TY - JOUR
T1 - New strategy to enhance catalytic performance of Escherichia coli whole cell biocatalysts harboring P450cam mutants
AU - Mouri, Tsuyoshi
AU - Kamiya, Noriho
AU - Goto, Masahiro
N1 - Funding Information:
We are grateful to Dr. Ichinose Hirofumi for providing pET22-P450 (Y96F) and pET22-P450cam (F87W-Y96F) plasmids. This research was supported by a Grant-in-Aid for Scientific Research ( 18686067 ) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to N.K.) and partly by a Grant-in-Aid for the Global COE Program, “Science for Future Molecular Systems” from the MEXT of Japan (to M.G.). T.M. was supported by Research Fellowships of the Japan Society for the Promotion of Science (JSPS) for Young Scientists .
PY - 2011/1/15
Y1 - 2011/1/15
N2 - Cytochrome P450cam mutants (Y96F and F87W-Y96F) can catalyze the oxidation of dichlorobenzene and other aromatic compound that wild type P450cam cannot catalyze. The expression of the single mutant Y96F in an Escherichia coli host resulted in indigo formation (approximately 23mg/l), of which productivity was comparable to the highest reported so far using other P450cam mutant cultures. On the other hand, recombinant E. coli that harbored F87W-Y96F exhibited little activity for indigo production. However, co-expression of E. coli glycerol dehydrogenase (GLD) with F87W-Y96F triggered a marked increase in the productivity of indigo (approximately 20mg/l) without external glycerol addition. These results indicate the potential of introducing GLD-mediated NADH regeneration that utilizes endogenous NAD+ and glycerol in E. coli to enhance markedly the catalytic performance of a low-activity recombinant P450cam system. The present E. coli whole-cell biocatalysts might be applicable to practical microbial indigo production.
AB - Cytochrome P450cam mutants (Y96F and F87W-Y96F) can catalyze the oxidation of dichlorobenzene and other aromatic compound that wild type P450cam cannot catalyze. The expression of the single mutant Y96F in an Escherichia coli host resulted in indigo formation (approximately 23mg/l), of which productivity was comparable to the highest reported so far using other P450cam mutant cultures. On the other hand, recombinant E. coli that harbored F87W-Y96F exhibited little activity for indigo production. However, co-expression of E. coli glycerol dehydrogenase (GLD) with F87W-Y96F triggered a marked increase in the productivity of indigo (approximately 20mg/l) without external glycerol addition. These results indicate the potential of introducing GLD-mediated NADH regeneration that utilizes endogenous NAD+ and glycerol in E. coli to enhance markedly the catalytic performance of a low-activity recombinant P450cam system. The present E. coli whole-cell biocatalysts might be applicable to practical microbial indigo production.
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U2 - 10.1016/j.bej.2010.09.016
DO - 10.1016/j.bej.2010.09.016
M3 - Article
AN - SCOPUS:78649923377
SN - 1369-703X
VL - 53
SP - 229
EP - 233
JO - Biochemical Engineering Journal
JF - Biochemical Engineering Journal
IS - 2
ER -