Polyketide Synthase Gene Responsible for Citrinin Biosynthesis in Monascus purpureus

• Published in: Applied and Environmental Microbiology 2005-07, Vol.71 (7), p.3453-3457
• Main Authors: Shimizu, Takeo, Kinoshita, Hiroshi, Ishihara, Shinji, Sakai, Kanae, Nagai, Shiro, Nihira, Takuya
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Bacteria; Bacteriology; Biological and medical sciences; biosynthesis; citrinin; Citrinin - biosynthesis; Cloning, Molecular; Culture Media; enzymes; Eurotiales; Fundamental and applied biological sciences. Psychology; Genes; Genes, Fungal; Genetics; introns; Microbiology; Molecular Sequence Data; Monascus - enzymology; Monascus - genetics; Monascus - growth & development; Monascus purpureus; Mycology; nucleotide sequences; pksCT gene; polyketide synthase; Polyketide Synthases - chemistry; Polyketide Synthases - genetics; Polyketide Synthases - metabolism; Recombination, Genetic; Sequence Analysis, DNA; transcription (genetics); Transcription, Genetic
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.71.7.3453-3457.2005

Citrinin produced by Aspergillus, Penicillium, and Monascus species is a polyketide compound that has nephrotoxic activity in mammals and is bactericidal toward gram-positive bacteria. To avoid the risk of citrinin contamination in other fermentation products produced by Monascus purpureus, knowledge of the citrinin biosynthetic genes is needed so that citrinin-nonproducing strains can be generated. We cloned a polyketide synthase (PKS) gene from M. purpureus with degenerate primers designed to amplify the conserved region of a ketosynthase domain of a fungal PKS. A 13-kb genomic DNA fragment was identified that contained a full-length PKS gene (pksCT) of 7,838 bp with a single 56-bp intron. pksCT encodes a 2,593-amino-acid protein that contains putative domains for ketosynthase, acyltransferase, acyl carrier protein (ACP), and a rare methyltransferase. There was no obvious thioesterase domain, which usually is downstream of the ACP domain in multi-aromatic-ring PKSs. pksCT transcription was correlated with citrinin production, suggesting that the pksCT gene product was involved in citrinin biosynthesis. Homologous recombination between the wild-type allele and a truncated disruption construct resulted in a pksCT-disrupted strain of M. purpureus. The disruptant did not produce citrinin, but a pksCT revertant generated by successive endogenous recombination events in the pksCT disruptant restored citrinin production, indicating that pksCT encoded the PKS responsible for citrinin biosynthesis in M. purpureus.