% IMPORTANT: The following is UTF-8 encoded. This means that in the presence
% of non-ASCII characters, it will not work with BibTeX 0.99 or older.
% Instead, you should use an up-to-date BibTeX implementation like “bibtex8” or
% “biber”.
@PHDTHESIS{Zou:758220,
author = {Zou, Zhi},
othercontributors = {Schwaneberg, Ulrich and Pich, Andrij},
title = {{D}irected sortase evolution for site-specific protein
engineering and surface functionalization},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
reportid = {RWTH-2019-02768},
pages = {1 Online-Ressource (146 Seiten) : Illustrationen},
year = {2019},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2019},
abstract = {Sortase-mediated ligation (SML) has emerged as a improtant
tool for site-specific bioconjugation in protein engineering
and material functionalization. Sortase A from
Staphylococcus aureus (Sa-SrtA) specifically recognizes an
LPxTG (in which x means any amino acid) motif in the target
protein 1 and cleaves the scissile amide bond between
threonine and glycine. The generated thioester intermediate
subsequently ligates to the target protein 2 with oligo
glycine at N-terminal. Despite many highlights in
applications, wild type Sa-SrtA suffers from several notable
limitations (e.g. a relatively low catalytic efficiency
(high Km (LPxTG) ≈ 6.5 mM), a strict specificity for the
LPxTG motif and dependency on calcium cofactor). Directed
evolution is a powerful tool to tailor enzyme properties
towards user-defined goals. Directed sortase A evolution
requires the development of a robust high-throughput assay
which directly detects the formed conjugated products.
Several conjugated product-based high-throughput screening
strategies (e.g. cell surface display and in vitro
compartmentalization) of Sa-SrtA have been established.
Variants with enhanced activities, altered substrate
specificities (rather than LPxTG motif) and
calcium-independence were identified. However, these
strategies are rather specific for engineering of one
property of sortase A and usually limited in versatilities.
For such purpose, it was essential to establish a general,
robust and reliable screening system in microtiter plate
(MTP) format which is applicable to perform directed Sa-SrtA
evolution campaigns for different properties (e.g.
thermos-stability and solvent resistance). In order to
advance research of sortase engineering, this thesis was
focused on the development of a general high-throughput
screening system of sortase A, directed evolution of sortase
A for efficient site-specific ligations in organic solvents,
and applications of sortase A for covalent immobilization of
multiple proteins on microgel. In the first section, a
general directed sortase evolution platform (SortEvolve) was
developed in in 96-well MTP made of polypropylene (PP-MTP).
Two applications were carried out for SortEvolve. In
Application 1, SortEvolve was validated for the directed
Sa-SrtA evolution. In Application 2, SortEvolve was
validated for the directed evolution of CueO laccase with
minimized background noise (20-fold decreased). SortEvolve
ensures a comparable amount/semi-purified enzyme through
immobilization in PP-MTP. The latter is beneficial to avoid
false positives during screening and also suited for
directed evolution campaigns in which background activity
(or noise) from crude lysate has to be minimized in order to
identify beneficial variants In the next section, directed
Sa-SrtA evolution campaign (KnowVolution) towards organic
solvents was implemented. Organic solvents (e.g. DMSO, DMF)
are routinely used to dissolve hydrophobic compounds.
Engineering of Sa-SrtA for improved resistance/activity in
organic co-solvents facilitates SML for more broad range of
substrates. A random mutagenesis library (SeSaM library) of
Sa-SrtA was screened in DMSO co-solvent by a modified
SortEvolve protocol. Sa-SrtA variant M1 (R159G) with
2.2-fold improved resistance and variant M3
(D165Q/D186G/K196V) with 6.3-fold catalytic efficiency in
$45\%$ (v/v) DMSO co-solvent were obtained when compared
with Sa-SrtA WT, respectively. Interactions of between
Sa-SrtA and DMSO were investigated by molecular dynamic (MD)
simulations. The MD simulations revealed that conformational
mobility of Sa-SrtA is important for the gained resistance
and activities in the co-solvent of DMSO. Application of
Sa-SrtA M3 has exploited in site-specific conjugation in
organic co-solvents. Versatility of SML in organic
co-solvents was demonstrated by generating peptide-amines
conjugates. Sa-SrtA M3 showed an up to 4.7-fold increased
specific activity (vs Sa-SrtA WT) for site-specific
conjugation of peptide/primary amines in DMSO and DMF
co-solvents. In the last section, a general covalent
immobilization platform of enzymes on the surface of Poly
(N-vinylcaprolactam)/Glycidyl methacrylate (PVCL/GMA)
microgel was developed using sortase-mediated ligation.
Versatility of the platform was proved by immobilization of
five enzymes (lipase A, phytase, laccase, cellulase, and
monooxygenase) with either N-terminal GGG motif or
C-terminal LPxTG on surface of pVCL/GMA microgel. The
kinetic parameters, solvents resistance, pH profile,
thermo-stability and reusability of immobilized CueO laccase
and P450-BM3 monooxygenase on PVCL/GMA microgel were
subsequently investigated. Impressively, immobilized CueO
and P450 BM3 showed an up to 4-fold improved resistance in
the co-solvent of DMSO in comparison to corresponding free
enzymes (e.g. P450 BM3 monooxygenase, CueO laccase). The
highly stable immobilized CueO was further exploited in
decolourization of aromatic dyes with high efficiency and
reusability.},
cin = {162610 / 160000},
ddc = {570},
cid = {$I:(DE-82)162610_20140620$ / $I:(DE-82)160000_20140620$},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2019-02768},
url = {https://publications.rwth-aachen.de/record/758220},
}
Gast ::