Biosynthesis of steroidal antibiotic fusidanes: functional analysis of oxidosqualene cyclase and subsequent tailoring enzymes from Aspergillus fumigatus.
Three putative oxidosqualene cyclase (OSC) genes exist in the genome of the fungus Aspergillus fumigatus that produces a steroidal antibiotic, helvolic acid. One of these genes, Afu4g14770, designated AfuOSC3, is clustered with genes of cytochrome P450 monooxygenases (P450s), a short-chain dehydrogenase/reductase (SDR), and acyltransferases, which presumably function in triterpene tailoring steps, suggesting that this gene cluster codes for helvolic acid biosynthesis. AfuOSC3 was PCR amplified from A. fumigatus IFO8866 genomic DNA and expressed in yeast. The yeast transformant accumulated protosta-17(20)Z,24-dien-3beta-ol, an established precursor for helvolic acid. Its structural isomer, (20R)-protosta-13(17),24-dien-3beta-ol, was also isolated from the transformed yeast. To further identify the function of triterpene tailoring enzymes, four P450 genes (CYP5081A1-D1) and a SDR gene (AfuSDR1) in the cluster were each coexpressed with AfuOSC3 in yeast. As a result, coexpression of AfuSDR1 gave a 3-keto derivative of protostadienol. On the other hand, coexpression with CYP5081A1 gave protosta-17(20)Z,24-diene-3beta,29-diol and protosta-17(20)Z,24-dien-3beta-ol-29-oic acid. These metabolites are in well accord with the oxidative modification involved in helvolic acid biosynthesis. AfuSDR1 and CYP5081A1 presumably function together to catalyze demethylation of C-29 methyl group. These results provided a firm ground for identification of the present gene cluster to be involved in helvolic acid biosynthesis.
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Hisashi
H
Mitsuguchi
Yasuyo
Y
Seshime
Isao
I
Fujii
Masaaki
M
Shibuya
Yutaka
Y
Ebizuka
Tetsuo
T
Kushiro
D000217:Acyltransferases; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001232:Aspergillus fumigatus / Q000201:enzymology*; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System; D005672:Fusidic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D010088:Oxidoreductases; D013256:Steroids; D015003:Yeasts / Q000235:genetics
D000900:Anti-Bacterial Agents; D013256:Steroids; D005672:Fusidic Acid; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D000217:Acyltransferases; D019751:Intramolecular Transferases; C021924:lanosterol synthase; C011267:helvolic acid