Improvement of P450BM-3 whole-cell biocatalysis by integrating heterologous cofactor regeneration combining glucose facilitator and dehydrogenase in E. coli
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- Schewe, H., Kaup, BA. & Schrader, J. Appl Microbiol Biotechnol (2008) 78: 55. doi:10.1007/s00253-007-1277-1
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Escherichia coli BL21, expressing a quintuple mutant of P450BM-3, oxyfunctionalizes α-pinene in an NADPH-dependent reaction to α-pinene oxide, verbenol, and myrtenol. We optimized the whole-cell biocatalyst by integrating a recombinant intracellular NADPH regeneration system through co-expression of a glucose facilitator from Zymomonas mobilis for uptake of unphosphorylated glucose and a NADP+-dependent glucose dehydrogenase from Bacillus megaterium that oxidizes glucose to gluconolactone. The engineered strain showed a nine times higher initial α-pinene oxide formation rate corresponding to a sixfold higher yield of 20 mg g−1 cell dry weight after 1.5 h. The initial total product formation rate was 1,000 μmol h−1 μmol−1 P450 leading to a total of 32 mg oxidized products per gram cell of dry weight after 1.5 h. The physiological functioning of the heterologous cofactor regeneration system was illustrated by a sevenfold increased α-pinene oxide yield in the presence of glucose compared to glucose-free conditions.