Protein library and functional engineering of fungal cytochromes P450

2014 Fiscal Year Final Research Report

• Project/Area Number: 24688034
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Partial Multi-year Fund
• Research Field: Boundary agriculture
• Research Institution: Kyushu University
• Principal Investigator: ICHINOSE Hirofumi 九州大学, (連合)農学研究科(研究院), 准教授 (00432948)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Keywords: シトクロムP450 / 真核微生物 / タンパク質 / 異種発現

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.

Free Research Field

生物工学