Identification and In Vivo Functional Analysis by Gene Disruption of ctnA , an Activator Gene Involved in Citrinin Biosynthesis in Monascus purpureus

Abstract

Citrinin, a secondary fungal metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. From the red-pigment producer Monascus purpureus, a 21-kbp region flanking pksCT, which encodes citrinin polyketide synthase, was cloned. Four open reading frames (ORFs) (orf1, orf2, orf3, and orf4) in the 5-flanking region and one ORF (orf5) in the 3-flanking region were identified in the vicinity of pksCT. orf1 to orf5 encode a homolog of a dehydrogenase (similarity, 46%), a regulator (similarity, 38%), an oxygenase (similarity, 41%), an oxidoreductase (similarity, 26%), and a transporter (similarity, 58%), respectively. orf2 (2,006 bp with four introns) encodes a 576-amino-acid protein containing a typical Zn(II)2Cys6 DNA binding motif at the N terminus and was designated ctnA. Although reverse transcriptase PCR analysis revealed that all of these ORFs, except for orf1, were transcribed with pksCT under citrinin production conditions, the disruption of ctnA caused large decreases in the transcription of pksCT and orf5, together with reduction of citrinin production to barely detectable levels, suggesting that these two genes are under control of the ctnA product. Complementation of the ctnA disruptant with intact ctnA on an autonomously replicating plasmid restored both transcription and citrinin production, indicating that CtnA is a major activator of citrinin biosynthesis.Filamentous fungi have a versatile ability to produce different kinds of secondary metabolites, including antibiotics, anticancer drugs, and pigments (3). During the biosynthesis of these secondary metabolites, many biosynthetic enzymes are required and should function coordinately in the synthesis of these structurally complex metabolites. The genes encoding these enzymes have often been reported to localize in an adjacent region or to form a gene cluster (6, 13), similar to the situation of biosynthetic gene clusters for secondary metabolites in prokaryotic actinomycetes. In addition to such structural genes, each gene cluster may contain a gene encoding a regulator that functions to coordinate expression of the structural genes in the cluster and a gene encoding a transporter that excludes harmful intracellular secondary metabolites as a self-defense mechanism (7, 22), respectively.Regulators possessing a Zn(II)2Cys6 binuclear motif represent one of the largest classes of transcriptional regulators in filamentous fungi. They generally act as transcriptional activators, as exemplified by GAL4 from Saccharomyces cerevisiae (21). A large number of Zn(II)2Cys6 regulators have been identified exclusively in fungi and characterized as regulators of primary metabolism, secondary metabolism, drug resistance, or meiotic development. Through its DNA binding motif (CX 2 CX 6 CX 6 CX 2 CX 6 C [C, cysteinyl residue; X, any amino acid]) located at the N terminus, the Zn(II)2Cys6 regulator binds to a specific binding site (CGGN 6/11 CCG [N, any nucleotide]) in the promoter regions of target genes. Previous research on secondary met...

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