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@PHDTHESIS{Srdic:1008313,
author = {Srdic, Matic},
othercontributors = {Schwaneberg, Ulrich and Elling, Lothar},
title = {{D}evelopment and application of the human cytochrome
{P}450 monooxygenase 3{A}4 for preparative scale
biocatalysis of valuable pharmaceutical compounds},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {RWTH Aachen University},
reportid = {RWTH-2025-02940},
pages = {1 Online-Ressource : Illustrationen},
year = {2024},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University 2025; Dissertation, RWTH Aachen University, 2024},
abstract = {Cytochromes P450 (CYPs) are a vital group of enzymes found
in all the kingdoms of life where they primarily function as
monooxygenases, incorporating hydroxyl groups into a variety
of substrates to detoxify xenobiotics or as part of a
synthetic pathway. In humans they are best known for
metabolizing drugs or other xenobiotics and synthesizing
essential compounds like steroid hormones. Their genetic
diversity and unique oxygenation capability make them
interesting targets for biotechnological research. Here they
are particularly prized for both the wide variety of
accepted substrates and capability for exceptional regio-
and stereoselectivity in production of valuable compounds
such as drugs or fine chemicals. As important mediators of
drug metabolism and clearance they are routinely utilized in
metabolite identification (MetID) studies and metabolite in
safety testing (MIST). One of the reasons for the high cost
of bringing a drug to market is the high attrition rate of
potential drug candidates due to drug safety failures or its
inability to be effective at safe doses. Developing a novel
biocatalyst based on the human CYP3A4, the enzyme involved
in the metabolism of over $50\%$ of all FDA approved drugs,
would assist in solving an important problem and be of
considerable benefit to early drug testing. Therefore, the
first part of this thesis focused on enhancing the
expression of the human CYP3A4 enzyme in the yeast
Komagataella phaffii (Pichia pastoris), with the goal of
offering a novel cost-effective biocatalyst for drug
metabolism research. Ideally this biocatalyst would be able
to produce detectable amount of less abundant metabolites
that are often missed in early development, due to the low
reaction volumes and high speeds used in high-throughput
screening systems, therefore a high specific activity of the
biocatalyst that was retained after both production and
storage was critical for this project. Central was also the
incorporation of the CYP3A4 enzyme and its redox partner
enzyme CPR into the K. phaffii BSYBG11 strain, using
innovative bidirectional promoters for balanced protein
expression. This approach mitigated transcriptional
collisions, optimizing enzyme production. The development of
a novel specialized high-performance liquid
chromatography-mass spectrometry (HPLC-MS) method was
necessary for rapid and accurate metabolite separation and
quantification in a way that mimics the HTS methods used in
ADME research, to enable faithful assessment and screening
of the developed biocatalyst. We chose testosterone as the
model substrate for the enzyme, and the development of the
HPLC-MS method, due to it being the standard substrate used
in CYP3A4 research as it is biotransformed into a number of
products. The developed screening method efficiently
separated testosterone and its hydroxylation products. The
research also explored storage conditions' effects on enzyme
activity, concluding that freezing cells in buffer at -80°C
was most effective, with lyophilization providing handling
convenience at higher temperatures. The developed novel
biocatalyst has already contributed to discovering never
before detected testosterone metabolites, verified by NMR,
such as 6-dehydro-15β-hydroxytestosterone and
6β,16β-dihydroxytestosterone as reported in a study by
Fessner et al. The second part of the thesis was designed to
leverage the generated CYP3A4 biocatalyst for a
proof-of-concept industrial application, starting by
screening polycyclic aromatic hydrocarbons (PAHs) to find
novel metabolites formed by human CYP3A4 and later progress
to their pilot scale functionalization. PAHs are important
environmental pollutants and appealing building blocks for
synthesizing pharmaceuticals and fine chemicals. CYPs can
functionalize PAHs via C-H activation, a process which we
hoped to elucidate further by enzymatic bioconversion of
PAHs and their N- and O- containing derivatives using the
biocatalyst developed in the first part of the thesis. This
work reports the first screening of PAHs by human CYP3A4 and
the first scale-up study of a PAH functionalization reaction
by CYP3A4. The results of the screening provided the
empirical evidence for substrate preference towards medium
and high molecular weight PAHs, which falls in line with the
well supported description of CYP3A4 as an enzyme that
accepts mainly large substrates due to its large active
site. To further explore the robustness of the biocatalytic
reaction we scaled up the reaction to a 10 L stainless steel
fermenter, we chose fluorene as our model substrate due to
its widespread presence in a variety of natural products and
as an investigation of a potential biocatalytic cascade
reaction. Using standard unoptimized cultivation conditions
we yielded 237 mg of fluorenol and 48 mg of fluorenone from
498 mg of fluorene. Our combined bioconversion yield was
therefore $57\%$ with a peak productivity of 27.7 μmol/L/h
for fluorenol and 5.9 μmol/L/h for fluorenone, these
activities confirm that the novel CYP3A4 whole cell
biocatalyst is an excellent biocatalyst for producing
high-value pharmaceutical compounds. As our experimental
design utilized only the most widely used bioreactor
conditions further optimizations are foreseeable.
Nevertheless, this was a demonstration of robustness and
scalability of this biocatalyst from a 96-well to a 10 L
preparative scale and validates the systems scientific and
commercialization potential.},
cin = {162610 / 160000},
ddc = {570},
cid = {$I:(DE-82)162610_20140620$ / $I:(DE-82)160000_20140620$},
pnm = {OXYTRAIN - Harnessing the power of enzymatic oxygen
activation (722390)},
pid = {G:(EU-Grant)722390},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2025-02940},
url = {https://publications.rwth-aachen.de/record/1008313},
}
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