| Budget Amount *help |
¥14,900,000 (Direct Cost: ¥14,900,000)
Fiscal Year 2002: ¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 2001: ¥10,500,000 (Direct Cost: ¥10,500,000)
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| Research Abstract |
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.
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