TY - JOUR
T1 - Biosynthesis of steroidal antibiotic fusidanes
T2 - Functional analysis of oxidosqualene cyclase and subsequent tailoring enzymes from aspergillus fumigatus
AU - Mitsuguchi, Hisashi
AU - Seshime, Yasuyo
AU - Fujii, Isao
AU - Shibuya, Masaaki
AU - Ebizuka, Yutaka
AU - Kushiro, Tetsuo
PY - 2009/5/13
Y1 - 2009/5/13
N2 - 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-3β-ol, an established precursor for helvolic acid. Its structural isomer, (20R)-protosta-13(17),24-dien-3β-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-3β,29- diol and protosta-17(20)Z,24-dien-3β-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.
AB - 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-3β-ol, an established precursor for helvolic acid. Its structural isomer, (20R)-protosta-13(17),24-dien-3β-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-3β,29- diol and protosta-17(20)Z,24-dien-3β-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.
UR - http://www.scopus.com/inward/record.url?scp=70149100848&partnerID=8YFLogxK
U2 - 10.1021/ja8095976
DO - 10.1021/ja8095976
M3 - Article
C2 - 19415934
AN - SCOPUS:70149100848
VL - 131
SP - 6402
EP - 6411
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
SN - 0002-7863
IS - 18
ER -