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120 5 Multi-Enzyme Systems and Cascade Reactions Involving Cytochrome P450 Monooxygenases

catalyze the oxidation of 3-hydroxypyrrolidine to the corresponding pyrrolidinone
[115, 116], was selected for the oxidation of N-benzyl-4-hydroxy-piperidine to N-
benzyl-4-piperidone. Complete conversion of 5 mM N-benzyl-4-piperidine and 80%
yield in the two-enzyme system were achieved, and this was accompanied with a
+
TTN value for NAD regeneration of 4000.
Both described biocatalytic routes employing monooxygenase-containing whole
cells and isolated and purified ADHs displayed excellent regioselectivities of >99%
and the intermediate alcohols were completely converted to the corresponding
ketones [110].

5.3.3.2 Artificial Multi-Enzyme Cascades In Vivo
An outstanding example of multi-enzyme in vivo cascades is the ‘‘Artemisinin
Success Story’’ representing the production of artemisinic acid, a precursor of
the antimalarial drug artemisinin. Artemisinin is a component of the so-called
artemisinin-based combination therapies (ACTs), which is the recommended
malaria therapy by the WHO [117]. Artemisinin can be isolated only from the sweet
wormwood plant mostly grown in China and Vietnam. However, the availability
and market supply of this drug is negatively affected by varying harvests and long
production periods (∼14 months) (www.rsc.org/chemistryworld/2013/04/sanofi-
launches-malaria-drug-production). In 2006, Keasling and coworkers described the
production of artemisinic acid by engineered yeast [118]. The farnesyl pyrophos-
phate (FPP) biosynthetic pathway of Saccharomyces cerevisiae was metabolically
engineered to increase the production of FPP, which was converted to the sesquiter-
pene amorpha-4,11-diene by coexpression of amorphadiene synthase. CYP71AV1
from A. annua was implemented into this pathway for subsequent amorpha-4,11-
diene oxyfunctionalization, yielding artemisinic acid. Interestingly, CYP71AV1 is
able to perform a three-step oxidation at C12 of amorpha-4,11-diene to artemisinic
acid via the intermediates artemisinic alcohol and artemisinic aldehyde. Using this
engineered whole-cell system, titers of up to 100 mg l −1 artemisinic acid could be
produced.
Very recently, a further improved yeast system was reported, which included,
among other optimizations and besides CYP71AV1, an alcohol and aldehyde [119]
dehydrogenases (ADH1 and ALDH1) from A. annua for artemisinic alcohol and
aldehyde conversion, respectively (Scheme 5.28). Artemisinic acid titers of up
to 25 g l −1 were achieved in fermentation set-up [120]. A process based on the
developed artemisinic acid-producing yeast strain is now used for the industrial
production of artemisinin at Sanofi (www.rsc.org/chemistryworld/2013/04/sanofi-
launches-malaria-drug-production).
The implementation of a P450 into a metabolically engineered pathway for
paclitaxel (referred to as taxol) precursor production was reported by the group of
Stephanopoulos [121]. Taxol (and derivatives thereof) are mitotic inhibitors used
as chemotherapeutic agents. It originates from the Pacific yew tree Taxus brev-
ifolia, and thus its cost-effective production is limited. The authors describe the
engineering of the methylerythritol-phosphate (MEP) pathway forming isopentenyl
pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) in E. coli. IPP and