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114 5 Multi-Enzyme Systems and Cascade Reactions Involving Cytochrome P450 Monooxygenases

5.3.2
Cofactor Regeneration in Whole-Cell Biocatalysts
An attractive strategy for overcoming cofactor limitation is the application of whole-
cell biocatalysts. As long as living cells are provided with all the required nutrients,
all endogenous cofactor recycling systems are functional and there is no need to
externally supplement cofactors. However, during whole-cell P450-based oxidation
processes, cofactor concentration within the cell may become a bottleneck for the
overall process if the concentration of recombinant P450 and its activity are high
[95]. In such cases, cofactor regenerating enzymes that are coexpressed with P450
systems in recombinant hosts can be applied.
For instance, a GLD mentioned in the previous paragraph was coexpressed
together with P450 cam and its physiological redox partners in E. coli. Interestingly,
even without addition of external glycerol, the efficiency of recombinant resting
E. coli cells harboring both P450 cam and GLD was about 10-fold higher than the
system without GLD (37% vs 4% conversion of 2 mM camphor). This indicates
that endogenous glycerol present in the cells was utilized by GLD. After the
addition of 10% glycerol, the conversion achieved 100% in an aqueous system with
ethanol as cosolvent. In addition, this whole-cell oxidation system was applied for
camphor hydroxylation in a biphasic system with isooctane as a second organic
phase. However, only 30% conversion relative to the amount of 5-hydroxycamphor
formed in the aqueous system was achieved [96]. The reason for lower productivity
in the biphasic system might be either the disadvantageous partition coefficient
of camphor between the two phases or reduced stability of the enzymes in this
system.
Another approach in this field is the construction of an E. coli whole-cell biocatalyst
with improved intracellular cofactor regeneration driven by external glucose [97].
In this system, additional recombinant intracellular NADPH regeneration occurs
through coexpression of a glucose facilitator from Zymomonas mobilis foruptakeof
+
non-phosphorylated glucose and an NADP -dependent GDH from B. megaterium
oxidizing glucose to gluconolactone (Figure 5.2). When a mutant of P450 that
BM3
oxyfunctionalizes α-pinene – a cheap waste product of wood industry – to yield
α-pinene oxide, verbenol, and myrtenol [98] was expressed in this system, a 9-times
higher initial α-pinene oxide formation rate and a seven-fold increased α-pinene
oxide yield was observed in the presence of glucose compared to glucose-free
conditions. Further bioprocess engineering addressed the low water solubility and
the toxicity of α-pinene by setting up an aqueous–organic two-phase bioprocess
with diisononyl phthalate as a biocompatible organic carrier solvent. With an
aqueous/organic phase ratio of 3 : 2 and 30% (v/v) of α-pinene in the organic phase,
a total product concentration of more than 1 g l −1 was achieved [99].
Also, the hydroxylation of several 3-keto-4-ene steroids by bacterial CYP106A2
from B. megaterium ATCC 13368 was carried out when supported by a cofactor
regeneration enzyme. CYP106A2 was the first bacterial P450 found to catalyze
the oxidation of steroids [100, 101]. Interestingly, bovine adrenodoxin (Adx) and
adrenodoxin reductase (AdR) have successfully been implemented for efficient