Journal of Proteomics 187 (2018) 243–259 Contents lists available at ScienceDirect Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot Catabolism of phenylacetic acid in Penicillium rubens. Proteome-wide analysis in response to the benzylpenicillin side chain precursor T ⁎ 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, Spain b Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran c Área de Microbiología, Departamento de Biología Molecular, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain d Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain ARTICLE INFO ABSTRACT Keywords: Biosynthesis of benzylpenicillin in filamentous fungi (e.g. Penicillium chrysogenum - renamed as Penicillium ru- Phenylacetic acid bens- and Aspergillus nidulans) depends on the addition of CoA-activated forms of phenylacetic acid to iso- Benzylpenicillin penicillin N. Phenylacetic acid is also detoxified by means of the homogentisate pathway, which begins with the 2-hydroxyphenylacetate hydroxylation of phenylacetic acid to 2-hydroxyphenylacetate in a reaction catalysed by the pahA-encoded Phenylacetate hydroxylase phenylacetate hydroxylase. This catabolic step has been tested in three different penicillin-producing strains of Proteomics P. rubens (P. notatum, P. chrysogenum NRRL 1951 and P. chrysogenum Wisconsin 54–1255) in the presence of Penicillium rubens sucrose and lactose as non-repressing carbon sources. P. chrysogenum Wisconsin 54–1255 was able to accumulate 2-hydroxyphenylacetate at late culture times. Analysis of the P. rubens genome showed the presence of several PahA homologs, but only Pc16g01770 was transcribed under penicillin production conditions. Gene knock-down experiments indicated that the protein encoded by Pc16g01770 seems to have residual activity in phenylacetic acid 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 positive effect on some proteins directly related to the benzylpenicillin biosynthetic pathway, the synthesis of amino acid precursors and other important metabolic processes. Significance: The adaptive response of Penicillium rubens to benzylpenicillin production conditions remains to be fully elucidated. This article provides important information about the molecular mechanisms interconnected with phenylacetate (benzylpenicillin side chain precursor) utilization and penicillin biosynthesis, and will contribute 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 antimicrobial agent penicillin was initially identified by Fleming and colleagues as Penicillium rubrum, which was later re-identified as Peni- Since Fleming's fortuitous discovery of penicillin ninety years ago, cillium notatum and finally placed in synonymy with Penicillium chry- constant efforts have been made by the scientific community from in- sogenum. However, this nomenclature has been recently reconsidered, dustry and academia in order to improve penicillin titers. This has been leading to the conclusion that Fleming's original strain, the full genome achieved mainly by industrial strain improvement programs, where sequenced strain P. chrysogenum Wisconsin 54–1255 and its ancestor selected strains have been subjected during the last decades to several strain P. chrysogenum NRRL 1951 (wild-type), are in fact Penicillium rounds of radioactive and chemical mutagenesis, thus reaching product rubens [2]. Although all these strains are now classified as P. rubens, for titers and productivities three orders of magnitude higher than those the sake of clarity the old names P. notatum, P. chrysogenum Wisconsin provided by the ancestor strains [1]. The fungal strain producing the 54–1255 and NRRL-1951 are also used in this article, since hundreds of ⁎ 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). https://doi.org/10.1016/j.jprot.2018.08.006 Received 14 May 2018; Received in revised form 17 July 2018; Accepted 4 August 2018 Available online 06 August 2018 1874-3919/ © 2018 Published by Elsevier B.V. M.-S. Jami et al. Journal of Proteomics 187 (2018) 243–259 Fig. 1. Metabolic routes of phenylacetic acid in P. rubens: penicillin biosynthetic pathway (upper chart) and homogentisate pathway (lower chart). references using the old names have been published. In the case of benzylpenicillin, the side chain precursor is phenylacetic Much of the efforts made by the scientific community have focused acid, which is activated in the form of phenylacetyl CoA. Replacement on the biochemical and genetic characterization of the penicillin bio- is catalysed by the penDE-encoded acyl-CoA: IPN acyltransferase (IAT), synthetic pathway [3](Fig. 1), which is compartmentalized between which is synthesized as a 40-kDa precursor protein (proIAT) that un- cytosol and peroxisomes (for reviews see [4, 5]). It starts with the non- dergoes self-processing between residues Gly102 and Cys103. There- ribosomal condensation of L-α-aminoadipic acid, L-cysteine and L-va- fore, the active protein is a heterodimer comprising two subunits: α line by means of the nonribosomal peptide synthetase L-δ(α-aminoa- (11 kDa, corresponding to the N-terminal fragment) and β (29 kDa, dipyl)-L-cysteinyl-D-valine (ACV) synthetase (ACVS), which is a very corresponding to the C-terminal region). Activation of the side chain large multifunctional protein (MW 426 kDa). This protein is encoded by precursor is achieved by means of aryl CoA-ligases. At least three aryl- the single structural 11-kbp pcbAB gene. Next, in a reaction catalysed by CoA ligases, encoded by the phl, phlB (aclA) and phlC genes, respec- the isopenicillin N (IPN) synthase or cyclase (encoded by the pcbC tively, have been reported to activate phenylacetic acid [6–9]. How- gene), the ACV undergoes the oxidative ring closure of the tripeptide. ever, direct contribution to penicillin biosynthesis has only been de- This leads to the formation of the bicyclic structure (penam nucleus) of scribed in the case of the phenylacetyl CoA ligase encoded by the phl IPN in the cytosol. In the last step of the penicillin biosynthetic gene [6]. pathway, the α-aminoadipyl side chain of IPN is replaced inside per- Research has also been focused on the characterization of the oxisomes by a hydrophobic side chain activated as thioester with CoA. modifications introduced by industrial strain improvement programs. 244 M.-S. Jami et al. Journal of Proteomics 187 (2018) 243–259 Amplification of the penicillin gene cluster is well documented in many For proteomics experiments, P. rubens (P. chrysogenum Wisconsin of the improved penicillin producers, which contain several copies of 54–1255) was grown as indicated above in defined medium with 1 g/L this cluster (e.g. the AS-P-78 strain contains 5 or 6 copies) [10]. Mi- potassium phenylacetate. Control cultures lacked the side-chain pre- crobodies (peroxisomes), the organelles where activation of the side cursor potassium phenylacetate. Cultures were incubated at 25 °C and chain and its incorporation to the IPN molecule occur, are more 250 rpm and samples (mycelia and culture medium) were collected abundant in high-producer strains [11, 12]. Genome and transcriptome after 60 h for intracellular and extracellular proteome analysis. analyses have also revealed that transcription of genes involved in the For expression analysis experiments, conidia were inoculated in biosynthesis of the penicillin amino acid precursors, as well as of those complex inoculum medium CIM [25] without phenylacetate. After in- genes encoding microbody proteins, was higher in the high-producer cubation at 25 °C for 20 h in an orbital shaker (250 rpm), aliquots (5%) strain DS17690 [12]. More recently, proteomics studies concluded that were inoculated in CP complex penicillin production medium [25] with the increase in penicillin production along the industrial strain im- 4 g/L potassium phenylacetate and incubated under the same condi- provement program was a consequence of complex metabolic re- tions for 48 h and 60 h. organizations, and suggested that energetic burden, redox metabolism For transformation experiments, conidia were inoculated into MPPY or the supply of precursors are crucial for the biosynthesis of this an- medium (40 g/L glucose, 3 g/L NaNO3, 2 g/L yeast extract, 0.5 g/L KCl, tibiotic [13]. 0.5 g/L MgSO4·7H2O, 0.01 g/L FeSO4·7H2O, pH = 6.0) and grown for Besides this background knowledge, it is well known that the 24 h at 25 °C and 250 rpm. homogentisate pathway for the catabolism of phenylacetic acid (the side chain precursor in the biosynthesis of benzylpenicillin) to fumarate 2.2. Plasmid constructions for gene silencing and acetoacetate (Fig. 1) is diminished in Wisconsin 54–1255, and presumably, in derived