'Royal College of Obstetricians & Gynaecologists (RCOG)'
Abstract
P450-dependent biotransformations in Escherichia
coli are attractive for the selective oxidation of organic
molecules using mild and sustainable procedures. The overall
efficiency of these processes, however, relies on how
effectively the NAD(P)H cofactors derived from oxidation
of the carbon source are utilized inside the cell to support the
heterologous P450-catalyzed reaction. In this work, we
investigate the use of metabolic and protein engineering
to enhance the product-per-glucose yield (Y_(PPG)) in wholecell
reactions involving a proficient NADPH-dependent
P450 propane monooxygenase prepared by directed evolution
[P450_(PMO)R2; Fasan et al. (2007); Angew Chem Int Ed
46:8414–8418]. Our studies revealed that the metabolism of
E. coli (W3110) is able to support only a modest propanol:
glucose molar ratio (Y_(PPG)~0.5) under aerobic, nongrowing
conditions. By altering key processes involved in
NAD(P)H metabolism of the host, considerable improvements
of this ratio could be achieved. A metabolically
engineered E. coli strain featuring partial inactivation of
the endogenous respiratory chain (Δndh) combined with
removal of two fermentation pathways (ΔadhE, Δldh) provided
the highest YPPG (1.71) among the strains investigated,
enabling a 230% more efficient utilization of the energy
source (glucose) in the propane biotransformation compared
to the native E. coli strain. Using an engineered
P450_(PMO)R2 variant which can utilize NADPH and NADH
with equal efficiency, we also established that dual cofactor specificity of the P450 enzyme can provide an appreciable improvement in Y_(PPG). Kinetic analyses suggest, however,
that much more favorable parameters (K_M, k_(cat)) for the
NADH-driven reaction are required to effectively compete
with the host’s endogenous NADH-utilizing enzymes. Overall,
the metabolic/protein engineering strategies described
here can be of general value for improving the performance
of NAD(P)H-dependent whole-cell biotransformations in
E. coli
