| Budget Amount *help |
¥26,910,000 (Direct Cost: ¥20,700,000、Indirect Cost: ¥6,210,000)
Fiscal Year 2014: ¥8,190,000 (Direct Cost: ¥6,300,000、Indirect Cost: ¥1,890,000)
Fiscal Year 2013: ¥7,930,000 (Direct Cost: ¥6,100,000、Indirect Cost: ¥1,830,000)
Fiscal Year 2012: ¥10,790,000 (Direct Cost: ¥8,300,000、Indirect Cost: ¥2,490,000)
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| Outline of Final Research Achievements |
We performed extensive heterologous expression of fungal cytochrome P450s in Escherichia coli using 304 isoforms from white-rot and brown-rot basidiomycetes. Based upon a large-scale screening, we confirmed that at least 27 P450s could be expressed with/without simple sequence deletion at the 5′ end of cDNAs, which encode the N-terminal hydrophobic domain of the enzyme. Moreover, we identified N-terminal amino acid sequences that can potentially be used to construct chimeric P450s, which could dramatically improve their expression levels even when the expression of the wild-type sequence was unpromising. So far, we constructed a protein library in which 60 fungal P450s were overexpressed in E. coli. In addition, catalytic potentials of Aspergillus oryzae P450s were improved by genetic engineering to show high activity against flavonoid compounds. The engineered P450s are potentially useful for production of bioactive compounds.
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