title: Delineating citrinin biosynthesis: Ctn-ORF3 dioxygenase-mediated multi-step methyl oxidation precedes a reduction-mediated pyran ring cyclization.
abstract: Citrinin (3) is a polyketide-derived mycotoxin, that is, produced by Monascus, Penicillium, and Aspergillus spp. and is a common contaminant in a number of agricultural products. ctPKS, a non-reducing type iterative polyketide synthase with a C-terminal reductive domain, is proposed to generate the polyketide backbone of 3. The targeted gene inactivation of ctn-orf1 or ctn-orf3 gene resulted in the accumulation of a benzaldehyde derivative 6, and the ectopic expression of ctPKS/ctnB in yeast produced 6, demonstrating that ctPKS generates 6 with the support of CtnB and suggesting that Ctn-ORF1/Ctn-ORF3 converts 6 into 3. The Δctn-orf1 mutant also produced a novel benzdialdehyde derivative 10. When either 6 or 10 was fed into a ΔctPKS mutant, 3 was readily detected, which confirms that both 6 and 10 are involved in the biosynthesis of 3. A bioconversion experiment of 6 in the ectopic expression system demonstrated that ctn-orf3 expression, but not ctn-orf1 expression, efficiently consumed 6. The resulting metabolite(s) of 6 could not be identified, however. A recombinant Ctn-ORF3 enzyme was demonstrated to convert 6 into 10 and a hypothetical carboxylic derivative 8, which substantiates that Ctn-ORF3 oxidizes the exocyclic methyl moiety of 6. Ctn-ORF1 is thus proposed to reduce 8 and the subsequent non-enzymatic reactions to complete the biosynthesis of 3. The present study delineates the biosynthetic route of 3, proposing the biochemical mechanism, that is, involved in producing the natural dihydropyranoquinone structure.
authors.affiliation: Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Republic of Korea.
authors.forename: Bijinu
authors.initials: B
authors.lastname: Balakrishnan
authors.affiliation: Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Republic of Korea.
authors.forename: Ramya
authors.initials: R
authors.lastname: Chandran
authors.affiliation: Department of Oriental Medicine Resources and Institute for Traditional Korean Medicine Industry, Mokpo National University, Muan 534-729, Republic of Korea.
authors.forename: Si-Hyung
authors.initials: SH
authors.lastname: Park
authors.affiliation: Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Republic of Korea. Electronic address: hjink@mju.ac.kr.
authors.forename: Hyung-Jin
authors.initials: HJ
authors.lastname: Kwon
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism; D001578:Benzopyrans / Q000378:metabolism; D053898:Biosynthetic Pathways; D002953:Citrinin / Q000378:metabolism*; D003500:Cyclization; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting; D032741:Monascus / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010084:Oxidation-Reduction; D010860:Pigments, Biological / Q000235:genetics / Q000378:metabolism; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001578:Benzopyrans; D005656:Fungal Proteins; D010860:Pigments, Biological; C494154:azaphilone; D002953:Citrinin; D048630:Polyketide Synthases; D049308:Dioxygenases
keywords: Azaphilone; Biosynthetic genes; Citrinin; Monascus purpureus; Non-reducing polyketide synthase