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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).

Journal of Proteomics 187 (2018) 243–259

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).

M.-S. Jami et al. Journal of Proteomics 187 (2018) 243–259

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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.


248

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).


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).


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).


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


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


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


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)


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


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


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


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


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


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


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).


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)


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

journal of proteomicseprints.skums.ac.ir/7009/1/2.pdfprecursor is achieved by means of aryl...

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