Abstract
Penicillium chrysogenum is an economically important ascomycete used as industrial producer of penicillin. However, with the exception of penicillin biosynthesis genes, little attention has been paid to the genetics of other aspects of the metabolism of this fungus. In this article we describe the first attempt of systematic analysis of expressed genes in P. chrysogenum, using a suppression subtractive hybridization approach to clone and identify sequences of genes differentially expressed in media with glucose or lactose as carbon source (penicillin-repressing or non-repressing conditions). A total of 167 clones were analysed, 95 from the glucose condition and 72 from the lactose condition. Genes differentially expressed in the glucose condition encode mainly proteins involved in the mitochondrial electron transport chain and primary metabolism. Genes expressed differentially in lactose-containing medium include genes for secondary metabolism (pcbC, isopenicillin N synthase), different hydrolases and a gene encoding a putative hexose transporter or sensor. The results provided information on how the metabolism of this fungus adapts to different carbon sources. The expression patterns of some of the genes support the hypothesis that glucose induces higher rates of respiration in P. chrysogenum while repressing secondary metabolism.
References
Aharanowitz Y, Cohen G, Martín JF (1992) Penicillin and cephalosporin biosynthetic genes: structure, organization, regulation, and evolution. Annu Rev Microbiol 46:461–495
Akopyants N, Fradkov A, Diatchenko L, Hill J, Siebert P, Lukyanov S, Sverdlov E, Berg D (1998) PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc Natl Acad Sci USA 95:13108–13113
Bañuelos O, Casqueiro J, Gutiérrez S, Martín JF (2000) Overexpression of the lys1 gene in Penicillium chrysogenum: homocitrate synthase levels, α-aminoadipic acid pool and penicillin production. Appl Microbiol Biotechnol 54:69–77
Barredo JL, Cantoral JM, Álvarez E, Díez B, Martín JF (1989a) Cloning, sequence analysis and transcriptional study of the isopenicillin N synthase of Penicillium chrysogenum AS-P-78. Mol Gen Genet 216:91–98
Barredo JL, van Solingen P, Díez B, Álvarez E, Cantoral JM, Kattevilder A, Smaal EB, Groenen MAM, Veenstra AE, Martín JF (1998b) Cloning and characterization of acyl-CoA:6-APA acyl-transferase gene of Penicillium chrysogenum. Gene 83:291–300
Brakhage AA (1998) Molecular regulation of β-lactam biosynthesis in filamentous fungi. Microbiol Mol Biol Rev 62:547–585
Cohen G, Argaman A, Schreiber R, Mislovati M, Aharonowitz Y (1994) The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis. J Bacteriol 176:973–984
Diatchenko L, Lau Y-FC, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93:6025–6030
Díez B, Gutiérrez S, Barredo J, van Solingen P, van der Vort LHM, Martín JF (1990) The cluster of penicillin biosynthetic genes. Identification and characterization of the pcbAB gene encoding the α-aminoadipyl-cysteinil-valine synthetase and linkage to the pcbC and penDE genes. J Biol Chem 265:16358–16365
Dilks DW, Ring RH, Khawaja XZ, Novak TJ, Aston C (2003) High-throughput confirmation of differential display PCR results using reverse Northern blotting. J Neurosci Methods 123:47–54
Feng B, Friedlin E, Marzluf GA (1994) A reporter gene analysis of penicillin biosynthesis gene expression in Penicillium chrysogenum and its regulation by nitrogen and glucose catabolite repression. Appl Environ Microbiol 60:4432–4439
Fierro F, Gutiérrez S, Díez B, Martín JF (1993) Resolution of four large chromosomes in penicillin producing filamentous fungi: the penicillin gene cluster is located on chromosome II (9.6 Mb) in Penicillium notatum and chromosome I (10.4 Mb) in Penicillium chrysogenum. Mol Gen Genet 241:573–578
Fierro F, Barredo JL, Díez B, Gutiérrez S, Fernández FJ, Martín JF (1995) The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences. Proc Natl Acad Sci USA 92:6200–6204
Fierro F, Montenegro E, Gutiérrez S, Martín JF (1996) Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence. Appl Microbiol Biotechnol 44:597–604
Fierro F, Martín JF, Kosalková K (2002) Sulfur-containing β-lactam antibiotics: enzymes, genes and regulation of the biosynthesis. In: Fierro F, Martín JF (eds) Microbial secondary metabolites: biosynthesis, genetics and regulation. Research Signpost, Trivandrum, pp 179–210
Gancedo JM (1998) Yeast carbon catabolite repression. Microb Mol Biol Rev 62:334–361
Gurskaya N, Diatchenko L, Chenchik A, Siebert P, Khaspekov G, Lukyanov K, Vagner L, Ermolaeva O, Lukyanov S, Sverdlov E (1996) Equalizing cDNA substraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal Biochem 240:90–97
Gutiérrez S, Díez B, Montenegro E, Martín JF (1991) Characterization of the Cephalosporium acremonium pcbAB gene encoding α-aminoadipyl-cysteinyl-valine synthetase, a large multidomain peptide synthetase: linkage to the pcbC gene as a cluster of early cephalosporin biosynthetic genes and evidence of multiple functional domains. J Bacteriol 173:2354–2365
Gutiérrez S, Velasco J, Fernández FJ, Martín JF (1992) The cefG gene of Cephalosporium acremonium is linked to the cefEF gene and encodes a deacetylcephalosporin C acetyltransferase closely related to homoserine O-acetyltransferase. J Bacteriol 174:3056–3064
Gutiérrez S, Marcos AT, Casqueiro J, Kosalkova K, Fernández FJ, Velasco J, Martín JF (1999) Transcription of the pcbAB, pcbC and penDE genes of Penicillium chrysogenum AS-P-78 is repressed by glucose and the repression is not reversed by alkaline pHs. Microbiology 145:317–324
Ji W, Wright MB, Cai L, Flament A, Lindpaintner K (2002) Efficacy of SSH PCR in isolating differentially expressed genes. BMC Genomics 3:12–18
Kosalková K, Marcos AT, Fierro F, Hernando-Rico V, Gutiérrez S, Martín JF (2000) A novel heptameric sequence (TTAGTAA) is the binding site for a protein required for high level expression of pcbAB, the first gene of the penicillin biosynthesis in Penicillium chrysogenum. J Biol Chem 275:2423–2430
Laich F, Fierro F, Martín JF (2002) Production of penicillin by fungi growing on food products: identification of a complete penicillin cluster in Penicillium griseofulvum and a truncated cluster in Penicillium verrucosum. Appl Environ Microbiol 68:1211–1219
Liu X, Thiele D (1997) Yeast metallothionein gene expression in response to metals and oxidative stress. Methods: a companion to Methods Enzymol 11:289–299
Liu G, Casqueiro J, Bañuelos O, Cardoza RE, Gutiérrez S, Martín JF (2001) Targeted inactivation of the mecB gene encoding cystathionine-γ-lyase shows that the transsulfuration pathway is required for high level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine-induction of the cephalosporin genes. J Bacteriol 183:1765–1772
Madi L, McBride SA, Bailey LA, Ebbole DJ (1997) rco-3, a gene involved in glucose transport and conidiation in Neurospora crassa. Genetics 146:499–508
Martín JF (2000) Molecular control of expression of penicillin biosynthesis genes in fungi: regulatory proteins interact with a bidirectional promoter region. J Bacteriol 182:2355–2362
Martín JF, Casqueiro J, Kosalková K, Marcos AT, Gutiérrez S (1999) Penicillin and cephalosporin biosynthesis: mechanism of carbon catabolite regulation of penicillin production. Antonie Van Leeuwenhoek 75:21–31
Moore A, Siedow J (1991) The regulation and nature of the cyanide-resistant alternative oxidase of plant mitochondria. Biochim Biophys Acta 1059:121–140
Naranjo L, Martín de Valmaseda E, Bañuelos O, López P, Riaño J, Casqueiro J, Martín JF (2001) The conversion of pipecolic acid into lisine in Penicillium chrysogenum requires pipecolate oxidase and saccharopine reductase: characterization of the lys7 gene encoding saccharopine reductase. J Bacteriol 183:7165–7172
Pao S, Paulsen I, Saier M Jr (1998) Major facilitator superfamily. Microbiol Mol Biol Rev 62:1–34
Raitt DC, Bradshaw RE, Pillar TM (1994) Cloning and characterisation of the cytochrome C gene of Aspergillus nidulans. Mol Gen Genet 242:17–22
Revilla G, López-Nieto MJ, Martín JF (1984) Carbon catabolite expresión of penicillin biosíntesis by Penicillium chrysogenum. J Antibiot 37:781–789
Revilla G, Ramos FR, López-Nieto MJ, Álvarez E, Martín JF (1986) Glucose represses formation of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum. J Bacteriol 168:947–952
Rolland F, Winderickx J, Thevelein JM (2001) Glucose-sensing mechanisms in eukaryotic cells. Trends Biochem Sci 26:310–317
Ronne H (1995) Glucose repression in fungi. Trends Genet 11:12–17
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Schmidt MC, McCartney R, Zhang X, Tillman T, Solimeo H, Wölfl S, Almonte C, Watkins S (1999) Std1 and Mth1 proteins interact with the glucose sensors to control glucose-regulated gene expression in Saccharomyces cerevisiae. Mol Cell Biol 19:4561–4571
Siedow J, Umback A (2000) The mitochondrial cyanide-resistant oxidase: structural conservation amid regulatory diversity. Biochim Biophys Acta 1459:432–439
Soares MB (1997) Identification and cloning of differentially expressed genes. Curr Opin Biotechnol 8:542–546
Somerson NL, Demain AL, Nunheimer TD (1961) Reversal of lysine inhibition of penicillin production by α-aminoadipic acid. Arch Biochem 93:238–241
Thykaer J, Nielsen J (2003) Metabolic engineering of β-lactam production. Metab Engineer 5:56–69
Von Stein O, Thies W-G, Hofmann M (1997) A high throughput screening for rarely transcribed differentially expressed genes. Nucleic Acids Res 25:2598–2602
Yao Y, Ni Z, Zhang Y, Chen Y, Ding Y, Han Z, Liu Z, Sun Q (2005) Identification of differentially expressed genes in leaf and root between wheat hybrid and its parental inbreds using PCR-based cDNA subtraction. Plant Mol Biol 58:367–384
Acknowledgements
This work was supported by grants of the CICYT (BIO2000-1726-C02-01) and the European Union (EUROFUNG Project: QLK3-1999-00729). We acknowledge the interest of ANTIBIÓTICOS, S.A. N. Castillo received a fellowship of the AECI, Ministry of Foreign Affaire, Madrid, Spain. We thank M. Mediavilla and M. Álvarez for excellent technical assistance.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by Joseph Heitman
Rights and permissions
About this article
Cite this article
Castillo, N.I., Fierro, F., Gutiérrez, S. et al. Genome-wide analysis of differentially expressed genes from Penicillium chrysogenum grown with a repressing or a non-repressing carbon source. Curr Genet 49, 85–96 (2006). https://doi.org/10.1007/s00294-005-0029-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-005-0029-y