53
Pc16
g084
60gi|211
5857
89strong
simila
rity
tosorbitol
dehy
drog
enasegu
tB-Ba
cillu
ssubtilis
−3.3
4.6E
-04
Carbo
hydrateMetab
olism
M.-S. Jami et al. Journal of Proteomics 187 (2018) 243–259
254
(Pc21g21370), which is directly involved in the last step of the peni-cillin biosynthetic pathway and catalyses the replacement of the IPNside chain by the aromatic aryl side chain provided by the activatedform of phenylacetic acid (i.e. phenylacetyl CoA) [39]. Other importantproteins are a hypothetical cystathionine beta synthase, which is in-volved in the biosynthesis of cystathionine, a precursor of the cysteinein the ACV tripeptide [40, 41], a probable ketol-acid reductoisomeraseilv-2 involved in step 2 of the subpathway that synthesizes L-valine (oneof the three amino acid precursors of penicillin) from pyruvate, and aprobable thioredoxin peroxidase (Pc22g04430), which together withthioredoxin and thioredoxin reductase, comprise the thioredoxinsystem involved in the reduction of bis-ACV (the oxidized disulfideform of ACV) and reincorporation of this molecule to the penicillinbiosynthetic pathway [42]. S-adenosylmethionine synthase also re-sulted overrepresented after phenylacetate addition. This protein hasbeen reported to coordinate fungal secondary metabolism and devel-opment [43] and its overexpression has been related to increased pro-ductivity of secondary metabolites in bacteria [44, 45].
When the extracellular protein fraction was analysed, we foundsome proteins lacking predicted signal sequences for secretion. Thisphenomenon has also been previously described in fungi [23, 46],pointing to these proteins as truly secreted multifunctional proteinswith different activities according to their intracellular or extracellularlocation, a fact that has been confirmed in other organisms [47].However, the extracellular presence of these proteins due to cell lysisevents cannot be completely ruled out. One important mechanism in-volved in penicillin production and triggered by phenylacetic acid canbe related to the induction and finding of the glutamate dehydrogenase(encoded by the gdhA gene) in the culture broths. Although this proteinlacks classical or non-classical signal sequences for secretion, an ex-tracellular form of glutamate dehydrogenase has been reported in other
microorganisms, such as Clostridium difficile, where it confers resistanceto hydrogen peroxide [48]. The protein encoded by the gdhA gene isNADPH-dependent and catalyses inside the cell the reductive aminationof 2-oxoglutarate, thus giving rise to glutamate by means of a ther-modynamically and energetically favored pathway for ammonium as-similation. Interestingly, the NADPH-dependent glutamate dehy-drogenase has been reported to be involved in regulation of β-lactamproduction in industrial strains of P. chrysogenum [49] and therefore, itspresence in the extracellular protein fraction in response to phenyla-cetic acid addition may represent important information for improvedproductivity.
5. Conclusions
These results provide important data about the fate of phenylaceticacid in P. rubens. In addition to being the side chain precursor of ben-zylpenicillin, this molecule also plays a positive role in penicillin pro-duction. This is achieved by means of the effect exerted on some pro-teins directly related to the biosynthesis of penicillin (IAT, thioredoxinperoxidase) and precursor amino acids (cystathionine beta synthase,ketol-acid reductoisomerase ilv-2), and other important proteins(NADPH-dependent glutamate dehydrogenase, S-adenosylmethioninesynthase). This information contributes to the knowledge of the mole-cular mechanisms interconnected with phenylacetate utilization andpenicillin biosynthesis in penicillin-producing strains of P. rubens.
Acknowledgments
This research was supported by Instituto de CompetitividadEmpresarial (ICE, formerly ADE) and Junta de Castilla y León. R.Domínguez-Santos was granted a fellowship from Junta de Castilla y
Fig. 7. Effect of phenylacetic acid (PAA) in the extracellular proteome of P. rubens (P. chrysogenum Wisconsin 54–1255). Extracellular proteins obtained from culturesupernatants of P. rubens (P. chrysogenum Wisconsin 54–1255) grown for 60 h in DP medium with and without 1 g/L potassium phenylacetate, were separated by 2-DE using 18-cm wide-range IPG strips (pH 4–7 NL) and 12.5% SDS-PAGE gels, which were stained with CC following the “Blue Silver” staining method. Those spotsoverrepresented without PAA (underrepresented with PAA) are designated as “S”, whereas the letter “P” was used to designate those spots overrepresented in thepresence of PAA. The spots differentially represented in each condition are numbered and correspond to those proteins listed in Tables 3 (“P” spots) and 4 (“S” spots)and Supplementary Tables S3 (“P” spots) and S4 (“S” spots).
M.-S. Jami et al. Journal of Proteomics 187 (2018) 243–259
255
Table3
Extracellularproteins
overrepresen
tedat
60hin
thepresen
ceof
phen
ylacetic
acid.F
oldincrease
andp-va
lueareindicated.
Proteins
that
areon
lyde
tected
afterthead
dition
ofph
enylacetic
acid
arede
notedas
N/A
.
Spot
ORF
Accession
No
Simila
rity
Fold
chan
geP-Value
Func
tion
Sign
alpe
ptide
Non
-classically
secreted
protein
P1Pc
18g0
2900
gi|211
5868
64lysoph
osph
olipaseph
osph
olipaseBplb1
-Pen
icillium
chrysoge
num
N/A
N/A
Glyceroph
osph
olipid
metab
olism
YES
P2Pc
22g0
6490
gi|211
5916
94strong
simila
rity
toalka
lineph
osph
atase-N
eurosporacrassa
N/A
N/A
Dep
hospho
rylation
YES
P3Pc
22g0
6490
gi|211
5916
94strong
simila
rity
toalka
lineph
osph
atase-N
eurosporacrassa
N/A
N/A
Dep
hospho
rylation
YES
P4Pc
22g0
2000
gi|211
5912
61strong
simila
rity
tomitoc
hond
rial
acon
itatehy
drataseAco
1-
Saccha
romyces
cerevisiae
N/A
N/A
Citricacid
cycle
NO
NO
P5Pc
22g0
2000
gi|211
5912
61strong
simila
rity
tomitoc
hond
rial
acon
itatehy
drataseAco
1-
Saccha
romyces
cerevisiae
N/A
N/A
Citricacid
cycle
NO
NO
P6Pc
22g0
9380
gi|211
5919
67strong
simila
rity
toglycosylph
osph
atidylinositol-anc
horedbe
ta(1–3
)glucano
syltransferase
gel3
-Aspergillu
sfumigatus
3.7
1.0E
-04
Cellwallmorph
ogen
esis
YES
P7Pc
22g0
2800
gi|211
5913
41strong
simila
rity
tocalcium-binding
proteinprecursorcn
x1p-
Schizo
saccha
romyces
pombe
N/A
N/A
Con
trol
ofcellu
larfunc
tion
sYES
Pc22
g165
10gi|211
5925
82strong
simila
rity
toisoa
myl
alco
holox
idasemreA
-Aspergillu
soryzae
N/A
N/A
Form
ationof
isov
aleralde
hyde
YES
P8Pc
22g0
2800
gi|211
5913
41strong
simila
rity
tocalcium-binding
proteinprecursorcn
x1p-
Schizo
saccha
romyces
pombe
N/A
N/A
Con
trol
ofcellu
larfunc
tion
sYES
P9Pc
22g0
2000
gi|211
5912
61strong
simila
rity
tomitoc
hond
rial
acon
itatehy
drataseAco
1-
Saccha
romyces
cerevisiae
N/A
N/A
Citricacid
cycle
NO
NO
P10
Pc22
g227
10gi|211
5931
82strong
simila
rity
todihy
drox
y-acid
dehy
drataseIlv3
-Sa
ccha
romyces
cerevisiae
N/A
N/A
Biosyn
thesis
ofbran
ched
-cha
inam
inoacids
NO
YES
P11
Pc22
g093
90gi|211
5919
68strong
simila
rity
toman
nitolde
hydrog
enasemtlD
-Pseudo
mon
asfluo
rescens
N/A
N/A
Fruc
tose
andman
nose
metab
olism
NO
NO
Pc18
g013
90gi|211
5867
17strong
simila
rity
toph
osph
ogluco
mutasepg
mB-Aspergillu
snidu
lans
N/A
N/A
Hexosemetab
olism
NO
YES
P12
Pc18
g009
80gi|211
5866
77strong
simila
rity
tohy
pothetical
trun
klateralcellspecificge
neHrTLC
1-Haloc
ynthia
roretzi
N/A
N/A
Red
oxmetab
olism
NO
YES
P13
Pc18
g009
80gi|211
5866
77strong
simila
rity
tohy
pothetical
trun
klateralcellspecificge
neHrTLC
1-Haloc
ynthia
roretzi
N/A
N/A
Red
oxmetab
olism
NO
YES
P14
Pc20
g047
20gi|211
5877
49strong
simila
rity
toprecursorof
dihy
drolipoa
midede
hydrog
enase
Lpd1
-Sa
ccha
romyces
cerevisiae
N/A
N/A
E3co
mpo
nent
ofthepy
ruva
te,α
-ketog
lutarate,a
ndbran
ched
-cha
inam
inoacid-deh
ydroge
nase
complexes
andtheglycinecleava
gesystem
NO
YES
P15
Pc18
g013
90gi|211
5867
17strong
simila
rity
toph
osph
ogluco
mutasepg
mB-Aspergillu
snidu
lans
2.6
1.3E
-06
Hexosemetab
olism
NO
YES
P16
Pc22
g175
60gi|211
5926
84glutam
atede
hydrog
enasegd
hA-Pen
icillium
chrysoge
num
N/A
N/A
Ammon
ium
utilization
.Reg
ulationof
beta-la
ctam
prod
uction
NO
NO
P17
Pc18
g013
90gi|211
5867
17strong
simila
rity
toph
osph
ogluco
mutasepg
mB-Aspergillu
snidu
lans
3.4
1.8E
-04
Hexosemetab
olism
NO
YES
P18
Pc14
g017
40gi|211
5847
89en
olaseBA
C82
549-Pe
nicillium
chrysoge
num
N/A
N/A
Glyco
lysis
NO
NO
P19
Pc20
g036
10gi|211
5876
47strong
simila
rity
toprecursorof
mitoc
hond
rial
isoc
itrate
dehy
drog
enaseicdA
-Aspergillu
snige
rN/A
N/A
Citricacid
cycle
NO
YES
P20
Pc16
g050
80gi|211
5854
78strong
simila
rity
toad
enosylho
moc
ysteinase-H
omosapien
sN/A
N/A
Methy
lation
cycle
NO
YES
P21
Pc12
g148
60gi|211
5829
09extracellularacid
phosph
atasePh
oA-Pen
icillium
chrysoge
num
16.1
3.5E
-04
Dep
hospho
rylation
YES
P22
Pc12
g043
10gi|211
5819
20strong
simila
rity
toacetate-indu
ciblege
neaciA
-Aspergillu
snidu
lans
N/A
N/A
Oxida
tion
ofform
ate.
Form
ationof
energy
NO
YES
P23
Pc12
g043
10gi|211
5819
20strong
simila
rity
toacetate-indu
ciblege
neaciA
-Aspergillu
snidu
lans
N/A
N/A
Oxida
tion
ofform
ate.
Form
ationof
energy
NO
YES
P24
Pc16
g027
90gi|211
5852
72strong
simila
rity
toaspa
rtatetran
saminaselik
eproteinAn0
8g01
000
-Aspergillu
snige
rN/A
N/A
Aminoacid
metab
olism
NO
YES
P25
Pc22
g048
50gi|211
5915
39strong
simila
rity
toD-arabino
sede
hydrog
enaseAra1-
Saccha
romyces
cerevisiae
N/A
N/A
Oxido
redu
ctaseactivity
NO
NO
P26
Pc21
g169
50gi|211
5904
06strong
simila
rity
totran
saldolaseTa
l1-Sacch
arom
yces
cerevisiae
N/A
N/A
Pentoseph
osph
atepa
thway
NO
NO
P27
Pc21
g169
50gi|211
5904
06strong
simila
rity
totran
saldolaseTa
l1-Sacch
arom
yces
cerevisiae
N/A
N/A
Pentoseph
osph
atepa
thway
NO
NO
P28
Pc20
g072
30gi|211
5879
83strong
simila
rity
toinorga
nicpy
roph
osph
ataseIpp1
-Sa
ccha
romyces
cerevisiae
N/A
N/A
Gen
eral
metab
olism
NO
NO
(con
tinuedon
next
page)
M.-S. Jami et al. Journal of Proteomics 187 (2018) 243–259
256
Table3(con
tinued)
Spot
ORF
Accession
No
Simila
rity
Fold
chan
geP-Value
Func
tion
Sign
alpe
ptide
Non
-classically
secreted
protein
P29
Pc18
g027
40gi|211
5868
48strong
simila
rity
toregu
calcin
also
know
nas
sene
scen
cemarke
rprotein-30
likeproteinAn0
4g03
420-Aspergillu
snige
rN/A
N/A
Con
trol
ofcellu
larfunc
tion
sNO
NO
Pc13
g087
30gi|211
5839
18strong
simila
rity
to1,3-be
ta-glucano
syltransferase
bgt1
-Aspergillu
sfumigatus
N/A
N/A
Cellwallmorph
ogen
esis
YES
P30
Pc12
g058
20gi|211
5820
45strong
simila
rity
toesterase
DES
D-Hom
osapien
sN/A
N/A
Gen
eral
metab
olism
NO
YES
P31
Pc13
g044
20gi|211
5834
97orotidine5-ph
osph
atede
carbox
ylasepy
rG-Pen
icillium
chrysoge
num
N/A
N/A
Uridine
biosyn
thesis
NO
YES
P32
Pc22
g254
70gi|211
5934
49strong
simila
rity
toSo
l1-Sa
ccha
romyces
cerevisiae
N/A
N/A
Gen
eral
metab
olism
NO
YES
P33
Pc12
g008
30gi|211
5816
03strong
simila
rity
tosorbitol
utilization
proteinsou2
-Can
dida
albicans
N/A
N/A
Carbo
hydrateMetab
olism.(R
elated
toshort-ch
ainalco
hol
dehy
drog
enases)
NO
NO
P34
Pc22
g222
90gi|211
5931
41strong
simila
rity
toIgE-bind
ingprotein-Aspergillu
sfumigatus
N/A
N/A
Unk
nown
YES
P35
Pc12
g008
30gi|211
5816
03strong
simila
rity
tosorbitol
utilization
proteinsou2
-Can
dida
albicans
N/A
N/A
Carbo
hydrateMetab
olism.(R
elated
toshort-ch
ainalco
hol
dehy
drog
enases)
NO
NO
P36
Pc12
g136
00gi|211
5827
86strong
simila
rity
tohy
pothetical
necrosis
andethy
lene
indu
cing
proteinBH
0395
-Ba
cillu
sha
lodu
rans
N/A
N/A
Prod
uces
Immun
erespon
sesan
dcellde
athin
plan
tsYES
Pc22
g222
90gi|211
5931
41strong
simila
rity
toIgE-bind
ingprotein-Aspergillu
sfumigatus
N/A
N/A
Unk
nown
YES
P37
Pc20
g048
10gi|211
5877
57strong
simila
rity
toestrog
enreceptor-binding
cyclop
hilin
cypD
-Bos
prim
igen
iustaurus
N/A
N/A
Con
trol
ofcellu
larfunc
tion
sNO
NO
P38
Pc21
g142
20gi|211
5901
48strong
simila
rity
tohy
pothetical
proteinco
ntig14
95_2.tfa_63
0cg-
Aspergillu
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