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Applied and Environmental Microbiology, August 2007, p. 5097-5103, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.01979-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Takeo Shimizu,1,{ddagger} Hiroshi Kinoshita,1 and Takuya Nihira1,2*

International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan,1 MU-OU Collaborative Research Center for Bioscience and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., 10400 Bangkok, Thailand2

Received 21 August 2006/ Accepted 9 June 2007

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.

* Corresponding author. Mailing address: International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-7452. Fax: 81-6-6879-7454. E-mail: nihira{at}icb.osaka-u.ac.jp

{triangledown} Published ahead of print on 22 June 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Department of Life Sciences, Faculty of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.

Applied and Environmental Microbiology, August 2007, p. 5097-5103, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.01979-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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