Targeted Pursuit of Bioactive Molecules That Show Inhibitory Activity Against Fungal Polyketide Synthases

有针对性地寻找对真菌聚酮化合物合成酶具有抑制活性的生物活性分子

• 批准号: 13480184
• 负责人: EBIZUKA Yutaka
• 金额: $ 9.54万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480184/
• 关键词: Filamentous Fungi; Polyketide; Polyketide Synthase; Mycotoxin; Mycosis; Inhibitor; 植物病原菌

Aspergillus fumigatus, a ubiquitous fungus, causes allergy, noninvasive colonization or life-threatening invasive pulmonary aspergillosis in human beings. Its bluish-green conidial pigmentation has been shown to be a critical virulence factor and is resulted by polymerization of 1,8-dihydroxynaphthalene. Alb1p, a poiyketide synthase of this fungus, is the initial enzyme of the biosynthetic pathway leading to the pigment and has been chosen as the target for screening of novel inhibitors from natural sources in this study. In the first year, construction of in vivo screening method for inhibitors was pursued with heterologous expression of Alb1 gene in fungal host, Asp. Oryzae. However, several drawbacks including poor reproducibility in growth of transformant, relatively long culture period, and etc, had hampered the use of this fungal transformant for the practical screening for large number of samples. In the second year, expression of the same enzyme gene in yeast was attempted. Yeast expression vector harboring Alb1 gene was introduced into yeast together with the second vector with phosphopanthethenyl transferase gene by the established procedures. The double transformant produced yellow pigment YWA1, the Alb1p reaction product, as confirmed by LC/APCIMS. With this transformant, test samples of some 240 kinds of microbial cultures were tested for their in vivo inhibitory activity against Alb1p reaction, which was estimated by the decrease of YWA1 production as monitored by absorption at 406 nm. Five of them showed potent inhibition comparable to that of a positive control, cerulenin. Two microbial starins were cultured in large scale to isolate active principles. Activity guided fractionation of the extracts, however, resulted in separation of the inhibitory activity in several fractions and so far none of the pure compound has been obtained yet.

烟曲霉是一种普遍存在的真菌，可引起人类过敏、非侵袭性定植或危及生命的侵袭性肺曲霉病。它的蓝绿色分生孢子色素沉着已被证明是一个关键的毒力因子，是由1，8-二羟基萘聚合。Alb 1 p是该真菌的聚酮合酶，是导致色素的生物合成途径的起始酶，并已被选为本研究中从天然来源筛选新型抑制剂的靶点。在第一年中，通过在真菌宿主Asp中异源表达Alb 1基因，构建了抑制剂的体内筛选方法。大米。但由于该菌株生长的重复性差、培养周期较长等缺点，限制了其在大批量样品筛选中的应用。第二年，尝试在酵母中表达相同的酶基因。将携带Alb 1基因的酵母表达载体和携带磷酸蒽基转移酶基因的酵母表达载体一起导入酵母中。通过LC/APCIMS证实，双结晶产生黄色颜料YWA 1，即Alb 1 p反应产物。利用该方法，测试了约240种微生物培养物的测试样品对Alb 1 p反应的体内抑制活性，通过406 nm处的吸收监测YWA 1产生的减少来评估。其中5个显示出与阳性对照浅蓝菌素相当的强效抑制作用。对两种微生物淀粉进行了大规模培养，以分离其有效成分。然而，活性导向分级的提取物，导致在几个馏分中的抑制活性的分离，到目前为止还没有得到纯的化合物。

项目成果

H.F.Tsai: "Pentrkctide-melanin Biosynthesis in Aspergilln, fnmigrtns Requires Chamlength Shortening of a Heptaketide precursor"J.Biol.Chem. 276. 29292-29298 (2001)

H.F.Tsai：“曲霉中的五肽-黑色素生物合成，需要缩短七肽前体的 Chamlength”J.Biol.Chem。

Akira Watanabe, Yutaka Ebizuka: "A Novel Hexaketide Naphthalene Synthesized by a Chimeric Polyketide Synthase Composed of Fungal Pentaketide and Heptaketide Synthases"Tetrahedron Letters. 43. 843-846 (2002)

Akira Watanabe、Yutaka Ebizuka：“由真菌五肽和七肽合成酶组成的嵌合聚酮化合物合成酶合成的新型六酮化合物萘”四面体字母。

S.Okamoto: "Inhibitory Effect of Aflastctin A on melanin Biosynthesis by Colitotrichnm lagenarium"Microbiology. 147. 2623-2628 (2001)

S.Okamoto：“Aflastctin A 对大肠菌黑色素生物合成的抑制作用”微生物学。

Huei-Fung Tsai, Isao Fujii, Akira Watanabe, Michael H. Wheeler, Yun C. Chang, Yoshinori Yasuoka, Yutaka Ebizuka, and K. J. Kwon-Chung: "Pentaketide-melanin Biosynthesis in Aspergillus fumigatus Requires Chain-length Shortening of a Heptaketide Precursor"J

Huei-Fung Tsai、Isao Fujii、Akira Watanabe、Michael H. Wheeler、Yun C. Chang、Yoshinori Yasuoka、Yutaka Ebizuka 和 K. J. Kwon-Chung：“烟曲霉中的五肽黑色素生物合成需要缩短七肽前体的链长

Akira Watanabe, and Yutaka Ebizuka: "A Novel Hexaketide Naphthalene Synthesized by a Chimeric Polyketide Synthase Composed of Fungal Pentaketide and Heptaketide Synthases"Tetrahedron Lett.. 43. 843-846 (2002)

Akira Watanabe 和 Yutaka Ebizuka：“由真菌五肽和七肽合成酶组成的嵌合聚酮化合物合成酶合成的新型六酮化合物萘”Tetrahedron Lett.. 43. 843-846 (2002)