University of Groningen. Genomic Wake-Up Call Samol, Marta

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1 University of Groningen Genomic Wake-Up Call Samol, Marta IMPORTNT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2015 Link to publication in University of Groningen/UMCG research database Citation for published version (P): Samol, M. (2015). Genomic Wake-Up Call: ctivating Silent Biosynthetic Pathways for Novel Metabolites in Penicillium chrysogenum [Groningen]: University of Groningen Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 PPENDIX Relation Between Penicillin and Siderophores Production in Penicillium chrysogenum.1 Methodology.1.1 Fungal Strains and Cultures Conditions Penicillium chrysogenum strains analyzed in this study are described in chapter 5 and 6, and listed in Tables 5.1 and 6.1. Primers designed for the gene deletion constructs are listed in Tables S5.1 and 6.2. Media were prepared as described in chapter 5. The medium was supplemented with 18.4 mm phenylacetic acid (P), when indicated. Cells were growing for 7 days in shaking flasks at 220 rpm and 25 C..1.2 qpcr nalysis and Gene Copy Number The penicillin biosynthetic gene copy number was determined for all studied strains as described (Nijland et al. [2010]) using primers directed against the genes of penicillin cluster as listed in Tables S5.3 and S Extracellular Metabolite Levels Extracellular concentrations of phenylacetic acid and penicillin G were determined by high-pressure liquid chromatography (HPLC-UV) using an isocratic flow of acetonitrile at 245 g/l, 640 mg/l KH 2 PO 4, and 340 mg/l H 3 PO 4. Separation was performed on Shim-pack XR-ODS C18 column ( mm, 2.2 µm, Shimadzu, Japan) at a flow rate of 0.5 ml/min at 40 C. Chromatograms were acquired at a wavelength

3 192. Relation Between Penicillin and Siderophores Production in P. chrysogenum of 254 nm (Harris et al. [2006]). Levels were corrected for growth differences by dry weight measurements..2 Results - Penicillin Production under Iron Limited Conditions The DS54465 strain of P. chrysogenum shows a high β-lactam antibiotics production yield (Newbert et al. [1997]). Isopenicillin N synthetase, one of the key enzymes in β-lactam production, requires Fe 2+ for functioning. Therefore, the role of the pss, psm, pst genes that are involved in siderophore production (see Chapter 5 and 6) was analyzed in relation to penicillin production. Strain DS54465 contains eight copies of the penicillin biosynthetic gene clusters and these were maintained in the various pss deletion strains (Tab. 5.1) and the strain in which the putative transport genes pst and psm (Chapter 6) were deleted (data not shown). Strain DS54465 strain was grown in penicillin production medium (PPM) with phenylacetic acid (P) at: a) standard iron concentration (+Fe), b) at standard iron concentration with BPS (+Fe, +BPS), c) in the absence of iron (-Fe), and d) in the absence of iron with BPS (-Fe, +BPS). For the parental strain, there was no drastic change in β-lactam titer when cells were grown in the presence of a standard concentration of iron with or without the BPS chelator. However, under iron depletion (-Fe) and chelating conditions (-Fe, +BPS) the penicillin titers were reduced to 18 and nearly 0%, respectively (Fig..1). To examine the impact of the pss gene deletion on penicillin production, iron standard (+Fe) and depletion (-Fe) conditions were examined. However, there was no major additional effect of the deletion of the pss, pssb and pssc genes on penicillin production even when cells were grown in the absence iron (Fig..1C). lso for the various putative siderophore transporters gene deletion, there was no major effect on penicillin production (Fig..1D). Finally, the expression of the pss genes was analyzed by qpcr in relation to penicillin production. The cultures were grown under standard penicillin producing conditions in the presence (+Fe, +P) or absence of iron (-Fe, +P), while control growth experiments were not supplemented with P. The expression of the pss genes was elevated in the iron depleted growth conditions, but was only marginally affected by the presence or absence of P (Fig..1B)..3 Discussion The formation of penicillin (Fig..1) (Leiter et al. [2001]) and other secondary metabolites (Yin et al. [2013]) can be affected by the iron content in the medium. The iron requirement for optimal penicillin production in Penicillium chrysogenum

4 .3. Discussion 193 exceeds that for growth by a factor 10 (Nielsen and Jørgensen [1995]). In addition to many iron-dependent proteins involved in crucial intracellular processes (Oberegger et al. [2002]), the iron concentration has to be sufficient to allow for full activity of the key enzyme of β-lactam biosynthesis Isopenicillin N-synthase. In vitro, the enzyme requires 100 µmol/l of Fe 2+ to reach its maximum activity (Ramos et al. [1985]). We have investigated the relation between the penicillin G production and iron, with a special emphasis on siderophores biosynthesis using a high β-lactam producing industrial strain of P. chrysogenum (Fig..1C,D). Previous observations have shown that the addition of iron and/or iron in combination with siderophores (ferrichrome and coprogen) did not influence the β-lactam production in lowiron minimal medium (Leiter et al. [2001]). lthough, in contrast to our experiment where the cultures were grown under iron-devoid conditions, Leiter et al. [2001] grew the P. chrysogenum cells on a complex medium and the amount of iron was enough for antibiotic biosynthesis even after the transfer to low-iron media. Pss is responsible for the production of coprogen (Chapter 5). The expression of pss has been shown to be slightly up-regulated when cells are grown in the presence of phenylacetic acid (P) a precursor of penicillin production (Berg et al. [2008]) contrasting with the marginal negative effect in iron depletion media (.1B). P has been earlier reported to reduce the biomass and penicillin production in early stage cultures (Leiter et al. [2001]). Our data demonstrated a reduced penicillin concentration in the pss mutant when cells are grown in iron replete conditions which was also apparent but to lesser degree when cells were grown in iron depleted medium (.1C). On the other hand, the double deletion mutant of pss and pssb did not show this phenomenon. t high concentration, P might be oxidized to p-hydroxy-p (Hockenhull et al. [1952]; White et al. [1999]). Deletion strains of orthologs of Pss in Magnaporthe grisea and Cochliobolus heterostrophus display hypersensitivity towards oxidative stress (Hof et al. [2009]; Gillian Turgeon et al. [2008]). Therefore, the mutant lacking coprogen biosynthesis might be more sensitive to the toxic effect of P, which is often associated with increased cellular autolysis and decreased penicillin production White et al. [1999].

5 194. Relation Between Penicillin and Siderophores Production in P. chrysogenum 10 Penicillin cluster gene copy number DKS54466 DpssC DpssB Dpss Dpss, DpssB DS47274 Figure.1: Penicillin cluster copy number analyzed by qpcr as described by (Nijland et al. [2010]) in the strains used in this study. The DS54465 is the high penicillin producer derivative strain and carries eight copies of penicillin cluster. The deletion of pss, pssb and double deletion ( pss, pssb) was performed in DS54465 strain as a parental strain, which resulted in the mutants containing eight-nine penicillin cluster amplicons. Genomic DN of DS47274 (one penicillin cluster) was used as a control in the procedure. Error bars presented standard error of the mean.

6 .3. Discussion 195 Penicillin production (mg/gdw) Fe + Fe, + BPS - Fe - Fe, + BPS B pss pssb pssc Relative fold change C Penicillin production (mg/gdw) DKS Dpss, DpssB Dpss DpssB DpssC + Fe - Fe D Penicillin production (mg/gdw) DKS DpsmB DpstB Dpst Dpsm Figure.2: Penicillin G production of P. chrysogenum DS54465 strain and deletion mutants of genes involved in siderophore biosynthesis under different iron availability conditions: +Fe (black), +Fe and +BPS (grey), -Fe (white). () Error bars represent standard deviation from two biological replicates and three technical replicates of DS54465 strain. (B) qpcr analysis of NRPS genes involved in siderophores biosynthesis: Pc16g03850 (pss), Pc22g20400 (pssb), Pc13g05250 (pssc) in DS54465 strain. Bars represent fold changes of transcript level relative to penicillin non-producing conditions (no P in the medium). (C,D) Penicillin G production in cultures growing at the presence of iron (black) and the absence of iron as trace element (white). Error bars represent standard deviation from three (C) and four biological replicates (D). The cultures were growing in the penicillin production medium (PPM) containing phenylacetic acid (P) for 7 days at rotary incubator at 200 rpm, at 25 C.

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8 References Berg, M.. v. d., lbang, R., lbermann, K., Badger, J. H., Daran, J.-M., Driessen,. J. M., Garcia-Estrada, C., Fedorova, N. D., Harris, D. M., Heijne, W. H. M., Joardar, V., Kiel, J.. K. W., Kovalchuk,., Martín, J. F., Nierman, W. C., Nijland, J. G., Pronk, J. T., Roubos, J.., van der Klei, I. J., van Peij, N. N. M. E., Veenhuis, M., von Döhren, H., Wagner, C., Wortman, J., and Bovenberg, R.. L. (2008). Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nat Biotech, 26(10): Gillian Turgeon, B., Oide, S., and Bushley, K. (2008). Creating and screening Cochliobolus heterostrophus non-ribosomal peptide synthetase mutants. Mycol. Res., 112(2): Harris, D. M., Diderich, J.., van der Krogt, Z.., Luttik, M.. H., Raamsdonk, L. M., Bovenberg, R.. L., van Gulik, W. M., van Dijken, J. P., and Pronk, J. T. (2006). Enzymic analysis of NDPH metabolism in beta-lactam-producing penicillium chrysogenum: Presence of a mitochondrial NDPH dehydrogenase. Metab. Eng., 8(2): Hockenhull, D. J. D., Walker,. D., Wilkin, G. D., and Winder, F. G. (1952). Oxidation of phenylacetic acid by Penicillium chrysogenum. Biochem J, 50(5): Hof, C., Eisfeld, K., ntelo, L., Foster,. J., and nke, H. (2009). Siderophore synthesis in Magnaporthe grisea is essential for vegetative growth, conidiation and resistance to oxidative stress. Fungal Genet. Biol., 46(4): Leiter, É., Emri, T., Gyémánt, G., Nagy, I., Pócsi, I., Winkelmann, G., and Pócsi, I. (2001). Penicillin v production by Penicillium chrysogenum in the presence of fe 3+ and in low-iron culture medium. Folia Microbiol, 46(2): Newbert, R. W., Barton, B., Greaves, P., Harper, J., and Turner, G. (1997). nalysis of a commercially improved Penicillium chrysogenum strain series: involvement of recombinogenic regions in amplification and deletion of the penicillin biosynthesis gene cluster. J Ind Microbiol Biotech, 19(1): Nielsen, J. and Jørgensen, H. S. (1995). Metabolic control analysis of the penicillin biosynthetic pathway in a high-yielding strain of Penicillium chrysogenum. Biotechnol Prog., 11(3): Nijland, J. G., Ebbendorf, B., Woszczynska, M., Boer, R., Bovenberg, R.. L., and Driessen,. J. M. (2010). Nonlinear biosynthetic gene cluster dose effect on penicillin production by Penicillium chrysogenum. ppl Env. Microbiol, 76(21): Oberegger, H., Schoeser, M., Zadra, I., Schrettl, M., Parson, W., and Haas, H. (2002). Regulation of fre, aco, lysf, and cyc expression by iron availability in spergillus nidulans. ppl Env. Microbiol, 68(11): Ramos, F. R., López-Nieto, M. J., and Martín, J. F. (1985). Isopenicillin n synthetase of Penicillium chrysogenum, an enzyme that converts delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine to isopenicillin n. ntimicrob gents Chemother, 27(3):

9 198 References White, S., Berry, D. R., and McNeil, B. (1999). Effect of phenylacetic acid feeding on the process of cellular autolysis in submerged batch cultures of Penicillium chrysogenum. J. Biotechnol., 75(2 3): Yin, W.-B., Baccile, J.., Bok, J. W., Chen, Y., Keller, N. P., and Schroeder, F. C. (2013). nonribosomal peptide synthetase-derived iron(iii) complex from the pathogenic fungus aspergillus fumigatus. J m Chem Soc, 135(6):