Balakrishnan Bijinu, Chandran Ramya, Park Si-Hyung, Kwon Hyung-Jin
Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Republic of Korea.
Department of Oriental Medicine Resources and Institute for Traditional Korean Medicine Industry, Mokpo National University, Muan 534-729, Republic of Korea.
Bioorg Med Chem Lett. 2016 Jan 15;26(2):392-396. doi: 10.1016/j.bmcl.2015.12.001. Epub 2015 Dec 10.
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.
桔霉素（3）是一种聚酮类衍生的霉菌毒素，即由红曲霉菌、青霉菌和曲霉菌属产生，是许多农产品中的常见污染物。ctPKS是一种具有C末端还原结构域的非还原型迭代聚酮合酶，被认为可生成3的聚酮骨架。ctn-orf1或ctn-orf3基因的靶向基因失活导致苯甲醛衍生物6的积累，ctPKS/ctnB在酵母中的异位表达产生了6，这表明ctPKS在CtnB的支持下生成6，并表明Ctn-ORF1/Ctn-ORF3将6转化为3。Δctn-orf1突变体还产生了一种新型苯二醛衍生物10。当将6或10添加到ΔctPKS突变体中时，很容易检测到3，这证实6和10都参与了3的生物合成。在异位表达系统中对6进行的生物转化实验表明，ctn-orf3的表达而非ctn-orf1的表达有效地消耗了6。然而，6产生的代谢产物无法鉴定。重组Ctn-ORF3酶被证明可将6转化为10和一种假定的羧酸衍生物8，这证实Ctn-ORF3氧化了6的环外甲基部分。因此，推测Ctn-ORF1还原8并通过随后的非酶反应完成3的生物合成。本研究描绘了3的生物合成途径，提出了其生化机制，即参与生成天然二氢吡喃醌结构。
