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124 5 Multi-Enzyme Systems and Cascade Reactions Involving Cytochrome P450 Monooxygenases

Later, the same group succeeded in engineering an even more complex
mammalian biosynthetic pathway in S. cerevisiae. An engineered recombinant
S. cerevisiae strain was able to produce hydrocortisone from simple carbon sources.
For this purpose, the previously constructed yeast strains producing ergosta-5-ene-
ol and ergosta-5,22-diene-ol was used [123]. Starting with prenenolone produced
by CYP11A1, further conversions to hydrocortisone via progesterone, 17-hydroxy-
progesterone, and 11-deoxycortisole were catalyzed by the mitochondrial forms
of Adx, 3β-HSD, CYP17A1, CYP21A1, and CYP11B1. The enzymes responsible
for the two main side reactions, the esterification of pregnenolone and the 20-
keto reduction of 17α-hydroxyprogesterone, were inactivated. Finally, an artificial
biosynthetic pathway of hydrocortisone from glucose was performed in a single
engineered yeast strain [124].

5.4
Conclusions and Outlook

To date, in contrast to other enzymes, the implementation of P450s in artificial cas-
cade reactions has been mainly limited to cofactor regeneration systems. One of the
main reasons for this restricted application is likely the complex multicomponent
nature of most P450s, meaning their requirement for additional redox partner pro-
teins for electron transfer. Moreover, most eukaryotic P450s are membrane-bound
and interact with membrane-associated reductases. Both factors make the han-
dling of such enzyme systems much more difficult compared to mono-component
enzyme types. A further issue might be the relatively low activity of P450s compared
to other enzymes, although in recent years substantial progress has been made
in the discovery of novel P450s [125] as well as in protein engineering of these
biocatalysts toward different target compounds [11].
P450s bear the enormous potential for converting (inert) hydrocarbons selec-
tively into high-value compounds in a single or multiple oxidation steps [22]. These
reactions are in many cases difficult to achieve by using chemical catalysts. In
particular, multi-enzyme cascades involving P450s become essential for whole-cell
applications when complex biologically active compounds, for instance, plant sec-
ondary metabolites, cannot be produced at reasonable costs by chemical methods.
The described ‘‘Artemisinin Success Story’’ highlights the importance of P450s in
complex and sustainable synthesis of essential pharmaceutical compounds of high
importance. Thus, the implementation of P450s into multi-enzyme cascades to
produce plant metabolites and their analogs in recombinant microbial cells seems
to be a straightforward strategy.
Besides, recent pioneering work regarding one-pot P450–ADH cascades leading
to ketones has been conducted with isolated enzymes. Although only at the
beginning of their development, such reaction concepts demonstrate their high
potential and can be extended to additional enzymes beside ADHs. Such multistep
one-pot routes offer sustainable and elegant alternatives to chemical multistep
syntheses. Moreover, as described in this book chapter, some P450s offer the
unique opportunity for multistep oxidations using only a single enzyme, yielding,