Transcriptional upregulation of four genes of the lysine biosynthetic pathway by homocitrate accumulation in Penicillium chrysogenum: homocitrate as a sensor of lysine-pathway distress

• Published in: Microbiology (Society for General Microbiology) Vol. 155; no. 12; pp. 3881 - 3892
• Main Authors: Teves, Franco, Lamas-Maceiras, Monica, Garcia-Estrada, Carlos, Casqueiro, Javier, Naranjo, Leopoldo, Ullan, Ricardo V, Scervino, Jose-Martin, Wu, Xiaobin, Velasco-Conde, Tania, Martin, Juan F
• Format: Journal Article
• Language: English
• Published: England Soc General Microbiol 01.12.2009
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Aspergillus nidulans; Cloning, Molecular; DNA Primers - genetics; DNA, Fungal - genetics; Fungal Proteins - genetics; Fungal Proteins - metabolism; Genes, Fungal; Genetic Complementation Test; Hydro-Lyases - genetics; Hydro-Lyases - metabolism; Lysine - biosynthesis; Models, Biological; Molecular Sequence Data; Penicillium chrysogenum; Penicillium chrysogenum - genetics; Penicillium chrysogenum - metabolism; Point Mutation; RNA, Fungal - genetics; RNA, Fungal - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Homology, Amino Acid; Transformation, Genetic; Tricarboxylic Acids - metabolism; Up-Regulation
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.031005-0

1 Área de Microbiología, Departamento de Biología Molecular, Facultad de CC. Biológicas y Ambientales, Universidad de León, Campus de Vegazana s/n, 24071 Leon, Spain 2 Instituto de Biotecnología de León (INBIOTEC), Parque Científico de León, Av. Real, 1, 24006 León, Spain The lysine biosynthetic pathway has to supply large amounts of -aminoadipic acid for penicillin biosynthesis in Penicillium chrysogenum . In this study, we have characterized the P. chrysogenum L2 mutant, a lysine auxotroph that shows highly increased expression of several lysine biosynthesis genes ( lys1 , lys2 , lys3 , lys7 ). The L2 mutant was found to be deficient in homoaconitase activity since it was complemented by the Aspergillus nidulans lysF gene. We have cloned a gene (named lys3 ) that complements the L2 mutation by transformation with a P. chrysogenum genomic library, constructed in an autonomous replicating plasmid. The lys3 -encoded protein showed high identity to homoaconitases. In addition, we cloned the mutant lys3 allele from the L2 strain that showed a G 1534 to A 1534 point mutation resulting in a Gly 495 to Asp 495 substitution. This mutation is located in a highly conserved region adjacent to two of the three cysteine residues that act as ligands to bind the iron–sulfur cluster required for homoaconitase activity. The L2 mutant accumulates homocitrate. Deletion of the lys1 gene (homocitrate synthase) in the L2 strain prevented homocitrate accumulation and reverted expression levels of the four lysine biosynthesis genes tested to those of the parental prototrophic strain. Homocitrate accumulation seems to act as a sensor of lysine-pathway distress, triggering overexpression of four of the lysine biosynthesis genes. Correspondence Juan F. Martín jf.martin{at}unileon.es Abbreviations: IRP, iron regulatory protein Present address: Unidad de Biotecnología del Petróleo, Centro de Biotecnología, Fundación Instituto de Estudios Avanzados (IDEA), Sartenejas, Caracas 1080, Venezuela. The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is EU264159.