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@PHDTHESIS{Krfer:661859,
author = {Körfer, Georgette Dorothea Johanna},
othercontributors = {Schwaneberg, Ulrich and Elling, Lothar},
title = {{D}evelopment of a flow cytometer-based in vitro
compartmentalization screening platform for directed protein
evolution},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
reportid = {RWTH-2016-06001},
pages = {1 Online-Ressource (X, 212 Seiten) : Illustrationen,
Diagramme},
year = {2016},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2016},
abstract = {Directed evolution is a powerful algorithm for tailoring
industrially important biocatalysts. Methodological
andconceptual advancements in directed evolution in the past
decades enabled engineering of enzymes towardsunnatural
substrates with catalytic efficiencies beyond natural
enzymes. Despite all progress in the field ofdirected
evolution the exploration of generated sequence space and
acceleration of iterative rounds ofmutagenesis and screening
is limited by throughput of commonly employed screening
technologies.Development of ultrahigh throughput screening
(uHTS) represents the main challenge in today’s
state-of-theartdirected evolution and ideally, in contrast
to medium screening formats, enables rapid analysis of up to
$10^7events$ per hour using flow cytometry. The possibility
to screen millions of variants within one day using
uHTSoffers the opportunity to generate libraries of high
mutational load to successfully identify and
investigatestructure-function relationships or combinatorial
effects within directed evolution campaigns. The latter is
upto now unobtainable employing standard screening formats
due to cost and time limitations of directedevolution
campaigns. Most of reported uHTS screening systems employ in
vivo screening where the bacterialhost is used as carrier of
enzyme variants. The main limitation of the cell-based
systems is the lowtransformation efficiency resulting in
reduced gene diversity. In this study we developed an uHTS
platform inwhich cells were replaced by cell-free
transcription-translation machinery encapsulated within
artificial cell-likecompartments (water-in-oil-in-water
(w/o/w) emulsions). Each emulsion compartment encapsulates a
singlegene copy of a highly diverse gene library $(10^8).$
Upon cell-free transcription and translation of genes,
encodingactive enzyme variants, compartments are labelled by
conversion of fluorogenic substrate into fluorescentproduct
and are analyzed by flow cytometry. In vitro
compartmentalization (IVC) enables genotype-phenotypelinkage
and isolation of genes encoding for active enzyme variants
from an excess of genes encoding inactiveenzymes. The uHTS
screening platform was developed for cellulase (CelA2) using
in vitro production of CelA2enzyme within (w/o/w) emulsions
and fluorescein-di-beta-D-cellobioside (FDC)-fluorescein
substrate-product pair(Ex.: 494 nm/Em.: 516 nm). The
developed uHTS cell-free compartmentalization platform was
validated byscreening of a random epPCR cellulase
mutagenesis library and yielded an improved cellulase
variant withinonly two weeks. The identified variant
CelA2-H288F-M1 (N273D/N468S) showed 5-fold reduced KM value
and13.3-fold increased specific activity (220.60 U/mg)
compared to cellulase wild type (16.57 U/mg). The uHTS
invitro based platform was employed to study differences
between three focused mutagenesis approaches(iterative
site-saturation mutagenesis, multiple site-saturation
mutagenesis, and simultaneous saturationmutagenesis of four
amino acid positions i.e. OmniChange) in order to explore
combinatorial effects. As anoutcome the best performing
variant contained only two amino acid substitutions (F288
and Q524) andshowed 8-fold improvement in specific activity
compared to parent.In order to expand the uHTS platform on
other enzymes, in this study in vitro based screening assay
for Yersiniamollaretii phytase wildtype (YmPhWT) was
optimized. Due to discrepancies between pH optimum for
cell-freeexpression machinery (pH 7) and acidic pH optimum
of YmPhWT, optimization of YmPhWT through a
directedevolution campaign was performed. The goal of the
campaign was to broaden the activity of the phytasetowards
neutral pH. Phytase libraries were generated via epPCR and
subsequent simultaneous site-saturationmutagenesis based on
identified beneficial positions in the epPCR library
screening. The variants YmPh-M10and -M16, showing 7-fold
improved specific activity at pH 6.6 towards the model
substrate4-methylumbelliferylphosphate (4-MUP) compared to
YmPhWT, were isolated.The flow cytometer-based uHTS-IVC
technology platform drastically reduces time requirements
for one roundof directed evolution from several months to
only one day and enables an efficient screening of
mutantlibraries $(10^10)$ with high diversity covering a
significant portion of the generated sequence space in a
timeefficient manner. Consequently, the flow cytometer-based
uHTS-IVC technology platform offers an efficientsolution to
tackle present challenges of directed evolution, like the
performance of multiple, iterative rounds ofdiversity
generation and screening of mutant libraries with unlimited
diversity, in order to successfully identifybeneficial
variants with altered selectivity, specificity or activity.
In addition, the flow cytometer-based uHTSIVCtechnology
platform has an impressive potential to study
demanding/challenging scientific questions e.g.the
exploration of combinatorial effects and answering
fundamental questions on structure-functionrelationships
within biocatalysts. Additionally, the uHTS technology
platform can - from our point of view - beadapted to other
enzyme classes since it uses a widely applicable
fluorescence sorting of cell-free expressedactive enzyme
variants within (w/o/w) emulsions compartments.},
cin = {162610 / 160000},
ddc = {570},
cid = {$I:(DE-82)162610_20140620$ / $I:(DE-82)160000_20140620$},
typ = {PUB:(DE-HGF)11},
urn = {urn:nbn:de:hbz:82-rwth-2016-060015},
url = {https://publications.rwth-aachen.de/record/661859},
}
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