Improvement of P450(BM-3) whole-cell biocatalysis by integrating heterologous cofactor regeneration combining glucose facilitator and dehydrogenase in E. coli.

Hendrik Schewe, Bjoern-Arne Kaup, Jens Schrader

Index: Appl. Microbiol. Biotechnol. 78(1) , 55-65, (2008)

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Abstract

Escherichia coli BL21, expressing a quintuple mutant of P450(BM-3), oxyfunctionalizes alpha-pinene in an NADPH-dependent reaction to alpha-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 alpha-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 micromol h(-1) micromol(-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 alpha-pinene oxide yield in the presence of glucose compared to glucose-free conditions.