TY - JOUR
T1 - High-level heterologous expression of fungal cytochrome P450s in Escherichia coli
AU - Ichinose, Hirofumi
AU - Wariishi, Hiroyuki
N1 - Funding Information:
This research was supported in part by a Grant-in-Aid (No. 24688034 ) for Young Scientists (A) (to H.I.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan .
PY - 2013/8/23
Y1 - 2013/8/23
N2 - A thorough understanding of the sequence-structure-function relationships of cytochrome P450 (P450) is necessary to better understand the metabolic diversity of living organisms. Significant amounts of pure enzymes are sometimes required for biochemical studies, and their acquisition often relies on the possibility of their heterologous expression. In this study, we performed extensive heterologous expression of fungal P450s in Escherichia coli using 304 P450 isoforms. Using 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. These findings will help increase the chance of heterologous expression of a variety of fungal and other eukaryotic membrane-bound P450s in E. coli.
AB - A thorough understanding of the sequence-structure-function relationships of cytochrome P450 (P450) is necessary to better understand the metabolic diversity of living organisms. Significant amounts of pure enzymes are sometimes required for biochemical studies, and their acquisition often relies on the possibility of their heterologous expression. In this study, we performed extensive heterologous expression of fungal P450s in Escherichia coli using 304 P450 isoforms. Using 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. These findings will help increase the chance of heterologous expression of a variety of fungal and other eukaryotic membrane-bound P450s in E. coli.
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U2 - 10.1016/j.bbrc.2013.07.057
DO - 10.1016/j.bbrc.2013.07.057
M3 - Article
C2 - 23886957
AN - SCOPUS:84882720783
VL - 438
SP - 289
EP - 294
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 2
ER -