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60 3 Monooxygenase-Catalyzed Redox Cascade Biotransformations

directly for the subsequent enzymatic oxidation or was epimerized via an acidic
resin to obtain the trans-ketone, a precursor of the isomeric unnatural Aerangis
lactone. In the last step, both ketones were converted, individually, by different
BVMOs in a ‘‘two-phase’’ liquid system (n-heptane and water). The oxidative
reactions were performed using two cell-free extracts containing, respectively, the
cis selective cyclododecane-monooxygenase (CDMO) from Rhodococcus ruber SC1
and the predominantly trans-selective cyclopentanone-monooxygenase (CPMO)
from Comamonas sp., which gave excellent kinetic resolution and enantioselectivity
(Scheme 3.20). Pure products were obtained by column chromatography. The
presented approach is a significant example of the advantages deriving from the
combination of different types of catalysis to achieve results that might be suitable
for industrial applications.

3.1.8
Conclusion and Outlook

The above examples provided an exemplary overview on the diverse application of
various monooxygenases in cascade biotransformations. While the utilization of
such enzymes together with other biocatalysts was recognized already in the early
days, their widespread application in multistep integrated processes just picked up
pace during the more recent years. This may be due to the fact that monooxygenases
represent a particularly ‘‘difficult’’ class of enzymes, often displaying limited
stability and strict cofactor requirements. Thanks to the significant progress
in molecular biology and enzyme engineering, these enzymes have received
the attention they deserve, as a result of the interesting and highly selective
transformations that they are capable of catalyzing, which are often unrivalled by
conventional chemistry.
Moreover, new and unexpected sources for monooxygenases are continuously
discovered. Lately, an increasing numbers of BVMOs have been identified related
to the biosynthesis of secondary metabolites. Very recently, a BVMO has been
incorporated into the polyketide synthase (PKS) machinery by Tang and coworkers
[41]. They biochemically characterized the domain catalyzing the Baeyer–Villiger
oxidation of an acyl carrier protein (ACP) tethered thioester to an ACP-linked thio-
carbonate. The putative monooxygenase-like domain was identified as a member
of type I BVMOs. It did not show any activity against classical BVMO substrates
(like cycloalkanones), but it acted as a tailoring domain within the PKS/NRPS
(nonribosomal peptide-synthetase) biosynthetic paradigm. As this represented the
first case of a BVMO integrated into an intricate machinery for a biosynthetic path-
way, it might certainly inspire future investigations toward new artificial cascade
processes.
It was shown that the incorporation of monooxygenases into cascade net-
works allows the efficient interconversion of diverse functional groups. Different
approaches have proved to be suitable for multistep biotransformations, ranging
from fusion enzyme strategies for facile cofactor recycling to artificial metal-
loenzymes. However, monooxygenases seem to perform preferably well within a