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The Inducers 1,3-Diaminopropane and Spermidine Cause The JOURNAL OF PROTEOMICS 85 (2013) 129– 159 Available online at www.sciencedirect.com www.elsevier.com/locate/jprot The inducers 1,3-diaminopropane and spermidine cause the reprogramming of metabolism in Penicillium chrysogenum, leading to multiple vesicles and penicillin overproduction Carlos García-Estradaa,⁎, Carlos Barreiroa, Mohammad-Saeid Jamia, Jorge Martín-Gonzáleza, Juan-Francisco Martínb,⁎⁎ aINBIOTEC, Instituto de Biotecnología de León, Avda. Real no. 1, Parque Científico de León, 24006 León, Spain bÁrea de Microbiología, Departamento de Biología Molecular, Universidad de León, Campus de Vegazana s/n; 24071 León, Spain ARTICLE INFO ABSTRACT Article history: In this article we studied the differential protein abundance of Penicillium chrysogenum in Received 21 February 2013 response to either 1,3-diaminopropane (1,3-DAP) or spermidine, which behave as inducers Accepted 15 April 2013 of the penicillin production process. Proteins were resolved in 2-DE gels and identified by Available online 30 April 2013 tandem MS spectrometry. Both inducers produced largely identical changes in the proteome, suggesting that they may be interconverted and act by the same mechanism. Keywords: The addition of either 1,3-DAP or spermidine led to the overrepresentation of the last 1,3-diaminopropane enzyme of the penicillin pathway, isopenicillin N acyltransferase (IAT). A modified form of Spermidine the IAT protein was newly detected in the polyamine-supplemented cultures. Both Penicillin inducers produced a rearrangement of the proteome resulting in an overrepresentation of Penicillium chrysogenum enzymes involved in the biosynthesis of valine and other precursors (e.g. coenzyme A) of Vesicles penicillin. Interestingly, two enzymes of the homogentisate pathway involved in the degradation of phenylacetic acid (a well-known precursor of benzylpenicillin) were reduced following the addition of either of these two inducers, allowing an increase of the phenylacetic acid availability. Both inducers produced also an increase in the intracellular content of vesicles that derived to vacuoles in late stages and promoted sporulation of P. chrysogenum in solid medium. Biological significance The analysis of global protein changes produced in response to polyamines 1,3-DAP and spermidine provides a valuable information for the understanding of the molecular mechanisms underlying the production of penicillin. This represents useful information to improve the production of this antibiotic and many other bioactive secondary metabolites not only in P. chrysogenum,butinotherfilamentousfungiaswell. © 2013 Elsevier B.V. All rights reserved. ⁎ Corresponding author. Tel.: +34 987210308; fax: +34 987210388. ⁎⁎ Corresponding author. Tel.: +34 987291505; fax: +34 987291409. E-mail addresses: [email protected] (C. García-Estrada), [email protected] (J.-F. Martín). 1874-3919/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2013.04.028 130 JOURNAL OF PROTEOMICS 85 (2013) 129– 159 (IPN). This reaction takes place in the cytosol and is catalyzed 1. Introduction by the 38-kDa IPN synthase or cyclase (encoded by the intronless pcbC gene). Once IPN is synthesized, it enters the The biosynthesis of penicillin in Penicillium chrysogenum is an peroxisome, where the L-α-aminoadipic side-chain of this excellent model to study the molecular mechanisms of intermediate is replaced by a hydrophobic acyl molecule. Side biosynthesis, control of gene expression and secretion of chain replacement is catalyzed by the peroxisomal acyl-CoA: IPN secondary metabolites in fungi [1–3] due to the excellent acyltransferase (IAT), which is encoded by the penDE gene. IAT is knowledge accumulated on the biosynthesis [4,5] and molec- synthesized as a preprotein of 40 kDa termed proacyltransferase ular genetics of this β-lactam compound [3,6,7]. The penicillin or proIAT. The proacyltransferase is autocatalytically self- biosynthetic pathway (Fig. 1) has been extensively reviewed processed into subunits α (11 kDa, corresponding to the [2–7]. Penicillin biosynthesis starts in the cytosol with the N-terminal fragment) and β (29 kDa, corresponding to the formation of the tripeptide δ-L-(α-aminoadipyl)-L-cysteinyl- C-terminal region), which constitute an active heterodimer. A D-valine (ACV) after the non-ribosomal condensation of two-step enzymatic process has been proposed for side chain L-α-aminoadipic acid, L-cysteine and L-valine. This reaction replacement reaction. During the first step, the amidohydrolase is catalyzed by the 426-kDa ACV synthetase (ACVS), which is activity removes the L-α-aminoadipate side chain of IPN, thus encoded by the 11-kbp intron-free pcbAB gene. ACVS is forming 6-aminopenicillanic acid (6-APA). Next, the acyl-CoA: synthesized as an inactive apoprotein that requires activation 6-APA acyltransferase activity introduces the new activated by means of the addition of a 4´-phosphopantetheine arm acyl side chain. Activation of the precursor acyl molecules as (derived from coenzyme A (CoA)) in a reaction carried out by a CoA thioesters (phenylacetyl-CoA from phenylacetate in the 4´-phosphopantetheinyl transferase (PPTase). During the next case of benzylpenicillin) is a prerequisite for the incorporation step, the oxidative ring closure of the tripeptide occurs, thus of acyl side chains during the biosynthesis of hydrophobic giving rise to the penam nucleus structure of isopenicillin N penicillins and peroxisomal acyl-CoA ligases are in charge of such activation [2,3]. L-α-aminoadipic L-cysteine L-valine The expression of the penicillin biosynthetic genes pcbAB, pcbC and penDE is greatly increased by the inducers 1,3- H N H N SH H2N 2 COOH 2 diaminopropane (1,3-DAP) and spermidine [8] that were H COOH COOH COOH initially identified in a screening of autoinducers synthesized by P. chrysogenum and Acremonium chrysogenum [9]. The inducing pcbAB ACV synthetase effect of 1,3-DAP and spermidine is not exerted by other H SH H2N N diamines (e.g. putrescine, cadaverine or the triamine spermine), H O NH suggesting that the inducing effect is specific of those COOH O COOH two compounds 1,3-DAP and spermidine that might be δ(-L-α-aminoadipyl)-L-cysteinyl-D-valine interconnected through conversion reactions. This induction is independent of the pH control mediated by the general pH regulator PacC. However, it appears to be mediated, at least pcbC Isopenicillin N synthase partially, by the LaeA regulatory protein, since 1,3-DAP and H spermidine restore the expression of penicillin biosynthesis H2N N S H O genes and, therefore, penicillin production in a laeA defective COOH N O mutant [8,10]. COOH The stimulatory effect of 1,3-DAP and spermidine is observed Isopenicillin N not only on penicillin biosynthesis, but also on the formation of the green spore pigment and the brown mycelia pigment [8].This effect is similar to that exerted by the LaeA regulatory protein L-α-aminoadipic Phenylacetyl-CoA both in P. chrysogenum [10] and in Aspergillus nidulans [11]. It is well known that the last two enzymes required for benzylpenicillin biosynthesis, namely phenylacetyl-CoA ligase penDE H N S 2 (Phl) and isopenicillin N acyltransferase (IAT) are located in the L-α-aminoadipic N – IsopenicillinN O peroxisome lumen [12 14]. Recent evidence in several filamen- HS-CoA acyltransferase COOH 6-APA tous fungi revealed an important role of endoplasmic reticulum (ER)-derived vesicles in the production of several secondary metabolites, including aflatoxins in Aspergillus species [15–17]. Phenylacetyl-CoA H Proteomics is an interesting tool to study global protein N S HS-CoA changes in response to different inducers or stressing factors O N [18]. Proteomics has been applied to investigate changes in the O COOH proteome of P. chrysogenum during the strain improvement program [19,20]. Therefore, it was interesting to apply a Benzylpenicillin combination of proteomics and other biochemical and micros- copy tools to elucidate the role of 1,3-DAP on the shift of Fig. 1 – Schematic representation of the benzylpenicillin P. chrysogenum metabolism that leads to penicillin over- biosynthetic pathway. production. In this article we report that both inducers JOURNAL OF PROTEOMICS 85 (2013) 129– 159 131 1,3-DAP and spermidine exert a strong effect on cellular than 10%, which may be due to experimental variability. Spot metabolism resulting in a rearrangement of pathways for the normalization (internal calibration to make the data indepen- synthesis of amino acids precursors of penicillin and an dent from experimental variations among gels) was made increase of the penicillin biosynthetic enzymes, in addition to using relative volumes to quantify and compare the gel spots. the previously reported transcriptional activation of the peni- Relative spot volumes correspond to the volume of each cillin biosynthetic genes pcbAB, pcbC and penDE [8].Both spot divided by the total volume of all the spots in the gel. inducers produce a significant increase of internal vesicles in Differentially expressed proteins between two conditions the cytoplasm of the supplemented cells and also promote were considered when the ratio of the relative volume average sporulation of P. chrysogenum. for one specific spot (present in the three biological replicates) was higher than 1.5 and the p-value was <0.05. 2. Materials and methods 2.4. Protein identification by tandem MS spectrometry 2.1. Strains and growth conditions The protein spots of interest were manually excised, digested and processed
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